INFLUÊNCIA DOS COMPOSTOS POLIFENÓLICOS NO … · Influência dos compostos ... a nível sensorial...
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INFLUÊNCIA DOS COMPOSTOS POLIFENÓLICOS NO SABOR DOS ALIMENTOS: RELAÇÃO ENTRE A SUA ESTRUTURA E A CAPACIDADE DE INTERAÇÃO COM PROTEÍNAS DA SALIVA E RECETORES DO SABOR
Susana Isabel Pinto T. P. SoaresPrograma Doutoral em QuímicaDepartamento de Química e Bioquímica2012
Orientador Victor de Freitas, Professor Catedrático, Faculdade de Ciências da Universidade do Porto
“A ciência nos traz conhecimento; a vida, sabedoria" Will Durant
“Não há nada que não se consiga com a força de vontade,…" Cícero
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de
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AGRADECIMENTOS
Em primeiro lugar gostava de agradecer à Fundação Para a Ciência e Tecnologia
(FCT) pelo financiamento deste trabalho na forma de uma bolsa de Doutoramento
(SFRH/BD/41946/2007) sem a qual a realização deste trabalho seria impossível.
Gostava de agradecer ao Departamento de Química e Bioquímica da Faculdade de
Ciências da Universidade do Porto e à Linha de Investigação 2 do Centro de
Investigação em Química (CIQ) pelos diversos apoios logísticos e financeiros
concedidos.
Gostava de agradecer ao meu orientador Professor Doutor Victor de Freitas sem o
qual este trabalho não seria possível. Todas as “discussões” científicas, todo o
interesse, disponibilidade e compreensão foram fundamentais. Gostava também de
agradecer ao Professor Doutor Nuno Mateus, “co-orientador”. Sempre disponível,
atento, interessado e principalmente com um humor característico. Para além da
excelente orientação científica, os professores foram excelentes “companheiros” e
conselheiros nas fases menos boas desta etapa e também a nível pessoal, e não há
palavras para expressar esse reconhecimento.
Gostava de agradecer a alguns colegas investigadores e professores do
Departamento de Química e Bioquímica, nomeadamente à Isabel Sousa pela
disponibilidade e ajuda na utilização do DLS, à Dra. Zélia pela ajuda técnica nas
análises de LC-MS e à Professora Doutora Maria José Feio pela ajuda inicial na
realização da electroforese.
Gostava de agradecer a todas as pessoas que trabalharam e conviveram comigo
diariamente na realização deste trabalho. Ana Luísa, André, Iva, Joana Azevedo,
Joana Oliveira, Luís, Natércia e Rui. Sem dúvida que o excelente ambiente deste
laboratório é uma das mais-valias e fundamental para o sucesso científico de todas as
pessoas que por aqui passam. Espero que assim continue!
Já agora obrigada pelas dádivas de saliva e pelo “grande” sacrifício de estarem 1h
sem comer e beber…
Uma pequena palavra de apreço também ao Frederico Nave. Uma pessoa única e um
poço de conhecimento. Obrigada pelas pequenas discussões científicas.
Apesar de mais recentes na equipa, mas não menos importantes, a Natércia Brás e a
Virgínia. Obrigada pela boa disposição, companheirismo e disponibilidade.
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Gostava de agradecer também à minha família, em particular aos meus queridos pais,
marido, irmão, sobrinhos e cunhada. Somos poucos, na verdade cada vez menos,
mas somos felizes e unidos. Vocês têm sido o meu corrimão na vida; quando a subida
está mais difícil tenho tido sempre em quem me apoiar! Não sei como será no dia em
que faltarem. Adoro-vos.
Uma palavra em especial ao meu marido porque é quem me atura todos os dias. Há
fases menos boas e sem dúvida que o que temos passado não tem sido fácil. A vida já
é complicada e cheia de contratempos e penso que conservar o bom humor é um
segredo de sobrevivência. E agradeço-te por isso, porque apesar de tudo estás
sempre bem disposto, tentas por-me bem disposta e dia-a-dia vamos caminhando
juntos e percorrendo a estrada da nossa vida. Obrigada pelas vezes em que levas ao
colo nessa estrada…
Gostava de agradecer a alguns amigos muito especiais. Debi, Ligia, Ricardo Lopes e
Raquel. Um bocado cliché e lamechas mas sem dúvida que os amigos, e vocês em
particular, são a família que me permitiram escolher. Nem toda a gente tem a sorte de
ser protegido e acarinhado pela família ou amigos sinceros, mas eu tive essa sorte.
Sem Deus não há vida, sem família não há base e sem amigos não há mundo
colorido. Obrigada por terem estado, por estarem e por “irem estar” presentes em
todas as fases cruciais da minha vida. Por todas as maluqueiras que já fizemos e as
que ainda vamos fazer. Sem dúvida que vocês são uma parte de mim.
Uma palavra especial para a Raquelita porque és sem dúvida a irmã que nunca tive e
estás no meu coração.
Gostava de agradecer a outras pessoas por também serem uma parte importante da
minha vida e pelos mais de 10 anos de amizade (em alguns casos já lá vão 20 anos!!)
e aventuras. Liliana, Carlos, Zé Manel, Cátia, Manuel André, Ricardo (Baroni) o que
nós temos é raro e precioso.
Gostava de agradecer a mais um grupo de amigos Iva, Ricardo, Joana (Pá), António
(Toli), Vanessa, Manuel, Rita (Mocas), João (Cucus) e Paulo (Xeno) por partilharem a
vossa vida comigo e fazerem parte da minha também.
Gostava de agradecer à Susie Kohl e Sophie Thallman pela enorme hospitalidade,
disponibilidade, simpatia e amizade aquando da minha estadia na Alemanhã. Apesar
de ter sido pouco tempo e corresponder a apenas uma pequena parte deste trabalho,
foi uma experiência muito enriquecedora quer a nível científico como a nível pessoal.
Sem dúvida que vocês deixaram saudade dos muitos bons momentos que passamos.
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Gostava de agradecer ao Professor Doutor Wolfgang Meyerhof do Deutsches Institut
für Ernährungsforschung Potsdam – Rehbrücke (DIFE) pela oportunidade que me
proporcionou de ir para o seu laboratório e crescer como investigadora. Uma pessoa
sempre extremamente disponível, interessada e atenciosa.
Gostava de agradecer ao Hugo Osório (IPATIMUP) pela incansável ajuda na
identificação das proteínas salivares. Uma excelente pessoa, sempre disponível,
interessada, muito trabalhadora e experiente. Gostava também de agradecer ao Rui
Vitorino (Universidade de Aveiro) pela valiosa e crucial ajuda na identificação das
proteínas salivares. Sempre disponível e interessado.
Gostava de agradecer ao Professor Massimo Castagnola pela valiosa contribuição na
interpretação e identificação das proteínas salivares por ESI-MS. Mostrou-se uma
pessoa sempre disponível e extremamente interessada.
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RESUMO
Os polifenóis são metabolitos secundários das plantas e, por isso, estão
presentes em alimentos e bebidas de origem vegetal (e.g. vinho tinto, cerveja,
chá, sumos de frutas, etc). Estes compostos têm recebido muita atenção nos
últimos anos devido às suas propriedades biológicas (antioxidantes,
anticancerígena, etc) e às propriedades organoléticas que conferem aos
alimentos. Os taninos são um grupo específico de polifenóis que têm a
capacidade de interagir com as proteínas, nomeadamente as proteínas
salivares. Esta interação é importante não só no contexto biológico, mas também
a nível sensorial uma vez que está na origem da sensação da adstringência nos
alimentos. De facto, é aceite pela comunidade científica que a adstringência se
deve à complexação e/ou precipitação das proteínas salivares ricas em prolina
(PRPs) pelos taninos.
Enquanto a adstringência é uma sensação tátil percecionada na cavidade oral, o
amargor é um gosto resultante da interação de compostos com os recetores de
sabor amargo. Alguns compostos polifenólicos de baixo peso molecular são
amargos.
Este trabalho tem como objetivo global a compreensão das propriedades
sensoriais associadas a polifenóis (amargor e a adstringência), e de que modo é
que diferentes carboidratos (usados na indústria alimentar) influenciam a
adstringência. Para isso, este trabalho focou-se em determinar: (a), as principais
famílias de proteínas salivares que apresentam mais afinidade para interagirem
com os taninos numa solução modelo e diretamente no vinho tinto; (b), como é
que o enriquecimento de um vinho tinto em taninos influencia a perceção da
adstringência; (c), como é que as caraterísticas (solubilidade e tamanho) dos
complexos formados entre as diferentes famílias de proteínas salivares e dois
tipos de taninos (condensados e hidrolisáveis) podem influenciar o
desenvolvimento da adstringência (d), de que modo certos carboidratos
comerciais (goma arábica, β-ciclodextrina, pectina e ácido poligalacturónico)
inibem a interação taninos/proteínas, particularmente com as proteínas salivares
(e), quais os recetores de sabor amargo que são ativados por alguns polifenóis
vulgarmente presentes em alimentos de origem vegetal e produtos derivados.
As proteínas salivares presentes na saliva humana foram caraterizadas por uma
abordagem proteómica (HPLC-DAD, ESI-MS, SDS-PAGE e MALDI-TOF). Estas
técnicas foram então adaptadas para estudar a afinidade entre as diferentes
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famílias de proteínas salivares e os taninos condensados. Os resultados
desmonstraram que os taninos condensados interagem inicialmente com as
PRPs acídicas e estaterina e só depois com as histatinas, PRPs glicosiladas e
PRPs básicas.
A análise da composição das proteínas salivares por HPLC após interação direta
com o vinho tinto, permitiu conhecer alguns fenómenos relacionados com a
adstringência de vinhos tintos enriquecidos em taninos condensados por
comparação com a análise sensorial dos mesmos vinhos. Os resultados
demonstraram que no caso de vinhos com concentrações baixas de taninos, a
complexação/precipitação de PRPs acídicas e estaterina está correlacionada
com a intensidade da adstringência. No entanto, para vinhos com concentrações
maiores, a adstringência parece correlacionar-se com a interação dos taninos
com as PRPs glicosiladas. Pela primeira vez, surgiram evidências de que as
diferentes proteínas salivares estão envolvidas em diferentes fases do fenómeno
fisiológico associado à perceção da adstringência.
Posteriormente, pretendeu-se comparar a interação das proteínas salivares com
um tanino hidrolisável (PGG) vs. tanino condensado (procianidina trimérica). Os
resultados obtidos por HPLC e dynamic light scattering (DLS) mostraram que
ambos os taninos interagem sobretudo com as proteínas aPRPs e estaterina
formando complexos insolúveis e solúveis. Por outro lado, as bPRPs interagem
pouco com a procianidina trimérica e as gPRPs só complexam com a PGG. Em
geral, a PGG apresentou mais tendência para a formação de complexos
insolúveis enquanto a procianidina trimérica mostrou mais tendência para a
formação de complexos solúveis (exceto com a estaterina). Os resultados
obtidos apresentam importantes evidências de que diferentes taninos e
proteínas salivares podem influenciar e ter mecanismos diferentes na sua
interação e consequentemente no desenvolvimento da adstringência.
O efeito dos carboidratos na interação taninos/proteínas foi estudado usando
dois modelos de proteínas diferentes: taninos condensados/α-amilase e taninos
condensados/proteínas salivares. No primeiro estudo, foram usadas as técnicas
de extinção da fluorescência, nefelometria e DLS; no segundo modelo,
monitorizou-se por HPLC as proteínas salivares que permaneciam solúveis na
presença dos taninos após a adição dos carboidratos e analisaram-se por SDS-
PAGE os precipitados formados.
Globalmente os resultados mostraram que os carboidratos estudados (goma
arábica, β-ciclodextrina, pectina e ácido poligalacturónico) reduziram a formação
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de agregados insolúveis e precipitação de complexos. A pectina foi sempre o
carboidrato mais eficiente, seguida da goma arábica. Na interação com a α-
amilase, os estudos por fluorescência permitiram conhecer os mecanismos mais
prováveis subjacentes à ação inibidora dos carboidratos: a goma arábica e a β-
ciclodextrina inibem a interação tanino/α-amilase pela sua associação aos
taninos, impedindo-os de interagir com a proteína (mecanismo de competição);
no caso da pectina observou-se a formação de um complexo ternário tanino/α-
amilase/pectina solúvel em meio aquoso (mecanismo complexo ternário). A
formação de complexos ternários envolvendo a goma arábica e a pectina
também foram evidenciadas nos estudos com as proteínas salivares.
Na parte final deste trabalho, estudou-se o sabor amargo de alguns polifenóis
pertencentes a diferentes classes [(-)-epicatequina, PGG, procianidinas dimérica
B3 e trimérica C2, malvidina-3-glucósido e cianidina-3-glucósido], identificando
quais dos 25 recetores de sabor amargo dos humanos (hTAS2Rs) são ativados
por estes compostos.
Os resultados obtidos mostraram que diferentes compostos ativam diferentes
hTAS2Rs. (-)-Epicatequina ativou três recetores hTAS2R4, hTAS2R5 e
hTAS2R39, enquanto a PGG só ativou dois recetores, hTAS2R5 e hTAS2R39.
Por outro lado, a malvidina-3-glucósido e o trímero C2 só ativaram um recetor,
hTAS2R7 e hTAS2R5, respetivamente. Um dos resultados mais notáveis é que
os taninos são os primeiros agonistas naturais encontrados para o hTAS2R5,
apresentando grande potência na ativação deste recetor. Para além disso, os
grupos catecol e/ou galhoilo parecem ser estruturalmente importantes na
mediação da interação dos polifenóis com este recetor.
Os valores obtidos de EC50 para os diferentes compostos variam cerca de 100-
vezes, sendo os valores mais baixos os da PGG e malvidina-3-glucósido. Isto
sugere que estes compostos podem ser significativamente importantes no
amargor de frutos, vegetais e produtos derivados, mesmo que estejam presentes
em concentrações muito baixas.
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ABSTRACT
Polyphenols compounds are secondary metabolites of plants being present in
food and beverages derived from plants (e.g., red wine, beer, tea, fruit juices,
etc). A lot of interest has been paid to these compounds because of their
biological properties (antioxidants, anticancer, etc) and because of the
organoleptic properties that they confere to food and their. Tannins are a group of
polyphenols that have the ability to interact with proteins, particularly salivary
proteins. This interaction is important both in a biological context and also in a
sensorial context, since it is at the origin of astringency development in food.
Indeed, it is widely accepted by the scientific community that astringency is due
to the complexation and/or precipitation of salivary proteins rich in proline (PRPs)
by tannins.
While astringency is a tactile sensation perceived in the oral cavity, bitterness is a
taste that results by the interaction between compounds and bitter taste
receptors. Some low molecular weight polyphenols are known to taste bitter.
The main goal of this work was to understand and have insights about the
sensorial properties of polyphenols (bitter taste ans astringency) and to study in
which way different carbohydrates commonly used in food industry influence
astringency. So, this work as focused in determine: (a), the main families of
salivary proteins that have more affinity to interact with tannins in a model
solution and directly in red wine; (b), how supplementation of red wine with
tannins influence the perception of astringency; (c), how the characteristics
(solubility and size) of the formed complexes between the different families of
salivary proteins and two types of tannins (condensed and. Hydrolysable) could
affect astringency; (d), how some commercial carbohydrates (arabic gum, β-
ciclodextrina, pectin and polygalacturonic acid) inhibit the interaction
tannins/proteins, in particular with salivary proteins; (e), which human bitter taste
receptors are activated by several polyphenols commonly present in vegetal food
and derived products.
The salivar proteins present in human saliva were identified by proteomic
approache (HPLC-DAD, ESI-MS, SDS-PAGE and MALDI-TOF). These
techniques were then adapted to study the interaction between different families
of salivary proteins and condensed tannins. The results showed that condensed
tannins interact firstly with acidic PRPs ans statherin and only then with histatins
and glycosylated and basic PRPs.
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The analysis of the salivary proteins by HPLC after interaction with red wine,
allowed to study some phenomena related to astringency of red wines enriched
with condensed tannins by comparison with the sensory analysis of the same
wines. The results showed that for red wines with low concentrations of
condensed tannins, the complexation/precipitation of acidic PRPs and statherin is
correlated with astringencency intensity. However, for red wines with higher
tannin concentrations, astringency seems to be correlated with tannins’
interaction with glycosylated PRPs. To our knowledge, for the first time there are
evidences that different families of salivary proteins are involved in different
stages of the physiological phenomena associated to astringency perception.
In the next step, it was compared the interaction between salivary proteins and a
hydrolizable tannin (PGG) vs. condensed tannin (procianidin trimer). The results
obtained by HPLC and dynamic light scattering (DLS) show that both tannins
interact mainly with aPRPS and statherin, forming both soluble as insoluble
complexes. On the other hand, bPRPs interact poorly with procyanidin trimer and
gPRPs only complex with PGG. In general, PGG showed more tendency to form
insoluble complexes while procyanidin trimer formed more soluble complexes
(except for statherin). The results present significant evidences that different
tannins and salivary proteins could influence and have different mechanisms in
their interactions, and consequently, in astringency development.
The effect of carbohydrates in tanins/proteins interaction was study based on two
models of different proteins: condensed tannins/α-amylase and condensed
tannins/salivary proteins. In the first study, it were used the following techniques
fluorescence, nephelometry and DLS; in the second model, the salivary proteins
that remain soluble in the presence of condensed tannins after addition of
carbohydrates were monitored by HPLC and the respective precipitates were
analyzed by SDS-PAGE.
Overall, the results showed that all the tested carbohydrates (arabic gum, β-
ciclodextrina, pectin and polygalacturonic acid) reduced the formation of insoluble
aggregates and complexes precipitation. Pectin was always the most efficient
carbohydrate followed by arabic gum. In the interaction with α-amylase,
fluorescence studies gave insights about the mechanism by which different
carbohydrates inhibit tannins/α-amylase interaction: arabic gum and β-
cyclodextrin inhibit this interaction competing with proteins for tannins association
(competition mechanism); pectin seems to inhibit this interaction by the formation
of a ternary complex tannin/α-amylase/pectin with increasing solublity in aqueous
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medium (ternary complex mechanism). The formation of ternary complexes
involving arabic gum and pectin was also suggested in the study with salivary
proteins.
In the final part of this work, the bitterness of several polyphenols from different
classes [(-)-epicatechin, PGG, procyanidin dimer B3 and trimer C2, malvidin-3-
glucoside and cyanidin-glucoside] was studied by identification of the hTAS2Rs
that were activated by these compounds.
The results show that diferente compounds activated different subsets of
hTAS2Rs. (-)-Epicatechin activated three receptors hTAS2R4, hTAS2R5 and
hTAS2R39 while PGG activated only two receptors, hTAS2R5 and hTAS2R39.
Otherwise, malvidin-3-glucoside and trimer C2 only activated one receptor,
hTAS2R7 and hTAS2R5, respectively. One of the most important result is that
tannins are the first natural agonists for hTAS2R5, having high potency in its
activation. Futhermore, the catechol and/or galloyl groups seem to be structural
important in mediating polyphenols interaction with this receptor.
The EC50 values obtained for the several tested compounds vary around 100-
times fold, being the lowest for PGG and malvidin-3-glucoside. This suggests that
these compounds could be significant in the bitterness of fruits, vegetables and
derived products, even if they are present in very low concentrations.
xiv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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ÍNDICE
Agradecimentos .................................................................................................. iii
Resumo .............................................................................................................. vii
Abstract ............................................................................................................... xi
Índice ................................................................................................................. xv
Lista de Quadros .............................................................................................. xvii
Lista de Figuras ................................................................................................. xix
Lista de Abreviaturas ....................................................................................... xxix
Publicações científicas e Organização da tese ............................................... xxxiii
Introdução Geral ................................................................................................ 35
1. Compostos não-flavonóides ....................................................................... 39
2. Compostos flavonóides .............................................................................. 39
2.1 Antocianinas ......................................................................................... 40
2.2 Flavan-3-óis ......................................................................................... 41
3. Taninos ...................................................................................................... 43
3.1 Taninos condensados (Proantocianidinas) ........................................... 44
3.2 Taninos hidrolisáveis ............................................................................ 46
4. Compostos polifenólicos nos alimentos. Ingestão e impacto na saúde humana ......................................................................................................... 47
5. Interação entre taninos e proteínas ............................................................ 52
5.1 Tipos de ligação envolvidos na interação tanino-proteína .................... 52
5.2 Modelos da interação tanino/proteína ................................................... 56
5.3 Fatores que influenciam a interação tanino/proteína ............................ 59
5.3.1 Estrutura dos taninos ..................................................................... 59
5.3.2 Estrutura das proteínas .................................................................. 62
5.3.3 Presença de carboidratos .............................................................. 64
5.3.4 Outros fatores (força iónica, pH, etanol e temperatura) .................. 70
6. Saliva humana ........................................................................................... 71
6.1 Proteínas ricas em prolina (PRPs) ....................................................... 71
6.1.1 PRPs acídicas (aPRPs) ................................................................. 73
6.1.2 PRPs básicas (bPRPs) e glicosiladas (gPRPs) .............................. 74
6.2 Histatinas ............................................................................................. 75
6.3 Estaterina ............................................................................................. 76
7. Polifenóis e sabor amargo ......................................................................... 76
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7.1 Relação entre a estrutura dos polifenóis e o sabor amargo .................. 77
7.2 Recetores do sabor amargo (hTAS2Rs) ............................................... 78
Objetivos ............................................................................................................ 83
Resultados ......................................................................................................... 87
Capítulo 1 ...................................................................................................... 89
Capítulo 2 .................................................................................................... 123
Capítulo 3 .................................................................................................... 143
Capítulo 4 .................................................................................................... 161
Capítulo 5 .................................................................................................... 183
Capítulo 6 .................................................................................................... 203
Conclusão e Perspetivas futuras ...................................................................... 227
Referências Bibliográficas ................................................................................ 233
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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LISTA DE QUADROS
Tabela 1. Teor em polifenóis de diversos alimentos. Adaptado de Scalbert e
Williamson (2000).............................................................................................. 47
Tabela 2. Distribuição e grau de polimerização de taninos condensados em
alimentos. Adaptado de Gu et al. (2004). .......................................................... 48
Tabela 3. Resumo de alguns trabalhos que evidenciam o tipo e caraterísticas
importantes das interações tanino/proteína, indicando os taninos e proteínas
estudados, bem como as metodologias utilizadas no estudo. ........................... 55
Capítulo 1:
Table 1.1. Experimental Masses (Da) Detected in the Twelve Chromatographic
Fractions by RP-HPLC-ESI-MS, and MALDI-TOF/TOFa .................................. 102
Capítulo 2:
Table 2.1. Tannin specific activity (TSA) of 50 µL of whole (WS) and acidic saliva
(AS) toward red wines supplemented with several concentrations of oligomeric
procyanidins (OPC). For the same concentration of OPC, the results of WS and
AS are significantly different (P<0.05), except for the 2.0 g.L-1 concentration
(athese results are statistically equal). Astringency rating from the sensorial
evaluation. The orders with different letters are significantly different (P<0.1). 137
Capítulo 3:
Table 3.1 Percentage of salivary proteins involved in the formation of soluble and
insoluble complexes formed by the interaction with 399 µM of each tannin. These
results represent the average of three independent experiments. ................... 156
Capítulo 4:
Table 4.1 Average size of aggregates (Z) present in procyanidin fractions and
PPA (1 µM) solutions (100 mM acetate buffer with 12% ethanol/water (v/v) pH
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5.0) and polydispersity in absence and presence of carbohydrates, measured by
dynamic light scattering. .................................................................................. 175
Capítulo 5:
Table 5.1 Half maximal effective concentration (EC50) in inhibition of
tannin/salivary protein precipitation by three carbohydrates: AG, PGA and pectin.
The values with equal letters are not significantly different (P<0.05). ............... 195
Capítulo 6:
Table 6. 1 EC50 values (µM) for the test compounds and respective receptors.
The values with superscript equal letters are not significantly different (P < 0.05).
Values with ≥ are estimates because the dose-response curves did not saturate.
........................................................................................................................ 215
Table 6.2 Threshold concentrations (µM), defined as the lowest concentration
that resulted in calcium signals in receptor-transfected cells, for the test
compounds...................................................................................................... 215
Table 6.3 Signal amplitudes (given as relative fluorescence changes ∆F/F) for
the test compounds. ........................................................................................ 216
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LISTA DE FIGURAS
Fig.1 Estrutura química dos maiores grupos de polifenóis e alguns exemplos de
alimentos ricos em cada classe de compostos. ................................................. 38
Fig.2 Estrutura química do núcleo flavânico. A e B – anéis aromáticos, C – anel
heterocíclico pirano. .......................................................................................... 39
Fig. 3 Estrutura química do catião flavílio (3-glicósido) e dos respetivos derivados
pirúvicos (DP) de antocianinas. Substituintes das antocianinas (e dos derivados
pirúvicos) mais comuns na natureza. Glc – glucose. ......................................... 41
Fig.4 Estrutura química dos flavan-3-óis mais comuns nas plantas. .................. 42
Fig.5 Reação de decomposição das proantocianidinas em meio ácido – Reação
de Bate-Smith (Bate-Smith 1954). ..................................................................... 44
Fig.6 Estrutura química dos dois tipos de procianidinas diméricas e substituintes
de cada procianidina dimérica tipo B. ................................................................ 45
Fig.7 Estrutura das moléculas base dos taninos hidrolisáveis (ácidos gálhico e
elágico) e exemplo de um tanino hidrolisável β-1,2,3,4,6-pentagalhoil-O-D-
glucopiranose (pentagalhoilglucose, PGG). ...................................................... 46
Fig.8 Caracterização da dieta alimentar da população portuguesa para o período
entre 2003 e 2008 (com base na Balança Alimentar Portuguesa). O gráfico
superior apresenta o consumo da população em função dos grupos de
alimentos, estando salientados por rectângulos laranja os grupos de alimentos
que contêm polifenóis; o gráfico inferior apresenta os alimentos mais
consumidos dentro de cada grupo de alimentos rico em polifenóis (Balança
Alimentar Portuguesa, Instituto Nacional de Estatística).................................... 49
Fig.9 Vias de absorção dos polifenóis. Adaptado de Visioli et al. (2011) ........... 52
Fig.10 Esquema representativo das interações entre taninos e proteínas,
evidenciando o tipo de ligações mais relevante bem como os grupos de cada
molécula (do tanino e da proteína) envolvidos em cada tipo de ligação. Adaptado
de Asano et al. (1982). ...................................................................................... 54
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Fig.11 Modelo da interação tanino/proteína. Os taninos são exibidos com duas
extremidades as quais podem ligar-se às proteínas. As proteínas são
apresentadas como possuindo um número fixo de locais de ligação. Adaptado
de Siebert et al. (1996). ..................................................................................... 56
Fig.12 Esquema de uma curva de nefelometria para uma concentração fixa de
tanino e concentrações crescentes de proteína. Adaptado de Hagerman e
Robbins (1987). ................................................................................................. 57
Fig.13 Mecanismo molecular proposto para a interação das PRPs com os
taninos. Na fase inicial (1ª fase) as proteínas compactam-se, devido a ligações
apertadas múltiplas com os taninos multidentados. Na 2ª fase forma-se um
dímero com outra proteína recoberta de taninos, tornando o complexo insolúvel.
Na 3ª fase, há uma maior complexação e ocorre precipitação do complexo.
Adaptado de Jöbstl et al. (2004). ....................................................................... 58
Fig.14 Conformações da (-)-epicatequina, dímero B2 e seus derivados
galhoilados, obtidos por mecânica molecular e RMN. Adaptado de de Freitas et
al. (1998). .......................................................................................................... 61
Fig.15 Representação no modo space filling da estrutura terciária da albumina
sérica bovina (BSA), mioglobina e gelatina as quais apresentam conformações
diferentes. A mioglobina e a BSA têm estrutura terciária globular, sendo a
estrutura da mioglobina mais fechada e aparecendo aqui apresentada com o
grupo heme (grupo prostético essencial para a sua função). O colagénio tem
uma estrutura enrolada em tripla hélice apresentando uma conformação mais
aberta que as outras proteínas. ......................................................................... 64
Fig.16 Representação esquemática da (A) estrutura da parede celular primária
evidenciando os carboidratos mas importantes e a lamela média, e da (B)
degradação da pectina e da hemicelulose durante o amadurecimento dos frutos.
A degradação destes carboidratos reduz a integridade das paredes celulares,
diminuindo a rigidez dos frutos (adaptado de Wakabayashi (2000)). ................. 65
Fig.17 Mecanismos possíveis de inibição da agregação dos taninos com as
proteínas pelos carboidratos. P: proteína, T: tanino, C: carboidrato. Adaptado de
Mateus et al. (2004). ......................................................................................... 67
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Fig.18 Percentagens aproximadas (p/p) das principais classes de proteínas e
péptidos salivares na saliva total. Adaptado de Messana et al. (2008). ASH,
albumina sérica humana; sIgA, imunoglobulina A secretora; IgG, imunoglobulina
G; aPRPs, proteínas-ricas em prolina acídicas; bPRPs, proteínas-ricas em
prolina básicas; gPRPs, proteínas-ricas em prolina glicosiladas. ...................... 71
Fig.19 Esquema representativo da estrutura das aPRPs com indicação da região
acídica responsável pela função atribuída a estas proteínas. ............................ 73
Fig.20 Esquema representativo da estrutura da estaterina com indicação das
diferentes regiões responsáveis pelas funções atribuídas a esta proteína. ....... 76
Fig.21 Representação esquemática dos hTAS2Rs. Os resíduos de aminoácidos
estão indicados por círculos coloridos. A similaridade da sequência entre os 25
hTAS2Rs está indicada pelo código de cores. Os círculos com a linha em pontos
correspondem aos resíduos que não estão presentes em todos os recetores. Os
quadrados magenta indicam as regiões que contêm resíduos adicionais em
alguns recetores (Meyerhof 2005). .................................................................... 80
Fig.22 Mecanismo proposto de transdução de sinal do sabor amargo para as
células recetoras de sabor. Os compostos amargos ativam os recetores de sabor
amargo, os quais ativam os heterotrimeros da gustaducina. A α-gustaducina
estimula a PDE (fosfodiesterase) a hidrolizar o cAMP (adenosina monofosfato
cíclica); a diminuição de cAMP pode disinibir os canais de iões inibidos por
nucleótidos, elevando o Ca2+. Por outro lado, as subunidades βγ-gustaducina
libertadas ativam a PLCβ2 (fosfolipase C2), gerando IP3 (inositol trisfosfato) que
leva à libertação de Ca2+ armazenado, contribuindo para o aumento do Ca2+
intracelular. Adaptado de Meyerhof (2005). ....................................................... 81
Capítulo 1:
Fig.1.1(A) Typical RP-HPLC profile detected at 214 nm of the acidic saliva (AS)
solution of whole human saliva. The pointed lines and numbers show the ranges
and names assigned to each HPLC fraction, with the outline of the main proteins
identified for each HPLC fraction (Table 1). At the bottom, there is the distribution
of the different families of salivary proteins along the chromatogram. *, fragments
of proteins; His, histatin; Stat, sthaterin; (di-, mono-, n-)-phosp, (di-, mono-, non-)-
phosphorylated; bPRP, basic proline-rich protein; gPRPs, glycosylated proline-
xxii FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
rich proteins, aPRPs, acidic proline-rich proteins. (B) Total ion current (TIC)
profile of AS solution collected by the ion-trap mass spectrometer (ESI-MS)... 100
Fig.1.2 Deconvolution process for fraction 10. (A) ESI mass spectrum obtained
by the average of 77 mass spectra collected in the 38.49-40.39 min range during
the HPLC separation reported in the TIC profile (Fig.1.1B). (B) The bottom panel
reports the deconvolution of the upper ESI-MS (A). ......................................... 101
Fig.1.3 SDS-PAGE of the 12 HPLC fractions isolated from the HPLC after the
injection of the AS solution. The molecular weight markers were substituted by
lines, and the molecular mass is expressed in kDa as marked on the left side.
The gels were stained by with Imperial Protein Stain, a Coomassie R-250 dye-
based reagent. ................................................................................................ 104
Fig.1.4 SDS-PAGE of each HPLC fraction isolated from the HPLC after the
injection of the AS solution. The molecular weight markers were substituted by
lines and the molecular mass is expressed in kDa. The gels were stained by the
periodic acid Schiff procedure in order to visualize glycoproteins. ................... 107
Fig.1.5 RP-HPLC profile detected at 214 nm of the AS solution before (AS
control) and after the interaction with increasing concentrations of grape seed
fraction (GSF). ................................................................................................. 108
Fig.1.6 Percentages of area decrease of each HPLC fraction after the interaction
of AS solution with increasing concentrations of GSF (A, B, and C). Percentages
of area decrease for each family of salivary proteins (D). ................................ 109
Fig.1.7 SDS-PAGE of the AS solution before and after the interaction with two
concentrations of grape seed fraction (GSF) (0.133 mM and 0.232 mM). The
molecular weight markers were substituted by lines, and the molecular mass is
expressed in kDa. ........................................................................................... 110
Fig.1.8 SDS-PAGE of HPLC fractions 6 to 11 isolated from the HPLC after the
injection of the AS solution before (C) and after the interaction with two
concentrations of grape seed fraction (0.133 and 0.232 mM). The molecular
weight markers were substituted by lines, and the molecular mass is expressed
in kDa. ............................................................................................................. 110
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Capítulo 2:
Fig.2.1 Typical Reverse Phase HPLC profile detected at 214 nm of the AS
solution of human saliva. The dotted lines and numbers show the ranges and the
main SP family assigned to each HPLC peptide region. .................................. 131
Fig.2.2 (A) HPLC profile detected at 214 nm of the AS solution before (control)
and after the interaction with increasing volumes of red wine supplemented with
1.5 g.L–1 of OPC (condensed tannins). (B) Percentages of area decrease of each
HPLC salivary peptide region after the interaction of AS solution with increasing
volumes of red wine supplemented with 1.5 g.L–1 OPC. Values are expressed as
the arithmetic means of 3 experiments ± standard deviation. .......................... 132
Fig.2.3 Reverse Phase HPLC profile detected at 214 nm of the AS solution
before (control) and after the interaction with 10 µL of each red wine
supplemented with different concentrations of OPC. ....................................... 133
Fig.2.4 Percentages of area decrease of each HPLC peptide regions after the
interaction of AS solution with (A) 10 and (B) 20 µL of red wine supplemented
with increasing concentrations of OPC. Values are expressed as the arithmetic
means of 3 experiments ± standard deviation. ................................................ 134
Fig.2.5 Percentages of area decrease of each HPLC peptide regions after the
interaction of AS solution with 20 µL of OPC solutions in acetate buffer matrix.
Values are expressed as the arithmetic means of 3 experiments ± standard
deviation. ......................................................................................................... 135
Fig.2.6 Reverse Phase HPLC profile detected at 214 nm of the precipitates
resultant from the interaction of 10 or 20 µL of red wine supplemented with 0.5,
2.0, and 3.0 g.L–1 of OPC with the AS. ............................................................ 136
Capítulo 3:
Fig.3.1 A. Adapted from Hagerman and Robbins (1987). Titration of a plant
extract containing tannin with protein (BSA): region A, excess tannin; region B,
equivalence point; region C, excess protein. B. Adapted from Siebert and co-
workers (Siebert, Troukhanova et al. 1996). Model for protein/polyphenol
interaction that explains the results observed in Fig. 3.1A. Polyphenols are
xxiv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
depicted as having two ends that can bind to protein. Proteins are depicted as
having a fixed number (three) of polyphenol binding sites. .............................. 146
Fig.3.2 Scheme of the experimental approach used to study protein/tannin
interaction indicating the supposed kind of complexes formed and removed in
each step. ....................................................................................................... 149
Fig.3.3 RP-HPLC profile detected at 214 nm of the AS solution before (control
saliva) and after the interaction with 133 and 399 µM of A, PGG and B,
procyanidin trimer. All these solutions were filtered (0.22 µm) prior to HPLC
analysis. .......................................................................................................... 151
Fig.3.4 RP-HPLC profiles detected at 214 nm of the AS solution after the
interaction with 399 µM of procyanidin trimer and 133 µM of PGG. These
solutions were non-filtered and filtered (0.22 µm) prior to HPLC analysis. ....... 153
Fig.3.5 The graphics represent the variation of the chromatographic peaks area
of the several salivary proteins studied with the increase in tannins concentration.
The results are expressed as percentage of the area of each protein relatively to
the control saliva (100%) analyzed by HPLC, before and after solutions filtration
(0.22 µm). Statherin graphic was used as an example indicating the percentage
of protein involved in the formation of soluble and insoluble complexes. These
results represent the average of three independent experiments. ................... 154
Fig.3.6 Influence of tannin concentration on the average size of salivary soluble
proteins/tannins complexes present in solution. These results represent the
average of three independent experiments. .................................................... 155
Capítulo 4:
Fig.4.1 Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM) and
procyanidin fraction IV (17 µM; mean MW = 2052) solution, in the absence (full
line) and presence of increasing concentrations of Arabic gum (0.1, 0.2, 0.3, 0.4,
0.5, 0.6 g.L–1). The experiments were performed in 100 mM acetate buffer (pH
5.0) with 12% ethanol/water (v/v). ................................................................... 169
Fig.4.2 A. Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM)
(solid spectra) and in the presence of the highest polysaccharide concentration
[arabic gum 0.6 g.L-1, ; β-cyclodextrin 4.4 g.L-1, - - -; and pectins 80 mg.L-1
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(27% MD, ; 58-63% MD, ; and 72% MD, ]. B. Several fluorescence
emission spectra of procyanidin fractions alone and in the presence of the
highest polysaccharide concentration (arabic gum 0.6 g.L-1, ; β-cyclodextrin
4.4 g L-1, ; and pectin 58-63% MD 80 mg.L-1, ). The experiments were
performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v). .... 170
Fig.4.3 Influence of increasing concentrations of arabic gum in (upper data)
variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution
and (lower data) percentage of procyanidin/PPA (1 µM) insoluble aggregates
measured by nephelometry. Mean MWs of fractions II, III, and IV are 950, 1512,
and 2052, respectively. All experiments were performed in 100 mM acetate buffer
(pH 5.0) with 12% ethanol/water (v/v). ............................................................. 171
Fig.4.4 Influence of increasing concentrations of β-cyclodextrin in (upper data)
variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution
and (lower data) percentage of procyanidin/PPA (1 µM) insoluble aggregates
measured by nephelometry. Mean MWs of procyanidin fractions II, III, and IV are
950, 1512, and 2052, respectively. All experiments were performed in 100 mM
acetate buffer (pH 5.0) with 12% ethanol/water (v/v). ...................................... 172
Fig.4.5 Percentage of procyanidin/PPA (1 µM) insoluble aggregates in the
presence of increasing concentrations of different pectins measured by
nephelometry. Mean MWs of procyanidin fractions II (a), III (b), and IV (c) are
950, 1512, and 2052, respectively. All experiments were performed in 100 mM
acetate buffer (pH 5.0) with 12% ethanol/water (v/v). ...................................... 173
Fig.4.6 Possible mechanisms (i and ii) involved in the inhibition of the
aggregation of tannins and proteins by polysaccharides. P, protein; T, tannin; C,
polysaccharide/carbohydrate. .......................................................................... 176
Capítulo 5:
Fig.5.1Typical RP-HPLC profile detected at 214 nm of the AS solution of human
saliva in the absence (— ) and presence (- - -) of 300 µM GSF. The vertical
dotted lines show the ranges and the main salivary proteins family assigned to
each HPLC peptide region: bPRPs, gPRPs, and aPRPs. ................................ 191
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Fig.5.2 Part of the chromatograms of the AS solution after the interaction with
GSF (300 µM) in the absence (AS solution + 300 µM GSF) and presence of the
several tested carbohydrates (pectin, 5.0 g.L–1; AG, 10.0 g.L–1; and PGA, 20.0
g.L–1). .............................................................................................................. 193
Fig.5.3 Influence of carbohydrate concentration on salivary proteins precipitation
by condensed tannins (GSF, 300 µM). (A) AG, (B) PGA, and (C) pectin. These
results represent the average of three independent experiments. ................... 193
Fig.5.4 SDS-PAGE of the pellets that resulted from the interaction between AS
and GSF in the absence (C) and presence of the several carbohydrates (pectin,
10.0 g.L−1; AG, 25.0 g.L−1; and PGA, 25.0 g.L−1). The molecular weight markers
were substituted by lines, and the molecular mass marked on the left side is
expressed in kDa. The gels were stained with Imperial Protein Stain, a
Coomassie R-250 dye-based reagent. The table shows the ratio between the
densitometry values obtained for total salivary proteins present in AS (control AS)
and in each experiment. The values with equal letters are not significantly
different (P < 0.05). ......................................................................................... 194
Fig.5.5 Possible mechanism (i and ii) involved in the inhibition of the aggregation
of tannins and proteins by carbohydrates. P, protein; T, tannin; and C,
carbohydrate.40 ................................................................................................ 196
Capítulo 6:
Fig.6.1 Chemical structures of the four polyphenol compounds studied. ......... 208
Fig.6.2 Fluorescence changes of Fluo4-AM loaded HEK293T-Gα16gust44 cells
expressing the TAS2Rs indicated in the graphs following administration of 10.0
mM (-)-epicatechin, 20.0 µM malvidin-3-glucoside, 100.0 µM procyanidin trimer or
10.0 µM PGG. Responses of mock-transfected cells (empty plasmid) are
indicated by the thinner solid line. Fluorescence changes of Fluo4-AM loaded
HEK293T-Gα16gust44 cells expressing the TAS2Rs indicated in the graphs
following administration of 10.0 mM (-)-epicatechin, 20.0 µM malvidin-3-
glucoside, 100.0 µM procyanidin trimer or 10.0 µM PGG. Responses of mock-
transfected cells (empty plasmid) are indicated by the thinner solid line. ......... 213
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Fig.6.3 Concentration-response curves for HEK293T-Gα16gust44 cells
transfected with DNA for the indicated TAS2R following stimulation with the
indicated test compound. Error bars represent the confidential interval (P = 0.05).
(a) Dose dependent activation was observed for TAS2R4, TAS2R5, and
TAS2R39 by (-)-epicatechin (a), for TAS2R5 and TAS2R39 by PGG (b), for
TAS2R7 by malvidin-3-glucoside (c), and for TAS2R5 by procyanidin trimer (d).
........................................................................................................................ 214
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LISTA DE ABREVIATURAS
aa – aminoácido
ACN – acetonitrilo
AG – Arabic gum (goma arábica)
AGPs – arabinogalactana-proteínas
aPRP(s) – proteínas ricas em prolina acídicas
AS – acidic saliva (saliva acídica)
ASH – albumina sérica humana
bPRP(s) – proteínas ricas em prolina básicas
BSA – bovine serum albumin (albumina sérica bovina)
cAMP – cyclic adenosine mono-phosphate (adenosina monofosfato cíclica)
cNMP – cyclic nucleoside 5’ mono-phosphate (nucleósido monofosfato cíclico)
DAG – diacilglicerol
DC – dicroísmo circular
DLS – dynamic light scattering
DP – derivado pirúvico
EC50 – half maximal effective concentration (concentração efectiva na inibição de
metade da actividade)
ECG – galhato de epicatequina
EGCG – galhato de epigalhocatequina
EGC – epigalhocatequina
ESI – electrospray ionization (ionização por “electrospray”)
ESI-MS – electrospray ionization-mass spectrometry (espectrometria de massa
com ionização por “electrospray”)
FDR – false discovery rate (taxa de descobertas falsas)
FRET – fluorescence resonance energy transfer (fluorescência por transferência
de energia por ressonância)
Glc – glucose
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gPRP(s) – proteínas ricas em prolina glicosiladas
GSF – grape seed fraction (fração extraída de grainhas de uvas)
HPLC-DAD ou RP-HPLC-DAD – reverse phase-high performance liquid
chromatography-diode array detector (cromatografia de fase reversa de elevada
eficiência com detector por díodo)
hTAS2Rs – human bitter taste receptors (recetors de sabor amargo do homem)
His – histatin (histatina)
IgG – imunoglobulina G
IP3 – inositol trisfosfato
LC-MS – liquid chromatography mass spectrometry (cromatografia liquída
seguida de análise por espectrometria de massa)
IUP – intrinsically unstructured proteins (proteínas não-estruturadas
intrinsecamente)
MALDI-TOF/TOF – matrix assisted light desorption ionization – time-of-
flight/time-of-flight (ionização e dessorção a laser assistida por matriz com
analisador do tempo de vôo)
MD – methoxylation degree (grau de metil esterificação)
MS – mass spectrometry (espectrometria de massa)
MW – molecular weight (peso molecular)
OPC – oligomeric procyanidins (procianidinas oligoméricas)
PAS – periodic acid Schiff’s staining (coloração de Schiff com ácido periódico)
PDE – phosphodiesterase (fosfodiesterase)
PGG – pentagalhoilglucose
PGA – polygalacturonic acid (ácido poligalacturónico)
pI – ponto isoeléctrico
PLCβ2 – phospholipase C2 (fosfolipase C2)
PMF – peptide mass fingerprint (identificação de proteínas pela identificação da
massa de péptidos característicos)
PPA – α -amylase from porcine pancreas (α-amilase de pâncreas de porco)
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PRP(s) – proteínas ricas em prolina
RMN ou NMR – ressonância magnética nuclear (nuclear magnetic ressonance)
RGII – ramnogalacturonanas do tipo II
SDS – sodium dodecylsulfate (dodecilsulfato de sódio)
SDS-PAGE – sodium dodecylsulfate-polyacrylamide gel electrophoresis
(electroforese em gel de dodecilsulfato de sódio)
sIgA – imunoglobulina A secretora
SNPs – single nucleotide polymorphisms (polimorfismos de um nucleótido)
SP – salivary proteins (proteínas salivares)
Stat – statherin (estaterina)
tetraGG - tetragalhoilglucose
TFA – trifluoroacetic acid (ácido trifluoracético)
TIC – total ion current (corrente iónica total)
TRC – taste receptor cells (células recetoras do sabor)
triGG - trigalhoilglucose
Tris – tris(hydroxymethyl)aminomethane [tris(hidroximetil)aminometano]
TSA – tannin-specific activity (actividade específica dos taninos)
UV – ultravioleta
UV-Vis – ultravioleta-visível
WS – whole saliva (saliva total)
XIC – extracted ion current (corrente iónica extraída)
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PUBLICAÇÕES CIENTÍFICAS E ORGANIZAÇÃO DA TESE
Esta tese de doutoramento foi organizada com base nestas seis publicações
científicas.
A parte inicial da tese é uma introdução global que visa elucidar o leitor acerca
dos compostos utilizados neste trabalho (polifenóis e proteínas, em particular as
proteínas salivares), bem como da relevância quer destes compostos quer do
objecto de estudo deste trabalho (interação tanino/proteína).
A segunda parte da tese inclui os resultados sob a forma de seis capítulos,
correspondendo cada capítulo a uma das publicações referida. A organização
dos resultados não foi efectuada por ordem cronológica das publicações mas foi
dividida em dois temas (interação tanino/proteína e efeito dos carboidratos na
interação tanino/proteína). Salvo pequenas excepções, toda a parte
experimental descrita foi realizada por mim.
Capítulo 1: Reactivity of Human Salivary Proteins Families Toward Food Polyphenols Soares, S., Vitorino, R., Osório, H., Fernandes, A., Venâncio, A., Mateus, N., Amado, F., de Freitas, V.
J. Agric. Food Chem. 2011, 59, 5535–5547
Com a exceção do manuseamento do MALDI e do LC-ESI-MS, e a análise de dados com o Global Protein Server Workstation, toda a parte experimental descrita neste artigo foi realizada por mim.
Capítulo 2: Effect of Condensed Tannins Addition on the Astringency of Red Wines
Soares, S., Sousa, A., Mateus, N., de Freitas, V.
Chem. Senses 2011, 37, 191-198
Toda a parte experimental descrita neste artigo foi realizada por mim.
Capítulo 3: Interaction of Different Classes of Salivary Proteins with Food Tannins Soares, S., Mateus, N., de Freitas V.
Food Research International, 2012, 49, 807-813
Com exceção da síntese da procianidina trimérica C2, toda a parte experimental descrita neste artigo foi realizada por mim.
xxxiv FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Capítulo 4: Mechanistic Approach by Which Polysaccharides Inhibit α-Amylase/Procyanidin Aggregation Soares, S., Gonçalves, R. M., Fernandes, I., Mateus, N., de Freitas, V.
J. Agric. Food Chem. 2009, 57, 4352–4358
Toda a parte experimental descrita neste artigo foi realizada por mim.
Capítulo 5: Carbohydrates Inhibit Salivary Proteins Precipitation by Condensed Tannins Soares, S., Mateus, N., de Freitas, V.
J. Agric. Food Chem. 2012, 60, 3966–3972
Com exceção da análise da pectina, toda a parte experimental descrita neste artigo foi realizada por mim.
Capítulo 6: Different Phenolic Compounds Activate Distinct Subsets of Human Bitter Taste Receptors Soares, S., Kohl, S., Thalmann, S., Mateus, N., Meyerhof, W., de Freitas, V.
Submetido ao J. Agric. Food Chem.
Com exceção da síntese da procianidina dimérica B3 e trimérica C2 e da construção dos constructos, toda a parte experimental descrita neste artigo foi realizada por mim.
INTRODUÇÃO GERAL
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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37
Os compostos polifenólicos (vulgo polifenóis, designação utilizada ao longo da
tese) são uma grande família de metabolitos secundários sintetizados pelas
plantas e, consequentemente, estão presentes em alimentos e bebidas de
origem vegetal. Actualmente, já foram identificados milhares de polifenóis
naturais, apesar de só alguns estarem presentes em níveis significativos na dieta
alimentar (Shahidi e Naczk 1995). Nas plantas, estes compostos têm diversas
funções biológicas, sendo responsáveis pela cor, protecção contra a radiação
ultra-violeta, defesa contra ataques microbiológicos e contra predadores, entre
outras. Nos alimentos e bebidas de origem vegetal, os polifenóis são
responsáveis pelas suas principais caraterísticas organoléticas, nomeadamente
propriedades do sabor (amargor e adstringência), cor, odor e estabilidade
oxidativa (Naczk e Shahidi 2006).
O interesse nos polifenóis existentes nos alimentos, nomeadamente em frutas,
vegetais e bebidas derivadas de plantas (e.g. cerveja, chá e vinho tinto), tem
aumentado exponencialmente ao longo dos últimos anos. Isto deve-se ao facto
destes compostos estarem associados aos benefícios para a saúde humana das
dietas ricas nesses alimentos, em particular a famosa dieta Mediterrânica. Na
verdade, os polifenóis têm sido alvo de inúmeros estudos biológicos e
epidemiológicos, tendo-lhes sido atribuídas várias propriedades biológicas
benéficas como ação anti-mutagénica (Shim et al. 1995, Morley et al. 2005),anti-
cancerígena (Khafif et al. 1998, Faria et al. 2006), anti-oxidante (Faria et al.
2006, Wolfe et al. 2008, Azevedo et al. 2010), anti-alérgica (Akiyama et al. 2000,
Singh et al. 2011) e anti-bacteriana (Funatogawa et al. 2004, Taguri et al. 2004).
De facto, os polifenóis são os antioxidantes naturais mais abundantes na nossa
dieta alimentar e quase diariamente surgem evidências do seu papel na
prevenção de doenças degenerativas como o cancro e doenças
cardiovasculares.
Quimicamente, os polifenóis apresentam um ou mais aneis aromáticos com um
ou mais grupos hidroxilo com uma grande diversidade estrutural que
compreende desde moléculas fenólicas simples até polímeros de elevado peso
molecular. Os polifenóis são geralmente divididos em duas grandes famílias: os
flavonóides e os não-flavonóides (Fig.1).
38 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.1 Estrutura química dos maiores grupos de polifenóis e alguns exemplos de alimentos ricos em cada classe de compostos.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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39
1. Compostos não-flavonóides
Os não-flavonóides são uma vasta família de compostos, sendo sobretudo
moléculas simples como ácidos benzóicos, ácidos cinâmicos e estilbenos (Fig.1),
mas incluem também moléculas mais complexas derivadas destes,
nomeadamente galhotaninos e elagitaninos. Os ácidos benzóicos e cinâmicos
são denominados ácidos fenólicos e encontram-se principalmente em formas
conjugadas. Estes ácidos existem numa variedade de produtos vegetais, desde
frutos a cereais.
2. Compostos flavonóides
A família de polifenóis mais relevante nos alimentos é a dos flavonóides, sendo
também a família mais diversificada estruturalmente. Atualmente já foram
descritos mais de 4000 flavonóides e esta listagem continua a crescer.
A estrutura básica dos flavonóides é o núcleo flavânico, que consiste numa
estrutura C6-C3-C6 constituída por dois anéis aromáticos (A e B) ligados por um
anel heterocíclico pirano (C) (Fig.2). As diferentes classes de flavonóides diferem
entre si no grau de oxidação e padrão de substituição do anel C. Para além
disso, dentro de uma classe, os compostos diferem entre si no número e posição
dos grupos hidroxilo, metoxilo e glicosilo (Fig.1).
Os flavonóides são divididos em várias classes, como apresentado na Fig.1. A
razão para uma diversidade tão elevada advém da ocorrência de numerosos
padrões de substituição em que os substituintes primários (e.g. grupos hidroxilo,
metoxilo ou glicosilo) podem ser adicionalmente substituídos (e.g. glicosilados ou
acilados), originando por vezes estruturas muito complexas.
Fig.2 Estrutura química do núcleo flavânico. A e B – anéis aromáticos, C – anel heterocíclico pirano.
Sendo a família de polifenóis mais abundante nos alimentos é também a família
de polifenóis mais conhecida pelas suas propriedades antioxidantes. Em 1990
(Bors et al. 1990), foram descritas pela primeira vez as caraterísticas químicas
O8
5
7
6
43
2
2'3'
4'
5'
6'
1'
A C
B
40 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
dos flavonóides que determinam o seu potencial de neutralizar radicais livres
e/ou a sua capacidade antioxidante: a estrutura o-di-hidroxilo (catecol) do anel B,
a conjugação da ligação dupla C2-C3 com o grupo 4-oxo do anel C e a presença
adicional de grupos hidroxilo nas posições 3 e 5.
2.1 Antocianinas
As antocianinas (do grego anthos que significa flor e kyanos que significa azul)
são os pigmentos mais abundantes e importantes das plantas, frutas e vegetais,
sendo responsáveis pelas variadíssimas cores apresentadas por estes produtos.
A estrutura das antocianinas deriva de glicósidos do catião flavílio poli-
hidroxilados e/ou metoxilados (Fig. 3). Nos frutos, as antocianinas encontram-se
na forma glicosilada, no entanto, noutros contextos podem existir sob a forma
não glicosilada denominando-se antocianidinas (agliconas). A diversidade
estrutural das antocianidinas depende do número e posição dos grupos hidroxilo
e metoxilo ligados aos anéis aromáticos e esta diversidade reflete-se na cor de
cada molécula. Para além da diversidade relacionada com os substituintes do
catião flavílio, as antocianinas podem diferir no tipo, número e posição dos
glicósidos ligados à molécula (pentoses, hexoses e metilpentoses; mono-, di- e
trissacáridos); e na presença e tipo de ácidos esterificados na molécula de
açúcar. Na maioria dos casos, os açúcares ligam-se na posição O-3, mas
também pode ocorrer esterificação nas posições O-5 e O-7. Os açúcares mais
comuns são a glucose, a ramnose, a galactose, a xilose e a arabinose. As
antocianidinas mais frequentemente encontradas em frutas são pelargonidina,
cianidina, delfinidina, peonidina, petunidina e malvidina, cujas estruturas estão
apresentadas na Fig. 3. Devido a todas estas possibilidades de substituição
estão identificadas na natureza um elevado número de antocianinas.
Dependendo da sua estrutura estes compostos exibem cores desde vermelha a
azul (Brouillard 1983). Por exemplo, a pelargonidina tem cor laranja-
avermelhada enquanto a delfinidina tem cor violeta-azulada a pH ácido.
Recentemente foram detectados no vinho novos pigmentos derivados de
antocianinas, em particular os derivados pirúvicos de antocianinas (Fig. 3)
(Fulcrand et al. 1998, Mateus et al. 2003). Estes compostos exibem uma cor
alaranjada e pela sua natureza química são muito estáveis, apresentando maior
intensidade de cor a pH mais elevado comparativamente aos seus derivados
antociânicos (Oliveira et al. 2006, Carvalho et al. 2010).
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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41
Catião flavílio Derivado pirúvico (DP)
Fig. 3 Estrutura química do catião flavílio (3-glicósido) e dos respetivos derivados pirúvicos (DP) de antocianinas. Substituintes das antocianinas (e dos derivados pirúvicos) mais comuns na natureza. Glc – glucose.
A diversidade de cores apresentada por estes pigmentos naturais e seus
derivados é a razão pela qual eles têm sido tão estudados, pois existe uma vasta
possibilidade de aplicação tecnológica, especialmente na indústria alimentar. No
entanto, um dos maiores obstáculos para a aplicação das antocianinas em
matrizes alimentares advém da sua instabilidade a variações de pH e
temperatura.
Dentro da família dos compostos flavonóides, as antocianinas são o grupo de
compostos que mais se destaca pela capacidade antioxidante devido à sua
biodisponibilidade (Rio et al. 2010), a qual será abordada em mais detalhe na
secção 4.
2.2 Flavan-3-óis
Os flavan-3-óis são dos grupos de flavonóides mais abundantes na natureza.
Estes compostos diferem entre si na estereoquímica do carbono 3 do anel C e
grau de hidroxilação do anel B (Fig.4). Têm uma unidade monomérica básica de
(+)-catequina ou (-)-epicatequina, e podem encontrar-se sob a forma de
monómeros ou polímeros (procianidinas). Ao contrário das outras classes de
flavonóides (que existem principalmente sob a forma de glicósidos), os flavanóis
encontram-se normalmente sob a forma de agliconas ou esterificados com o
OOH
OH
R1
R2
OH
O-Glc
+OOH
R1
R2
OH
O-Glc
O
COOH
+
Antocianina R1 R2 Malvidina-3-glucósido OCH3 OCH3
Cianidina-3-glucósido OH H
Delfinidina-3-glucósido OH OH
Pelargonidina-3-glucósido H H
Petunidina-3-glucósido OCH3 OH
42 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
ácido gálhico, formando por exemplo, epigalhocatequina e epigalhocatequina
galhato.
Fig.4 Estrutura química dos flavan-3-óis mais comuns nas plantas.
Têm uma unidade monomérica básica de (+)-catequina ou (-)-epicatequina, e
podem encontrar-se sob a forma de monómeros ou polimeros (procianidinas).
Ao contrário das outras classes de flavonóides (que existem principalmente sob
a forma de glicósidos), os flavanóis encontram-se normalmente sob a forma de
agliconas ou esterificados com o ácido gálhico, formando por exemplo,
epigalhocatequina e epigalhocatequina galhato.
Os carbonos C2 e C3 desta unidade são assimétricos constituindo centros
quirais. Nos isómeros (+)-catequina e (-)-epicatequina o grupo 3,4-hidrofenilo
ligado ao carbono C2 e o grupo hidroxilo no carbono C3 surgem em posição
trans (2R, 3S) e nos restantes isómeros em posição cis (2R, 3R).
Tal como a maioria dos compostos polifenólicos, vários estudos mostram que os
flavanóis têm propriedades antioxidantes devido a várias caraterísticas
importantes, nomeadamente grande estabilização das formas oxidadas devido a
uma deslocalização eletrónica extensa, quelatação de metais e captura de
radicais livres e espécies reativas de oxigénio (Faria et al. 2006, De La Iglesia et
al. 2010). Estes compostos podem também inibir a agregação plaquetária (Jin et
al. 2008), a inflamação vascular (Oizumi et al. 2010, Hu et al. 2011) e proteger
contra a neurodegeneração (Guo et al. 1996, Bastianetto et al. 2006,
Ramassamy 2006).
A importância desta classe de compostos reside também no facto de serem a
unidade estrutural constituinte das procianidinas (vulgo taninos condensados)
muito abundantes na natureza.
Flavan-3-ol R1 R2 R3 (+)-catequina OH H H
(-)-epicatequina H OH H
(+)-galhocatequina OH H OH
(-)-epigalhocatequina H OH OH
(-)-epigalhocatequina galhato H O-galhoilo OH
O
OH
OH
R1R2
OH
OH
R3A C
B
2
3
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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43
3. Taninos
Os taninos representam um grupo de compostos polifenólicos muito
heterogéneo, sendo difícil uma definição química rigorosa. O termo tanino foi
inicialmente usado para descrever compostos de origem vegetal responsáveis
pela conversão das peles de animais em couro, através da formação de
complexos estáveis entre esses compostos e o colagénio da pele animal. No
entanto, em 1962, foi proposta por Bate-Smith e Swain (1962) a definição
química de tanino, que diz que os taninos são “todos os compostos fenólicos
solúveis em água, com um peso molecular situado entre 500 e 3000 Dalton, cuja
principal propriedade (para além das reacções caraterísticas dos compostos
fenólicos) é a de formarem complexos insolúveis com os alcalóides, gelatina e
outras proteínas”.
Desde então, têm vindo a ser descobertos taninos com pesos moleculares muito
elevados na ordem das dezenas de milhares de Dalton (Prieur et al. 1994,
Souquet et al. 1996, Guyot et al. 1997).
Como referido anteriormente, uma das principais caraterísticas dos taninos é a
capacidade para complexar e precipitar proteínas. Esta propriedade é
duplamente a origem de atributos positivos e negativos atribuídos a estes
compostos. Por um lado, é aceite que a interação dos taninos com as proteínas
salivares está na origem da sensação de adstringência, que poderá ser, numa
certa dose, atributo positivo da qualidade de certas bebidas, como o vinho tinto,
cerveja e chá. A adstringência traduz-se numa sensação equilibrada de secura,
constrição e aspereza percebida na cavidade oral aquando da ingestão de
certos alimentos (Mcrae e Kennedy 2011). Por outro lado, a interação dos
taninos com enzimas digestivas pode inibi-las originando problemas
gastrointestinais e pode levar à diminuição do ganho de peso corporal (Griffiths
1986, Goncalves et al. 2007).
Classicamente os taninos são divididos em dois grandes grupos: os taninos
condensados (proantocianidinas), que são oligómeros de catequinas ligados por
ligações C-C; e os taninos hidrolisáveis, que são ésteres de monossacáridos
com ácido gálhico ou oligómeros de ácido gálhico/elágico.
44 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
3.1 Taninos condensados (Proantocianidinas)
Os taninos condensados são mais frequentes na alimentação do que os taninos
hidrolisáveis, estando presentes em concentrações significativas no chocolate,
frutos (uvas, maçãs, entre outros) e bebidas derivadas (Santos-Buelga e
Scalbert 2000).
Estruturalmente, os taninos condensados são polímeros de unidades flavanol, e
a sua estrutura varia de acordo com o número de grupos hidroxilo no anel B e
com a estereoquímica do carbono 3 do heterociclo C. Estes compostos dividem-
se globalmente em dois grandes grupos: as procianidinas, que são polímeros de
catequinas ((+)-catequina ou (-)-epicatequina); e as prodelfinidinas, que são
derivadas de galhocatequina e epigalhocatequina. As prodelfinidinas distinguem-
se das procianidinas por apresentarem um grupo OH na posição 5 do anel B. A
designação de proantocianidinas resulta do facto destes compostos libertarem
antocianidinas por decomposição em meio ácido – reação de Bate-Smith (Bate-
Smith 1954) (Fig.5).
Fig.5 Reação de decomposição das proantocianidinas em meio ácido – Reação de Bate-Smith (Bate-Smith 1954).
As unidades fundamentais das proantocianidinas estão geralmente ligadas entre
si por ligações do tipo C-C (ligação interflavânica que resulta do acoplamento
oxidativo entre monómeros) entre o carbono C4 de um flavanol e os carbonos
C8 (4→8) ou C6 (4→6) do flavanol seguinte. Consoante a ligação interflavânica,
as proantocianidinas diméricas são divididas em tipo A e tipo B (Fig.6). As de
tipo A possuem uma ligação 4→8 acrescida de uma ligação éter entre o grupo
hidroxilo do carbono C5 ou C7 do anel A de uma unidade e o carbono C2 do
anel pirânico da outra unidade. As de tipo B possuem apenas ligações
O
OH
OH
OH
ROH
OH
O
OH
OH
OH
ROH
OH
O
OH
OH
OH
ROH
OH
+
O
OH
OH
OH
OH
OH
R
H / Qcal
Oxigénio
+R=H CianidinaR=OH Delfinidina
R=H CatequinaR=OH Galhocatequina
+
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45
interflavânicas 4→8 ou 4→6. Os dois tipos de proantocianidinas diméricas são
classificados numericamente, de acordo com a estereoquímica do carbono C3
do anel C de cada unidade. As proantocianidinas triméricas pertencem ao tipo C
e as de tipo D são as proantocianidinas triméricas que têm uma ligação de tipo A
e outra de tipo B.
Tipo A Tipo B Procianidina A2 Dímero C4-C8 Dímero C4-C6 (C4-C8 e ligação éter C2-O7)
R1 R2 R3 R4 Dímeros Tipo B C4-C8 C4-C6
OH H H OH B1 B5 OH H OH H B2 B6 H OH H OH B3 B7 H OH OH H B4 B8
Fig.6 Estrutura química dos dois tipos de procianidinas diméricas e substituintes de cada procianidina dimérica tipo B.
O grau de polimerização dos taninos condensados pode variar, podendo-se
encontrar oligómeros (dímeros a hexâmeros) e polímeros (que podem atingir
graus de polimerização muito elevados) (Prieur et al. 1994). Na verdade, já
foram identificados nas uvas taninos condensados com diferentes graus de
polimerização. Nas grainhas, o grau de polimerização situa-se entre 2 e 16,
enquanto na película é superior, entre 3 e 83 unidades de flavanol (Souquet et
al. 1996).
Em suma, os taninos condensados podem diferir nas unidades constituintes,
posições das ligações interflavânicas, comprimento da cadeia e presença de
substituintes (e.g. grupos galhoilo ou glicosilo) o que se traduz numa diversidade
muito elevada de compostos.
O
O
OH
OH
OH
OH
OOH
OH
OH
OH
OH
4
2
8
O
R4R3
OH
OH
OH
OH
O
R2R1
OH
OH
OH
OH
4
8
O
R2R1
OH
OH
OH
OH
O
OH
OH
R3R4
OHOH6
4
46 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
3.2 Taninos hidrolisáveis
Os taninos hidrolisáveis estão presentes sobretudo nas partes não comestíveis
das plantas. No entanto, estes compostos podem ser introduzidos na dieta
alimentar devido a operações tecnológicas de transformação dos alimentos. No
caso do vinho tinto, por exemplo, estes compostos podem passar da madeira
para o vinho durante o envelhecimento em barrica.
Este grupo de taninos tem como moléculas base o ácido gálhico (taninos
gálhicos) e o ácido elágico (taninos elágicos). Os taninos gálhicos estão quase
sempre na forma de ésteres múltiplos de D-glucose e podem formar estruturas
complexas como a pentagalhoilglucose (β-1,2,3,4,6-pentagalhoil-O-D-
glucopiranose) (PGG) (Fig.7). A PGG tem cinco ligações éster idênticas que
envolvem os grupos hidroxilo alifáticos da molécula de açúcar. Tal como a maior
parte dos galhotaninos, a PGG tem muitos isómeros entre os quais variam as
propriedades (bio)químicas, nomeadamente susceptibilidade de hidrólise e
capacidade para precipitar proteínas.
Ácido gálhico Ácido elágico β-1,2,3,4,6-pentagalhoil-O-D-glucopiranose (Pentagalhoilglucose, PGG)
Fig.7 Estrutura das moléculas base dos taninos hidrolisáveis (ácidos gálhico e elágico) e exemplo de um tanino hidrolisável β-1,2,3,4,6-pentagalhoil-O-D-glucopiranose (pentagalhoilglucose, PGG).
Nos taninos isolados das árvores ou produtos derivados de sumagre e de
carvalho, são frequentemente encontrados galhotaninos contendo até 12 grupos
galhoilo esterificados com uma glucose central. Por exemplo, o ácido tânico
comercial (utilizado na indústria alimentar, cosméticos, bebidas e tintas) é
composto por uma mistura de galhotaninos destas árvores.
OH
OH
OHO
OH
O
O
OH
OH
O
OH
OH
O OH
OHOH
OHOH OH
OHOH
OH
O
O
O
O
O
OHOH
OHO
O
OH
OHOH
OOO
O
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4. Compostos polifenólicos nos alimentos. Ingestão e impacto na saúde
humana
Diversos alimentos de origem vegetal são ricos em polifenóis. Como tem vindo a
ser referido ao longo do texto, os alimentos e bebidas conhecidos pelo seu
elevado teor em polifenóis incluem frutos, legumes, cereais e produtos
derivados, como cerveja, sumos de fruta, vinho, chá e chocolate. A Tabela 1
apresenta o teor em polifenóis de alguns alimentos frequentemente ingeridos na
dieta alimentar.
Tabela 1. Teor em polifenóis de diversos alimentos. Adaptado de Scalbert e Williamson (2000).
Alimento (100 g ou mL)
Família de polifenóis (mg) Fenóis totais
(mg) Ácidos fenólicos
Flavanóis
Antocianinas Outros Monómeros catequina Proantocianidinas
Batata 14,5 14,5 - 28,5 Tomate 8 0,5 8 - 37 Alface 8 1 9 - 23 Maçã 5,5 10,5 100 7 239 - 440 Chocolate preto 80 430 510 - 840 Sumo laranja 22 22 - 75 Café 75 2 75 – 89,5 Vinho tinto 9 27,2 36 3 77,6 - 180 Chá preto 65 4 69 - 100
A maioria dos estudos sobre a composição polifenólica das plantas ou alimentos
foca-se apenas em monómeros e estruturas oligoméricas de pequena dimensão,
devido à ausência de técnicas eficazes na separação de estruturas mais
complexas. Geralmente, os polímeros são negligenciados, apesar de serem
frequentemente os polifenóis principais das plantas. Assim, a maior parte das
quantificações em polifenóis diz respeito a um número reduzido de compostos
e/ou a estimativas aproximadas do conteúdo total de polifenóis.
No entanto, apesar destas limitações, é de salientar o trabalho de Gu e seus
colaboradores (Gu et al. 2004) que determinaram a concentração e o grau de
polimerização de taninos condensados de vários alimentos e bebidas
usualmente ingeridos (Tabela 2).
48 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Tabela 2. Distribuição e grau de polimerização de taninos condensados em alimentos. Adaptado de Gu et al. (2004).
Grau de polimerização 1 2 3 4-6 7-10 > 10 Total Alimento (mg/100 g peso fresco ou mg/L de bebida) Maçã 9,6 ± 0,9 13,8 ± 0,6 9,3 ± 0,4 30,2 ± 1,2 25,4 ± 1,2 37,6 ± 2,6 125,8 ± 6,8 Morango 4,2 ± 0,7 6,5 ± 1,3 6,5 ± 1,2 28,1 ± 6,5 23 ,9 ± 3,5 75,8 ± 13,4 145,0 ± 24,9 Pêra 2,7 ± 1,5 2,8 ± 1,3 2,3 ± 0,9 6,5 ± 1,9 4,6 ± 1,0 13,1 ± 11,3 31,9 ± 7,8 Chocolate preto
31,4 ± 0,2 31,2 ± 0,9 21,1 ± 0,8 55,5 ± 3,5 38,5 ± 3,0 68,2 ± 8,8 246,0 ± 0,3
Cerveja 4,0 11,0 ± 1,0 3,0 4,0 ND ND 23,0 ± 2,0 Vinho tinto 20,0 ± 1,0 40,0 ± 1,0 27,0 ± 1,0 67,0 ± 2,0 50,0 ± 1,0 110,0 ± 2,0 313,0 ± 5,0 Feijão vermelho
10,6 ± 0,0 19,4 ± 0,8 18,1 ± 0,6 80,0 ± 2,7 75,7 ± 2,4 252,9 ± 0,8 456,6 ± 7,5
Canela 23,9 ± 1,3 256,3 ± 11,3 1252,2 ± 62,2 2608,6 ± 140,3 1458,3 ± 116,1 2508,8 ± 92,9 8108,2 ± 424,2 Pistachio 10,9 ± 4,3 13,3 ± 1,8 10,5 ± 1,2 42,2 ± 5,2 37,9 ± 4,9 122,5 ± 37,1 237,3 ± 52,0
ND – Não detectado
Dependendo dos hábitos alimentares, uma pessoa pode consumir desde 100 ng
a vários gramas de compostos polifenólicos diariamente, principalmente se tiver
uma alimentação rica em frutas e legumes (Macheix et al. 1990).
Em Portugal, de acordo com a Balança Alimentar Portuguesa, no período entre
2003 e 2008 acentuaram-se os desequilíbrios da dieta alimentar portuguesa em
comparação com a década de 90. Verificou-se um consumo excessivo de
calorias e gorduras saturadas, disponibilidades deficitárias em frutos, hortícolas
e leguminosas secas e recurso excessivo aos grupos alimentares de carne,
pescado e de óleos e gorduras. A caracterização da alimentação da população
portuguesa nesse período está apresentada na Fig.8. Os grupos de alimentos
que contêm polifenóis estão salientados com rectângulos laranja e o gráfico
inferior discrimina os alimentos que são consumidos em maior quantidade em
cada grupo salientado.
Apesar de, tal como referido, se acentuarem os desequilíbrios da dieta alimentar
portuguesa, verifica-se que os grupos de alimentos ricos em polifenóis
constituem ainda uma importante parcela da dieta portuguesa. O vinho, a
cerveja, os frutos e as hortícolas ainda são ingeridos em quantidades
importantes. Em última instância, esta monitorização da dieta alimentar e as
acções sociais de incentivo ao consumo destes grupos de alimentos residem
numa questão de saúde pública.
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49
Fig.8 Caracterização da dieta alimentar da população portuguesa para o período entre 2003 e 2008 (com base na Balança Alimentar Portuguesa). O gráfico superior apresenta o consumo da população em função dos grupos de alimentos, estando salientados por rectângulos laranja os grupos de alimentos que contêm polifenóis; o gráfico inferior apresenta os alimentos mais consumidos dentro de cada grupo de alimentos rico em polifenóis (Balança Alimentar Portuguesa, Instituto Nacional de Estatística).
Há muito que a associação da dieta mediterrânica a uma baixa incidência de
doenças cardiovasculares é conhecida e suportada por vários estudos
epidemiológicos (Hertog et al. 1993, Hollman e Katan 1999, Kaur e Kapoor
2001). Esta dieta carateriza-se pelo baixo consumo de alimentos ricos em
gordura saturada e de grande valor calórico, e consumo elevado de alimentos
ricos em hidratos de carbono complexos, fibras, vitaminas, minerais e
numerosos antioxidantes, nomeadamente, alimentos ricos em polifenóis. Deste
Bebidas n
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50 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
modo, neste tipo de alimentação predominam os cereais e seus derivados,
legumes, hortaliças, frutos e azeite.
O Paradoxo Francês é também um exemplo clássico desta associação: a
população de uma região francesa apresentava uma baixa incidência de
doenças cardiovasculares, apesar do elevado consumo de gorduras saturadas e
hábitos tabágicos. Este facto foi atribuído ao consumo moderado e regular de
vinho tinto (Renaud e De Lorgeril 1992).
Do total de polifenóis ingeridos diariamente estima-se que 1/3 é composto por
ácidos fenólicos e 2/3 por flavonóides (Scalbert e Williamson 2000). Os
flavonóides consumidos em maior extensão são os taninos condensados e as
antocianinas existindo então um grande interesse nestes compostos.
Relativamente à ação benéfica dos polifenóis na saúde humana uma das
questões principais é a sua biodisponibilidade. Isto porque um composto pode
ter grandes capacidades antioxidantes e outras atividades biológicas importantes
in vitro, as quais não serão significativas in vivo se pouco ou nenhum composto
chegar aos tecidos/órgãos alvo. Assim, após a estimativa da ingestão de
alimentos ricos em polifenóis, é importante estimar e estudar a sua
biodisponibilidade.
Dos diversos grupos de polifenóis, a biodisponibilidade das antocianinas tem
sido das mais estudadas (Fig.9) e actualmente sabe-se que a absorção de
alguns destes compostos ocorre no intestino delgado (Rio et al. 2010). Tem sido
aceite que a absorção das antocianinas ocorre na forma de agliconas, após
hidrólise dos glicósidos. Para explicar esta hidrólise e o aparecimento das
agliconas nas células epiteliais, têm sido propostas duas vias, a ‘difusão passiva
após hidrólise pela lactase floridizina hidrolase’ e ‘hidrólise pela β-glicosidade
citosólica’. No caso da primeira via, a hidrólise das antocianinas em agliconas
torna as moléculas mais apolares, aumentando o seu carácter lipofilico e a sua
proximidade com a membrana celular, levando a uma difusão passiva das
moléculas (Day et al. 2000). A última via pressupõe o transporte dos glicósidos
polares para as células, envolvendo possivelmente o transportador dependente
de Na+ GLUT-1, e a hidrólise ocorre após as antocianinas chegarem ao
citoplasma das células (Gee et al. 2000). Antes de passarem para a corrente
sanguínea, as agliconas são metabolizadas por enzimas sulfotransferases, UDP-
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51
glucuronosiltransferases e catecol-O-metiltransferases originando
respetivamente metabolitos sulfatados, glucurinados e/ou metilados.
Os compostos que não são absorvidos no intestino delgado podem ser
absorvidos no intestino grosso onde a microflora intestinal pode clivar as
moléculas conjugadas. As agliconas resultantes podem sofrer ainda abertura
dos anéis, levando à formação de ácidos fenólicos e hidroxicinâmicos os quais
podem ser absorvidos e sujeitos ao metabolismo de fase II no fígado antes de
serem excretados.
Relativamente à biodisponibilidade das proantocianidinas a literatura existente
não é tão vasta, mas é um pouco controversa. Alguns estudos sugerem que
apenas os oligómeros de baixo peso molecular e grau de polimerização
(monómeros, dímeros e trimeros) são absorvidos intatos no trato gastrointestinal
(Holt et al. 2002, Deprez et al. 2004, Rasmussen et al. 2005) e as estruturas
mais complexas seriam degradadas nas condições do estômago. No entanto
outros estudos suportam que as proantocianidinas poliméricas passariam
inalteradas pelo intestino delgado, sendo depois degradadas em ácidos fenólicos
pela microflora do intestino grosso (Déprez et al. 2000, Manach et al. 2004). Os
produtos de degradação incluiriam os ácidos fenilacético, fenilpropiónico e
fenilvalérico.
Após a passagem da barreira intestinal pelas proantocianidinas ou seus
derivados originados na fermentação, estes compostos chegam ao fígado pela
veia porta, onde são metabolizados, provavelmente hidroxilados, metilados ou
conjugados a ésteres de sulfato ou glucuronilados tal como acontece com outros
flavonóides (e.g. antocianinas).
52 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.9 Vias de absorção dos polifenóis. Adaptado de Visioli et al. (2011)
Relativamente às acções benéficas para a saúde humana já foram reportados
diversos estudos que atestam esta relação. As capacidades antioxidante e
anticancerígena dos polifenóis já foram comprovadas inúmeras vezes (Faria et
al. 2006, Fernandes et al. 2009, Faria et al. 2010). Para além disto já foi
observada a diminuição do risco de doenças cardiovasculares (pela redução da
oxidação das LDL e diminuição do colesterol plasmático (Green e Jucha 1986,
Porto et al. 2003), redução do risco de desenvolvimento de doenças
neurodegenerativas, em particular doença de Alzheimer e Parkinson (Ehrnhoefer
et al. 2008, Wang et al. 2012) e retardamento do envelhecimento celular (Howitz
et al. 2003).
5. Interação entre taninos e proteínas
Uma das propriedades mais importantes dos taninos é, como já foi referido
anteriormente, a capacidade de interação e precipitação de proteínas. Esta
propriedade está na origem de várias atividades associadas a estes compostos,
nomeadamente as suas atividades biológica e organolética, quer positivas quer
nocivas. Devido à sua relevância, esta propriedade tem sido o âmbito de estudo
de diversos grupos de investigação (De Freitas e Mateus 2012).
5.1 Tipos de ligação envolvidos na interação tanino -proteína
O núcleo polifenólico tem uma estrutura molecular favorável à interação com
proteínas (Fig.10), pois apresenta:
ConjugaçãoMetilaçãoSulfatação
Glucuronilação
Células e tecidos
FÍGADO
Excreção urinária
VEI
A P
OR
TA
Glicósidos AgliconasÁcidos
fenólicos
CÓLON
INTE
STIN
O D
ELG
AD
OAgliconas
Glicósidos
ESTÔMAGO
Polifenóis oligoméricos
Monómeros
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53
⇒ Regiões hidrofóbicas (apolares) que podem interagir com regiões
hidrofóbicas das proteínas, tais como cadeias laterais dos resíduos de
aminoácidos alanina, leucina, isoleucina e prolina, entre outros;
⇒ Regiões hidrofílicas que podem estabelecer pontes de hidrogénio com os
grupos carbonilo e amina das proteínas.
Apesar da estrutura dos taninos permitir a existência de vários tipos de ligações
com as proteínas, grande parte da literatura sugere que a maioria das interações
tanino/proteína são governadas por ligações não-covalentes, em particular
ligações hidrofóbicas (Oh et al. 1980, Baxter et al. 1997, Jöbstl et al. 2006) e
pontes de hidrogénio (Hagerman e Butler 1980, Hagerman e Klucher 1986)
(Fig.10). As ligações iónicas são as menos significativas pois os polifenóis são
ácidos fracos, com pKa de 9-10, e praticamente não apresentam grupos
carregados a valores de pH ácidos e neutros.
Diversos autores verificaram a existência de interações hidrofóbicas em
complexos entre taninos e proteínas, em particular em proteínas ricas em prolina
(PRPs) ou péptidos similares, sendo também as ligações por pontes de
hidrogénio apontadas como ligações importantes (Tabela 3). Na verdade, vários
estudos sugerem que as interações tanino/proteína são inicialmente governadas
por interações hidrofóbicas e os complexos são, então, estabilizados por pontes
de hidrogénio (Oh et al. 1980, Haslam 1996). As ligações de hidrogénio são
forças que, apesar de serem relativamente fracas individualmente, no seu
conjunto são importantes para a estabilização dos complexos tanino/proteína.
54 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.10 Esquema representativo das interações entre taninos e proteínas, evidenciando o tipo de ligações mais relevante bem como os grupos de cada molécula (do tanino e da proteína) envolvidos em cada tipo de ligação. Adaptado de Asano et al. (1982).
.
O
OH
OH
OH
OH
OH
Região
hidrofílica
Região
hidrofóbica
Tanino
NH3 CH
O
N CH
HCH3 CH2
N
O
CH
COO
CHCH3 CH3
CH2 CH2
CH2
H+ _
Regiões
hidrofóbicas
Regiões
hidrofílicas
Proteína
N
NHCO
CO
NHC O
NHCO
NCO
COO
NH3
OOH
OH
OHOH
HOH
OOH
OH
OHOH
HOH
+
_
OOH
OH
OHOH
HOH
OOH
OH
OHOH
HOH
NH
N
O
C
NHC
O
O
NC
NHC
O
O
NH3
COO
+
_TaninoProteína Proteína
Pontes de hidrogénio Ligações hidrofóbicas
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55
Tabela 3. Resumo de alguns trabalhos que evidenciam o tipo e caraterísticas importantes das interações tanino/proteína, indicando os taninos e proteínas estudados, bem como as metodologias utilizadas no estudo.
Tanino Proteína/Técnica Conclusão Referência Taninos condensados de grainhas de uva
Poli-aa de Pro, hidroxiprolina e Lys, BSA, gelatina, citocromo c / Cromatografia
Interações hidrofóbicas entre a gelatina ou a poli-prolina e os taninos
Oh et al. (1980)
PGG Dois péptidos (19 e 22 aa) com sequência repetitiva rica em Pro, Gli e Glu / RMN
Locais de ligação maioritários são, não só os resíduos de prolina, mas também a ligação amida e o aa precedentes
Murray e Williamson (1994)
Catequina e seu dimero
Poli-Pro / RMN Interações hidrofóbicas entre os anéis benzénicos da catequina e os resíduos hidrofóbicos conformacionalmente acessíveis, mas sem preferência para a prolina
Hatano e Hemingway (1996)
(-)-EC, PGG, tetraGG, triGG
PRP salivar humana (IB-5) / RMN
A IB-5 enrola-se no tanino aumentando as interações cooperativas intramoleculares. A rigidez das moléculas é importante para estas interações, sendo maior a interação para moléculas relativamente rígidas
Charlton et al. (1996)
(-)-Epicatequina, B2, galhoilglucoses, galhato de propilo
Péptido (19 aa) igual a um fragmento de PRP salivar de rato / RMN
Interações hidrofóbicas do tipo “stacking” do anel benzénico do polifenol com a face do anel pirrolidina da prolina
Baxter et al. (1997)
EGCG Heptapéptido rico em Pro / RMN As prolinas estão quase sempre envolvidas em interações hidrofóbicas do tipo “stacking” com preferência para envolver a primeira prolina de um grupo de duas
Charlton et al. (2002)
B3 Fragmento de 14 aa de PRP salivar humana (IB-7) / RMN em fase sólida, MS em MALDI e ESI, DC
A B3 liga-se à região hidrofílica do péptido, sugerindo que a interação é dominada por pontes de hidrogénio entre os grupos carbonilo dos resíduos de prolina e os grupos OH fenol e catecol
Simon et al. (2003)
EGCG β-caseína / Single Molecule Force Microscopy
Interações hidrofóbicas multivalentes Jöbstl et al. (2006)
EGC, ECG, EGCG, B2 e derivado galhato
PRP salivar humana (IB-5) / ESI-MS e tandem MS
Na fase gasosa, as interações são governadas por pontes de hidrogénio
Canon et al. (2010)
aa – aminoácidos, BSA – albumina sérica sovina, CD – dicroismo circular, ECG – galhato de epicatequina, EGC – galhato de epigalhocatequina, ESI – electrospray ionization, MALDI – matrix assisted laser desorption ionization, MS – espectroscopia de massa, PGG – pentagalhoilglucose, PRP – proteína rica em prolina, RMN – ressonância magnética nuclear, tetraGG – tetragalhoilglucose, triGG - trigalhoilglucose
Em suma, estes estudos indicam que tanto as interações hidrofóbicas como as
ligações por pontes de hidrogénio podem estar presentes entre taninos e
proteínas, fortalecendo-se mutuamente e sendo afetadas por diversos fatores,
em particular a estrutura da proteína e do tanino, e as condições do meio
(solvente, pH, entre outros).
As interações hidrofóbicas incluem interações de Van der Waals ou π-π stacking
entre o anel B dos taninos rico em electrões ou o grupo galhoilo e a face planar
pro-S das ligações amida heterocíclicas da prolina.
Nas PRPs, os principais locais de ligação são os resíduos de prolina, sendo um
local particularmente favorável à ligação o primeiro resíduo de prolina de uma
sequência Pro-Pro. O resíduo de prolina pode ser considerado como um bom
56 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
local de ligação já que proporciona uma superfície plana, rígida e hidrofóbica
favorável para interações com outras superfícies planas e hidrofóbicas como os
anéis benzénicos. As principais caraterísticas e propriedades dos resíduos de
prolina e das PRPs serão abordadas mais à frente.
5.2 Modelos da interação tanino/proteína
Estudos da interação da BSA (Hagerman e Robbins 1987) e de um fragmento
sintético de 19 resíduos de uma PRP com taninos (Baxter et al. 1997) sugerem
que os taninos se comportam como ligandos multidentados, isto é, diferentes
anéis fenólicos da mesma molécula podem interagir com a proteína. Isto permite
aos taninos estabelecerem ligações cruzadas entre duas ou mais moléculas
proteicas, ou ligar-se a mais do que um ponto da cadeia peptídica de uma
proteína. Deste modo, a estequiometria e tamanho dos complexos
tanino/proteína dependem das suas concentrações relativas, i. e., da razão
tanino/proteína.
Hagerman e Robbins (1987) verificaram que adicionando quantidades
crescentes de BSA a uma quantidade fixa de tanino, existia uma razão
tanino/proteína óptima para a qual ocorria precipitação máxima (concentração
estequiométrica). Para razões tanino/proteína superiores ou inferiores, a
quantidade de complexos insolúveis diminuía. Um modelo proposto para explicar
estas observações surgiu mais tarde (Siebert et al. 1996) e está esquematizado
na Fig.11.
Fig.11 Modelo da interação tanino/proteína. Os taninos são exibidos com duas extremidades as quais podem ligar-se às proteínas. As proteínas são apresentadas como possuindo um número fixo de locais de ligação. Adaptado de Siebert et al. (1996).
Considerando que cada molécula de tanino e de proteína têm um número fixo de
locais de ligação, a situação em que ocorre a maior interação possível é aquela
em que o número de locais de ligação dos taninos iguala o número de locais de
ligação da proteína, resultando na formação de agregados maiores e um
máximo de turvação (plateau). Se houver excesso de proteína relativamente ao
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tanino, cada tanino é capaz de estabelecer pontes entre duas moléculas de
proteína, mas será improvável que estas proteínas formem mais pontes com
outras proteínas. Isto resultará principalmente em dímeros proteicos, ou seja,
agregados mais pequenos. Com tanino em excesso, todos os locais de ligação
da proteína estarão ocupados, mas a probabilidade de se formarem pontes será
baixa porque cada extremidade livre do tanino terá poucas hipóteses de
encontrar um local de ligação de uma proteína livre. Isto também resultará em
agregados mais pequenos (Fig.12). Este modelo faria prever o aumento de
agregação à medida que se aumentam simultaneamente as concentrações de
proteína e tanino, o que na prática é o que é observado (Siebert et al. 1996).
Deste modo, na maioria dos casos, a agregação está hiperbolicamente
relacionada tanto com a concentração de proteína como com a de tanino
(Fig.12). O tipo de agregados tanino-proteína formado depende das suas
concentrações relativas. Nas concentrações estequiométricas (plateau), os
taninos são capazes de atuar como ligandos multidentados, estabelecendo
pontes entre proteínas ou complexos tanino-proteína, correspondendo a um
máximo de agregação com agregados enormes. Para razões tanino-proteína
mais baixas ou mais elevadas, formam-se agregados mais pequenos.
Fig.12 Esquema de uma curva de nefelometria para uma concentração fixa de tanino e concentrações crescentes de proteína. Adaptado de Hagerman e Robbins (1987).
No entanto, aparentemente, este modelo pode não se aplicar a todas as
proteínas. Por exemplo, para o caso das PRPs o comportamento parece ser
distinto. A adição de quantidades crescentes de uma PRP salivar a uma
quantidade fixa de tanino levou à formação de complexos insolúveis
tanino/proteína, os quais permaneceram insolúveis independentemente da
Tur
vaçã
o
Concentração de proteína
Excesso de tanino
Excesso de proteína
Estequiometria Plateau
58 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
quantidade de proteína adicionada. Deste modo, foi proposto um mecanismo
molecular alternativo para a interação dos taninos com as PRPs (Fig.13).
PRP Tanino
1ª fase 2ª fase 3ª fase
Proteína com Compactação da Uma segunda Agregação das enrolamento proteína após proteína é revestida proteínas revestidas aleatório ligação aos por taninos formando complexos
(PRP) taninos extensos
Fig.13 Mecanismo molecular proposto para a interação das PRPs com os taninos. Na fase inicial (1ª fase) as proteínas compactam-se, devido a ligações apertadas múltiplas com os taninos multidentados. Na 2ª fase forma-se um dímero com outra proteína recoberta de taninos, tornando o complexo insolúvel. Na 3ª fase, há uma maior complexação e ocorre precipitação do complexo. Adaptado de Jöbstl et al. (2004).
Os princípios gerais deste mecanismo são similares ao mecanismo
anteriormente descrito. A associação dos taninos com as proteínas é um
fenómeno principalmente superficial. Numa primeira fase, para concentrações
baixas de tanino, este associa-se à superfície das PRPs ligando-se
simultaneamente várias moléculas de tanino a uma molécula de proteína e o
mesmo tanino a vários locais da proteína. A proteína, que inicialmente
apresentava uma conformação aleatória, envolve os taninos enrolando-se e
tornando a sua estrutura mais compacta, ficando como que revestida pelos
taninos. Com o aumento da concentração de tanino (2ª fase), os taninos
estabelecem ligações cruzadas entre diferentes moléculas proteicas, tornando o
complexo insolúvel e a solução coloidal. Na terceira fase, ocorrem mais ligações
cruzadas aumentando a agregação e levando à precipitação (Luck et al. 1994,
Jobstl et al. 2004).
A observação de que o comportamento dos taninos com as PRPs é distinto do
comportamento com as outras proteínas tem implicações interessantes na
função defensiva proposta para as PRPs (Luck et al. 1994), o que será abordado
na secção 6.1.
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5.3 Fatores que influenciam a interação tanino/prot eína
A interação tanino/proteína depende de vários fatores e, obviamente das
caraterísticas do tanino e da proteína. O tipo de tanino e proteína (tamanho
molecular, grupos funcionais, conformação, entre outros), bem como as
condições do meio (solvente, força iónica, pH, temperatura e presença de outros
compostos, entre outros) afetam esta interação. A influência dos parâmetros
mais importantes para este trabalho vai ser discutida nas próximas secções.
5.3.1 Estrutura dos taninos
Vários aspetos estruturais dos taninos influenciam a capacidade de ligação às
proteínas, nomeadamente o peso molecular e a estereoquímica da molécula, o
tipo de ligação interflavânica, projeção dos grupos hidroxilo e presença de
grupos galhoilo (Okuda et al. 1985, Ricardo-Da-Silva et al. 1991, Kawamoto et
al. 1995, Baxter et al. 1997, De Freitas e Mateus 2001).
De uma forma geral, a interação dos taninos com as proteínas aumenta com o
grau de polimerização, peso molecular e extenção de galhoilação, resultando
provavelmente da presença de mais anéis fenólicos e grupos o-fenólicos.
A influência do peso molecular do tanino é evidenciada por vários estudos
(Baxter et al. 1997, Sarni-Manchado et al. 1999, De Freitas e Mateus 2001,
Canon et al. 2010). Baxter e os colaboradores (1997) estudaram por RMN a
interação de vários taninos (e.g procianidina dimérica B2, PGG e (-)-
epicatequina propil galhato) com um fragmento sintético (19 resíduos) de uma
PRP e concluíram que os compostos maiores e mais complexos interagem mais
com o péptido pela seguinte ordem: (-)-epicatequina propil galhato << PGG <
dímero B2. Os compostos mais pequenos podem ligar apenas um anel fenólico
a um resíduo de prolina enquanto os compostos maiores ocupam dois ou três
resíduos de prolina consecutivos. Os polifenóis mais complexos interagem como
ligandos multidentados.
Sarni-Machado e seus colaboradores (1999) obtiveram também a mesma
tendência estudando a interação de taninos condensados isolados de grainhas
de uvas e proteínas salivares; eles analisaram o sobrenadante e o precipitado
resultante da interação por SDS-PAGE e tiólise ácida. Neste estudo observaram
que as procianidinas de elevado peso molecular, o que neste caso correspondia
a um elevado grau de polimerização, foram selectivamente precipitadas pelas
60 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
proteínas salivares, enquanto as procianidinas diméricas e triméricas
permaneceram em solução.
De Freitas e Mateus (2001) estudaram a interação de taninos condensados (e.g.
(+)-catequina, procianidinas diméricas e triméricas, galhato de (-)-epicatequina,
procianidina B2 galhato) com proteínas salivares humanas, nomeadamente α-
amilase e PRPs, por nefelometria. Neste estudo observaram o efeito do peso
molecular, bem como o efeito da extenção de galhoilação e da estereoquímica.
Relativamente ao efeito do peso molecular os resultados obtidos são
concordantes com os restantes estudos, sendo os dímeros e os trímeros mais
reativos que os monómeros. Relativamente à estereoquímica observaram que a
(+)-catequina era mais reativa com as PRPs do que a (-)-epicatequina e que os
dímeros com ligação interflavânica C4-C8 eram mais reactivos que os
correspondentes dímeros com ligação interflavânica C4-C6. Isto possivelmente
deve-se a restrições conformacionais impostas pelas ligações do tipo C4-C6.
A presença de grupos galhoilo também afeta a interação dos taninos com as
proteínas pois induz um aumento de locais no tanino aptos a ligarem-se à
mesma proteína ou a proteínas diferentes. De facto, de Freitas e Mateus (2001)
observaram que a presença de um grupo galhoilo aumentava a reatividade
comparativamente com os correspondentes não galhoilados. No entanto, esse
aumento de reatividade foi maior no caso dos monómeros do que no caso dos
dímeros. Este facto foi explicado com base na estrutura fechada que o dímero
B2 -3”-O-galhato apresenta como resultado de uma possível interação “π-π
stacking” entre os anéis benzénicos do grupo galhoílo e catecol (Fig.14).
Também Okuda e seus colaboradores (1985) observaram o efeito da presença
de grupos galhoilo na interação tanino/proteína. Eles determinaram
colorimetricamente a ligação da hemoglobina e azul de metileno a diversos
taninos isolados de plantas (e.g. ácido gálhico, PGG, (-)-epicatequina,
procianidina dimérica B2 e derivados galhoilados) de peso molecular e grau de
galhoilação diferentes. Neste estudo observaram que os compostos de baixo
peso molecular tinham uma afinidade de ligação nula ou muito baixa, e que o
aumento do peso molecular, sobretudo induzido pela extensão de galhoilação,
levou a um aumento da ligação de 35%.
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Fig.14 Conformações da (-)-epicatequina, dímero B2 e seus derivados galhoilados, obtidos por mecânica molecular e RMN. Adaptado de de Freitas et al. (1998).
Este efeito da presença de grupos galhoilo também é visível nos taninos
hidrolisáveis. Kawamoto e seus colaboradores (1995) observaram que a
afinidade de uma série de galhoilglucoses para a BSA aumenta com a extensão
da galhoilação: monogalhoilglucose < di- < tri- < tetra- < penta. Para além do
número de grupos galhoilo também a sua posição nos taninos hidrolisáveis afeta
a interação com proteínas. Os mesmos autores observaram que a afinidade da
2,3,6- é superior à da 2,3,4-tri-O-galhoilglucose e a afinidade da 4,6- é superior à
da 2,3-di-O-galhoilglucose.
De acordo com alguns autores, os taninos hidrolisáveis interagem com as
proteínas de modo similar aos taninos condensados regendo-se pelos mesmos
princípios gerais (Hagerman et al. 1998, Tang et al. 2003, Gyémánt et al. 2009) e
revelando, em alguns casos, mais afinidade para as proteínas (Bacon e Rhodes
1998, 2000, Soares et al. 2007, Obreque-Slier et al. 2010).
Por exemplo, Gyémánt e colaboradores (2009) demonstraram que a PGG inibe
a amilase salivar humana e esta interação pode envolver os anéis aromáticos da
PGG, sugerindo um stacking com as cadeias laterais aromáticas dos resíduos
da proteína. Este tipo de interação é semelhante às ligações stacking sugeridas
para a interação dos taninos condensados com as proteínas.
Bacon e Rhodes (2000) observaram que as várias famílias de PRPs (acídicas,
básicas e glicosiladas) apresentavam elevada afinidade para os taninos
62 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
hidrolisáveis (afinidade igual ou superior a monómeros de taninos condensados
estudados anteriormente (Bacon e Rhodes 1998)) e que essa afinidade estava
relacionada com a estrutura dos taninos, em particular com a extenção de
galhoilação e grau de polimerização.
5.3.2 Estrutura das proteínas
Outro fator que influencia a interação tanino/proteína é a estrutura das proteínas.
A estrutura primária, i.e., a sequência de resíduos de aminoácidos, bem como a
estrutura terciária das proteínas, o tamanho e a carga da proteína são aspectos
importantes na interação com os taninos.
Uma das primeiras observações de que a capacidade para os taninos
precipitarem proteínas diferentes varia consideravelmente foi feita por Hagerman
e Butler (1981). Estes autores estudaram a especificidade desta interação por
um ensaio de competição em que mediram a percentagem de precipitação de
uma proteína (BSA) marcada radioativamente na presença e ausência de outra
proteína (competidora). Estes autores observaram que a afinidade relativa das
proteínas para os taninos condensados pode variar mais de 100 vezes e que é
uma interação muito específica. As proteínas globulares com enrolamento mais
compacto têm afinidades muito inferiores para os taninos do que as proteínas
conformacionalmente mais abertas.
Um dos fatores que afecta a interação tanino/proteína é a estrutura primária das
proteínas. Wroblewski e seus colaboradores (2001) verificaram por RMN que a
ligação de taninos a histatinas modificadas (i.e., com os mesmos resíduos de
aminoácidos, mas em que os resíduos de aminoácidos foram dispostos
aleatoriamente) foi significativamente diminuída. Esta observação é suportada
por evidências recentes de que os taninos se ligam a locais muito específicos
das proteínas (Cala et al. 2010), os quais são alterados se houver modificação
da sequência.
Uma caraterística comum às proteínas e péptidos com elevada afinidade para os
taninos parece ser um elevado teor em resíduos de prolina. A gelatina contém
18% de prolina e de hidroxiprolina e a PRP da parótida de rato contém 40% de
prolina e ambas as proteínas são conhecidas por uma elevada afinidade para os
taninos. No entanto, a afinidade das proteínas não é intrínseca aos anéis
heterocíclicos de prolina, já que a prolina e seus derivados apresentam
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afinidades para os taninos semelhantes à mostrada por outros aminoácidos
acíclicos como a glicina ou a alanina (Hagerman e Butler 1981).
Para além disto, o trabalho de Richard e seus colaboradores (2005) apresenta
evidências de que pode não existir interação entre os polifenóis e os resíduos de
prolina se o ambiente circundante a esses resíduos não for adequado à
interação. Richard e seus colaboradores estudaram a interação entre polifenóis
(trans-resveratrol e PGG) e a neurotensina (um péptido biológico de 13 resíduos
de aminoácidos com conformação semelhante às PRPs) por RMN estrutural e
modelação molecular. Estes autores demonstraram que a sequência primária
tem um papel importante na interação com os polifenóis. Neste trabalho, os
autores não observaram stacking hidrofóbico entre os polifenóis e os resíduos de
prolina, mas em estudos anteriores com complexos de bradiquinina-PGG (Vergé
et al. 2002) e noutro trabalho com um péptido rico em prolina (Charlton et al.
2002) essas interações foram observadas. Com base nisto, Richard e seus
colaboradores sugeriram que o ambiente estereoquímico dos resíduos de prolina
é muito importante. No caso da neurotensina, os resíduos de prolina estão
desarranjados e desorientados pelos resíduos adjacentes de arginina, tirosina e
lisina e as suas cadeias laterais volumosas previnem a aproximação dos anéis
do polifenol à prolina. Por outro lado, no caso da bradiquinina e do péptido rico
em prolina, a presença de dois resíduos consecutivos de prolina liberta do
esqueleto peptídico os anéis da prolina, permitindo assim uma aproximação
mais fácil dos anéis fenólicos. Esta aproximação é ainda reforçada pela
presença de um resíduo de glicina em ambos os casos, dado que este
aminoácido não é volumoso e confere um certo grau de flexibilidade ao
esqueleto peptídico. Deste modo, o ambiente que envolve os resíduos de prolina
parece ter um papel determinante na interação destes resíduos com os
polifenóis. Aparentemente, os resíduos de prolina têm de estar posicionados
correctamente na sequência concedendo uma certa flexibilidade para dar origem
a uma interação forte com os polifenóis.
Como tem vindo a ser referido, os resíduos de prolina, para além de poderem
ser um local de ligação, são resíduos que induzem aos péptidos uma
conformação aberta, levando a uma maior exposição de locais de ligação aos
taninos comparativamente a proteínas com uma estrutura mais compata e
globular. Proteínas que têm estruturas globulares compactas, tais como a
ribonuclease A, lisozima e mioglobina têm afinidades inferiores para os taninos
do que proteínas globulares menos fechadas e estruturadas como a BSA e a
64 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
histona F1 (Fig.15). Isto ocorre provavelmente por causa da acessibilidade
acrescida do esqueleto peptídico nas proteínas menos compactas. Por exemplo,
a ribonuclease A tem metade da afinidade para os taninos que a mesma
proteína oxidada pelo ácido perfórmico, porque esta última tem uma estrutura
com enrolamento aleatório enquanto a proteína nativa tem uma estrutura mais
compacta (Hagerman e Butler 1981).
BSA Mioglobina Colagénio
Fig.15 Representação no modo space filling da estrutura terciária da albumina sérica bovina (BSA), mioglobina e gelatina as quais apresentam conformações diferentes. A mioglobina e a BSA têm estrutura terciária globular, sendo a estrutura da mioglobina mais fechada e aparecendo aqui apresentada com o grupo heme (grupo prostético essencial para a sua função). O colagénio tem uma estrutura enrolada em tripla hélice apresentando uma conformação mais aberta que as outras proteínas.1
Este conjunto de dados permite afirmar que a estrutura primária, a qual se reflete
na estrutura terciária das proteínas, são fatores importantes e determinantes na
interação com os taninos.
5.3.3 Presença de carboidratos
A interação tanino/proteína pode ser influenciada pela presença de outros
compostos, nomeadamente, carboidratos.
As primeiras conceções de que a presença de carboidratos poderia inibir esta
interação surgiram no âmbito da compreensão do amadurecimento dos frutos,
que se traduz por uma importante diminuição da sensação de adstringência.
1 As imagens da estrutura terciária das proteínas foram retiradas de: BSA: http://www.pdb.org/pdb/education_discussion/molecule_of_the_month/images/1e7i.gif Mioglobina: http://courses.washington.edu/conj/protein/myo.gif Gelatina: http://www.chm.bris.ac.uk/pt/polymer/images/johnmarshall/gelatin.gif
Grupo heme
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5.3.3.1 Amadurecimento de frutos
Durante o amadurecimento dos frutos surgem várias alterações de natureza
físico-químicas a sensoriais. Duas das principais alterações consistem na
diminuição da adstringência e na alteração da textura, com os frutos a tornarem-
se mais moles e macios.
A alteração da textura tem sido explicada com base na degradação enzimática
de carboidratos estruturais da parede celular (pectina, hemicelulose e celulose) e
de carboidratos de armazenamento (Wakabayashi 2000, Prasanna et al. 2007).
Neste processo têm particular importância as pectinas dado que são os
constituintes principais da parede celular e praticamente os únicos constituintes
da lamela média (Fig.16).
Para além da degradação de carboidratos, o amolecimento dos frutos é
acompanhado de uma redução da adesão celular, como resultado da
degradação da lamela média. Portanto, globalmente, a degradação dos
carboidratos leva a uma diminuição da integridade da parede celular que por fim
resulta na perda de firmeza dos tecidos da fruta, como esquematizado na Fig.16.
A
B
Fig.16 Representação esquemática da (A) estrutura da parede celular primária evidenciando os carboidratos mas importantes e a lamela média2, e da (B) degradação da pectina e da hemicelulose durante o amadurecimento dos frutos. A degradação destes carboidratos reduz a integridade das paredes celulares, diminuindo a rigidez dos frutos (adaptado de Wakabayashi (2000)).
2 Adaptado de http://upload.wikimedia.org/wikipedia/commons/thumb/5/53/Plant_cell_wall_diagram.svg/250px-Plant_cell_wall_diagram.svg.png
Lamela média
Parede celular
primária
Membrana
plasmática
Pectina
Microfibrila
Celulose
Hemicelulose
Proteínas solúveis
Enzimas que hidrolisam hemicelulose
HemicelulosePectina
Amadurecimento
Microfibrila de celulose
Enzimas que hidrolisam pectina
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Relativamente à diminuição da adstringência, duas linhas de pensamento
principais foram propostas ao longo dos anos para explicar esta alteração.
Uma das hipóteses consiste na modificação da composição polifenólica dos
frutos ao longo do amadurecimento, em particular na alteração do peso
molecular/grau de polimerização dos taninos (Goldstein e Swain 1963, Haslam
1998). Alguns estudos associam a diminuição de adstringência à diminuição da
concentração em taninos nos frutos. De acordo com Goldstein e Swain (1963) só
os polifenóis de tamanho médio são eficazes na interação com proteínas: os de
baixo peso molecular são muito pequenos e os de elevado peso molecular ou
não são suficientemente solúveis ou têm uma estrutura muito grande impedindo-
os de encaixar na matriz das proteínas salivares. Estes autores observaram que
na maturação de alguns frutos houve um declínio destes polifenóis. Estes
mesmos autores também avançaram a hipótese de que a perda de adstringência
se deve a um aumento do grau de polimerização dos taninos condensados,
afectando a sua solubilidade e extração. Para além disso, outros estudos sobre
a evolução da composição polifenólica de uvas tintas ao longo do processo de
maturação mostram que o teor em proantocianidinas totais diminui (Mateus et al.
2001).
No entanto, apesar de alguns estudos suportarem esta teoria, em alguns frutos
não há qualquer alteração da sua composição polifenólica e portanto a
diminuição de adstringência tem de ser explicada por outros factos.
Outra linha de pensamento para explicar a diminuição da adstringência durante
o amadurecimento reside na capacidade dos carboidratos presentes ou
libertados da parede celular interagirem com as proteínas salivares e/ou com os
taninos. Esta hipótese tem as suas origens há aproximadamente 100 anos e tem
vindo a ser suportada pela literatura até aos dias de hoje (Joslyn e Goldstein
1964, Ozawa et al. 1987, Haslam et al. 1992, Luck et al. 1994, Carvalho et al.
2006). As primeiras observações apontavam para que os taninos dos frutos se
ligassem à celulose. Mas, foi com o trabalho de Ozawa e colaboradores que
apareceram as evidências que suportaram esta teoria. Ozawa e seus
colaboradores (Ozawa et al. 1987) averiguaram a capacidade dos polifenóis
inibirem a enzima β-glucosidase na presença de outros compostos,
nomeadamente dos carboidratos poligalacturonato de sódio e ciclodextrinas.
Este trabalho demonstrou que estes compostos foram capazes de inibir a
interação do polifenol com a proteína e assim recuperar a actividade enzimática
da β-glucosidase. Os autores justificaram a ação destes compostos pela
capacidade dos carboidratos formarem bolsas hidrofóbicas em solução capazes
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de encapsular e complexar os polifenóis, impedindo-os de interagir com a
enzima.
5.3.3.2 Mecanismos de inibição da interação tanino/proteína pelos
carboidratos
Com base nestes trabalhos e nas suas próprias observações, Haslam e seus
colaboradores (1992, Luck et al. 1994) propuseram dois mecanismos pelos
quais os carboidratos poderiam inibir a interação tanino/proteína (Fig.17):
(a) Os carboidratos são geralmente polielectrólitos (macromoléculas que
contêm grupos carregados) e, portanto, podem formar complexos
ternários com o complexo tanino/proteína, tornando-o solúvel em meio
aquoso – mecanismo do complexo ternário;
(b) Os carboidratos, devido à estrutura terciária (tipo gel) adquirida em
solução, têm capacidade para encapsular os polifenóis, ou total ou
parcialmente e, assim inibir a sua interação com as proteínas –
mecanismo de competição.
Fig.17 Mecanismos possíveis de inibição da agregação dos taninos com as proteínas pelos carboidratos. P: proteína, T: tanino, C: carboidrato. Adaptado de Mateus et al. (2004).
Esta capacidade dos carboidratos inibirem a interação tanino/proteína tem sido
documentada por diversos trabalhos, apesar de não ser uma investigação fácil
devido à escassez de carboidratos solúveis em água com estrutura e peso
molecular bem definidos. A grande maioria dos trabalhos foca-se principalmente
na ligação de polifenóis a carboidratos das paredes celulares das uvas pois este
é um aspecto muito importante no processo vinícola e que afeta a extração dos
Complexo ternário solúvel carboidrato/tanino/proteína
Complexo insolúvel tanino/proteína
Complexo solúvel carboidrato/tanino proteína
+
(a)
(b)
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taninos das uvas. Na verdade, as evidências principais que suportam a interação
dos taninos com os carboidratos da parede celular surgiram em experiências
utilizando carboidratos modelo como a ciclodextrina, pectina, xantana, entre
outros e géis de carboidratos (Mcmanus et al. 1985, Luck et al. 1994, De Freitas
et al. 2003, Carvalho et al. 2006, Hanlin et al. 2010).
Luck e seus colaboradores (1994) verificaram a existência de diversos
carboidratos que inibiram a interação entre a gelatina e um tanino hidrolisável, a
PGG. Estes autores observaram que a eficiência de inibição varia com as
caraterísticas do carbohidrato, sendo a pectina, a goma arábica, a xantana e a
gelana os mais eficazes a prevenir a interação.
De Freitas e seus colaboradores (2003) também observaram a importância da
estrutura dos carboidratos na inibição da interação tanino/proteína. Estes
autores estudaram a influência de vários carboidratos na interação da BSA com
procianidinas oligoméricas por nefelometria. No geral, todos os carboidratos
testados foram eficientes em inibir esta interação, sendo observado um
comportamento diferente para os carboidratos iónicos e neutros. Os carboidratos
aniónicos, como a pectina, a xantana, o ácido poligalacturónico e a goma
arábica, foram muito mais eficientes na sua ação inibidora, o que sugere que as
interações hidrofílicas são dominantes na sua ação. Por outro lado, os
carboidratos neutros, como a glucose, dextrana, β-ciclodextrina e
arabinogalactana, foram menos eficientes na inibição da interação
tanino/proteína. Isto foi explicado com base no baixo carácter iónico destes
carboidratos, o que leva a uma baixa afinidade para eles complexarem com os
polifenóis.
Também Carvalho e seus colaboradores (2006) observaram que o carácter
iónico dos carboidratos é importante para a sua ação. Estes autores estudaram
o efeito de carboidratos isolados do vinho tinto (arabinogalactana-proteinas,
AGPs e ramnogalacturonanas do tipo II, RGII) na interação de uma PRP ou da
α-amilase com procianidinas oligoméricas. Este trabalho mostrou que a inibição
da agregação tanino/proteína com ambas as proteínas estava relacionada com
as cargas dos carboidratos: os mais eficientes eram os que apresentavam maior
carácter iónico (AGPs).
Com estes trabalhos verifica-se que é importante a interação entre os taninos e
os carboidratos.
Deste modo, torna-se relevante que os carboidratos têm de ter uma estrutura e
composição (carácter iónico) adequadas, bem como tamanho molecular e
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flexibilidade capazes de complexar os polifenóis ou de interagir com os
complexos tanino/proteína aumentando a sua solubilidade.
Alguns estudos indicam que a estrutura do polifenol também é importante para o
efeito dos carboidratos. McManus e seus colaboradores (1985) estudaram a
interação entre os taninos e carboidratos por cromatografia usando gel de
dextrana. Estes autores observaram que os compostos aromáticos, em particular
os taninos, tinham uma grande afinidade para o gel de dextrana, a qual foi
aumentada pela presença de grupos hidroxilo na estrutura aromática. Apesar do
mecanismo pelo qual esta associação acontece ser ainda desconhecido, foi
proposto que os taninos são adsorvidos nos poros do gel devido a interações
entre os grupos hidroxilo dos taninos e os átomos de oxigénio envolvidos nas
ligações glicosídicas.
O efeito da pectina na adstringência de catequinas também já foi estudado em
soluções modelo. Hayashi e seus colaboradores (2005) estudaram o efeito da
pectina na adstringência de catequinas por espectroscopia de RMN e por um
“sensor de adstringência” (medição de diferenças de potencial entre a solução
de tanino e uma solução de referência). Estes autores observaram que a pectina
diminuiu a adstringência de catequinas com grupos galhoilo (EGCG e ECG)
enquanto nas catequinas não galhoiladas (EGC e epicatequina) não foi
observado nenhum efeito. Estes autores também observaram com base nas
alterações do deslocamento químico nos espetros de RMN que as catequinas
galhoiladas têm maior afinidade do que as não galhoiladas para interagir com a
pectina. Estes resultados apontam que a formação de complexos catequina-
pectina pode ser um fator importante na redução da adstringência.
Em suma, as conclusões principais dos diversos trabalhos apresentados são
que os polifenóis e os carboidratos podem formar complexos, sendo esta
interação governada pelos mesmos princípios que a interação com as proteínas.
A interação dos carboidratos com os polifenóis pode inibir a interação destes
com as proteínas ou pode ocorrer quando estes já formaram complexos com as
proteínas. A adsorção dos polifenóis aos carboidratos é mediada por interações
hidrofóbicas e pontes de hidrogénio, sendo a primeira favorecida pela existência
de cavidades hidrofóbicas, como por exemplo a cavidade interna das
ciclodextrinas. A afinidade é fortemente influenciada pelo peso molecular e
flexibilidade conformacional do polifenol. Para além disto, o estado e
conformação dos carboidratos também são importantes: os polifenóis ligam-se a
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géis de dextrana mas não se ligam a oligómeros ou pequenos polímeros de
dextrana (Williamson et al. 1995).
5.3.4 Outros fatores (força iónica, pH, etanol e temperatura)
Para além dos carboidratos, vários outros fatores afetam a interação
tanino/proteína, nomeadamente a força iónica, o pH, o etanol e a temperatura.
Carvalho e seus colaboradores (2006) observaram um efeito oposto da força
iónica para a interação de duas proteínas diferentes com um tanino; enquanto
para uma PRP o aumento da força iónica levou a um grande aumento da
interação, para a α-amilase levou a uma ligeira diminuição da interação com o
tanino. A explicação sugerida assentou no ponto isoelétrico (pI) das proteínas;
ao pH a que se efetuaram as experiências (pH 5) a α-amilase provavelmente
estaria menos carregada positivamente que a PRP e por isso a força iónica não
provocou nenhuma alteração. Por outro lado, no caso da PRP houve
provavelmente uma maior hidratação dos iões com o aumento da força iónica, o
que removeu a água da estrutura dos agregados promovendo as interações
tanino/proteína.
No entanto, os estudos nesta área são controversos e aparentemente o efeito da
força iónica também depende da proteína e tanino em questão (Oh et al. 1980,
Siebert et al. 1996, De Freitas et al. 2003, Rawel et al. 2005).
O pH do meio está diretamente relacionado com a interação tanino/proteína
dado que ele afeta o grau de ionização de ambos os componentes. Em geral, a
interação e precipitação diminuem a pH baixos (<2) e elevados (>8) e é máxima
a pH próximo do pI da proteína onde as repulsões entre as proteínas são
minimizadas (Hagerman e Butler 1981, Yan e Bennick 1995, Naczk et al. 1996).
Relativamente ao efeito da temperatura, a literatura existente também não é
coerente. Por exemplo, Hagerman e seus colaboradores (1998) observaram que
a precipitação da BSA pela PGG aumenta com a temperatura. Também Siebert
e seus colaboradores (1996) estudaram a formação de precipitados entre
diversas proteínas (e.g. gelatina, lisozima, péptido rico em prolina, gliadina) e
taninos observaram que o aumento de temperatura conduzia a um aumento de
precipitação. Por outro lado, Rawel e seus colaboradores (2005) observaram em
estudos de ligação de diferentes taninos à BSA e HSA que o aumento da
temperatura levou a uma diminuição da interação. Portanto, também este fator
parece depender quer da proteína quer do tanino em questão.
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6. Saliva humana
A saliva humana é uma mistura aquosa complexa de proteínas e minerais e tem
diversas funções fisiológicas. Devido às suas ações de lubrificação,
antibacteriana e tamponante contribui para a manutenção da integridade da
cavidade oral, mas também funciona como primeira etapa da digestão devido à
presença das enzimas α-amilase e lipase. A saliva é também importante na
perceção e sabor dos alimentos, dado que é o meio através do qual estas
propriedades são percebidas.
O fluído oral, vulgarmente referido como “saliva total (whole saliva)”, é composto
maioritariamente pelas salivas das glândulas maiores (parótida, submandibular,
sublingual), das glândulas salivares menores e também pelos fluidos das
gengivas.
A composição proteica da saliva tem sido alvo de interesse nos últimos anos, e o
conhecimento do proteoma salivar tem avançado muito, em particular na última
década com o desenvolvimento de técnicas de proteómica novas e potentes. O
avanço e o recurso a estas técnicas permitiu identificar diferentes classes de
proteinas e péptidos, muitas delas específicas da cavidade oral. As principais
classes estão apresentadas na Fig.18 e as mais importantes para este trabalho
serão abordadas nas secções seguintes.
Fig.18 Percentagens aproximadas (p/p) das principais classes de proteínas e péptidos salivares na saliva total. Adaptado de Messana et al. (2008). ASH, albumina sérica humana; sIgA, imunoglobulina A secretora; IgG, imunoglobulina G; aPRPs, proteínas-ricas em prolina acídicas; bPRPs, proteínas-ricas em prolina básicas; gPRPs, proteínas-ricas em prolina glicosiladas.
6.1 Proteínas ricas em prolina (PRPs)
As PRPs são, como o próprio nome indica, ricas no aminoácido prolina e, como
observado na Fig.18, são das classes mais abundantes na saliva humana.
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Recentemente, estas proteínas têm sido consideradas uma classe da família das
proteínas intrinsecamente desestruturadas (intrinsically unstructured proteins,
IUP) (Boze et al. 2010). Estas proteínas caracterizam-se pela ausência de uma
estrutura terciária, permanecendo no entanto funcionais apesar da ausência de
uma estrutura bem definida.
Aproximadamente 25 a 42% dos resíduos de aminoácidos das PRPs são
prolina. Para além deste resíduo estas proteínas têm também um elevado
conteúdo em glutamina e glicina. Em conjunto, estes três resíduos
correspondem a cerca de 70 a 88% do total de resíduos destas proteínas
(Bennick 1975, Levine e Keller 1977) .
O aminoácido prolina é um aminoácido particular pois a sua cadeia lateral é
cíclizada com a amida do esqueleto peptídico principal. Esta caraterística tem
três consequências importantes (Williamson 1994): primeiro, a conformação do
próprio esqueleto da prolina é restrita (MacArthur, M. W. and Thornton, J. M.
1991 J. Mol. Biol. 218, 397; Nicholson, H., Tonrad, H., Becktel, W. J. and
Matthews, B. W. 1992 Biopolymers 32, 1432); segundo, o volume do grupo N-
metileno dá origem a restrições na conformação do resíduo de aminoácido
anterior à prolina; terceiro, dado que o azoto do grupo amida não tem um
hidrogénio é incapaz de atuar como dador de pontes de hidrogénio. Para além
disto, a prolina também é única na sua capacidade para formar ligações
peptídicas cis.
Estas caraterísticas conferem algumas restrições nas conformações acessíveis a
um dipéptido X-Pro, que como resultado tem tendência a ter uma conformação
bastante rígida e extendida. Um dipéptido Pro-Pro é mais restrito e existem
várias evidências de que uma sequência de quatro ou mais resíduos de prolina
em solução adoptam uma conformação de hélice poliprolina II. Tem sido
amplamente assumido que o elevado nível de resíduos de prolina nas PRPs é
apenas importante porque se traduz em estruturas abertas e flexíveis nestas
proteínas correspondendo a um potencial para complexação mais elevado.
Globalmente, as PRPs salivares são classificadas em dois grandes grupos: as
acídicas (aPRPs), e as básicas (bPRPs). Esta classificação reflete apenas a
carga global das proteínas, mas no final também se reflete em diferenças
funcionais entre as duas classes. Para além disto, as bPRPs ainda são
tipicamente divididas em PRPs glicosiladas (gPRPs) e não-glicosiladas.
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6.1.1 PRPs acídicas (aPRPs)
As aPRPs são codificadas pelos genes PRH1-2 que estão no cromossoma
12p13.2 e a sua tradução resulta em proteínas que apresentam dois domínios
estruturais distintos. A região C-terminal é estruturalmente equivalente às bPRPs
e compreende cerca de 70-80% da molécula. Por outro lado, a região N-terminal
contêm resíduos fortemente acídicos (ácidos aspártico e glutâmico) e poucos
resíduos de prolina (Fig.19). A existência desta região acídica traduz-se num
ponto isoeléctrico (pI) baixo (4-5).
Na saliva humana podem ser detetadas 5 isoformas desta classe de proteínas.
Todas apresentam um N-terminal piroglutâmico e são normalmente di-
fosforiladas nas Ser-7 e Ser-22, apesar de serem também detetadas
quantidades menores de aPRPs mono- e não-fosforiladas. Também já foram
detetadas menores percentagens de isoformas tri-fosforiladas (Ser-17). Destas
isoformas, quatro (PRP-1, PRP-2, PIF-s e Dbs) podem ser parcialmente clivadas
próximo do C-terminal libertando um péptido comum de 44 resíduos (péptido P-
C) e quatro isoformas truncadas chamadas PRP-3, PRP-4, PIF-f e Db-f. A
isoforma Pa não é clivada, mas encontra-se frequentemente na saliva na forma
dimérica devido à presença específica de um resíduo de cisteína (C-103) na sua
estrutura.
Relativamente à função destas proteínas, está descrito que contribuem
principalmente para a manutenção da homeostasia do cálcio e seus sais na
cavidade oral. Na verdade, a atividade e os locais de ligação associados à
manutenção do equilíbrio do cálcio em solução está confinada à região acídica
N-terminal destas proteínas.
Fig.19 Esquema representativo da estrutura das aPRPs com indicação da região acídica responsável pela função atribuída a estas proteínas3.
3 Este esquema foi adaptado da base de dados UniprotKB
Péptido sinal
PRP1/PRP2
PRP3/PRP4
Péptido P-C
Região responsável pela inibiação da formação de hidroxiapatite; liga-se à hidroxiapatite e ao cálcio
N-terminal acídico C-terminal ~bPRPs
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6.1.2 PRPs básicas (bPRPs) e glicosiladas (gPRPs)
As PRPs básicas e glicosiladas são os grupos mais complexos das PRPs, sendo
expressas por quatro genes diferentes (PRB1-PRB4) que estão agrupados no
cromossoma 12p13.2 perto dos genes PRH1-2. Devido há existência de uma
grande homologia e de um crossing-over desigual entre as repetições do terceiro
exão, isto produz frequentemente polimorfismos. Para além disto, as múltiplas
PRPs podem ser produzidas a partir do mesmo gene através de variações
alélicas, splicing diferencial e modificações pós-tradução.
Ao contrário das aPRPs, que estão contemporaneamente presentes na saliva
como isoformas completas ou truncadas, todas as bPRPs são detetadas apenas
sob a forma de múltiplos fragmentos peptídicos originados de maiores pro-
proteínas. A estrutura de vários péptidos já foi estabelecida [IB1, IB4 (P-H), IB5
(P-D), IB6, IB7, IB8a, IB8b, IB8c (P-F), IB9 (P-E), II-1 e II-2] mas ainda faltam
muitas peças para a definição total desta classe. Por exemplo, no perfil de LC-
MS da saliva é sempre detectado um péptido designado por P-J com massa de
5945 Da, mas que ainda não foi caracterizado definitivamente. Para além disso,
pelo menos três potenciais bPRPs de estrutura não conhecida, com massas de
10434, 23462 e 29415 Da, são recorrentemente detetadas. Uma propriedade
bastante interessante desta classe de proteínas é a elevada homologia das
sequências das diferentes proteínas.
As bPRP são compostas por resíduos que não têm carga a pH básico ou neutro.
Deste modo, o seu pI (pI > 9) é bastante mais elevado do que para as aPRPs. A
sua função ainda não é totalmente compreendida apesar de existirem evidências
que apontam para funções protetoras. Mehansho e seus colaboradores (1987)
propuseram que uma das suas funções seria ligar os taninos, prevenindo assim
os seus efeitos tóxicos no trato gastro-intestinal. Mais recentemente, outros
autores descobriram que estas proteínas ligam-se a vírus, em particular a uma
proteína do HIV-1, tendo assim uma ação anti-vírica (Robinovitch et al. 2001).
Relativamente às gPRPs, como o próprio nome indica, esta classe de proteínas
caracteriza-se pela presença de glicósidos na sua estrutura. Devido à
complexidade destes substituintes, não existem muitos dados na literatura
acerca destas proteínas. Atualmente, começam a surgir as primeiras
informações no que diz respeito ao tipo de açúcar e à posição de glicosilação
destas proteínas (Vitorino et al. 2011). Na verdade, estes dados são
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fundamentais na análise destas proteínas por espectrometria de massa, pois só
sabendo a sua estrutura global é possível saber o seu peso molecular.
A função principal atribuída às gPRPs é a de lubrificação. Na verdade, as gPRPs
têm uma actividade lubrificante superior às formas não-glicosiladas (Hatton et al.
1985). No entanto, esta propriedade é afetada pelo tipo de carboidratos, bem
como pela extenção da glicosilação (Mcarthur et al. 1995). A glicosilação das
PRPs ocorre por ligação dos açúcares aos resíduos de asparagina (Hatton et al.
1985), treonina e serina (Oppenheim et al. 1985) o que permite frequentemente
antecipar os potenciais locais de glicosilação.
Para além desta função está também descrito que estas proteínas ligam-se às
bactérias da cavidade oral (Carpenter e Proctor 1999).
6.2 Histatinas
As histatinas são proteínas salivares ricas em histidina. Na verdade, este
aminoácido constitui cerca de 18-29% destas proteínas o que é invulgar pois a
histidina é, em conjunto com a prolina, um dos aminoácidos mais raros de
encontrar nas proteínas (Oppenheim et al. 1988).
Até à data, foram identificados dois genes responsáveis pela síntese das
histatinas, HTN1 (HIS1) e HTN2 (HIS2), os quais estão localizados no
cromossoma 4q13 (Vanderspek et al. 1989). Os produtos destes genes são a
histatina 1 e 3, respetivamente. Enquanto a primeira é um péptido de 38
resíduos de aminoácidos, fosforilado na Ser-2, a última apesar de ter uma
sequência semelhante à histatina 1, tem apenas 32 resíduos e não é fosforilada
(Oppenheim et al. 1988). Para além destas histatinas, existem muitas outras que
resultam maioritariamente de clivagem proteolítica da histatina 3 (Castagnola et
al. 2004).
Estas proteínas são consideradas como os principais percursores da estrutura
proteica que protege a superfície dos dentes (esmalte). Para além disto, as
histatinas têm actividade antibacteriana e anti-fúngica. A histatina 5 é
especialmente eficaz contra C.albicans (Oppenheim et al. 1988) e também é um
inibidor potente das metaloproteinases MMP2 e MMP9 (Gusman et al. 2001).
76 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
6.3 Estaterina
A estaterina é um fosfopéptido salivar de 43 resíduos de aminoácidos muito
específico e multifuncional (Fig.20). Este péptido acídico é secretado por várias
glândulas salivares e tem um invulgar conteúdo elevado em resíduos de tirosina,
prolina e glutamina (Schlesinger et al. 1989). O seu gene (STATH) está também
localizado no cromossoma 4q13.3 (Sabatini et al. 1987).
Esta proteína contribui sobretudo para o equilíbrio dos minerais na superfície dos
dentes. Na verdade, esta proteína tem uma grande afinidade para o fosfato de
cálcio, inibindo a sua precipitação de soluções supersaturadas (Schlesinger et al.
1989). Deste modo, a estaterina mantém a supersaturação da saliva humana em
cálcio, inibindo o depósito de minerais na superfície dos dentes e contribuindo,
em conjunto com outras proteínas salivares, para a estabilização do esmalte
(Hay et al. 1984). Para além disto, a estaterina contribui para a colonização
bacteriana (Gibbons e Hay 1988) e atua como lubrificante (Douglas et al. 1991).
A região N-terminal da estaterina é muito negativa, sendo o domínio responsável
pela ligação aos sais de cálcio, mas o nível de fosforilação da proteína é também
crucial para esta função (Raj et al. 1992).
Fig.20 Esquema representativo da estrutura da estaterina com indicação das diferentes regiões responsáveis pelas funções atribuídas a esta proteína4.
7. Polifenóis e sabor amargo
Muitos dos polifenóis presentes na nossa dieta alimentar, tais como as
antocianinas e os flavan-3-óis, são amargos, tornando o sabor das plantas e
produtos derivados desagradável (Drewnowski e Gomez-Carneros 2000). Na
verdade, entre as cinco sensações de sabor básicas, o sabor amargo é
particularmente importante no sentido de evitar substâncias perigosas (Ames et
al. 1990). Numerosas substâncias venenosas têm sabor amargo sendo repelidas
por várias espécies do reino animal. A rejeição instintiva do sabor amargo tem
4 Este esquema foi adaptado da base de dados UniprotKB
Péptido sinal
Estaterina
Região de ligação da hidroxiapatite;
Inibe o crescimento de cristais
Região hidrofóbica;
Inibe a precipitação dos
sais de fosfato de cálcio
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sido crucial para a sobrevivência das espécies, apesar de não estar estabelecida
uma correlação evidente entre o amargor e a toxicidade (Glendinning 1994,
Hladik e Simmen 1996).
Por causa desta rejeição instintiva do sabor amargo, há muito tempo que a
remoção dos compostos responsáveis por este sabor dos alimentos é um
desafio sensorial crucial na indústria alimentar (Drewnowski e Gomez-Carneros
2000). Para isso, é importante a identificação dos compostos que tornam os
alimentos e bebidas derivadas de plantas amargos.
7.1 Relação entre a estrutura dos polifenóis e o sa bor amargo
A adstringência e o amargor são dois atributos sensoriais importantes no vinho
tinto, para os quais certos polifenóis têm um papel importante (Delcour et al.
1984, Noble 1998, Drewnowski e Gomez-Carneros 2000, Hufnagel e Hofmann
2008, Rudnitskaya et al. 2010). Tal como já foi referido anteriormente, enquanto
a adstringência aparenta ser uma sensação tátil de secura, constrição e
aspereza percecionada na cavidade oral, o amargor é um sabor que se
desenvolve pela ativação dos respetivos recetores de sabor.
Apesar de os taninos serem bem conhecidos pela indução da adstringência, eles
também têm sido frequentemente associados ao sabor amargo do vinho tinto.
No entanto, a literatura disponível relativamente à relação estrutura/amargor dos
taninos é inconsistente (Arnold et al. 1980, Robichaud e Noble 1990, Kallithraka
e Bakker 1997, Noble 1998, Kielhorn e Thorngate Iii 1999, Peleg et al. 1999).
Em geral, o número reduzido de estudos que avaliaram o amargor de vários
polifenóis, tais como frações poliméricas de ácido tânico e taninos, bem como de
monómeros, dímeros e trímeros de flavan-3-óis, demonstraram que as
moléculas maiores tendem a ser menos amargas e mais adstringentes. Peleg e
seus colaboradores (1999) observaram que a (-)-epicatequina era mais amarga
que o seu estereoisómero (+)-catequina e que ambos eram mais amargos que
os trímeros de procianidinas, catequina-(4-8)-catequina-(4-8)-catequina e
catequina-(4-8)-catequina-(4-8)-epicatequina. Robichaud e Noble (1990)
observaram que o ácido tânico, um tanino hidrolisável comercial rico em
pentagalhoilglucose, era mais amargo do que a (+)-catequina e do que um
extrato de grainhas de uva rico em procianidinas poliméricas. No entanto, outros
estudos mostraram que o amargor dos polifenóis aumenta com o peso
molecular.
78 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Um outro estudo recente (Hufnagel e Hofmann 2008) demonstrou claramente
que concentrações inferiores ao limite de deteção de ácidos fenólicos de ésteres
etílicos e dos flavan-3-óis [(+)-catequina, (-)-epicatequina, procianidinas B1,B2,
B3 e C1] são responsáveis pelo amargor do vinho tinto estudado (Amarone della
Valpolicella DOC). Estes autores identificaram 82 compostos presentes nesse
vinho tinto (incluindo açúcares, polifenóis, aminoácidos e sais) e fizeram diversas
análises sensoriais para determinar a intensidade do amargor de misturas
incluindo todos os compostos ou frações de um tipo de compostos. O objetivo foi
determinar que combinação de compostos apresentava a mesma intensidade de
amargor e de adstringência que o vinho original. Noutro trabalho, os mesmos
autores (Hufnagel e Hofmann 2008), eles observaram que o limite de deteção do
amargor das procianidinas diméricas era aproximadamente metade dos
respetivos monómeros, ou seja, estas últimas mesmo em concentrações baixas
serão mais importantes pelo sabor amargo do vinho tinto.
Para além das inconsistências referidas, não há conhecimento suficiente acerca
do sabor amargo de muitas famílias de polifenóis, tais como as antocianinas.
Apenas Vidal e seus colaboradores (2004) observaram que as antocianinas
monoglicosiladas e respetivos derivados cumaroílo, não influenciavam a
adstringência e o amargor. Para além disso, estes autores observaram que os
polifenóis pigmentados semelhantes a taninos isolados do vinho apresentaram
resultados semelhantes às antocianinas.
Deste modo, torna-se importante a avaliação do amargor de diversos compostos
polifenólicos por um método fidedigno, reprodutível e objectivamente mensurável
por forma a esclarecer que compostos são responsáveis pelo amargor do vinho
tinto.
7.2 Recetores do sabor amargo (hTAS2Rs)
Os compostos amargos são detectados por um conjunto específico de células
recetoras de sabor (taste receptor cells, TRC) localizadas na cavidade oral em
grupos de células chamadas corpúsculo gustativo, as quais se encontram no
epitélio das papilas gustativas da língua e palato. Estas TRC caracterizam-se
pela expressão de membros da família do gene TASTE 2 Receptor (TAS2R) que
codifica recetores do sabor amargo (Adler et al. 2000, Chandrashekar et al.
2000, Matsunami et al. 2000, Meyerhof et al. 2009, Shi e Zhang 2009). Nos
seres humanos, esta família genética codifica cerca de 25 recetores de sabor
amargo (hTAS2Rs).
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Os hTAS2Rs são proteínas com aproximadamente 290–330 resíduos de
aminoácidos. Apesar de serem estruturalmente diversos, os genes paralogos5
apresentam aproximadamente 17-90% de similaridade na sequência de
aminoácidos, sugerindo que diferentes membros da família podem reconhecer
compostos com estruturas muito diferentes (Matsunami et al. 2000). Na prática é
realmente necessário que assim seja, dado que os seres humanos têm apenas
25 hTAS2Rs para a detecção de um número enorme de compostos amargos
(Meyerhof 2005). Os compostos amargos são não só numerosos mas também
estruturalmente muito diversos, incluindo péptidos, aminoácidos, lactonas,
terpenóides, fenóis e flavonóides, entre outros, o que torna a previsão do
amargor difícil. Até à data, já foram identificados compostos amargos que
ativavam 20 destes recetores (Behrens e Meyerhof 2006).
Em geral, estes recetores têm em comum uma estrutura em hepta-hélice e
outros motivos sequenciais, que os classifica como membros da subfamília dos
recetores acoplados à proteína G.
A Fig.21 mostra que, em geral, a região citoplasmática dos segmentos putativos
transmembranares e os loops intracelulares são bem conservados. Os 20
aminoácidos que estão presentes em quase todos os hTAS2Rs (assinalados a
vermelho) estão, ou nos domínios intracelulares ou região inferior dos
segmentos transmembranares, provavelmente envolvidos no acoplamento à
proteína G, ativação do recetor e no enrolamento tri-dimensional dos recetores.
Os loops extracelulares e as partes superiors dos segmentos transmembranares
são menos conservados. Isto não é imprevisível dado que são estas regiões que
provavelmente formam os motivos heterogéneos de ligação aos inúmeros
compostos amargos. A região C-terminal dos hTAS2Rs também é muito variável,
o que reflecte possíveis diferenças na regulação dos recetores e no tráfego
celular (Meyerhof 2005).
É também interessante que a existência de numerosos polimorfismos de um
nucleótido (single nucleotide polymorphisms, SNPs) não-sinónimos nos genes
TAS2Rs, origine a codificação de variantes dos recetores (haplotipos), as quais
são funcionalmente distintas e são a base da alteração da perceção do amargor
na população (Drayna 2005, Kim et al. 2005, Pronin et al. 2007).
5 i.e. dois genes existentes em cromossomas diferentes do mesmo organismo mas que têm similaridades estruturais o que indica que derivaram do mesmo gene ancestral.
80 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.21 Representação esquemática dos hTAS2Rs. Os resíduos de aminoácidos estão indicados por círculos coloridos. A similaridade da sequência entre os 25 hTAS2Rs está indicada pelo código de cores. Os círculos com a linha em pontos correspondem aos resíduos que não estão presentes em todos os recetores. Os quadrados magenta indicam as regiões que contêm resíduos adicionais em alguns recetores (Meyerhof 2005).
Os hTAS2Rs, tal como referido anteriormente, pertencem à família dos recetores
acoplados à proteína G. Esta família compreende uma família de recetores
transmembranares que ao serem ativados por moléculas extracelulares,
desencadeiam uma cascata de transdução de sinal (a qual ativa a proteína G)
que resulta no final numa resposta celular.
No caso dos hTAS2Rs a proteína G associada à sua ativação é a gustaducina
(Margolskee 2002), nomeadamente a subunidade α e mais particularmente
serão os últimos 37 resíduos do C-terminal desta subunidade que interagem
com os hTAS2Rs (Ueda et al. 2003). Como qualquer proteína G, a gustaducina
é uma proteína heterotrimérica com as subunidades α, β e γ (Fig.22).
Inicialmente, após a ativação dos recetores ocorre a dissociação das
subunidades da gustaducina do recetor; a α-gustaducina induz uma diminuição
no cNMPs (nucleósido 5’-monofosfato cíclico) e o dímero βγ-gustaducina eleva o
IP3 (inositol trisfosfato)/DAG(diacilglicerol). As etapas seguintes da cascata de
sinalização não estão confirmadas, mas suspeita-se que a diminuição do cNMPs
pode atuar nas proteínas cinases as quais podem regular a atividade dos canais
iónicos das células recetoras do sabor. A cascata do dímero continua com a
ativação dos recetores do IP3, levando à libertação de Ca2+ intracelular seguida
pela libertação de neurotransmissores.
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Fig.22 Mecanismo proposto de transdução de sinal do sabor amargo para as células recetoras de sabor. Os compostos amargos ativam os recetores de sabor amargo, os quais ativam os heterotrimeros da gustaducina. A α-gustaducina estimula a PDE (fosfodiesterase) a hidrolizar o cAMP (adenosina monofosfato cíclica); a diminuição de cAMP pode disinibir os canais de iões inibidos por nucleótidos, elevando o Ca2+. Por outro lado, as subunidades βγ-gustaducina libertadas ativam a PLCβ2 (fosfolipase C2), gerando IP3 (inositol trisfosfato) que leva à libertação de Ca2+ armazenado, contribuindo para o aumento do Ca2+ intracelular. Adaptado de Meyerhof (2005).
Para compreender melhor a relação estrutura/amargor é necessário um método
objetivo, robusto e fiável que permita analizar o amargor de vários compostos.
Nesse sentido, surgiu recentemente uma metodologia que consiste na medição
da ativação dos hTAS2Rs pelos compostos a testar, após a expressão
heteróloga dos recetores em células humanas, normalmente em células
embriónicas de rim (HEK-293). Esta metodologia baseia-se precisamente na
deteção do aumento do cálcio intracelular que ocorre após a ativação dos
recetores (Fig.22). Esta metodologia tem sido usada para testar compostos
pressupostamente amargos presentes em alimentos e bebidas, e.g., vegetais,
produtos de soja e cerveja (Bufe et al. 2002, Maehashi et al. 2008, Intelmann et
al. 2009, Roland et al. 2011).
Um ponto importante nesta metodologia, é a correlação dos dados
experimentais obtidos com os recetores in vitro, com os dados sensoriais obtidos
por experiências in vivo. Este aspecto foi considerado por Bufe e seus
colaboradores (2002), os quais observaram que as propriedades dos recetores
estudadas in vitro se aproximavam bastante dos resultados obtidos nos estudos
psicofísicos com humanos. Deste modo, a expressão heteróloga dos hTAS2Rs
aparenta ser uma metodologia in vitro viável e fidedigna para obter dados
relevantes do sabor amargo de composto.
TAS2R
Extracelular
Intracelular
PLC β2
DAG IP3α-gustaducina
GDP
α-gustaducina
GTP
Gβ1/3Gγ13
Gβ1/3Gγ13
Ca2+
ArmazénsInternos Ca2+
IP3
Ca2+
Catiões
Despolarizaçãocelular
Libertaçãoneurotransmissores
PDE
cNMP NMP
?
82 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
OBJETIVOS
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Perante o que foi introduzido acerca da interação entre os taninos e as
proteínas, em particular as proteínas salivares, várias questões/objetivos foram
abordadas ao longo deste trabalho:
Capítulo 1
A interação tanino/proteína tem sido objecto de estudo de inúmeros grupos de
investigação, resultando na publicação de numerosos artigos científicos. No
entanto, em geral estes estudos debruçam-se em apenas uma ou duas
proteínas salivares as quais são postas a reagir individualmente com um dado
tanino. Deste modo, a questão inicial que se coloca é:
a) Qual/quais das principais famílias de proteínas salivares têm mais afinidade
para os taninos condensados, quando estas estão presentes em simultâneo?
Capítulo 2
É conhecido que a interação tanino/proteína é afetada por diversos fatores,
nomeadamente, pH, etanol, carboidratos, força iónica, entre outros. Para além
disto, é também conhecido que são várias as práticas vinícolas usadas no
sentido de aumentar a concentração de taninos no vinho tinto. Deste modo, vai-
se procurar saber:
a) De que forma é afetada a interação dos taninos condensados com as
proteínas salivares numa matriz complexa de vinho tinto.
b) De que forma é que o enriquecimento do vinho em taninos condensados
influencia a adstringência percebida.
Capítulo 3
Como já foi referido nos capítulos anteriores é conhecido que a interação
tanino/proteína é afetada por diversos fatores, nomeadamente, a estrutura do
polifenol. Para além disto, a interação entre taninos condensados e/ou
hidrolisáveis e as proteínas salivares também está pouco caracterizada, em
particular no que diz respeito à afinidade da interação, solubilidade e tamanho
dos complexos formados. Deste modo, várias questões se levantam:
86 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
a) Qual dos taninos (condensado vs. hidrolisável) tem mais afinidade para as
diferentes famílias de proteínas salivares, quando estas estão presentes em
simultâneo?
b) Qual o tipo de agregados (solúvel e/ou insolúvel) e tamanho dos complexos
formados entre os taninos e os diferentes tipos de proteínas salivares?
c) De que forma, a interação entre os taninos e as proteínas salivares se podem
correlacionar com a adstringência de vinhos tintos?
Capítulo 4
A capacidade dos carboidratos inibirem a interação tanino/proteína tem sido
documentada por diversos trabalhos. No entanto, existe pouca informação sobre
o mecanismo desta inibição. Assim, pretende-se saber:
a) De que forma carboidratos frequentemente utilizados na indústria alimentar
afectam a interação, a nível molecular, dos taninos com proteínas?
b) Qual o mecanismo pelo qual diferentes carboidratos exercem o seu efeito
inibidor da interação tanino/proteína?
Capítulo 5
No seguimento do Capítulo 4, mas com o foco na interação taninos/proteínas
salivares, a principal questão que se pretende estudar neste capítulo é:
a) De que forma carboidratos frequentemente utilizados na indústria alimentar
afectam a interação de taninos condensados com as proteínas salivares?
Capítulo 6
Vários estudos de análise sensorial têm atribuído o sabor amargo como
caraterística sensorial associada a vários polifenóis. No entanto, os resultados
dos vários estudos são contraditórios, e até à data não existem dados referentes
de quais dos 25 recetores de sabor amargo humanos são ativados por alguns
polifenóis. Assim, neste trabalho vai-se procurar saber:
a) Quais os recetores de sabor amargo que são ativados por alguns compostos
polifenólicos pertencentes a diferentes classes?
b) Quais os compostos que ativam mais eficientemente os recetores?
RESULTADOS
Capítulo 1
Reactivity of Human Salivary Proteins Families Toward Food Polyphenols
Soares, S., Vitorino, R., Osório, H., Fernandes, A., Venâncio, A., Mateus, N., Amado, F., de Freitas, V.
J. Agric. Food Chem. 2011, 59, 5535–5547
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Reactivity of Human Salivary Proteins Families Towa rd Food Polyphenols
Susana Soares,† Rui Vitorino,‡ Hugo Osório,§ Ana Fernandes,† Armando
Venâncio,|| Nuno Mateus,† Francisco Amado,‡ and Victor de Freitas†
†Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
‡Department of Chemistry, University of Aveiro, Aveiro, Portugal
§Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465 Porto, Portugal
||IBB, Center of Biological Enginneering, University of Minho, Campus de Gualtar, Braga, Portugal
Tannins are well-known food polyphenols that interact with proteins, namely,
salivary proteins. This interaction is an important factor in relation to their
bioavailability and is considered the basis of several important properties of
tannins, namely, the development of astringency. It has been generally accepted
that astringency is due to the tannin-induced complexation and/or precipitation of
salivary proline-rich proteins (PRPs) in the oral cavity. However, this
complexation is thought to provide protection against dietary tannins.
Neverthless, there is no concrete evidence and agreement about which PRP
families (acidic, basic, and glycosylated) are responsible for the interaction with
condensed tannins. In the present work, human saliva was isolated, and the
proteins existing in saliva were characterized by chromatographic and proteomic
approaches (HPLC-DAD, ESI-MS, sodium dodecyl sulfatepolyacrylamide gel
electrophoresis (SDS-PAGE), and MALDI-TOF). These approaches were also
adapted to study the affinity of the different families of salivary proteins to
condensed tannins by the interaction of saliva with grape seed procyanidins. The
results obtained when all the main families of salivary proteins are present in a
competitive assay, like in the oral cavity, demonstrate that condensed tannins
interact first with acidic PRPs and statherin and thereafter with histatins,
glycosylated PRPs, and bPRPs.
KEYWORDS: Astringency, condensed tannins, proline-rich proteins, salivary proteins
92 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
� INTRODUCTION
Tannins are polyphenols commonly found in plant-derived foods. Tannins are
classically divided into two major classes: condensed tannins
(proanthocyanidins), which are polymers of catechin, and hydrolyzable tannins,
which are gallic or ellagic esters of glucose. Although tannins have benefits for
human health regarding their antioxidant properties,1-3 they can have a number of
harmful effects including decrease in growth and body weight gain,4 and inhibition
of digestive enzymes.5 Regarding their antioxidant properties, an in vitro study1
showed that independently of the degree of polymerization, condensed tannins
provide protection of lipidic peroxidation, but this effect is increased for the less
complex structures. The same behavior was shown for condensed tannins
against tumor cell viability and proliferation.
One of the characteristics of tannins is their ability to precipitate certain proteins,
and this ability has been considered the basis of the sensation of astringency on
the human palate. Astringency has been defined as a complex group of
sensations involving dryness and tightening of the oral surface and puckering
sensations of the oral cavity.6,7 It was proposed by Bate-Smith7,8 that astringency
results from the interaction of tannins with salivary proteins (SP) in the mouth,
and since then, it has been generally accepted and supported by the literature9-12
that astringency is due to the tannin-induced interaction and/or precipitation of
salivary proline-rich proteins (PRPs) in the oral cavity. However, astringency is a
very complex sensory experience, and the possible mechanisms for its
development are presently controversially discussed in the scientific
community.13 Some studies point to the importance of the interaction of salivary
glycoproteins with tannins with consequent modifications of their viscous
properties and oral cavity delubrication,14,15 and also, the salivary flow rate
appears to be correlated with astringency intensity.16 The involvement of the cells
of the oral cavity in the development of astringency has also been suggested.17
Whole saliva represents a mixture of the secretions of the major (submandibular,
sublingual, and parotid) and minor salivary glands, together with the crevicular
fluid, bacteria, and cellular debris. The secretions from the different glands have
been shown to differ considerably and to be affected by different forms of
stimulation, day time, diet, age, gender, several disease states, and
pharmacological agents.18
In general, saliva is composed of proteins, electrolytes, and small organic
compounds. With respect to the proteinaceous component, saliva similarly to
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other bodily fluids, presents a wide range of small molecular weight components
that are assigned by several authors as salivary peptides, all species with a m/z
below 20 kDa. Salivary peptides have been grouped into six structurally19 related
major classes, namely, histatins, basic proline- rich proteins (bPRPs), acidic
proline-rich proteins (aPRPs), glycosylated proline-rich proteins (gPRPs),
statherin, and cystatins. These peptides have important biological functions in
saliva associated with calcium binding to enamel, maintenance of ionic calcium
concentration (PRPs and statherin), associated with antimicrobial action
(histatins and cystatins), or protection of oral tissues against degradation by
proteolytic activity such as cystatins.20-28
There is a lot of detailed information for all these families of SP. The family of
PRPs is divided into three classes: acidic, basic, and glycosylated. More than 11
human basic-PRPs and five acidic PRP isoforms have been identified.29,30
Several closely related acidic proline-rich phosphoproteins (aPRPs) were
identified by isolation (proteins A and C,31 PRP1, 2, 3, and 432) or by studies of
protein polymorphism (identified as PIF-s, PIF-f, Db-s, Db-f, and Pa). Although
the nomenclature of this family of proteins is somehow confusing, in general, all
these proteins are isoforms.33 In general, the designations PRP1, 2, 3, and 4 will
be used here including the following alternative names: PRP1/PRP2-PIF-s, Pa,
and Db-s; PRP3/PRP4-PIF-f and Db-f. Histatins are a family of small, histidine-
rich proteins secreted by the parotid and submandibular glands.21 Statherin is
secreted by the parotid gland and is abundant in tyrosine residues.20 Cystatins
are natural inhibitors of cysteine proteinases.34
In the past years, several research groups studied the molecular basis of the
development of astringency using models bioassays with pure/isolated PRPs (or
similar proteins) and tannins.35-43 However, only few works had used whole saliva
to study the onset of astringency.44,45
Several studies have been carried out to evaluate interactions between
polyphenols and SP using different techniques, namely, SDS-PAGE,35,42,46
spectrophotometry,37,43 nephelometry,38,40,47 NMR,36,48 DLS (dynamic light
scattering),15,49 and mass spectrometry.50 Despite the several techniques applied,
there are some difficulties in correlating the perceived astringency to a single
physicalchemical phenomenon. To our knowledge, there are no experimental
evidence about the relative affinity of different PRP families (acidic, basic, and
glycosylated) individually or in a competitive/associative medium such as whole
saliva. In fact, the first studies that analyzed salivary protein (SP) interaction with
94 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
polyphenols by HPLC were done by Kallithraka and co-workers.44,51 They
analyzed human saliva before and after the interaction with polyphenols ((+)-
catechin, (-)-epicatechin, procyanidin B2, or procyanidin C1) by HPLC. However,
the SP involved in the interaction were not identified. In the present work,
chromatographic and proteomic approaches (HPLC-DAD, ESI-MS, sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and MALDI-
TOF-MS) were developed in order to study the affinity of different families of SP
by the interaction of saliva with grape seed procyanidins.
� MATERIALS AND METHODS
Materials. All reagents used were of analytical grade or better.
Hydrochloric acid and acetonitrile were purchased from Panreac Quimica, acetic
acid was purchased from Carlo Erba Reagents, sodium acetate and trifluoracetic
acid (TFA) were purchased from Fluka Biochemica, and ethanol was purchased
from AGA, Álcool e Géneros Alimentares, SA. Trizma base, glycerol, sodium
dodecyl sulfate, β-mercaptoethanol, acrylamide, N’,N’-methylenebisacrylamide,
tricine, tetramethylethylenediamine, ammonium persulfate, ammonium
bicarbonate, formic acid, SigmaMarker Wide Range, molecular weight 6.5-200.0
kDa, and sodium thiosulfate were purchased from Sigma. Bromophenol blue,
periodic acid, Schiff’s reagent, and Fuchsin-sulfite reagent were purchased from
Sigma-Aldrich. Imperial Protein Stain was purchased from Thermo Scientific
(United Kingdom). Potassium metabissulfite was from BDH Chemicals Ltd.
Sequencing-grade modified trypsin (porcine) was from Promega (Portugal). R-
Cyano-4-hydroxycinnamic acid was purchased from Applied Biosystems
(Germany). Silver nitrate, sodium carbonate, and formaldehyde were purchased
from Merck (Germany).
Grape Seed Fraction (GSF) Isolation. Condensed tannins were
extracted from Vitis vinifera grape seed extract. This extract was fractionated
through a TSK Toyopearl HW-40(s) gel column (100 mm x 10 mmi.d., with 0.8
mL.min-1 methanol as eluent), yielding two fractions according to the method
described in the literature.52 The first fraction was obtained after elution with
99.8% (v/v) methanol during 5 h (240 mL) and the second one after elution with
methanol/5% (v/v) acetic acid during the next 14 h (670 mL). Both fractions were
mixed with deionized water, and the solvent was eliminated using a rotary
evaporator under reduced pressure at 30°C and then freeze-dried. The
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procyanidin composition of fractions was determined by direct analysis by ESI-
MS (Finnigan DECA XP PLUS). The first fraction contains mainly catechins (m/z
= 290), procyanidin dimers, and their galloyl derivatives, and the second fraction
contains procyanidin dimers galloylated, procyanidin trimers, and their galloyl
derivatives and procyanidin tetramers. The latter has a mean MW of 936 and a
polymerization degree average of 3.2. Only the second fraction named grape
seed fraction (GSF) was used because it is composed of more polymerized and
galloylated procyanidins, which are known to be more reactive toward
proteins.38,53
Saliva Collection, Treatment, and Analysis. Whole human saliva was
always collected freshly at 2 pm from a healthy, nonsmoking female volunteer.
The saliva samples were taken under unstimulated conditions and after at least 1
h without ingestion of food or beverages. Collection time was standardized in
order to reduce concentration variability connected to circadian rhythms of
secretion.30 As the objective of this work was to establish a relationship between
different families of PRPs (basic, acidic, and glycosylated PRPs) saliva was
supplied only by one volunteer in order to simplify the proteomic analysis and
chromatogram division.
TFA solution (10% aqueous TFA) was immediately added to 900 µL of collected
saliva (1:90 v/v), and the solution was centrifuged at 8000g for 5 min. After
centrifugation, the supernatant (acidic saliva, AS) was separated from the
precipitate, and 90 µL was immediately analyzed on a HPLC-DAD Elite Lachrom
system (L-2130) equipped with a Vydac C8 column, with 5 µm particle diameter
(column dimensions 150 x 2.1 mm); detection was carried out at 214 to 280 nm,
using a diode array detector (L-2455). The HPLC solvents were (eluent A) 0.2%
aqueous TFA and (eluent B) 0.2% TFA in ACN/water 80/20 (v/v). The gradient
applied was linear from 10 to 40% (eluent B) in 60 min, at a flow rate of 0.30
mL/min. After this program, the column was washed with 100% eluent B for 20
min in order to elute S-type cystatins and other late-eluting proteins. After
washing, the column was stabilized in the initial conditions.
Twelve selected salivary fractions containing different families of salivary proteins
were obtained during the HPLC analysis by separate collection of the eluent
deriving from the diode array detector.
96 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
SDS-PAGE. The 12 selected salivary fractions were dried in a SpeedVac
and dissolved in 25 µL of 1x electrophoresis sample buffer (50 mM Tris-HCl, pH
6.8, 12% v/v glycerol, 4% SDS, 2.5% v/v β-mercaptoethanol, and 0.01%
bromophenol blue) and heated at 60°C for 1 h with shaking. The 12 samples
were analyzed by SDS-PAGE in a tris-tricine buffer system according to the
method of Schägger54 using 16% acrylamide resolving gel. The stacking gel was
5% acrilamide. The cathode buffer was 0.1 M Tris, 0.1 M tricine, and 0.1% SDS,
and the anode buffer was 0.2 M Tris-HCl, pH 8.9. Electrophoresis was performed
on a Bio-Rad MiniProtean Cell electrophoresis apparatus (Bio-Rad). After
electrophoresis, the gels were stained with Imperial Protein Stain, a Coomassie
R-250 dye-based reagent, or silver stained. The staining with Imperial Protein
Stain was done according to the supplier's instructions. The destaining step was
done by washing the gels with water until the bands were visible.
Molecularweights were estimated by comparison with the migration rates of
standard proteins. The silver staining procedure was done according to
O’Connell and Stults.55
Periodic Acid Schiff’s (PAS) Staining. The PAS staining was done
according to Zacharius.56 After electrophoresis, the gels were fixed overnight
(40% ethanol and 7% acetic acid) and then immersed in a solution of 1% periodic
acid for 60 min. The gels were washed with water and incubated in the dark with
Schiff’s reagent for 60 min. The gels were then washed three times (0.58%
potassium metabissulfite and 3% acetic acid).
Tryptic Digestion. The protocol used for tryptic digestion was according
to Vitorino et al.57 After electrophoresis, the bands of interest were excised from
the gel and transferred to a rack. The gel pieces were washed twice with 25 mM
ammonium bicarbonate/50% ACN, one time with 100% ACN, and after the
washes, the gel pieces were dried in a SpeedVac (Thermo Savant). Twenty
microliters of 10 µg/mL trypsin in 50 mM ammonium bicarbonate was added to
the dried residue, and the samples were incubated overnight at 37°C. After the
incubation, the extraction of tryptic peptides was performed by the addition of
10% formic acid/50% ACN three times, followed by lyophylization in a SpeedVac
(Thermo Savant). Tryptic peptides were resuspended in 10 µL of a 50%
ACN/0.1% formic acid solution.
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Mass Spectrometry Analysis. The AS and the 12 HPLC salivary
fractions were analyzed by LC-ESI-MS and MALDI-TOF/TOF, respectively. The
AS was analyzed by LC-ESI-MS with the HPLC analysis performed on a liquid
chromatograph (Hewlett-Packard 1100 series) equipped with the same column
referred previously. The solvents and the HPLC gradient used were the same as
those reported above for the HPLC analysis. Double online detection was done in
a photodiode spectrophotometer and by mass spectrometry. The mass detector
was a Finnigan LCQ Deca (Finnigan Corporation, San Jose, CA) equipped with
an API source, using electrospray ionization (ESI) interface. Both the auxiliary
and the sheath gases were a mixture of nitrogen and helium. The capillary
voltage was 15 V and the capillary temperature 325°C. Spectra were recorded in
positive ion mode between m/z 250 and 2000 Da. The 12 HPLC salivary fractions
were analyzed by MALDI-TOF/TOF, using a 4800 MALDI-TOF/TOF analyzer
(Applied Biosystems, Foster City, CA) in the linear mode to obtain the molecular
weight of larger species and in the reflectron mode to obtain the peptide
sequence of small species. In linear mode, all samples were mixed (1:1) with a
matrix solution (3 mg/mL) of α-cyano-4-hydroxycinnamic acid matrix prepared in
50% ACN/0.1% TFA. Aliquots of samples (0.35 µL) were spotted onto the MALDI
sample target plate, and spectra were obtained in the mass range between 1500
and 70000 Da with ca. 1000 laser shots.
Top Down Analysis. Characterization of smaller species present in each
fraction was performed in the positive ion reflector mode using the above matrix
composition. Each fraction was applied in triplicate and MS spectra were
obtained in the mass range between 800 and 4500 Da with ca. 1200 laser shots.
A fragmentation voltage of 2 kV was used for MS/MS analysis. Automated
acquisition of MS and MS/MS data in the batch mode employed an interpretation
method with the following settings: number of shots per spot =10; minimum S/N
filter =50 to select peaks for MS/MS analyses, chromatogram peak width =3, and
fraction resolution of precursor exclusion window =200 fwhm.
Peptide Mass Fingerprint (PMF). SDS-PAGE bands were digested with
trypsin, and the generated tryptic peptides were analyzed. Peptide mass spectra
were obtained on a MALDI-TOF/-TOF mass spectrometer (4800 Analyzer;
Applied Biosystems, Foster CA) in conditions similar to those in the top-down
analysis in the positive ion reflector mode automated acquisition of MS and
98 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
MS/MS data. In this case, the interpretation method excludes the trypsin
autolysis peaks.
Data Analysis. The spectra were processed and analyzed by the Global
Protein Server Workstation (Applied Biosystems, Foster City, CA, USA), which
uses internal Mascot software (v.2.1.0.4, Matrix Science Ltd., U.K.) for
protein/peptide identification based on peptide mass fingerprints and MS/MS
data. The search was performed against the SwissProt protein database (march
2009, 428650 entries) for Homo sapiens. A MS tolerance of 30 ppm was found
for precursor ions and 0.3 Da for fragment ions, as well as two missed cleavages.
In the case of top-down, no enzyme was selected, and in the case of PMF,
trypsin was selected. Protein identifications were considered as reliable when the
MASCOT score was >70 (the MASCOT score was calculated as – 10 x log P,
where P is the probability that the observed match is a random event). This is the
lowest score indicated by the program as significant (P < 0.05) and indicated by
the probability of incorrect protein identification. In order to estimate the false
discovery rate (FDR) and considering the repetitive PRP motif, a random decoy
database was created for all SwissProt and internal database entries resulting in
10% of FDR (false positive peptides/(false positive peptides + total peptides)) x
100. Unique peptides retrieved from the FDR search were considered.
Protein-Tannin Interaction. The AS sample was analyzed by HPLC-
DAD before and after the interaction with increasing concentrations of GSF. The
control condition was a mixture of AS (150 µL) and acetate buffer 0.1 M, pH 5.0,
and 12% ethanol (50 µL) (final volume 200 µL). For the experiments with GSF,
the necessary volume (10 to 39 µL) of a GSF stock solution (2.66 mM) was
added to AS (150 µL) to obtain the desired final concentration, plus the volume of
acetate buffer to make the final volume 200 µL. GSF was tested in the following
final concentrations: 0.133, 0.306, 0.399, 0.505, 0.599, 1.300 mM, and each
concentration was an independent experiment. After shaking, the mixture reacted
at room temperature (20°C) for 5 min and then was centrifuged (8000 g, 5 min).
The supernatant was injected into the HPLC-DAD. These experiments were also
made in large scale with two different concentrations of GSF 0.133 and 0.232
mM to do semipreparative HPLC-DAD. The 12 fractions were collected by
recovering of the eluent deriving from the diode array detector. The 12 fractions
were dried in a SpeedVac and analyzed by SDS-PAGE.
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� RESULTS AND DISCUSSION
In order to observe in vitro the effect of condensed tannins in quantitative and
qualitative changes of the HPLC profile of human saliva, the first major task is the
identification of the proteins corresponding to the HPLC profile. The HPLC
method and identification described herein were based on the work developed by
Messana et al.30
Several HPLC analytical problems may appear related to the quantity of mucins
and other high molecular weight glycoproteins present in saliva. Indeed, these
proteins can block and contribute to the degeneration of HPLC columns and poor
reproducibility. In order to overcome these problems, saliva samples were mixed
immediately after collection with aqueous TFA (final concentration 0.1%). This
acidic treatment causes the precipitation of several high molecular weight SP
(such as α-amylases, mucins, carbonic anhydrase, and lactoferrin) contributing to
a decrease of the viscosity and also preserves sample protein composition since
TFA partially inhibits intrinsic protease activity.30,58 Peptides and proteins such as
histatins, basic and acidic proline-rich proteins (PRPs), statherin, cystatins, and
defensins are soluble in acidic saliva (AS) solution and may be directly analyzed
by RP-HPLC. The use of TFA provides a satisfactory compromise between high
ion-pairing strength for chromatographic separation and protein ionization for ESI
analysis. The HPLC chromatogram of this AS solution at 214 nm is presented in
Fig.1.1A, and the profile was similar to the one previously described in the
literature by Messana et al.30
On the basis of the identification of the different salivary proteins, the HPLC
chromatogram of the AS solution was roughly divided into 12 peptide salivary
fractions.
Identification of Salivary Proteins. In order to identify the main SP of
each HPLC fraction, the AS solution was analyzed by several techniques such as
RP-HPLC-ESI-MS, MALDI-TOF/TOF, and SDS-PAGE-MALDI-TOF/TOF. The
total ion current (TIC) chromatogram profile obtained by HPLC-ESI-MS (Fig.1.1B)
is similar to the chromatogram obtained at 214 nm (Fig.1.1A).
These identifications were first achieved by LC-MS analysis and deconvolution of
the averaged ESI mass spectra. An example of the deconvolution process is
presented in Fig.1.2 for the peak eluted at 40 min in Fig.1.1B.
100 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
A
B
Fig.1.1(A) Typical RP-HPLC profile detected at 214 nm of the acidic saliva (AS) solution of whole human saliva. The pointed lines and numbers show the ranges and names assigned to each HPLC fraction, with the outline of the main proteins identified for each HPLC fraction (Table 1). At the bottom, there is the distribution of the different families of salivary proteins along the chromatogram. *, fragments of proteins; His, histatin; Stat, sthaterin; (di-, mono-, n-)-phosp, (di-, mono-, non-)-phosphorylated; bPRP, basic proline-rich protein; gPRPs, glycosylated proline-rich proteins, aPRPs, acidic proline-rich proteins. (B) Total ion current (TIC) profile of AS solution collected by the ion-trap mass spectrometer (ESI-MS).
15 20 25 30 35 40 45 50 55 60 650
5
10
15
20
25
x 1041 2 3 5 6 7 10 124 8 9 11
bPRPs gPRPs aPRPs Statherin
IB-8
bP-
J, IB
-8c,
His
3*,
bPR
P2*,
bPR
P3*
IB-9
, IB
-4, H
is 7
, 8 a
nd 9
, His
3*
IB-6
, His
3*
His
5, H
is 3
*, P
-B*
bPR
P3
bPR
P3, I
I-2, I
B-1
His
3
PRP3
, PR
P1
His
1
Sta
t (di
-, m
ono-
, n-p
hosp
), P
eptid
e P-
B
IB-6
, IB
-4, I
B-9
, His
3*
t (min)
Ab
s (m
Au
)
Legend:* - fragments of proteinsHis - histatinbPRP - basic Proline-rich ProteinaPRP - acidic Proline-rich ProteingPRP - glycosylated Proline-rich ProteinStat - Statherindi-, mono-, n-phop - di-, mono-, n-phosphorilated
15 20 25 30 35 40 45 50 55 600
20
40
60
80
100
t (min)
Re
lativ
e A
bund
ance
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Fig.1.2 Deconvolution process for fraction 10. (A) ESI mass spectrum obtained by the average of 77 mass spectra collected in the 38.49-40.39 min range during the HPLC separation reported in the TIC profile (Fig.1.1B). (B) The bottom panel reports the deconvolution of the upper ESI-MS (A).
In general, the deconvolution of the average TIC spectra of each area allowed
the identification of 27 peptides shown in Table 1.1
Table 1.1. Experimental Masses (Da) Detected in the Twelve Chromatographic Fractions by RP-HPLC-ESI-MS, and MALDI-TOF/TOFa
HPLC fraction Peptide Theoric
mass Exp. ESI Exp. MALDI*
1 IB-8b 4371 4369.9 4369.1
bPRP2 (379-407,379-408,379-412)
- - 2817.3,2874.1,3271.1
bPRP4(261-305)
- - 4336.7
bPRP1(236-279)
- 4392.9 4392.5
2 His 3(33-43,24-36,20-36)
- - 1434.5,1750.8,2161.1
bPRP2(379-412,368-407,368-408)
- - 3271.3,3963.6,4020.6
bPRP3(268-288,219-261)
- - 2027.8,4396.5
P-J 5945 5942.9 5949.3
IB-8c 5843 5841.5 5847.7
bPRP1(94-139,235-261)
- - 2692.3,4376.5
IB-8b 4371 4369.6 4369.2
bPRP4(263-307)
- - 4398.5
His 8 1562 1561.7 1562.7
His 10 1719 - 1718.8
3 IB-9 6023 6023.3 6029.9
IB-4 5590 5589.6 5593.5
His 7 1718 - 1718.8
His 3(42-51,30-43,20-36,20-37,20-39)
- - 1264.5,1846.9,2161.1,2298.0,2522.1
His 8 1562 1561.7 1562.7
His 9 1875 - 1874.9
4 His 3(24-33,20-33,30-43)
- - 1356.7,1766.8,1846.9
IB-5 6950 6949.1 6957.4
IB-9 6023 6022.7 6028.2
IB-4 5590 - 5593.1
5 His 3(20-33,37-51,29-43,26-43,24-
- - 1766.9,1918.8,2009.9,2341.1,2405.2,2815.3
IB-6 11517 11515.6 -
Peptide P-B (57-67)
- - 1161.5
HPLC fraction Peptide Theoric
mass Exp. ESI Exp. MALDI*
6 His 5 3036 3036.6 3035.3
His 3(24-43, 34-51, 24-44)
- 2625.0 2625.1, 2312.8, 2781.2
Peptide P-B(55-67)
- - 1315.6
His 6 3192 3192.0 3191.4
7 bPRP3 - - -
Unknown - 23467.0 -
His 3(55-69, 33-51)
- 1920.8, 2459.9
Peptide P-B(55-67, 55-69)
- 1315.7, 1469.7
8 bPRP3 - - -
Unknown - 23467.0 -
II-2 (phos.) 7608 7607.1 7613.7
IB-1 9590 9590.7 -
His 3(33-51)
- - 2459.9
Peptide P-B(68-79)
- - 1200.6
9 His 3 4061 4061.0 4061.0
10 PRP1 (di-phos) 15514 15512.0 -
PRP3 (di-phos) 11161 11160.0 -
Cys S 14347.0 -
Cys SN - -
11 His 1 4926 4927.3 4932.1
His 1(37-57, 34-57, 33-57, 32-57)
- 2617.9,3012.1,3159.2,3287.3
His 2(31-57)
3443 3441.4 3443.6
12 Statherin (di-phos) 5378 5378.7 5381.5
Statherin (mono-phos) 5299 - 5299.9
Statherin (n-phos) 5219 - 5216.9
Peptide P-B 5789 5791.2 5796.3 a The experimental masses obtained by ESI-MS were compared to the theoretical masses of human salivary proteins reported in international data banks,and the experimental masses obtained by MALDI-TOF/TOF were identified as referred to in the Materials and Methods section. The superscriptednumbers are the residue numbers for the peptides identified for each protein; the proteins in bold are the main proteins for each fraction. Exp. ESI,experimental masses obtained in ESI analysis; * masses obtained by MS/MS and linear MALDI. Swiss-prot code: IB-8b, PRP1 and PRP3 (P02810); IB-8c and bPRP2 (P02812); bPRP1, P-J, IB-4, IB-6 and II-2 (P04280); bPRP3 (Q04118); bPRP4 and IB-5 (P10163); IB-9 (P02811); IB-1 (P04281); peptide P-B (P02814); His 3, 5, 6, 7, 8, 9 and 10 (P15516); His 1 (P15515); Cys S (P010136); Cys SN (P01037); statherin (P02808).
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Table 1.1 reports all the proteins/peptides identified in the 12 HPLC fractions of
the AS solution. It also indicates the experimental masses detected by RP-HPLC-
ESI-MS, MS/MS, and MALDI-TOF (linear or after trypsin digestion). Using these
different techniques, we were able to identify several peptides/small proteins.
From the results presented in Table 1.1, it can be observed that the different
techniques are complementary to identifying the major SP, probably because the
peptides/proteins ionized differently according to the technique used.
The assignment of experimental masses to peptides/proteins was performed
against the SwissProt protein database for Homo sapiens and also taking into
account the previous identifications and experimental masses of these SP by
ESI-MS and MALDI-TOF approaches by other groups, using similar experimental
conditions.30,59
In order to confirm the assignment of some peptides performed by ESI-MS data
and to increase the information of each HPLC fraction, the 12 chromatographic
fractions displayed in Fig.1.1A were isolated individually by HPLC and analyzed
by different approaches: (a) sequencing of peptides with MW below 5000 Da by
direct MALDI-TOF MS/MS of each fraction, (b) SDS-PAGE of each fraction using
different staining procedures, (c) identification of peptides/proteins with MW
above 5000 Da by trypsin digestion of the bands after the SDS-PAGE analysis,
and (d) linear MALDI-TOF analysis of each fraction. This strategy has been used
by expert groups in this area.30,59
It is also important to refer that for glycosylated PRPs (gPRPs) the identification
is even more difficult. The lack of the “total” molecular weight of these proteins
including the sugar moiety makes the direct identification by ESI more
complicated. Nevertheless, the digestion with trypsin and analysis by MALDI-TOF
as well as Schiff’s staining of the SDS-PAGE gels (see below) make it possible to
identify a protein (bPRP3) belonging to the gPRPs class in HPLC fractions 7 and
8.
It is interesting to notice that the deconvolution of the ESI average spectra for
HPLC fractions 7 and 8 gives a protein with a mass of 23467 Da, which could not
be identified since there is no report in the databases or elsewhere in the
literature. Messana et al. 30 have also identified a SP with this mass, but they
were not able to identify this protein. However, they suggested that this unknown
protein may be a putative basic PRP candidate since that protein was resistant to
trypsin cleavage, which is a main characteristic of this kind of proteins.
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The relative quantification of the proteins identified in each fraction based on the
intensities of the peaks is inaccurate due to differences in ionization ability of the
different proteins but could be a good approximative approach.
For the peptides identified by ESI-MS/MS, the extracted ion current (XIC)
strategy was applied, which compares the ion currents originated by the
peptides. The deconvolution process considers the major ions of ESI spectra
giving the relative quantity of each protein identified. On the basis of that
approach, the major proteins of each HPLC fraction were identified and are
indicated with bold text in Table 1.1.
From the results presented, it is possible to observe that several proteins were
found, namely, IB-1, IB-8b, IB-4, IB-9, IB-5, IB-6, bPRP3, II-2, PRP1, PRP3,
histatin 5, 7, 8, and several forms of statherin; small fragments were assigned as
histatin 3, bPRP1, bPRP2, and bPRP3 fragments. These observations had been
previously reported by Vitorino et al.5
In summary, the chromatogram is roughly divided into four regions corresponding
to the different families of SP. The first zone (1 to 6) comprises proteins that
belong to the classes of bPRPs and His. The second region (7 and 8) comprises
mainly a gPRP, the bPRP3. The third region (10) has as main proteins the
aPRPs. The last region (12) has phosphorylated and nonphosphorylated forms of
statherin.
SDS-PAGE. In order to obtain additional information and to do trypsin
digestion of the SP of each HPLC fraction, SDS-PAGE of each fraction was
carried out, and the results are presented in Fig.1.3.
Fig.1.3 SDS-PAGE of the 12 HPLC fractions isolated from the HPLC after the injection of the AS solution. The molecular weight markers were substituted by lines, and the molecular mass is expressed in kDa as marked on the left side. The gels were stained by with Imperial Protein Stain, a Coomassie R-250 dye-based reagent.
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Although SP can be separated and analyzed by SDS-PAGE, there are some
particular aspects that have to be considered, especially regarding PRPs. PRPs
stain poorly with conventional Coomassie Blue and even with silver staining
procedures. However, when Coomassie Blue R-250 is used and organic solvents
are omitted from the destain solution, PRPs stain pink-violet; regarding silver
staining, some improved staining can be achieved by a modified silver staining
procedure. Nevertheless, for PRPs the sensitivity is lower than that for other
proteins.60 From the results presented in Fig.1.3, it is possible to observe that for
fractions 1 (F1) and 12 (F12) there were no bands detected, even after testing
different sample concentrations and different staining methods with these
fractions.
It is also necessary to be cautious with the apparent molecular weight of this kind
of proteins on SDS-PAGE gels; several reports have described that PRPs do not
migrate in SDS-PAGE at the expected molecular weight.61,62 The high proline
content presumably increases the rigidity of the protein, which makes the protein
migrate slower in SDS-PAGE than globular proteins with the same molecular
weight (MW).
Unusual mobility on SDS-PAGE is one of the characteristics of intrinsically
unstructured proteins. Because of their unusual amino acid composition, PRPs
bind less SDS than usual, and their apparent MW on SDS-PAGE gel is often 1.2-
1.8 times higher (MW factor) than the real one.61
For fraction 2 (F2), amajor band smaller than 6.5 kDa is visible, which could
correspond to several peptides previously identified by ESI-MS and MALDI-TOF-
MS for this fraction, peptides deriving from histatin 3, bPRP2, and bPRP3 with a
molecular weight between 1.4 and 4.4 kDa. Besides these, peptides P-J and IB-
8c with a molecular weight of 5.9 and 5.8 kDa, respectively, were also identified
for this fraction. These peptides could correspond to a small smear just below the
6.5 kDa marker.
For fraction 3, it is possible to observe a significant smear at the bottom of the gel
and a high molecular weight band. The smear at the bottom of the gel spreads
below the 6.5 kDa. The upper part of the smear could correspond to IB-9 and IB-
4 proteins. These proteins have molecular weights of 6.0 and 5.5 kDa,
respectively, and they could migrate in the area of the 6.5-7.0 kDa (in this case,
the MW factor is 1.2). However, the other identified peptides, histatins 7, 8, 9,
and fragments of histatin 3, have lower molecular weight around 2.1 kDa, and
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therefore, they will migrate at the bottom of the gel. Regarding the band at the top
of the gel, its identification was not possible.
For fraction 4 (F4), the results presented are quite similar to F3. There is a
significant smear at the bottom of the gel. At the top of the smear, there is
probably IB-5, IB-9, and IB-4 with molecular weights of 6.9, 6.0, and 5.6 kDa,
respectively. These proteins could migrate in the area of 6.7 to 8.0 kDa (MW
factor of 1.2).
For fraction 5 (F5), it is possible to observe a pink-violet band in the region of 20
kDa. This band could be IB-6 protein, which was identified in this fraction by
deconvolution of the average ESI. Indeed, the real MW is 11.5 kDa, but as
referred to above, it could migrate with an apparent MW of 11.5 x 1.8 (MW factor)
= 20.7 kDa. This identification is further supported by the pink-violet color
(characteristic of PRPs) displayed by this band. This fraction presents one smear
at the bottom of the gel. At the bottom of this smear could be the several
fragments of histatin 3 previously identified (MW around 2.0 kDa), but there were
no identified peptides/proteins around 6.5 kDa. Therefore, the upper part of the
smear, as well as the band at 66 kDa, was not identified.
Fraction 6 (F6) presents two important bands. The analysis of the intense low
molecular weight band revealed the presence of histatin 5 (3.0 kDa) and several
fragments of histatin 3 at 2.5 kDa. The other band at the region of 45 kDa is
probably one protein that appears in the ESI analysis with 23.4 kDa (will migrate
with an apparent MW of 42 kDa). This protein seems also to be eluted in fraction
7 collected from the HPLC. Messana et al.30 have also verified the existence of
that protein, and they suggested that it could correspond to basic PRPs.
For fractions 7 (F7) and 8 (F8), there is a main band at the top of the gel with an
apparent MW of around 66 kDa. The analysis of this band by MALDI-TOF after
trypsin digestion allowed the identification of bPRP3 protein, a glycosylated
bPRP. The presence of sugars in protein structure was confirmed by periodic
acid schiff (PAS) staining, which is a basic procedure for the analysis of
glycoproteins (Fig.1.4). Basically, this procedure stains the sugar moieties of
glycoproteins yielding magenta bands with a colorless background.
Only these two fractions gave positive results to the PAS staining, which
indicates that glycosylated proteins are eluted only in those two fractions
(Fig.1.4).
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Fig.1.4 SDS-PAGE of each HPLC fraction isolated from the HPLC after the injection of the AS solution. The molecular weight markers were substituted by lines and the molecular mass is expressed in kDa. The gels were stained by the periodic acid Schiff procedure in order to visualize glycoproteins.
Although two other proteins, namely, II-2 and IB-1, have been identified in F8 by
ESI deconvolution, they do not appear in the SDS-PAGE gel probably because
their quantity is below the detection limit of the Coomassie stain.
For fraction 9 (F9), only histatin 3 (4.0 kDa) was identified in the SDS-PAGE gel.
Fraction 10 (F10) presents one band with an apparent MW of 24-29 kDa. The
previous analysis of this fraction by ESI-MS indicated the presence of two
aPRPs, PRP1 and PRP3 (15.5 and 11.2 kDa, respectively). Considering the
factor 1.8 into the apparent MW, we determined that these proteins would appear
in the SDS-PAGE gel in the zone of 20-27 kDa, which is in agreement with the
results obtained.
The band observed at the bottom of the SDS-PAGE gel of fraction 11 (F11) could
be attributed to the identified histatin 1 (4.9 kDa).
For fraction 12 (F12), different isoforms of statherin were identified, namely, di
(5.4 kDa)-, mono (5.3 kDa)-, and nonphosphorylated (5.2 kDa) forms. The
peptide P-B (5.8 kDa) was also identified, but surprisingly no bands were
detected in the SDS-PAGE gel stained with a Coomassie based dye. However,
the silver staining revealed the presence of a smear in the 6.5 kDa region;
probably this smear corresponds to the several isoforms of statherin and to the
peptide P-B since that these proteins have approximately the same molecular
weight.
Interaction between Salivary Proteins and Condense d Tannins. The
experiments in this study were all performed in buffer with 12% ethanol to mimic
a model wine and at pH of 5.0 as has already been referred to as a pH at which
salivary proteins strongly interact with condensed tannins63 and correspond to an
intermediary pH between wine pH (3.4) and saliva pH (7.0). Indeed, salivary pH
drops with the ingestion of acidic drinks, andthe degree of acidity in saliva
depends on the sampled volume, buffering capacity, and mode of drinking.64,65 In
108 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
order to compare the reactivity of the identified proteins with condensed tannins,
different concentrations of grape seed fraction (GSF) were mixed with acidic
saliva (AS) solution, and after centrifugation, the supernatant was analyzed by
HPLC. The profiles of AS solution before and after the interaction with GSF are
shown in Fig.1.5.
Fig.1.5 RP-HPLC profile detected at 214 nm of the AS solution before (AS control) and after the interaction with increasing concentrations of grape seed fraction (GSF).
The results presented in Fig.1.5 show that the HPLC profile of the AS solution is
interestingly affected by the interaction with GSF. Effectively, GSF interacts in a
different way with the different groups of proteins. While the areas of HPLC
fractions 10 and 12 declines significantly with the lowest GSF concentration, the
other fractions' areas remain relatively constant. It is important to mention that the
mixture of AS solution with GSF always resulted in the formation of insoluble
precipitates, which increased along with GSF concentration.
The decrease of percentage area for each HPLC fraction after the AS solution
interaction with increasing concentrations of GSF is summarized in Fig.1.6.
From the results presented in Fig.1.6, it is possible to observe that fractions 10,
11, and 12 interact more importantly with GSF (Fig.1.6C). For the lowest GSF
concentration (0.133 mM) assayed, the area of these fractions was much
reduced to 20% for fractions 10 and 12 and to 40% for fraction 11. With the
increase in GSF concentration (0.505 mM), fractions 5 and 6 were also greatly
reduced to 30% (Fig.1.6B). However, regarding the fractions 1 to 4 and 9, only
higher GSF concentrations significantly reduced their areas (Fig.1.6A e C). The
results were gouped by SP families (Fig.1.6D), and it is possible to observe that
the major SP families that interact with GSF were aPRPs and statherin. For the
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SP families gPRPs and bPRPs, their interaction depends on the concentration of
GSF. For the lowest concentration of GSF, the family that interacts less with GSF
is gPRPs. However, increasing the concentration of GSF leads to a more
significant interaction with gPRPs. Among the three main families of SP, for the
highest GSF concentration, bPRPs are the ones that interact less with GSF.
A B
C D
Fig.1.6 Percentages of area decrease of each HPLC fraction after the interaction of AS solution with increasing concentrations of GSF (A, B, and C). Percentages of area decrease for each family of salivary proteins (D).
In order to obtain additional information about which SP of each HPLC fraction is
interacting with tannins, SDS-PAGE of AS before and after the interaction with
GSF was carried out. The resulting precipitate was also analyzed by SDS-PAGE,
which is widely used for the analysis of human fluids containing proteins as well
as for the study of tannin protein interactions.41 In fact, gel electrophoresis is a
useful tool for assessing tannin binding proteins in human saliva because the
proteins dissociate from the insoluble tannin/protein complexes in the presence
of SDS and can be visualized and identified in the gel.
0.00 0.25 0.50 0.75 1.00 1.25 1.500
20
40
60
80
100
1
2
3
4
% area
HPLC pool:
GSF (mM)
0.00 0.25 0.50 0.75 1.00 1.25 1.500
20
40
60
80
100 5
6
7
8
% area HPLC pool:
GSF (mM)
0.00 0.25 0.50 0.75 1.00 1.25 1.500
20
40
60
80
100
9
10
11
12
% area
HPLC pool:
GSF (mM)
0.00 0.25 0.50 0.75 1.00 1.25 1.500
20
40
60
80
100
bPRP
gPRP
aPRP
statherin
% area
Salivary protein family:
His 1
GSF (mM)
110 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.1.7 presents the results of the SDS-PAGE of AS before and after the
interaction with 0.133 and 0.232 mM of GSF.
The results presented in Fig.1.7 clearly show that the proteins existing in the
control AS precipitate by GSF. It is also perceptible that increasing GSF
concentration also increases the amount of SP that appears in the respective
precipitate.
Fig.1.7 SDS-PAGE of the AS solution before and after the interaction with two concentrations of grape seed fraction (GSF) (0.133 mM and 0.232 mM). The molecular weight markers were substituted by lines, and the molecular mass is expressed in kDa.
The HPLC fractions that previously showed a significant interaction with GSF
(Fig.1.5 and Fig.1.6), namely, fractions 5 to 12, were also analyzed by SDS-
PAGE after the interaction with GSF. The same saliva sample was incubated with
two different concentrations of GSF (0.133 and 0.232 mM) and centrifuged, and
the supernatant was analyzed by HPLC. After HPLC collection, the fractions
were dried in a SpeedVac, dissolved in the same control volume, and analyzed
by SDS-PAGE. The results are presented in Fig.1.8. The gels for F5 and F12 are
absent because it was not possible to see any band in the control assay.
Fig.1.8 SDS-PAGE of HPLC fractions 6 to 11 isolated from the HPLC after the injection of the AS solution before (C) and after the interaction with two concentrations of grape seed fraction (0.133 and 0.232 mM). The molecular weight markers were substituted by lines, and the molecular mass is expressed in kDa.
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From the results presented, it is possible to see that for F6 the band that appears
at 45 kDa, probably corresponding to a bPRP as previously referred, and
disappears with the increase in GSF concentration. Similar behavior was
observed for the same protein that also appears in F7.
The intensity of the band of bPRP3 (near the 66 kDa) in F7 decreases slightly
with increasing GSF concentration. However, the effect of GSF in this
protein/band is more visible in F8. In general, these results are in agreement with
the ones obtained in the HPLC analysis (Fig.1.5); for F7 and F8, only with GSF
concentration above 0.5 mM is the profile greatly reduced. For the lowest GSF
concentrations, the HPLC profile of these fractions is not very affected.
The band corresponding to aPRPs in F10 disappear after 0.232 mM GSF
concentration. This result is in agreement with the results obtained from the
HPLC analysis (Fig.1.5F). Furthermore, the band's intensity corresponding to
histatin 3 and 1 in F9 and F11, respectively, is reduced importantly with the
increase in GSF concentration. Overall, the main family of SP that disappears
considerably after GSF incubation is the aPRPs (PRP1 and PRP3) present in
HPLC fraction 10.
These results seem to indicate that statherin and especially acidic PRPs (aPRPs)
have a high relative affinity toward condensed tannin complexation compared to
that of the other SP, in a competitive assay at pH 5.0. The acidic proteins PRP1
and PRP3 have 150 and 106 aminoacid residues, respectively. The first 106
residue sequence from PRP1 corresponds to PRP3. The acidic character of
these proteins is confined roughly to the first 30 amino acids at the N-terminal
due to the presence of many aspartic and glutamic acid residues. The remaining
part is basic and, similarly to basic PRPs, shows repeated sequences of proline
and glutamine.22
In general, PRPs have open randomly coiled structures. These open structures
allow the exposure of peptide carbonyl groups to hydrogen bonding as well as
the exposure of proline residues to act as binding sites for tannin by hydrophobic
interaction of the aromatic portion of tannin with the pyrrolidine structure of
proline. Nevertheless, in the case of aPRPs, previous studies on calcium binding
to aPRPs pointed to an interaction between the N- and C-terminal regions of the
proteins (possibly by electrostatic forces) indicating that the structures are not so
open when compared to those of other PRPs.66,67 However, at pH 5.0 as studied
herein, which is close to the isoelectric point (pI) of PRP1 and PRP3 (4.63 and
4.14, respectively), several acidic amino acids in the N- terminal part are not
112 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
expected to be charged, reducing the interaction with C-terminal and opening the
structure of aPRPs.42 Consequently, proline residues are readily accessible to
promote hydrophobic interaction with tannins. However, the presence of an
important number of carboxyl groups in aspartic and glutamic amino acid
residues in the N-terminal part may contribute importantly to strengthen the
interaction with tannins through hydrogen bonds.
Concerning the basic PRPs (bPRPs), these results generally show a significant
decrease of their area but only for the highest GSF concentrations (0.599 and 1.3
mM) and when almost all the other proteins have been depleted. These results
seem to indicate that the bPRPs have a low relative affinity toward condensed
tannin complexation compared to that of the other SP, in a competitive assay at
pH 5.0. Not totally in agreement with this, Lu and Bennick35 have measured the
amount of condensed tannins (crude quebracho tannin) and tannic acid
precipitated by pure bPRPs (IB-1, IB-4, IB-8b, and IB-6), aPRPs (PIF-s), and
gPRPs (gPRPs) at pH 7.4. These authors have observed that each bPRP
precipitated a higher quantity of tannins compared to that of the other PRPs.
Also, they showed that there were only small differences in the tannin-
precipitating ability of various bPRPs of different sizes or sequences, indicating
that, although there is considerable phenotypic variation of PRPs, it is not likely to
cause marked individual variation in tannin-binding ability. However, it is difficult
to compare the results obtained by these authors with the ones obtained herein
because first of all, the tannins used are completely different from ours, being a
complex mixture and not properly characterized. Also, these authors measured
the quantity of tannin precipitated, which depends not only on the PRP affinity but
also on the stoichiometry of the complexes. However, the pH used by these
authors (pH 7.4) and herein (pH 5.0) are substantially different, which will
probably differently affect the interaction for the reasons mentioned above.
The interaction of GSF with phosphorylated forms of statherin (HPLC fraction 12)
is also important. The results yielded by the HPLC analysis show a significant
interaction with statherin being one of the main SPs that interacts with condensed
tannins in the conditions described here. According to our knowledge, only Nayak
and Carpenter68 showed that statherin is precipitated by polyphenols, namely, tea
polyphenols. Statherin is abundant in tyrosine residues and is phosphorylated at
Ser2 and Ser3. Several variants have been identified and are derived both by
alternative splicing and posttranslational modifications. The presence of protic
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113
groups (phosphates) with acidic character may favor the hydrogen bond
interactions which probably explain statherin's high affinity to tannins.
Regarding the glycoproteins, bPRP3 did not show a high interaction at lowest
GSF concentration. Nevertheless, increasing GSF concentration leads to a
significant decrease of that protein. For GSF concentration of 1.3 mM, the area of
HPLC fractions 7 and 8 has been decreased about 60%, while other HPLC
fractions (bPRPs 1 to 6) only decreased about 40%. As previously mentioned,
bPRP3 is a glycosylated protein that contains about 50% carbohydrate, which is
composed of highly fucosylated N-linked saccharides.69 Glycosylated PRPs
(gPRPs), which have been implicated in the oral epithelium lubrication,70 were
shown here to resist precipitation compared to that of aPRPs, histatins, and
statherin. Sarni-Machado et al.46 have also observed that low concentrations of
condensed tannins (mainly (+)-catechin, (-)-epicatechin, and epicatechin gallate)
first precipitate lower molecular weight SP and only for higher concentrations
precipitate glycosylated PRPs. More recently, the same authors14 have observed
that the glycosylation of human PRPs favors the formation of soluble complexes
and reduces tannin precipitation with regard to tannin amounts. This is in
agreement with Pascal et al.15 who have stated that protein glycosylation (gPRP,
similar to II-1 herein) prevented PRP condensed tannin precipitation when
compared to that of a nonglycosylated PRP (IB-5). These authors have
concluded that gPRPs are effective in binding tannins but that these interactions
do not necessarily result in precipitation. In conclusion, although gPRPs are not
readily precipitated by condensed tannins compared with other SP, they can form
complexes with tannins modifying the rheological properties of saliva such as
viscosity and consequently astringency.15
The GSF also interacts importantly with histatin 1 (HPLC fraction 11), as shown
both by HPLC analysis and by SDS-PAGE. The results also showed an
interaction of tannins with histatins 3 and 5 (HPLC fractions 6 and 9), although
not as significant as the one with histatin 1. Contrary to these results, Naurato
and co-workers71 have found that His 1 bound only about half the amount of
condensed tannin (crude quebracho tannin and epigallocatechin gallate) than
histatins 3 and 5, and there was no difference between these two latter in their
ability to precipitate condensed tannins. Moreover, they found that histatins 3 and
5 share the same condensed tannin-binding region, with more tannin binding to
the C-terminal region. However, all the experimental conditions and methodology
114 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
used were quite different, namely, pH (7.4), buffer (isotonic barbital buffer), and
temperature of interaction (37°C).
Regarding the histatin (His) group, their interaction with tannins is well known.42,71
They comprise a group of structurally related, small histidine-rich proteins found
only in the saliva of humans and some monkeys. Twelve His, named His 1 to 12,
have been isolated from human saliva and their primary structures determined.21
The most prominent members are His 1, 3, and 5, and they account for 85-90%
of this family. Histidine is the prominent amino acid, accounting for about 25% of
all residues and, together with basic amino acids, makes up 30% to 75% of total
amino acids. In contrast to PRPs, they contain no proline residues except for a
single residue in His 1. It is interesting that, among the identified histatins, it was
His 1 that was the most effective in binding polyphenols.
In conclusion, regarding the interaction between tannins and different families of
SP, the published data at present is somehow controversial. Some authors have
stated that all salivary PRP families have similar affinities toward different
condensed tannins at a molecular level by means of a competition assay, while
others stated that basic PRPs are the main family of SP that interact with
condensed tannins. However, the experimental conditions described in the
literature are often different, and the results should be analyzed carefully.
The results present herein provide important insights concerning the influence of
the different families of SP in the development of astringency. In fact, when all
the main families of SP are present in a competitive assay, like in the oral cavity,
they demonstrate that tannins interact first with aPRPs and statherin and also
interact significantly with histatins and gPRPs. For future experiments, it would be
interesting to evaluate the interaction between condensed tannins and saliva
from different donors and also to evaluate the interaction with hydrolysable
tannins.
� AUTHOR INFORMATION
Corresponding Author
*Department of Chemistry, Faculty of Sciences, University of Porto, Rua do
Campo Alegre 687, 4169-007 Porto, Portugal. Phone: +351 220402558. Fax:
+351 220402659. E-mail: [email protected].
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
115
Funding Sources
This work was supported by Fundação para a Ciência e Tecnologia by one Ph.D.
grant [SFRH/BD/41946/2007] and one project grant [PTDC/AGR-
ALI/67579/2006].
� ACKNOWLEDGMENT
We thank Professor Massimo Castagnola for his help, knowledge, and
advice in the interpretation of ESI-MS spectra of salivary proteins.
� ABBREVIATIONS USED
PRPs, proline-rich proteins; bPRPs, basic proline-rich proteins; aPRPs, acidic
proline-rich proteins; gPRPs, glycosylated proline-rich proteins; SP, salivary
proteins; GSF, grape seed fraction; AS, acidic saliva; DLS, dynamic light
scattering; PMF, peptide mass fingerprint; TFA, trifluoracetic acid;MW, molecular
weight; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
� LITERATURE CITED
(1) Faria, A.; Calhau, C.; De Freitas, V.; Mateus, N. Procyanidins as antioxidants
and tumor cell growth modulators. J. Agric. Food Chem. 2006, 54, 2392–2397.
(2) Koleckar, V.; Kubikova, K.; Rehakova, Z.; Kuca, K.; Jun, D.; Jahodar, L.;
Opletal, L. Condensed and hydrolysable tannins as antioxidants influencing the
health. Mini-Rev. Med. Chem. 2008, 8, 436–447.
(3) De La Iglesia, R.; Milagro, F. I.; Campión, J.; Boqué, N.; Martínez, J. A.
Healthy properties of proanthocyanidins. Biofactors 2010, 36, 159–168.
(4) Jambunathan, R.; Mertz, E. T. Relation between tannin levels, rat growth, and
distribution of proteins in sorghum. J. Agric. Food Chem. 1973, 21, 692–696.
(5) Goncalves, R.; Soares, S.; Mateus, N.; De Freitas, V. Inhibition of trypsin by
condensed tannins and wine. J. Agric. Food Chem. 2007, 55, 7596–7601.
(6) McManus, J. P.; Davis, K. G.; Lilley, T. H.; Haslam, E. The association of
proteins with polyphenols. J. C. S. Chem. Commun. 1981, 309–311.
(7) Bate-Smith, E. C. Astringency in foods. Food 1954, 23, 124.
116 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(8) Goldstein, J. L.; Swain, T. Changes in tannins in ripening fruits.
Phytochemistry 1963, 2, 371–383.
(9) Dinnella, C.; Recchia, A.; Fia, G.; Bertuccioli, M.; Monteleone, E. Saliva
characteristics and individual sensitivity to phenolic astringent stimuli. Chem.
Senses 2009, 34, 295–304.
(10) Schwarz, B., Hofmann, T. Is there a direct relationship between oral
astringency and human salivary protein binding? Eur. Food Res. Technol. 2008.
(11) Obreque-Slier, E.; López-Solís, R.; Peña-Neira, Á.; Zamora-Marín, F.
Tanninprotein interaction is more closely associated with astringency than
tanninprotein precipitation: Experience with two oenological tannins and a gelatin.
Int. J. Food Sci. Technol. 2010, 45, 2629–2636.
(12) Dinnella, C.; Recchia, A.; Vincenzi, S.; Tuorila, H.; Monteleone, E.
Temporary modification of salivary protein profile and individual responses to
repeated phenolic astringent stimuli. Chem. Senses 2010, 35, 75–85.
(13) Bajec, M. R.; Pickering, G. J. Astringency: Mechanisms and perception. Crit.
Rev. Food Sci. Nutr. 2008, 48, 858 –875.
(14) Sarni-Manchado, P.; Canals-Bosch, J. M.; Mazerolles, G.; Cheynier, V.
Influence of the glycosylation of human salivary proline-rich proteins on their
interactions with condensed tannins. J. Agric. Food Chem. 2008, 56, 9563–9569.
(15) Pascal, C.; Poncet-Legrand, C.; Cabane, B.; Vernhet, A. Aggregation of a
proline-rich protein induced by epigallocatechin gallate and condensed tannins:
Effect of protein glycosylation. J. Agric. Food Chem. 2008, 56, 6724–6732.
(16) Fischer, U.; Boulton, R. B.; Noble, A. C. Physiological factors contributing to
the variability of sensory assessments: Relationship between salivary flow rate
and temporal perception of gustatory stimuli. Food Qual. Pref. 1994, 5, 55–64.
(17) Payne, C.; Bowyer, P. K.; Herderich, M.; Bastian, S. E. P. Interaction of
astringent grape seed procyanidins with oral epithelial cells. Food Chem. 2009,
115, 551–557.
(18) Mandel, I. D. Relation of saliva and plaque to caries. J. Dent. Res. 1974, 53,
246–266.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
117
(19) Helmerhorst, E. J.; Oppenheim, F. G. Saliva: A dynamic proteome. J. Dent.
Res. 2007, 86, 680–693.
(20) Schlesinger, D. H.; Hay, D. I.; Levine, M. J. Complete primary structure of
statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva of
the monkey, macaca arctoides. Int. J. Pept. Protein. Res. 1989, 34, 374–380.
(21) Oppenheim, F. G.; Xu, T.; Mcmillian, F. M.; Levitz, S. M.; Diamond, R. D.;
Offner, G. D.; Troxler, R. F. Histatins, a novel family of histidine-rich proteins in
human parotid secretion. Isolation, characterization, primary structure, and
fungistatic effects on candida albicans. J. Biol. Chem. 1988, 263, 7472–7477.
(22) Hay, D. I.; Bennick, A.; Schlesinger, D. H.; Minaguchi, K.; Madapallimattam,
G.; Schluckebier, S. K. The primary structures of six human salivary acidic
proline-rich proteins (prp-1, prp-2, prp-3, prp-4, pif-s and pif-f). Biochem. J. 1988,
255, 15–21.
(23) Shomers, J. P.; Tabak, L. A.; Levine, M. J.; Mandel, I. D.; Ellison, S. A.
Characterization of cysteine-containing phosphoproteins from human
submandibular-sublingual saliva. J. Dent. Res. 1982, 61, 764–767.
(24) Kauffman, D.; Wong, R.; Bennick, A.; Keller, P. Basic proline-rich proteins
from human parotid saliva: Complete covalent structure of protein IB-9 and partial
structure of protein IB-6, members of a polymorphic pair. Biochemistry 1982, 21,
6558–6562.
(25) Isemura, S.; Saitoh, E.; Sanada, K. Fractionation and characterization of
basic proline-rich peptides of human parotid saliva and the amino acid sequence
of proline-rich peptide p-e. J. Biochem. 1982, 91, 2067–2075.
(26) Isemura, S.; Saitoh, E.; Sanada, K. The amino acid sequence of a salivary
proline-rich peptide, p-c, and its relation to a salivary proline-rich phosphoprotein,
protein c. J. Biochem. 1980, 87, 1071–1077.
(27) Bennick, A. Chemical and physical characteristics of a phosphoprotein from
human parotid saliva. Biochem. J. 1975, 145, 557–567.
(28) Azen, E. A.; Oppenheim, F. G. Genetic polymorphism of proline-rich human
salivary proteins. Science 1973, 180, 1067–1069.
118 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(29) Inzitari, R.; Cabras, T.; Onnis, G.; Olmi, C.; Mastinu, A.; Sanna, M. T.;
Pellegrini, M. G.; Castagnola, M.; Messana, I. Different isoforms and post-
translational modifications of human salivary acidic proline-rich proteins.
Proteomics 2005, 5, 805–815.
(30) Messana, I.; Cabras, T.; Inzitari, R.; Lupi, A.; Zuppi, C.; Olmi, C.; Fadda, M.
B.; Cordaro, M.; Giardina, B.; Castagnola, M. Characterization of the human
salivary basic proline-rich protein complex by a proteomic approach. J. Proteome
Res. 2004, 3, 792–800.
(31) Bennick, A.; Connell, G. E. Purification and partial characterization of four
proteins from human parotid saliva. Biochem. J. 1971, 123, 455–464.
(32) Oppenheim, F. G.; Hay, D. I.; Franzblau, C. Proline-rich proteins from human
parotid saliva. I. Isolation and partial characterization. Biochemistry 1971, 10,
4233–4238.
(33) Amado, F.; Lobo,M. J. C.; Domingues, P.; Duarte, J. A.; Vitorino, R. Salivary
peptidomics. Expert Rev. Proteomics 2010, 7, 709–721.
(34) Henskens, Y. M. C.; Veerman, E. C. I.; Mantel, M. S.; Van Der Velden, U.;
Nieuw Amerongen, A. V. Cystatins s and c in human whole saliva and in
glandular salivas in periodontal health and disease. J. Dent. Res. 1994, 73,
1606–1614.
(35) Lu, Y.; Bennick, A. Interaction of tannin with human salivary proline-rich
proteins. Arch. Oral Biol. 1998, 43, 717–728.
(36) Pascal, C.; Paté, F.; Cheynier, V.; Delsuc, M.-A. Study of the interactions
between a proline-rich protein and a flavan-3-ol by NMR: Residual structures in
the natively unfolded protein provides anchorage points for the ligands.
Biopolymers 2009, 91, 745–756.
(37) Soares, S.; Mateus, N.; De Freitas, V. Interaction of different polyphenols
with bovine serum albumin (BSA) and human salivary α-amylase (HSA) by
fluorescence quenching. J. Agric. Food Chem. 2007, 55, 6726–6735.
(38) De Freitas, V.; Mateus, N. Structural features of procyanidin interactions with
salivary proteins. J. Agric. Food Chem. 2001, 49, 940–945.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
119
(39) Monteleone, E.; Condelli, N.; Dinnella, C.; Bertuccioli, M. Prediction of
perceived astringency induced by phenolic compounds. Food Qual. Pref. 2004,
15, 761–769.
(40) Asquith, T. N.; Uhlig, J.; Mehansho, H.; Putman, L.; Carlson, D. M.; Butler, L.
Binding of condensed tannins to salivary proline-rich glycoproteins: The role of
carbohydrate. J. Agric. Food Chem. 1987, 35, 331–334.
(41) Gambuti, A.; Rinaldi, A.; Pessina, R.; Moio, L. Evaluation of aglianico grape
skin and seed polyphenol astringency by SDS-PAGE electrophoresis of salivary
proteins after the binding reaction. Food Chem. 2006, 97, 614–620.
(42) Yan, Q.; Bennick, A. Identification of histatins as tannin-binding proteins in
human saliva. Biochem. J. 1995, 311, 341–347.
(43) Rawel, H. M.; Frey, S. K.; Meidtner, K.; Kroll, J.; Schweigert, F. J.
Determining the binding affinities of phenolic compounds to proteins by
quenching of the intrinsic tryptophan fluorescence. Mol. Nutr. Food Res. 2006,
50, 705–713.
(44) Kallithraka, S.; Bakker, J.; Clifford, M. N. Evidence that salivary proteins are
involved in astringency. J. Sens. Stud. 1998, 13, 29–43.
(45) Rossetti, D.; Yakubov, G. E.; Stokes, J. R.; Williamson, A. M.; Fuller, G. G.
Interaction of human whole saliva and astringent dietary compounds investigated
by interfacial shear rheology. Food Hydrocolloids 2008, 22, 1068–1078.
(46) Sarni-Manchado, P.; Cheynier, V.; Moutounet, M. Interactions of grape seed
tannins with salivary proteins. J. Agric. Food Chem. 1999, 47, 42–47.
(47) Horne, J.; Hayes, J.; Lawless, H. T. Turbidity as a measure of salivary
protein reactions with astringent substances. Chem. Senses 2002, 27, 653–659.
(48) Murray, N. J.; Williamson, M. P.; Lilley, T. H.; Haslam, E. Study of the
interaction between salivary proline-rich proteins and a polyphenol by 1H-NMR
spectroscopy. Eur. J. Biochem. 1994, 219, 923–935.
(49) Pascal, C.; Poncet-Legrand, C.; Imberty, A.; Gautier, C.; Sarni-Manchado,
P.; Cheynier, V.; Vernhet, A. Interactions between a nonglycosylated human
proline-rich protein and flavan-3-ols are affected by protein concentration and
polyphenol/protein ratio. J. Agric. Food Chem. 2007, 55, 4895–4901.
120 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(50) Canon, F.; Paté, F.; Meudec, E.; Marlin, T.; Cheynier, V.; Giuliani, A.; Sarni-
Manchado, P. Characterization, stoichiometry, and stability of salivary protein-
tannin complexes by ESI-MS and ESI-MS/MS. Anal. Bioanal. Chem. 2009, 1–11.
(51) Kallithraka, S.; Bakker, J.; Clifford, M. N. Interaction of (+)-catechin, (-)-
epicatechin, procyanidin B2 and procyanidin C1 with pooled human saliva in
vitro. J. Sci. Food Agric. 2001, 81, 261–268.
(52) Soares, S. I.; Gonçalves, R. M.; Fernandes, I.; Mateus, N.; De Freitas, V.
Mechanistic approach by which polysaccharides inhibit α-amylase/procyanidin
aggregation. J. Agric. Food Chem. 2009, 57, 4352–4358.
(53) Kawamoto, H.; Nakatsubo, F.; Murakami, K. Quantitative determination of
tannin and protein in the precipitates by high-performance liquid-chromatography.
Phytochemistry 1995, 40, 1503–1505.
(54) Scheägger, H.; Von Jagow, G. Tricine-sodium dodecyl sulfatepolyacrylamide
gel electrophoresis for the separation of proteins in the range from 1 to 100 kda.
Anal. Biochem. 1987, 166, 368–379.
(55) O’connell, K. L.; Stults, J. T. Identification of mouse liver proteins on two-
dimensional electrophoresis gels by matrix-assisted laser desorption/ionization
mass spectrometry of in situ enzymatic digests. Electrophoresis 1997, 18, 349–
359.
(56) Zacharius, R. M.; Zell, T. E.; Morrison, J. H.; Woodlock, J. J. Glycoprotein
staining following electrophoresis on acrylamide gels. Anal. Biochem. 1969, 30,
148–152.
(57) Vitorino, R.; Lobo, M. J. C.; Ferrer-Correira, A. J.; Dubin, J. R.; Tomer, K. B.;
Domingues, P. M.; Amado, F. M. L. Identification of human whole saliva protein
components using proteomics. Proteomics 2004, 4, 1109–1115.
(58) Castagnola, M.; Congiu, D.; Denotti, G.; Di Nunzio, A.; Fadda, M. B.; Melis,
S.; Messana, I.; Misiti, F.; Murtas, R.; Olianas, A.; Piras, V.; Pittau, A.; Puddu, G.
Determination of the human salivary peptides histatins 1, 3, 5 and statherin by
high-performance liquid chromatography and by diode-array detection. J.
Chromatogr., B 2001, 751, 153–160.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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121
(59) Vitorino, R.; Barros, A.; Caseiro, A.; Domingues, P.; J., D.; Amado, F.
Towards defining the whole salivary peptidome. Proteomics Clin. Appl. 2009, 3,
528–540.
(60) Beeley, J. A.; Sweeney, D.; Lindsay, J. C. B.; Buchanan, M. L.; Sarna, L.;
Khoo, K. S. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of
human parotid salivary proteins. Electrophoresis 1991, 12, 1032–1041.
(61) Pascal,C.; Bigey, F.; Ratomahenina,R.; Boze,H.; Moulin,G.; Sarni-
Manchado, P. Overexpression and characterization of two human salivary proline
rich proteins. Protein Expression Purif. 2006, 47, 524–532.
(62) Kirkland, T. N.; Finley, F.; Orsborn, K. I.; Galgiani, J. N. Evaluation of the
proline-rich antigen of coccidioides immitis as a vaccine candidate in mice. Infect.
Immun. 1998, 66, 3519–3522.
(63) De Freitas, V.; Mateus, N. Nephelometric study of salivary protein-tannin
aggregates. J. Sci. Food Agric. 2002, 82, 113–119.
(64) Johansson, A.-K.; Lingström, P.; Imfeld, T.; Birkhed, D. Influence of drinking
method on tooth-surface ph in relation to dental erosion. Eur. J. Oral Sci. 2004,
112, 484–489.
(65) Brand, H.; Tjoe Fat, G.; Veerman, E. The effects of saliva on the erosive
potential of three different wines. Aust. Dent. J. 2009, 54, 228–232.
(66) Bennick, A.; Mclaughlin, A. C.; Grey, A. A.; Madapallimattam, G. The
location and nature of calcium-binding sites in salivary acidic proline-rich
phosphoproteins. J. Biol. Chem. 1981, 256, 4741–4746.
(67) Hay, D. I.; Moreno, E. C.; Schlesinger, D. H. Phosphoprotein inhibitors of
calcium phosphate precipitation from human salivary secretions. Inorg. Perspect.
Biol. Med. 1979, 2, 271–285.
(68) Nayak, A.; Carpenter, G. H. A physiological model of tea induced
astringency. Physiol. Behav. 2008, 95, 290–294.
(69) Gillece-Castro, B. L.; Prakobphol, A.; Burlingame, A. L.; Leffler, H.; Fisher, S.
J. Structure and bacterial receptor activity of a human salivary proline-rich
glycoprotein. J. Biol. Chem. 1991, 266, 17358–17368.
122 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(70) Hatton, M. N.; Loomis, R. E.; Levine, M. J.; Tabak, L. A. Masticatory
lubrication. The role of carbohydrate in the lubricating property of a salivary
glycoprotein-albumin complex. Biochem. J. 1985, 230, 817–820.
(71) Naurato, N.; Wong, P.; Lu, Y.; Wroblewski, K.; Bennick, A. Interaction of
tannin with human salivary histatins. J. Agric. Food Chem. 1999, 47, 2229–2234.
Capítulo 2
Effect of Condensed Tannins Addition on the Astringency of Red Wines
Soares, S., Sousa, A., Mateus, N., de Freitas, V.
Chem. Senses 2011, 37, 191-198
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Effect of Condensed Tannins Addition on the Astring ency of Red Wines
Susana Soares, André Sousa, Nuno Mateus and Victor de Freitas
Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of
Sciences, University of Porto, 4169-007 Porto, Portugal
Correspondence to be sent to: Victor de Freitas, Department of Chemistry,
Faculty of Sciences, University of Porto, Rua do Campo Alegre 687, 4169-007
Porto, Portugal. e-mail: [email protected]
Accepted August 11, 2011
Astringency has been defined as a group of sensations involving dryness,
tightening, and shrinking of the oral surface. It has been accepted that
astringency is due to the tannin-induced interaction and/or precipitation of the
salivary proline-rich proteins (PRPs) in the oral cavity, as a result of the ingestion
of food products rich in tannins, for example, red wine. The sensory evaluation of
astringency is difficult, and the existence of fast and reliable methods to its study
in vitro is scarce. So, in this work, the astringency of red wine supplemented with
oligomeric procyanidins (condensed tannins), and the salivary proteins (SP)
involved in its development were evaluated by high-performance liquid
chromatography analysis of human saliva after its interaction with red wine and
by sensorial evaluation. The results show that for low concentration of tannins,
the decrease of acidic PRPs and statherin is correlated with astringency intensity,
with these families having a high relative complexation and precipitation toward
condensed tannins comparatively to the other SP. However, for higher
concentrations of tannins, the relative astringency between wines seems to
correlate’s to the glycosylated PRPs changes. This work shows for the first time
that the several families of SP could be involved in different stages of the
astringency development.
KEYWORDS: human saliva, oligomeric procyanidins, proline-rich proteins, sensory analysis
126 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
� INTRODUCTION
The astringency perception on the human palate has been defined as a complex
group of sensations involving dryness, tightening and shrinking of the oral
surface, and puckering sensations of the oral cavity (ASFTTO Materials 1989).
Astringency sensation results from the ingestion of some food products rich in
tannins, namely tea and red wine. Since 1954, when Bate-Smith (1954) proposed
that astringency results from the interaction of tannins with salivary proteins (SP)
in the mouth, it has been generally accepted and supported by recent literature
(De Freitas and Mateus 2001; Kallithraka et al. 2001; Mateus et al. 2004;
Hofmann et al. 2006; Preys et al. 2006) that astringency is due to the tannin-
induced interaction and/or precipitation of salivary proline-rich proteins (PRPs) in
the oral cavity. Although many research groups and literature support this
mechanism for astringency development, astringency is a very complex sensory
experience, and actually, the possible mechanisms for its development are
controversially discussed by the scientific community. Schwarz and Hofmann
(2008) proposed that astringency sensory perception is related to the ‘‘nonbound
free’’ astringent stimulus present in saliva and also suggested the involvement of
laminin receptor in its development; others suggested that modifications of the
viscous and lubrication properties of saliva are important, either by the
precipitation of several proteins (Rossetti et al. 2008) and increased friction
(Green 1993) or by interaction with glycosylated PRPs (gPRPs) modifying their
rheological properties (Pascal et al. 2008).
SP have been grouped into 6 structurally related major classes namely, histatins,
basic PRPs (bPRPs), acidic PRPs (aPRPs), gPRPs, statherin, and cystatins
(Oppenheim et al. 1971, 1988; Bennick 1982; Shomers et al. 1982; Hay et al.
1988; Schlesinger et al. 1989; Helmerhorst and Oppenheim 2007). All these
peptides, except bPRPs, have well-defined important biological functions in
saliva, including maintenance of ionic calcium concentration (aPRP and
statherin), antimicrobial action (histatins and cystatins), or protection of oral
tissues against degradation by proteolytic activity (cystatins). For bPRPs, it has
been proposed (Mehansho et al. 1983, 1985; Lu and Bennick 1998) that one of
their functions is to bind tannins, preventing their toxic effects in the
gastrointestinal tract.
One well-known beverage rich in tannins is red wine. Red wine quality takes into
account the fine balance between traditional parameters, such as acidity, sugar,
color, bitterness, and astringency, some of which are related to anthocyanins and
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tannins. Anthocyanins and tannins are polyphenolic compounds responsible for
the color and astringency attributes, respectively.
The composition of wines in tannins from grapes (condensed tannins) changes
drastically between wines depending on variety, wine-making procedures, region,
and among other factors. These aspects influence importantly the wines
sensorial characteristics, mainly the flavor. So, it is of fundamental importance to
understand the effect of tannins on wine astringency, in order to not compromise
the overall wine quality. The sensory evaluation of astringency is difficult, time
consuming, and expensive. Moreover, it is often wrongly associated with
bitterness, being sometimes difficult for a panel to distinguish different
astringency attributes (Peleg et al. 1999), and the existence of fast and reliable
methods to study the astringency in vitro is scarce. So, in this work, the
astringency of red wine supplemented with oligomeric procyanidins (OPC,
condensed tannins isolated from grape seeds), and the SP involved in its
development were evaluated by chromatographic analysis of human saliva after
its interaction with red wine and also by sensorial evaluation by a trained panel.
This approach also allowed to study the effect of the red wine matrix in the
reactivity of condensed tannins toward human SP.
� MATERIALS AND METHODS
Reagents. All reagents used were of analytical grade or better.
Acetonitrile (ACN) and hydrochloric acid were purchased from Panreac Quimica;
acetic acid (HOAc) was purchased from Carlo Erba Reagents; Folin-Ciocalteu,
sodium acetate, and trifluoroacetic acid (TFA) were purchased from Fluka
Biochemica; ethanol was purchased from AGA, Álcool e Géneros Alimentares,
SA.; ethyl acetate was purchased from Valente e Ribeiro, Lda; chloroform was
purchased from Pronalab, José M. Vaz Pereira, Lda; sodium hydroxide was
purchased from LaboratórioMaialab, Lda; sodium carbonate was purchased from
Sigma-Aldrich; and tartaric acid was purchased from Aldrich.
Isolation of OPC from grape seeds. OPC (condensed tannins) were
extracted from Vitis vinifera grape seeds with an ethanol/water/chloroform
solution (1:1:2, v/v/v), and the chloroform phase, containing chlorophylls and
lipids, was rejected. Then, the hydroalcoholic phase was extracted with ethyl
acetate. The organic solvent was removed using a rotary evaporator (30°C)
yielding a residue (OPC) that corresponds to catechin monomers and
128 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
procyanidinoligomers (Darné and Madero 1979; De Freitas et al. 1998). This
residue was characterized by high-performance liquid chromatography (HPLC)
regarding the composition in procyanidins, accordingly to De Freitas and Glories
(1999). Briefly, 2 Lichrospher (C18) ODS (250 x 4.6 mm id) columns placed in
line were used for all analysis. The chromatograms were monitored at 280 nm
using an ultraviolet (UV) detector.The elution system consisted of 2 solvents, A:
2.5% HOAc and B: 80% ACN + 20% A. The gradient applied was linear from 7%
to 20% (eluent B) in 90 min at a flow rate of 1.0 mL.min–1. The procyanidins
identified and quantified were: (+)-catechin (56.0 mg.g–1), (–)-epicatechin (50.0
mg.g–1), dimers B1–B3 (270.0 mg.g–1), B2 (138.0 mg.g–1), B4 (38.0 mg.g–1), B5
(26.0 mg.g–1), B2-gallate (151.0 mg.g–1), and epicatechin gallate (16.0 mg.g–1).
Total of catechins and procyanindins correspond to 745.0 mg.g–1 and remaining
255.0 correspond to others high molecular weight (MW) OPC (255.0 mg.g–1).
Red wine supplementation with grape seed OPC. A red wine (V.
vinifera, Touriga nacional, and T. Franca cv.) from the Douro demarked region
was provided by Lavradores de Feitoria, S. A. This wine has been determined to
have 0.993 ± 0.006 g catechin equivalents.L–1 of condensed tannins, determined
based on the Folin-Ciocalteu method described by Singleton and Rossi (1965).
Red wine was also characterized by HPLC regarding the composition in
procyanidins, accordingly to De Freitas and Glories (1999), as described above.
The procyanidins identified and quantified were: (+)-catechin (2.51 ± 0.11 mg.L–
1), (–)-epicatechin (1.57 ± 0.02 mg.L–1), dimers B1–B3 (109.63 ± 10.37 mg.L–1),
B2 (50.40 ± 5.86 mg.L–1), B4 (3.38 ± 0.70 mg.L–1), B5 (20.95 ± 0.63 mg.L–1), B6
(10.33 ± 0.63 mg.L–1), B2-gallate (42.64 ± 2.35 mg.L–1), and epicatechin gallate
(2.35 ± 0.21 mg.L–1). The total quantity of procyanidins is 243.76 mg.L–1. Different
red wines with different concentration in OPC were prepared by adding
increasing quantities of OPC extract to the selected wine (control wine): 0.5, 1.0,
1.5, 2.0, 2.5, and 3.0 g.L–1. The resulting wines were bottled in triplicate
(triplicates of 50 mL) and kept in the dark.
Human saliva collection. Saliva was collected from 6 healthy
nonsmoking volunteers, and 2 mL of saliva from each volunteer were used to
make a saliva pool (whole saliva, WS). Collection time was standardized at 2 PM
in order to reduce concentration variability connected to circadian rhythms of
secretion (Messana et al. 2004). The saliva pool was mixed with 10% TFA (final
concentration 0.1%), mixed and centrifuged at 8000g for 5 min. After the
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centrifugation, the supernatant (acidic saliva, AS) was separated from the
precipitate and used for the following experiments.
Protein–tannin interaction. The proteins in AS sample were analyzed by
HPLC before and after the interaction with increasing volumes of red wines
enriched with different concentration of OPC. The control condition was a mixture
of AS (150 µL) and acetate buffer 0.1 M, 12% ethanol, and pH 5.0 (50 µL) in
order to maintain the final volume constant (200 µL) between the several tested
volumes of red wine. The control condition was done in buffer with 12% ethanol
to simulate a model wine and at pH 5.0 because it is the average pH between
wine (3.4) and saliva (7.0). The first experiments were made with increasing
volumes (10, 20, 30, 40, and 50 µL) of red wine supplemented with 1.5 g.L–1 of
OPC, and acetate buffer was added to make the final volume 200 µL. Each
volume tested of red wine was an independent experiment. After shaking, the
mixture reacted at room temperature for 5 min and then was centrifuged (8000g,
5 min). The reaction time (5 min) was established as the minimal time to have
stable interactions and good reproducible results. The supernatant was analyzed
by HPLC. The precipitate resultant from some experiments was resolubilized in
110 µL of eluent A used for the HPLC analysis (see below) and analyzed by
HPLC. The same experiments were done for the other red wines supplemented
with different concentrations of OPC (control, 0.5, 1.0, 2.0, 2.5, and 3.0 g.L–1),
using 10 or 20 µL of wines in the reaction with saliva.
Negative control experiments were made with OPC dissolved in acetate buffer.
OPC solutions were made in increasing concentrations (the same concentrations
referred above), and 20 µL of these solutions were used to react with saliva in the
same experimental conditions described above.
HPLC analysis. Ninety microliters of each solution was injected on an
HPLC Lachrom system (L-7100) equipped with a Vydac C8 column, with 5 µm
particle diameter (column dimensions 150 x 2.1 mm); detection was carried out at
214 nm, using a UV-Vis detector (L-7420). The HPLC solvents were (eluent A)
0.2% aqueous TFA and (eluent B) 0.2% TFA in ACN/water 80/20 (v/v). The
gradient applied was linear from 10% to 40% (eluent B) in 60 min, at a flow rate
of 0.30 mL.min–1. After this program, the column was washed with 100% eluent B
for 20 min in order to elute S-type cystatins and other late-eluting proteins. After
washing, the column was stabilized with the initial conditions (Messana et al.
2004; Soares et al. 2011).
130 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Tannin-specific activity. The tannin-specific activity (TSA) toward WS
and AS was determined by nephelometry as described by De Freitas and Mateus
(2001). This method is based on the characteristic property of tannins to interact
and precipitate proteins. The red wines supplemented with condensed tannins
were diluted 50 times with filtered (0.45 µm) model solution (12% ethanol, 5.0
g.L–1 tartaric acid, pH 3.20). About, 4.0 mL of this solution were transferred to a
test tube and mixed with 50 µL of WS or AS. The test tube was kept in the dark
for 30 min, and after this time, the maximum turbidity was measured in a
turbidimeter HACH 2100 N adapted for cells of 100 x 12 mm. The TSA is
expressed in turbidity units NTU/mL of wine and is determined by the following
expression, where 0.08 corresponds to the dilution factor of wine:
Turbidity �NTU/mL� = Turbidity �����
0.08
Sensory evaluation. Red wines (control and supplemented wines with
OPC) were rated for astringency by a 6-member trained sensory panel. The
wines were presented to the panel members in glass cups (at room temperature,
20°C) in random order. The panel members were asked to rate the intensity of
the perceived astringency for each sample in each series on a 1–7 score scale.
Water was used for mouth rinsing between consecutive samples.
Data and statistical analysis. Values are expressed as the arithmetic
means ± standard deviation. Statistical significance of the difference between the
TSA of WS and AS was evaluated by t-test unparametric. Differences were
considered to be statistically significant when P < 0.05. Statistical significance of
the difference between the perceived astringency by the sensory panel was
evaluated by one-way analysis of variance, followed by the Bonferroni test.
Differences were considered to be statistically significant when P < 0.1.
� RESULTS
The reactivity of red wines supplemented with condensed tannins (OPC) toward
SP was evaluated by chromatographic analysis of human saliva after its
interaction with red wine and also by sensorial analysis.
The initial acidic treatment of saliva with TFA is used to precipitate several high
MWSP (such as α-amylases, mucins, carbonic anhydrase, and lactoferrin) and to
preserve sample protein composition because TFA partially inhibits intrinsic
protease activity (Messana et al. 2004). However, peptides and proteins like
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histatins, bPRPs and aPRPs, statherin, cystatins, and defensins are soluble in
AS solution and may be directly analyzed by Reverse Phase HPLC, as
previously described (Messana et al. 2004; Soares et al. 2011).
The HPLC chromatogram of this AS solution at 214 nm is presented in Fig.2.1,
and the profile was similar to the one previously described in the literature
(Messana et al. 2004; Soares et al. 2011). The top of the figure shows the
distribution of the different families of SP along the chromatogram that were
established previously by proteomic approaches, namely ESI-MS and MALDI-
TOF/TOF (Messana et al. 2004; Soares et al. 2011).
Fig.2.1 Typical Reverse Phase HPLC profile detected at 214 nm of the AS solution of human saliva. The dotted lines and numbers show the ranges and the main SP family assigned to each HPLC peptide region.
The HPLC chromatogram of the AS solution is roughly divided into 4 salivary
peptide family regions: The first Region 1 comprises proteins that belong to the
classes of bPRPs and histatins. The bPRPs identified in this region include IB-8b,
IB-8c, IB-9, IB-4, and P-J; and the histatins include histatins 3, 5, 7, 8, and 9.
Region 2 comprises mainly a gPRPs, the bPRP3. Region 3 corresponds entirely
to aPRPs, namely PRP1 and PRP3, and the last Region 4 has phosphorylated
and nonphosphorylated forms of statherin and peptide P-B (Soares et al. 2011).
The peak between regions 2 and 3 has not been previously assigned to any
family of SP, and in this work, it has shown a weak reactivity toward OPC. So,
this peak was not considered in the results analysis and discussion.
A red wine enriched with increasing concentrations of OPC was used in order to
compare the reactivity of the several SP families toward wine tannins directly in a
wine matrix. The wines were mixed with AS, and the insoluble aggregates were
removed by centrifugation. The supernatant was analyzed by HPLC.
15 20 25 30 35 40 45 50 55 60 650
2
4
6
8
101 3 4
bPRPs gPRPs aPRPs statherin
2
t (min)
Ab
s (A
u)
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The first experiments were done with the red wine supplemented with 1.5 g.L–1 of
OPC, which is the middle concentration of OPC used. These experiments were
done with increasing volumes of red wine from 10–50 µL in order to study if the
interaction with the different families of SP was affected by the wine volume.
Some examples of the HPLC profiles of AS solution before and after the
interaction with different volumes of this red wine arepresented in Fig.2.2A.The
percentage of area decreases for each HPLC region after the AS solution
interaction with increasing volumes of red wine supplemented with 1.5 g.L–1 of
OPC are summarized in Fig.2.2B. It was also perceptible that the amount of
precipitate increased with the volume of red wine added to AS.
A
B
Fig.2.2 (A) HPLC profile detected at 214 nm of the AS solution before (control) and after the interaction with increasing volumes of red wine supplemented with 1.5 g.L–1 of OPC (condensed tannins). (B) Percentages of area decrease of each HPLC salivary peptide region after the interaction of AS solution with increasing volumes of red wine supplemented with 1.5 g.L–1 OPC. Values are expressed as the arithmetic means of 3 experiments ± standard deviation.
20 25 30 35 40 45 50 55 60 65 700
2
4
6
8
10
12
t (min)
Ab
s (A
u)
0 (control)
10 µµµµL
20 µµµµL
30 µµµµL
Volume of red wine supplementedwith 1.5 g.L -1:
0 10 20 30 40 500
20
40
60
80
100
Red wine supplementedwith 1.5 g.L -1of OPC (µµµµL)
Are
a (
%)
1
2
3
HPLC peptide regions:
bPRPs
gPRPs
aPRPs
4 statherin
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The results displayed in Fig.2.2 show that the HPLC profile of the AS solution
and, therefore, the area of each different family of proteins are interestingly
affected in a different way by the interaction with increasing volumes of red wine
HPLC regions 3 (aPRPs) and 4 (statherin) decline significantly (to 50% and 20%,
respectively) with the lowest red wine volume added (10 µL), the area of the
other regions remains relatively constant (Fig.2.2B). Indeed, only the highest
volume added (50 µL) decrease the bPRPs to about 40% and gPRPs to 30%.
Volumes of 10 and 20 µL were chosen for further experiments with the other
wines supplemented with different concentrations of OPC. These volumes were
chosen because they correspond to the lowest volumes that affected differently
the several regions of the chromatogram and allowed comparing the reactivity of
several wines toward different families of SP. Indeed, for volumes of wine of 30
µL and higher, statherin and aPRPs precipitated almost completely (Fig.2.2B).
Fig.2.3 presents the HPLC profile of AS solution before and after the interaction
with 10 µL of red wine supplemented with increasing concentrations of OPC (1.0,
2.0, and 3.0 g.L–1). Only some chromatograms representative of the overall
tendency are presented.
Fig.2.3 Reverse Phase HPLC profile detected at 214 nm of the AS solution before (control) and after the interaction with 10 µL of each red wine supplemented with different concentrations of OPC.
From the results presented in Fig.2.3, it is possible to observe that for the same
volume of wine (10 µL) added to AS solution, the resulting decrease of SP is
markedly different. The addition of 10 µL of red wine supplemented with 3.0 g.L–1
of OPC, results in an important decrease of almost all regions. On the other
hand, for the red wine supplemented with 1.0 g.L–1 of OPC, only the areas of
aPRPs and statherin decrease importantly. These experiments were done with 2
15 20 25 30 35 40 45 50 55 60 650
2
4
6
8
101 3 42
t (min)
Abs
(Au
)
0 (control)
1.0 g.L -1 3.0 g.L -1
2.0 g.L -1
Red wine supplemented with OPC:
134 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
different volumes of wine (10 and 20 µL), and the decrease of percentage area is
summarized in Fig.2.4.
A – 10 µL B – 20 µL
Fig.2.4 Percentages of area decrease of each HPLC peptide regions after the interaction of AS solution with (A) 10 and (B) 20 µL of red wine supplemented with increasing concentrations of OPC. Values are expressed as the arithmetic means of 3 experiments ± standard deviation.
The results presented in Fig.2.4 clearly indicate that the interaction of SP present
in the AS solution with red wine is affected by the volume of red wine used and
also by the quantity of condensed tannins present in the red wines. While the
aPRPs and statherin are significantly affected for the lowest volume used
(Fig.2.4A) and with the increasing in the concentration of condensed tannins in
wines, bPRPs are practically not precipitated. Increasing the volume of red wine
to 20 µL leads to a significant decrease of gPRPs and a slight decrease of
bPRPs.
The area of statherin is reduced below 20% with addition of control red wine
(without OPC supplementation). This means that the tannins present in the
control red wine are enough to complex and precipitate statherin in the
concentration that it is present in saliva, and the presence of higher quantities of
condensed tannins leads to a total depletion of this protein.
For aPRPs, the behavior is similar: 20 µL of control red wine is enough to reduce
its area to 20%. However, for the experiments with 10 µL, it is possible to
observe that the area of aPRPs is reduced importantly for wine enriched in OPC.
For gPRPs, only the experiments with 20 µL showed a significant decrease of
their area. Indeed, increasing the volume to 20 µL and increasing the
0.0 0.5 1.0 1.5 2.0 2.5 3.00
20
40
60
80
100
Red wine suplementedwith OPC (g.L -1)
Are
a (
%)
0.0 0.5 1.0 1.5 2.0 2.5 3.00
20
40
60
80
100
Red wine suplementedwith OPC (g.L -1)
Are
a (
%)
1
2
3
HPLC region:
bPRPs
gPRPs
aPRPs
4 statherin
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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135
concentration of condensed tannins lead to a decrease of their area up to 20%.
For bPRPs, neither increasing the volume nor increasing the concentration of
condensed tannins, at least at the concentrations used, reduce significantly its
area.
In order to study the contribution of OPC added to supplemented wines in the SP
precipitation, blank experiments were done in which the interaction with SP was
made using OPC solutions made in acetate buffer (pH = 5.0), with the same
range of concentrations used previously (Fig.2.5).
Fig.2.5 Percentages of area decrease of each HPLC peptide regions after the interaction of AS solution with 20 µL of OPC solutions in acetate buffer matrix. Values are expressed as the arithmetic means of 3 experiments ± standard deviation.
From these results, it is possible to observe that aPRP and statherin families
decrease significantly along with the concentration of OPC as it was observed in
red wines (Fig.2.4B, 20 µL), whereas gPRP are practically not affected by OPC in
blank experiments. The concentration of OPC (catechin and dimers) in original
wines (244 mg.L–1, see the experimental section) probably do not explain the
high precipitation observed in wines (Fig.2.4B, 20 µL). Indeed, in Fig.2.5, for the
concentration around 244 mg.L–1, only a small percentage of SP precipitates.
This means that in original wine, already exists other components (e.g., tannin-
like structures) that interact strongly with SP. However, the slight increase of
tannins concentration in wines with OPC addition has an important contribution to
discriminate wines concerning their ability to precipitate SP.
It is important to refer that the mixture of AS solution withred wine always resulted
in the formation of insoluble precipitates that perceptibly increased along with the
added volume of wine and also with the concentration in OPC.
0.0 0.5 1.0 1.5 2.0 2.5 3.00
20
40
60
80
100
120
Acidic buffer with OPC (g.L -1)
Are
a (
%)
bPRP
gPRP
aPRP
Statherin
HPLC peptide regions:
136 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
The HPLC analysis of the precipitates resulting from the interaction of 10 or 20
µL of red wine supplemented with 0.5, 2.0, and 3.0 g.L–1 of OPC with the AS are
shown in Fig.2.6.
Fig.2.6 Reverse Phase HPLC profile detected at 214 nm of the precipitates resultant from the interaction of 10 or 20 µL of red wine supplemented with 0.5, 2.0, and 3.0 g.L–1 of OPC with the AS.
It is possible to observe that several SP families are effectively precipitated by
wine tannins. Indeed, in the HPLC chromatogram of the solubilized precipitate,
aPRPs (region 3) and statherin (region 4) appear in the precipitate formed by the
addition of 10 µL of red wine supplemented with 0.5 g.L–1 of condensed tannins.
Increasing the concentration in condensed tannins (red wine supplemented with
2.0 g.L–1 of OPC) also increases the percentage of those proteins in the
precipitate. The difference between the percentages of those proteins in the
precipitates obtained with 10 µL of wines supplemented with 0.5 and 2.0 g.L–1 of
OPC is not very high because the lowest concentration of condensed tannins
leads to depletion of almost all these 2 families of proteins.
For the highest volume of the more concentrated wine (20 µL of red wine
supplemented with 3.0 g.L–1 of OPC), it is possible to observe the appearance of
other proteins belonging to gPRPs in the precipitate. However, regarding bPRPs,
small peaks start to be detected in the insoluble aggregates resulting from the
interaction with 20 µL of red wine supplemented with 3.0 g.L–1 of OPC. The
appearance of these groups of proteins in the precipitate is in agreement with
their disappearance in the respective supernatant described previously. In
general, the first proteins to be precipitated by condensed tannins are aPRPs and
statherin, followed by gPRPs.
Overall, these results seem to indicate that statherin and aPRPs have a high
relative affinity toward condensed tannins complexation and precipitation
comparatively to the other SP, in a competitive assay in a wine matrix. On the
2
15 20 25 30 35 40 45 50 55 60 65 700
4
8
12
161 3 4
t (min)
Abs
(A
u)
10 µµµµL red wine supplemented with 2.0 g.L -1 of OPC
20 µµµµL red wine supplemented with 3.0 g.L -1 of OPC
10 µµµµL red wine supplement w ith 0.5 g.L -1 of OPC
Precipitates:
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137
other hand, these results seem to indicate that bPRPs, when present in a wine
matrix, have a low relative tannin affinity.
In order to study if the proteins present in WS and that are precipitated with TFA
are important for the interaction with condensed tannin in wines, the TSA of WS
and AS toward the several enriched red wines was also measured (Table 2.1).
Table 2.1. Tannin specific activity (TSA) of 50 µL of whole (WS) and acidic saliva (AS) toward red wines supplemented with several concentrations of oligomeric procyanidins (OPC). For the same concentration of OPC, the results of WS and AS are significantly different (P<0.05), except for the 2.0 g.L-1 concentration (athese results are statistically equal). Astringency rating from the sensorial evaluation. The orders with different letters are significantly different (P<0.1).
Concentration of OPC / g.L -1 0 0.5 1.0 1.5 2.0 2.5 3.0
TSA / NTU.mL -1
Whole saliva (WS) 66.6 ± 0.7 83.8 ± 7.3 90.5 ± 1.4 95.0 ± 3.1 86.6 ± 1.5a 103.7 ± 3.2 114.2 ± 4.4
Acidic saliva (AS) 77.8 ± 2.9 98.8 ± 6.5 103.8 ± 0.3 119.4 ± 7.1 97.5 ± 10.7a 129.7 ± 11.7 141.3 ± 9.4
Astringency rating 1.2 ± 0.4b 2.3 ± 1.4b,c 3.0 ± 0.0 c,d 4.0 ± 0.0 c,d,e 4.8 ± 1.6 e ,f 6.0 ± 0.0 f,g 6.7 ± 0.8 g
From the results presented in Table 2.1, it is possible to observe that the
aggregation is higher with AS comparatively to WS. However, these differences
are more significant for the higher supplemented red wines. Proteins present in
WS, such as α-amylase and mucins, that were removed with TFA, seem to affect
negatively the formation of aggregates. These results suggest that the AS, which
has been analyzed by HPLC, contains the most important proteins that react with
condensed tannins and that may contribute to astringency sensation.
As expected, the TSA increased regularly with the concentration of OPC added
to wine. This behavior concurs with the perceived astringency of those wines
(Table 2.1). The analysis of the SP families of this AS showed that the different
families could have different potentialities in developing astringency. From
Fig.2.4, it is possible to conclude that for low wine volume and therefore lower
concentration of tannins (10 µL of wine), the decrease of aPRPs is correlated
with astringency intensity. However, for higher concentrations of tannins (20 µL of
wine), the relative astringency between wines seems to be correlated to the
gPRPs changes.
� CONCLUSION
Astringency is a very complex sensation with different descriptives very hard to
define by sensorial analysis. Those descriptives could be related with tannins’
structure, concentration, wine medium, and class of SP precipitated.
138 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
The results presented show that human AS could be used for an in vitro
evaluation of the astringency of a sample because both HPLC and TSA results
using AS are in agreement with the results obtained by the sensory panel.
Moreover, this work shows for the first time that increasing the volume of red
wine, as well as OPC concentration in red wine matrix, affects differently the
several families of SP, with aPRPs and statherin being the most affected either
for low volumes as for low concentrations of OPC. Nevertheless, for higher
volumes of red wine and higher concentration of OPC, gPRPs are also
significantly affected and precipitated. So, it seems that the several SP families
have relative discriminatory functions in rating the wine astringency depending on
the concentrations of condensed tannins and the volume of wines. In summary,
the several families of SP could be involved in different stages of the
development of astringency.
The future work stands in studying the interaction of different human saliva
proteins with different classes of tannins.
� FUNDING
This work was supported by Fundação para a Ciência e Tecnologia by one phD
grant [SFRH/BD/41946/2007] and one project grant [PTDC/AGR-
ALI/67579/2006].
� REFERENCES
ASTM Materials. 1989. Standard terminology relating to sensory evaluation of
materials and products. Annual book of ASTM standards. Philadelphia (PA):
American Society of Testing and Materials.
Bate-Smith EC. 1954. Astringency in foods. Food. 23:124.
Bennick A. 1982. Salivary proline-rich proteins. Mol Cell Biochem. 45:83–99.
Darné G, Madero TJ. 1979. Mise au point d’une me´ thode d’extraction des
lipides solubles totaux, des glucides solubles totaux et des composes
phénoliques solubles totaux des organes de la vigne. Vitis. 18:221–228.
De Freitas V, Mateus N. 2001. Structural features of procyanidin interactions with
salivary proteins. J Agric Food Chem. 49:940–945.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
139
De Freitas V, Glories Y. 1999. Concentration and compositional changes of
procyanidins in grape seeds and skin of white Vitis vinifera varieties. J Sci Food
Agric. 79:1601–1606.
De Freitas V, Glories Y, Laguerre M. 1998. Incidence of molecular structure in
oxidation of grape seed procyanidins. J Agric Food Chem. 46:376–382.
Green BG. 1993. Oral astringency: a tactile component of flavor. Acta Psychol.
84:119–125.
Hay DI, Bennick A, Schlesinger DH, Minaguchi K, Madapallimattam G,
Schluckebier SK. 1988. The primary structures of six human salivary acidic
proline-rich proteins (prp-1, prp-2, prp-3, prp-4, pif-s and pif-f). Biochem J.
255:15–21.
Helmerhorst EJ, Oppenheim FG. 2007. Saliva: a dynamic proteome. J Dent Res.
86:680–693.
Hofmann T, Glabasnia A, Schwarz B, Wisman KN, Gangwer KA, Hagerman AE.
2006. Protein binding and astringent taste of a polymeric procyanidin, 1,2,3,4,6-
penta-o-galloyl-a-d-glucopyranose, castalagin, and grandinin. J Agric Food
Chem. 54:9503–9509.
Kallithraka S, Bakker J, Clifford MN, Vallis L. 2001. Correlations between saliva
protein composition and some t-i parameters of astringency. Food Qual Prefer.
12:145–152.
Lu Y, Bennick A. 1998. Interaction of tannin with human salivary proline-rich
proteins. Arch Oral Biol. 43:717–728.
Mateus N, Pinto R, Ruão P, De Freitas V. 2004. Influence of the addition of grape
seed procyanidins to port wines in the resulting reactivity with human salivary
proteins. Food Chem. 84:195–200.
Mehansho H, Clements S, Sheares BT, Smith S, Carlson DM. 1985. Induction of
proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and
by tannins. J Biol Chem. 260:4418–4423.
Mehansho H, Hagerman AE, Clements S, Butler LG, Rogler J, Carlson DM.
1983. Modulation of proline-rich protein biosynthesis in rat parotid glands by
sorghums with high tannin levels. Proc Natl Acad Sci U S A. 80:3948–3952.
140 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Messana I, Cabras T, Inzitari R, Lupi A, Zuppi C, Olmi C, Fadda MB, Cordaro M,
Giardina B, Castagnola M. 2004. Characterization of the human salivary basic
proline-rich protein complex by a proteomic approach. J Proteome Res. 3:792–
800.
Oppenheim FG, Hay DI, Franzblau C. 1971. Proline-rich proteins from human
parotid saliva. I. Isolation and partial characterization. Biochemistry. 10:4233–
4238.
Oppenheim FG, Xu T, Mcmillian FM, Levitz SM, Diamond RD, Offner GD, Troxler
RF. 1988. Histatins, a novel family of histidine-rich proteins in human parotid
secretion. Isolation, characterization, primary structure, and fungistatic effects on
Candida albicans. J Biol Chem. 263:7472–7477.
Pascal C, Poncet-Legrand C, Cabane B, Vernhet A. 2008. Aggregation of a
proline-rich protein induced by epigallocatechin gallate and condensed tannins:
effect of protein glycosylation. J Agric Food Chem. 56:6724–6732.
Peleg H, Gacon K, Schlich P, Noble AC. 1999. Bitterness and astringency of
flavan-3-ol monomers, dimers and trimers. J Sci Food Agric. 79:1123–1128.
Preys S, Mazerolles G, Courcoux P, Samson A, Fischer U, Hanafi M, Bertrand D,
Cheynier V. 2006. Relationship between polyphenolic composition and some
sensory properties in red wines using multiway analyses. Anal Chim Acta.
563:126–136.
Rossetti D, Yakubov GE, Stokes JR, Williamson AM, Fuller GG. 2008. Interaction
of human whole saliva and astringent dietary compounds investigated by
interfacial shear rheology. Food Hydrocolloids. 22:1068–1078.
Schlesinger DH, Hay DI, Levine MJ. 1989. Complete primary structure of
statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva of
the monkey, Macaca arctoides. Int J Pept Protein Res. 34:374–380.
Schwarz B, Hofmann T. 2008. Is there a direct relationship between oral
astringency and human salivary protein binding? Eur Food Res Technol.
227:1693–1698.
Shomers JP, Tabak LA, Levine MJ, Mandel ID, Ellison SA. 1982.
Characterization of cysteine-containing phosphoproteins from human
submandibular-sublingual saliva. J Dent Res. 61:764–767.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
141
Singleton VL, Rossi JAJr. 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic. 16:144–158.
Soares S, Vitorino R, Osório H, Fernandes A, Venâncio A, Mateus N, De Freitas
V. Forthcoming 2011. Reactivity of human salivary proteins families toward food
polyphenols. J Agric Food Chem. 59(10):5535–5547.
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Capítulo 3
Interaction of Different Classes of Salivary Proteins with Food Tannins
Soares, S., Mateus, N., de Freitas, V.
Food Res. Int. 2012, 49, 807-813
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145
Interaction of different classes of salivary protei ns with food tannins
Susana Soares, Nuno Mateus and Victor de Freitas*
Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
Tannins are polyphenolic compounds commonly present in vegetables, fruits and
derived products that interact with proteins, namely salivary proline-rich proteins
(PRPs). This interaction forms (in)soluble aggregates that are supposed to be at
the origin of the astringency sensation but that also influences tannins
bioavailability. Therefore, this interaction was studied herein by HPLC and
dynamic light scattering (DLS) using procyanidin trimer and PGG
(pentagalloylglucose), to obtain information about the relative affinity of the
interaction, solubility (both by HPLC) and size of the complexes formed (by DLS).
The results show that mainly acidic PRPs (aPRPs) and statherin interact with
both tannins, forming a significant quantity of complexes (either insoluble or
soluble), while bPRPs (basic PRPs) interact poorly with procyanidin trimer and
gPRPs (glycosylated PRPs) only complex with PGG. In general, PGG formed a
high amount of insoluble complexes with salivary proteins while procyanidin
trimer formed soluble complexes (except for statherin). For PGG, the maximum
aggregation and complexes size (~200 nm) corresponds to the stoichiometric
concentration (232 µM). For procyanidin trimer, the concentrations used did not
allow to reach the maximum of aggregation. These results highlight the influence
and mechanisms that different tannins and salivary proteins could present in their
interaction, and consequently in the development of the astringency sensation.
KEYWORDS: proline-rich proteins, condensed tannins, hydrolyzable tannins, soluble complexe
� INTRODUCTION
Tannins are a complex group of polyphenolic compounds that can be found in
vegetal foodstuffs, particularly in fruits, cereal grains and beverages (red wine,
tea and beer). These compounds are structurally divided in condensed (polymers
of catechin) and hydrolysable tannins (esters of glucose with gallic acid). During
foodstuff consumption, these polyphenols interact with salivary proteins,
especially with proline-rich proteins (PRPs), forming insoluble aggregates that
are supposed to be at the origin of astringency sensation (Kallithraka, Bakker et
146 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
al. 1998; Lu and Bennick 1998; Bacon and Rhodes 2000; Soares, Vitorino et al.
2011). Astringency has been defined has a dryness of the oral surface, puckering
and tightening sensations of the oral mucosa typically experienced during the
ingestion of tannin-rich food, in particular red wine (ASTM 1989). It is considered
to be a tactile, diffuse and poorly localized sensation.
Although there are a few theories about the origin of astringency sensation, the
most widely accepted one relies on the interaction between tannins and salivary
proteins. In general, the nature of tannin/protein interactions can be described as
covalent or non-covalent based on whether the molecules are irreversible bound
to each other or not, and which could result in the formation of soluble or
insoluble complexes. Non-covalent interactions are thought to involve the cross-
linking of separate protein molecules by the tannin, which acts as a polydentate
ligand on the protein surface involving hydrophobic and hydrogen bonds (Asano,
Shinagawa et al. 1982; Siebert, Troukhanova et al. 1996; de Freitas and Mateus
2001; Bennick 2002; Cala, Pinaud et al. 2010; Canon, Giuliani et al. 2010).
From tannin/protein interaction it is also known that there is an optimum
tannin/protein ratio for which maximum precipitation occurs (Fig.3.1A). In fact,
Hagerman and Robbins (1987) verified this fact with the addition of increasing
concentrations of BSA to a fixed amount of tannin. For higher or lower
tannin/protein ratios the quantity of insoluble precipitates was lower (hyperbolic
behavior, Fig.3.1A). The mechanism to explain these experimental observations
was proposed later by Siebert and co-workers (Siebert, Troukhanova et al. 1996)
(Fig.3.1B).
Fig.3.1 A. Adapted from Hagerman and Robbins (1987). Titration of a plant extract containing tannin with protein (BSA): region A, excess tannin; region B, equivalence point; region C, excess protein. B. Adapted from Siebert and co-workers (Siebert, Troukhanova et al. 1996). Model for protein/polyphenol interaction that explains the results observed in Fig. 3.1A. Polyphenols are depicted as having two ends that can bind to protein. Proteins are depicted as having a fixed number (three) of polyphenol binding sites.
If a protein is considered as having a fixed number of sites to which a tannin can
bind and a tannin is thought as having two (or more) ends that can bind to
protein, then the situation where the total concentration of tannin’s ends is
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147
roughly equal to the number of binding sites in the protein will result in a large
network, corresponding to large colloidal particles and maximum precipitation. On
the other hand, if there is a large excess of protein to tannin, each tannin
molecule is able to find binding sites in two proteins to attach to. However, it is
unlikely that there will be sufficient additional tannin molecules to bridge many of
these “protein dimers” together. This results in smaller particles and less haze.
The same is true if there is a large excess of tannin to protein, because nearly all
the sites in proteins would be occupied. This would make difficult for tannin
attached at one end to find an available site on another protein to bridge to. The
result would again be smaller particles and less precipitation.
As referred previously, astringency is thought to result from tannin/salivary
proteins interaction. The main salivary proteins have been grouped into six
structurally related major classes, namely histatins, basic PRPs (bPRPs), acidic
PRPs (aPRPs), glycosylated PRPs (gPRPs), statherin, and cystatins (Humphrey
and Williamson 2001; Huq, Cross et al. 2007). The differences between the
several families of PRPs depend on their charge and presence or absence of
carbohydrate. All these proteins have important biological functions in saliva
associated with calcium binding to enamel, maintenance of ionic calcium
concentration (PRPs and statherin), antimicrobial action (histatins and cystatins),
or protection of oral tissues against degradation by proteolytic activity (cystatins)
(Hay, Bennick et al. 1988; Oppenheim, Xu et al. 1988; Schlesinger, Hay et al.
1989; Kauffman, Keller et al. 1993; Helmerhorst and Oppenheim 2007).
Despite the knowledge about tannin/protein interactions, there is no much
information about the interaction of different families of salivary proteins with
different classes of tannins. Therefore, this work aimed to study the interaction
between different families of salivary proteins with condensed and hydrolysable
tannins, to obtain information about the relative affinity of the interaction, solubility
and size of the complexes formed.
� MATERIALS AND METHODS
Reagents. All reagents used were of analytical grade.
Procyanidin trimer C2 (condensed tannin) synthesis. Procyanidin
trimer (catechin-(4-8)-catechin-(4-8)-catechin) was synthesized according to the
procedure described in literature (Bras, Goncalves et al. 2010). Briefly, (+)-
taxifolin (Extrasynthèse) and (+)-catechin (ratio 1:3) were dissolved in ethanol
148 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
and the mixture was treated with sodium borohydride (in ethanol). The pH was
then lowered to 4.5 by addition of CH3COOH/H2O 50% (v/v), and the mixture
stood under argon atmosphere for 30 min. The reaction mixture was extracted
with ethyl acetate. After evaporation of the solvent, water was added and the
mixture was passed through C18 gel, washed with water, and recovered with
methanol. After evaporation of methanol, the fraction was passed through a TSK
Toyopearl HW-40(s) gel column (300 mm × 10 mm i.d., 0.8 mL.min-1, coupled to
a UV-Vis detector, using methanol as eluent). Several fractions were recovered
and analyzed by ESI-MS (Finnigan DECA XP PLUS) yielding procyanidins
dimers (B3 and B6) and trimer (catechin-(4-8)-catechin-(4-8)-catechin). The
structure was elucidated by HPLC-MS and NMR analysis (Tarascou, Barathieu et
al. 2006; Absalon, Fabre et al. 2011).
β-1,2,3,4,6-Penta-O-galloyl-d-glucopyranose (PGG) (h ydrolysable
tannin) synthesis. PGG was synthesized from tannic acid according to Chen
and Hagerman (2004). Tannic acid (5.0 g) was dissolved (“methanolyzed”) in
70% methanol in acetate buffer (0.1 M, pH 5.0) at 65 °C for 15 h. After this time,
the pH of the mixture was immediately adjusted to 6.0 with NaOH. Methanol was
then removed from the mixture by evaporation under reduced pressure at <30
°C, with addition of water to maintain the volume constant. The resulting solution
was extracted with 3 volumes of diethyl ether and 3 volumes of ethyl acetate. The
diethyl ether extracts were rejected while the ethyl acetate extracts were
combined and evaporated, with addition of water to maintain the volume. The
resulting suspension was centrifuged, and the precipitate was redissolved by
heating in 2% methanol solution. PGG precipitated as the solution cooled to room
temperature and was collected by centrifugation. PGG was washed twice with an
ice-cold 2% methanol solution and once with ice-cold distilled water. The final
material was lyophilized to yield a white powder with an overall mass yield of
23%. The purity of the obtained PGG was assessed by HPLC analysis and 1H
NMR spectroscopy (Fernandes, Fernandes et al. 2009).
Saliva Collection. Saliva was collected from six healthy non-smoking
volunteers and 2 mL of saliva from each volunteer were used to make a saliva
pool (whole saliva). Collection time was standardized at 2 p.m. in order to reduce
concentration variability connected to circadian rhythms of secretion (Messana,
Cabras et al. 2004). The saliva pool was mixed with 10% TFA (final concentration
0.1%) to precipitate several high molecular weight salivary proteins (such as α-
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amylases, mucins, carbonic anhydrase and lactoferrin) and to preserve sample
protein composition, since TFA partially inhibits intrinsic protease activity. After
centrifugation (8000g for 5 min), supernatant (acidic saliva, AS) was separated
from the precipitate and used for the following experiments.
Protein and Tannin Interaction. The AS sample was analyzed by HPLC
before and after interaction with increasing concentrations of PGG or procyanidin
trimer. The control condition was a mixture of AS (150 µL) and 0.1 M acetate
buffer (pH 5.0, 12% ethanol) (50 µL) (final volume 200 µL). For the experiments
with tannins, the necessary volume (8 to 60 µL) of tannin stock solution (2.66
mM) was added to AS (150 µL) to obtain the desired final concentration, plus the
volume of acetate buffer to make the final volume 200 µL. Tannins were tested in
the following final concentrations: 53.0, 133.0, 232.0, 299.0 and 399.0 µM, and
each concentration was an independent experiment. After shaking, the mixture
reacted at room temperature (20 ⁰C) for 5 min and was then centrifuged (8000g,
5 min). Supernatant was recovered for subsequent HPLC-DAD and DLS analysis
(Fig.3.2).
Fig.3.2 Scheme of the experimental approach used to study protein/tannin interaction indicating the supposed kind of complexes formed and removed in each step.
HPLC analysis. Half of the supernatant was directly injected into the
HPLC-DAD and the other half was filtered (cellulose acetate 0.22 µm) and only
then injected into the HPLC-DAD. 90 µL of each solution were injected on a
HPLC Lachrom system (Merck Hitachi, L-7100) equipped with a Vydac C8
column, with 5 µm particle diameter (column dimensions 150 x 2.1 mm);
detection was carried out at 214 nm, using a UV-Vis detector (L-7420). The
HPLC solvents were 0.2% aqueous TFA (eluent A) and 0.2% TFA in ACN/water
80/20 (v/v) (eluent B). The gradient applied was linear from 10 to 40% (eluent B)
in 60 min, at a flow rate of 0.30 mL.min-1. After this program the column was
washed with 100% eluent B for 20 min in order to elute S-type cystatins and
other late-eluting proteins. After washing, the column was stabilized with the
initial conditions (Messana, Cabras et al. 2004; Soares, Vitorino et al. 2011).
150 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Dynamic Light Scattering (DLS) Measurements. The size of the
aggregates present in the supernatant was determined by DLS. Half of the
supernatant was directly analyzed by DLS and the other half was filtered
(cellulose acetate 0.22 µm) and only then analyzed by DLS (Fig.3.2). The size of
procyanidin trimer/salivary proteins and PGG/salivary proteins aggregates in
filtered and non-filtered solutions was measured using the Zetasizer Nano ZS
Malvern instrument. This technique measures the time-dependent fluctuations in
the intensity of scattered light that occur because particles undergo Brownian
motion. The analysis of these intensity fluctuations enables determination of the
diffusion coefficients of particles, which are converted into a size distribution. The
sample solution was illuminated by a 633 nm laser, and the intensity of light
scattered at an angle of 173 was measured by an avalanche photodiode. This
analysis provides information concerning particle size (obtained by average size
parameter) and polydispersity. In general, Z average size results were
considered valid when the polydispersity of solutions was below 0.5.
Statistical Analysis. All assays were performed in n = 3 repetitions. The
mean values and standard deviations were evaluated using analysis of variance
(ANOVA); all statistical data were processed using GraphPad Prism version 5.0
for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).
� RESULTS
This work aimed to provide new insights about the influence of structural factors
of polyphenols and salivary proteins on the development of astringency
sensation. The interaction between different families of salivary proteins with
condensed and hydrolysable tannins was studied in order to have some new
information about the relative affinity of the interaction, solubility and size of the
complexes formed. This information was achieved by HPLC analysis of human
saliva before and after interaction with the referred tannins and by DLS
measurements of the complexes formed.
Salivary proteins
The initial acidic treatment of human saliva with TFA is used to precipitate several
high molecular weight salivary proteins (such as α-amylases, mucins, carbonic
anhydrase and lactoferrin) and to preserve sample protein composition, since
TFA partially inhibits intrinsic protease activity (Messana, Cabras et al. 2004).
However, peptides and proteins like histatins, basic, acidic and glycosylated
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PRPs, statherin, cystatins, and defensins are soluble in acidic saliva (AS)
solution and were thus directly analyzed by RP-HPLC, as previously described
(Messana, Cabras et al. 2004; Soares, Vitorino et al. 2011). The HPLC
chromatogram of this AS solution (at 214 nm) is displayed in Fig.3.3. The top of
the first HPLC chromatogram shows the distribution of different families of
salivary proteins along the chromatogram that were established previously by
proteomic approaches, namely ESI-MS and MALDI-TOF/TOF (Messana, Cabras
et al. 2004; Soares, Vitorino et al. 2011).
Fig.3.3 RP-HPLC profile detected at 214 nm of the AS solution before (control saliva) and after the interaction with 133 and 399 µM of A, PGG and B, procyanidin trimer. All these solutions were filtered (0.22 µm) prior to HPLC analysis.
The HPLC chromatogram of the AS solution is roughly divided into four salivary
proteins family regions: the first region comprises proteins that belong to bPRPs
and histatins classes. The bPRPs identified in this region include IB-8b, IB-8c, IB-
9, IB-4 and P-J and the histatins include histatins 3, 5, 7, 8 and 9. Second region
comprises mainly a gPRP, bPRP3. The next region corresponds entirely to
152 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
aPRPs, namely PRP1 and PRP3, and last region contains phosphorylated and
non-phosphorylated forms of statherin and peptide P-B (Soares, Vitorino et al.
2011).
Interaction of salivary proteins with tannins
The experiments in this study were all performed in buffer with 12% ethanol to
mimic a model wine and at pH 5.0. At this pH, salivary proteins have showed to
strongly interact with tannins (de Freitas and Mateus 2002) and it corresponds to
an intermediary pH between wine pH (3.4) and saliva pH (7.0). Indeed, salivary
pH drops with the ingestion of acidic drinks, and the degree of acidity in saliva
depends on the sampled volume, buffering capacity, and mode of drinking
(Johansson, Lingström et al. 2004; Brand, Tjoe Fat et al. 2009). In order to
compare the reactivity of salivary proteins with tannins, different concentrations of
procyanidin trimer (condensed tannins) and PGG (hydrolysable tannins) were
mixed with AS solution, and after removal of insoluble complexes by
centrifugation, half of the supernatant was directly analyzed by HPLC and the
other half was filtered (0.2 µm) prior to the analysis by HPLC. Some HPLC
profiles of AS solution after interaction with both tannins (filtered) are shown in
Fig.3.3.
In Fig.3.3, the area of each peak represents the amount of protein that did not
precipitate with tannins and eventually the amount of protein involved in
formation of soluble complexes with tannins that were dissociated by HPLC
conditions analysis.
The HPLC profile of the AS solution is interestingly affected by interaction with
both tannins. Effectively, each tannin interacts differently with each group of
proteins. In general, PGG was much more efficient than procyanidin trimer in
precipitating all salivary proteins, especially for higher concentrations. For
instance, for the same tannin concentration (133 µM) both tannins affected
differently several families of salivary proteins. In fact, while both tannins
precipitated much more efficiently aPRPs and statherin proteins, practically no
bPRPs and gPRPs were precipitated. However, for 399 µM concentration, PGG
practically precipitated all salivary proteins including bPRPs and gPRPs, while
procyanidin trimer only precipitated significantly aPRPs and statherin. So, both
protein and tannin structures are crucial features that govern tannin/protein
interactions.
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Formation of soluble and insoluble salivary protein/tannin
complexes
Protein/tannin complexes can be soluble and insoluble, depending among other
things on protein and tannin structure, concentration and protein/tannin ratio. The
presence of insoluble salivary protein/tannin complexes was visually perceived
and easily removed by centrifugation. However, the formation of soluble
complexes is more difficult to assess and was estimated herein by filtration
comparing filtered and non-filtered supernatants (Fig.3.4) and by DLS
measurements. Insoluble complexes were removed previously by centrifugation.
The HPLC chromatogram of the filtered and non-filtered AS solutions, without
tannins, were exactly the same (data not shown), indicating that the filtration
process does not retain proteins from AS.
Fig.3.4 RP-HPLC profiles detected at 214 nm of the AS solution after the interaction with 399 µM of procyanidin trimer and 133 µM of PGG. These solutions were non-filtered and filtered (0.22 µm) prior to HPLC analysis.
From Fig.3.4 it is possible to observe that filtration induced a decrease of some
chromatographic peaks of salivary proteins, in particular for aPRPs and statherin
proteins. That difference could be explained by retention of salivary protein/tannin
soluble complexes by the filter. In the case of the non-filtered solutions, the
observed higher area of some chromatographic peaks may be justified by
dissociation of these soluble complexes by the HPLC solvents into their
precursors (tannin and protein). This is particular evident in the PGG
experiments, were the chromatographic peak of PGG appear only in the non-
filtered solution, which may result from dissociation of soluble salivary
proteins/PGG complexes during HPLC analysis. In fact, the efficiency of HPLC
solvents in dissociate salivary protein/tannin complexes was tested with the
insoluble complexes formed and initially removed by centrifugation. These
154 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
experiments revealed that the HPLC solvents dissolve, at least partially, the
formed precipitates because the salivary proteins have appeared in the
chromatogram after dissolution (data not shown).
The variations in the chromatographic peaks of each family of salivary proteins
after interaction with both tannins before and after filtration are presented in
Fig.3.5. The results are expressed as percentage of the area of these proteins
relatively to the control saliva (without tannin). The results were divided by family
of salivary proteins. bPRPs peak areas were not considered in non-filtered
solutions because of the co-elution with PGG peak (shown in Fig.3.4) which
difficult its analysis.
Fig.3.5 The graphics represent the variation of the chromatographic peaks area of the several salivary proteins studied with the increase in tannins concentration. The results are expressed as percentage of the area of each protein relatively to the control saliva (100%) analyzed by HPLC, before and after solutions filtration (0.22 µm). Statherin graphic was used as an example indicating the percentage of protein involved in the formation of soluble and insoluble complexes. These results represent the average of three independent experiments.
From the results presented in Fig.3.5 it is possible to observe systematically a
reduction of proteins’ area comparing non-filtered and filtered solutions. Once
again, it is possible to observe that PGG is much more efficient than procyanidin
trimer in reducing almost all salivary proteins, especially for higher
concentrations.
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The observed differences between non-filtered and filtered solutions point to the
existence of soluble complexes that are retained by the filter and that could
explain the proteins’ area reduction upon filtration, as explained previously.
The differences between non-filtered and filtered solutions were also visible in the
size experiments measurements by DLS. The size of salivary protein/tannin
(soluble) complexes present in the centrifuged solution of AS and tannins that
was analyzed by HPLC, was determined by DLS and the results are presented in
Fig.3.6. This DLS analysis comprises all the salivary proteins together present in
AS solution and not the different families of salivary proteins separately, which
are only possible to separate from AS by HPLC.
Fig.3.6 Influence of tannin concentration on the average size of salivary soluble proteins/tannins complexes present in solution. These results represent the average of three independent experiments.
The presented results show unequivocally the existence of soluble salivary
protein/tannin complexes that are retained by the filter (0.22 µm). Although these
complexes present a lower size than the cut-off of the filter used in the
experiment, they are almost totally retained by filtration. This fact could be
explained by the presence of some higher complexes that are firstly retained by
the filter and that could act as adjuvants in the filtration of smaller complexes.
For procyanidin trimer, soluble complexes size increase along with its
concentration (240 nm for the highest concentration). However, the behavior for
PGG is not entirely similar. The size of the soluble complexes that result from
salivary proteins interaction with PGG first increased with PGG concentration
until it reaches a maximum (200 nm) and then it was reduced.
156 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
� DISCUSSION
In order to highlight the differences between non-filtered and filtered solutions
and facilitate discussion, the percentages of proteins involved in the formation of
soluble and non-soluble complexes for the highest tannin concentration are
shown in Table 3.1.
As schematically presented in Fig.3.5 for statherin, the percentage of insoluble
complexes was obtained subtracting the area of the chromatographic peaks of
each family obtained in the HPLC analysis of the non-filtered solution to the
respective area obtained in the control saliva (AS without tannin). The
percentage of soluble complexes was calculated in the same way but subtracting
the data obtained in the HPLC analysis of the non-filtered solution to the
respective area obtained in the filtered solution.
Table 3.1 Percentage of salivary proteins involved in the formation of soluble and insoluble complexes formed by the interaction with 399 µM of each tannin. These results represent the average of three independent experiments.
Tannin Complexes Salivary protein families (%)
bPRPs gPRPs aPRPs statherin
PGG Insoluble - 59.3 ± 1.7 65.6 ± 4.5 70.1 ± 1.7
Soluble - 35.5 ± 3.6 30.9 ± 4.0 28.1 ± 4.5
Procyanidin trimer Insoluble ~0 ~0 19.3 ± 2.6 72.1 ± 1.5
Soluble 14.3 ± 3.1 ~0 29.0 ± 12.4 24.9 ± 0.3
The results show that most of the salivary protein families’ studied interact with
both tannins, specially aPRPs and statherin. In fact, it is possible to observe that
these two families are involved in the formation of a significant quantity of
complexes (either insoluble or soluble) with both tannins, while bPRPs and
gPRPs interact poorly and practically do not complex with procyanidin trimer in
these conditions.
On the other hand, the amount of salivary proteins involved in the formation of
soluble and insoluble complexes is in general higher for PGG with the exception
of statherin for which the amount of complexes is practically the same for both
tannins. This shows that statherin is the only salivary protein that presents no
selectivity towards tannin structures.
In general, PGG interacted with all salivary proteins forming high amount of
insoluble complexes that precipitate. Oppositely, procyanidin trimer tends to form
soluble complexes with the exception of statherin.
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157
Comparing the different families of salivary proteins, the difference in the
percentage of proteins involved in the formation of complexes, especially the
insoluble ones, seem to be more affected by the protein structure for the
procyanidin trimer in comparison with PGG. In fact, for PGG it seems that protein
structure only affects the efficiency of the interaction. PGG seems to be more
reactive towards salivary proteins while procyanidin trimer is more selective. It is
also interesting to observe that gPRPs only complex with PGG.
Regarding the size measurements interpretation, it is interesting to notice that
PGG behavior is in agreement with the protein/tannin interaction model most
widely accepted and presented in Fig.3.1, in which the maximum aggregation
and complexes size corresponds to the stoichiometric concentration (in PGG
case 232 µM). For lower and higher PGG concentration the soluble complexes
formed have a lower size because there are fewer and higher PGG molecules
respectively, to occupy proteins binding sites, forming smaller complexes.
Regarding the procyanidin trimer, the used concentrations did not allow to reach
the stoichiometric maximum of aggregation for this tannin, although the size
seemed to begin to stabilize for the higher concentrations.
In order to interpret the size measurements in the light of the results observed for
the HPLC experiments, it is important to notice that the salivary proteins that are
probably involved in the formation of soluble complexes with the highest size
(~200 nm) at 232 µM of PGG (Fig.3.5) were mainly aPRPs and statherin.
� CONCLUSION
In summary, these results suggest that the kind (soluble or insoluble) and size of
complexes formed depends not only on tannin class/structure but also on protein
structure and on protein:tannin ratio. PGG originates mainly insoluble complexes
with salivary proteins (mainly composed by aPRPs or statherin proteins) with the
soluble complexes having a maximum size about 200 nm at near 200 µM, while
procyanidin trimer originates mainly soluble complexes (in particular for PRPs)
but also insoluble complexes (mainly composed by statherin) with maximum size
higher than 240 nm at a concentration superior to 400 µM.
These results highlight the diverse influence and mechanisms that different
tannins and salivary proteins could present in the development of the astringency
sensation.
158 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
� AKNOWLEDGMENTS
The authors thank the financial support by Fundação para a Ciência e
Tecnologia by one phD grant [SFRH/BD/41946/2007] and one project grant
[PTDC/AGR-ALI/67579/2006].
� REFERENCES
Absalon, C., Fabre, S. Tarascou, I., Fouquet, E., & Pianet, I. (2011). New
strategies to study the chemical nature of wine oligomeric procyanidins.
Analytical and Bioanalytical Chemistry, 401, 1489-1499.
Asano, K., Shinagawa, K., & Hashimoto, N. (1982). Characterization of haze-
forming proteins of beer and their roles in chill haze formation. Journal of the
American Society of Brewing Chemists, 40, 147-154.
ASTM (1989). Standard Terminology to Sensory Evaluation of Materials and
Products. Philadelphia, PA, American Society of Testing and Materials.
Bacon, J. R., & Rhodes, M. J. C. (2000). Binding Affinity of Hydrolyzable Tannins
to Parotid Saliva and to Proline-Rich Proteins Derived from It. Journal of
Agricultural and Food Chemistry, 48, 838-843.
Bennick, A. (2002). Interaction of plant polyphenols with salivary proteins. Critical
Reviews in Oral Biology & Medicine, 13, 184-196.
Brand, H., Tjoe Fat, G., & Veerman, E.. (2009). The effects of saliva on the
erosive potential of three different wines, Australian Dental Journal, 54, 228-232.
Bras, N. F., Goncalves, R., Fernandes, P. A., Mateus, N., Ramos, M. J., & de
Freitas, V. (2010). Understanding the Binding of Procyanidins to Pancreatic
Elastase by Experimental and Computational Methods, Biochemistry, 49, 5097-
5108.
Cala, O., Pinaud, N., Simon, C., Fouquet, E., Laguerre, M., Dufourc, E. J., &
Pianet, I. (2010). NMR and molecular modeling of wine tannins binding to saliva
proteins: revisiting astringency from molecular and colloidal prospects, The
FASEB Journal, 24, 4281-4290.
Canon, F., Giuliani, A., Pate, F., & Sarni-Manchado, P. (2010). Ability of a
salivary intrinsically unstructured protein to bind different tannin targets revealed
by mass spectrometry, Analytical and Bioanalytical Chemistry, 398, 815-822.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
159
Chen, Y., & Hagerman, A. E. (2004). Characterization of soluble non-covalent
complexes between bovine serum albumin and β-1,2,3,4,6-penta-O-galloyl-d-
glucopyranose by MALDI-TOF MS, Journal of Agricultural and Food Chemistry,
52, 4008-4011.
de Freitas, V., & Mateus, N. (2001). Structural features of procyanidin interactions
with salivary proteins, Journal of Agricultural and Food Chemistry, 49, 940-945.
de Freitas, V., & Mateus, N. (2002). Nephelometric study of salivary protein-
tannin aggregates. Journal of the Science of Food and Agriculture, 82, 113-119.
Fernandes, A., Fernandes, I., Cruz, L., Mateus, N., Cabral, M., & de Freitas, V.
(2009). Antioxidant and Biological Properties of Bioactive Phenolic Compounds
from Quercus suber L. Journal of Agricultural and Food Chemistry, 57, 11154-
11160.
Hagerman, A. E., & Robbins, C. T. (1987). Implications of soluble tannin-protein
complexes for tannin analysis and plant defense mechanisms. Journal of
Chemical Ecology, 13, 1243-1259.
Hay, D. I., Bennick, A., Schlesinger, D. H., Minaguchi, K., Madapallimattam, G., &
Schluckebier, S. K. (1988). The primary structures of six human salivary acidic
proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f).
Biochemical Journal, 255, 15-21.
Helmerhorst, E. J., & Oppenheim, F. G. (2007). Saliva: a Dynamic Proteome.
Journal of Dental Research, 86, 680-693.
Humphrey, S. P., & Williamson, R. T. (2001). A review of saliva: normal
composition, flow, and function. Journal of Prosthetic. Dentistry, 85, 162-169.
Huq, N., Cross, K., Ung, M., Myroforidis, H., Veith, P., Chen, D., Stanton, D., He,
H., Ward, B., & Reynolds, E. (2007). A review of the salivary proteome and
peptidome and saliva-derived peptide therapeutics. International Journal of
Peptide Research and Therapeutics, 13, 547-564.
Johansson, A.-K., Lingstrom, P., Imfeld, T., & Birkhed, D. (2004). Influence of
drinking method on tooth-surface pH in relation to dental erosion. European
Journal Of Oral Sciences, 112, 484-489.
Kallithraka, S., Bakker, J., & Clifford, M. N. (1998). Evidence that salivary proteins
are involved in astringency. Journal Sensory Studies 13, 29-43.
160 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Kauffman, D. L., Keller, P. J., Bennick, A., & Blum, M. (1993). Alignment of amino
acid and DNA sequences of human proline-rich proteins. Critical Reviews in Oral
Biology & Medicine, 4, 287-292.
Lu, Y., & Bennick, A. (1998). Interaction of tannin with human salivary proline-rich
proteins. Archives of Oral Biology, 43, 717-728.
Messana, I., Cabras, T., Inzitari, R., Lupi, A., Zuppi, C., Olmi, C., Fadda, M. B.,
Cordaro, M., Giardina, B., & Castagnola, M. (2004). Characterization of the
human salivary basic proline-rich protein complex by a proteomic approach.
Journal of Proteome Research, 3, 792-800.
Oppenheim, F. G., Xu, T., McMillian, F. M., Levitz, S. M., Diamond, R. D., Offner,
G. D., & Troxler, R. F. (1988). Histatins, a novel family of histidine-rich proteins in
human parotid secretion. Isolation, characterization, primary structure, and
fungistatic effects on Candida albicans. Journal of Biological Chemistry, 263,
7472-7477.
Schlesinger, D. H., Hay, D. I., & Levine, M. J. (1989). Complete primary structure
of statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva
of the monkey, Macaca arctoides. International Journal of Peptide and Protein
Research, 34, 374-380.
Siebert, K. J., Troukhanova, N. V., & Lynn, P. Y. (1996). Nature of polyphenol-
protein interactions. Journal of Agricultural and Food Chemistry, 44, 80-85.
Soares, S., Vitorino, R., Osorio, H., Fernandes, A., Venancio, A., Mateus, N.,
Amado, F., & de Freitas, V. (2011). Reactivity of human salivary proteins families
toward food polyphenols. Journal of Agricultural and Food Chemistry, 59, 5535-
5547.
Tarascou, I., Barathieu, K., Andre, Y., Pianet, I., & Dufourc, E. J. (2006). An
Improved Synthesis of Procyanidin Dimers: Regio- and Stereocontrol of the
Interflavan Bond. European Journal of Organic Chemistry, 2006, 5367-5377.
Capítulo 4
Mechanistic Approach by Which Polysaccharides Inhibit α-Amylase/Procyanidin Aggregation
Soares, S., Gonçalves, R. M., Fernandes, I., Mateus, N., de Freitas, V.
J. Agric. Food Chem. 2009, 57, 4352–4358
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Mechanistic Approach by Which Polysaccharides Inhib it α-Amylase/Procyanidin Aggregation
Susana Soares, Rui M. Gonçalves, Iva Fernandes, Nuno Mateus and Victor de
Freitas
Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
The present work studies the inhibition of aggregation of α-amylase and
procyanidin fractions by different polysaccharides (arabic gum, β-cyclodextrin,
and pectins). Several analytical approaches, namely, fluorescence quenching,
nephelometry, and dynamic light scattering (DLS), were used. In general,
nephelometry showed that the presence of the polysaccharides in solution
reduced the formation of insoluble aggregates. The fluorescence quenching
measurements showed two effects: arabic gum and β-cyclodextrin reduce the
quenching effect of procyanidin fractions on α-amylase fluorescence, whereas
pectins do not affect the quenching of α-amylase fluorescence by procyanidin
fractions. DLS measurements have revealed that the polysaccharides studied
induce a decrease in aggregates size, which probably is due to the formation of
smaller aggregates resulting from the disruption and reorganization of the
procyanidin fractions/α-amylase aggregates. Overall, the results obtained for
arabic gum and β-cyclodextrin strongly suggest that the main mechanism by
which these two compounds inhibit protein/polyphenol aggregation is by
molecular association between these polysaccharides and polyphenols,
competing with protein aggregation. In the case of pectins, the results obtained
provide evidence that the main mechanism by which they reduce
protein/polyphenol aggregation is by forming a protein/polyphenol/polysaccharide
complex, enhancing its solubility in aqueous medium.
KEYWORDS: Aggregation; α-amylase; fluorescence; polysaccharides; tannin
� INTRODUCTION
Tannins are phenolic compounds, yielded from the secondary metabolism of
higher plants, being found worldwide in many different families of plants (1).
164 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Bate-Smith and Swain defined the plant tannins as water-soluble phenolic
compounds with molecular masses between 300 and 3000 Da, displaying the
usual phenolic reactions [e.g., blue color with iron(III) chloride] and precipitating
alkaloids, gelatins, and other proteins (2, 3). However, this definition does not
include all tannins. Indeed, more recently, molecules with a molecular mass of up
to 20000 Da have been isolated and should also be classified as tannins on the
basis of their phenolic reactions. High concentrations of tannins can be found in
nearly every part of the plant, such as in wood, leaves, fruit, roots, and seed.
Usually, tannins are divided in two major classes: condensed (proanthocyanidins)
and hydrolyzable tannins. The first ones are polymers of catechin, and the latter
are gallic or ellagic esters of glucose. It is assumed that these compounds have a
biological role in the plant related to protection against infection and herbivores
(1).
From a nutritional point of view, the interaction between tannins and enzymes,
such as α-amylase or trypsin, has been shown to have harmful effects, with the
inhibition of these enzymes and decrease in body weight gain (4-6).
On the other hand, the interaction of α-amylase and other salivary proteins such
as proline-rich proteins (PRPs) and histatins with tannins is thought to be
responsible for the astringency sensation. This event is thought to result from the
formation of protein/tannin insoluble aggregates that precipitate, reducing the
palate lubrication and causing an unpleasant sensation of roughness, dryness,
and constriction (7, 8). Astringency is often perceived as a negative attribute as in
dairy products, nuts, and juices (9). However, in some beverages such as tea,
beer, and red wine, astringency could be perceived as a positive quality factor, if
not too intense.
The affinities of salivary proteins to complex tannins depend on many factors and
mainly on their chemical structures (10, 11). Previous works have shown that a
PRP (IB8c) binds to condensed tannins much more effectively than α-amylase
(12). This can be explained not only by their primary structure but also by the
three-dimensional structure of these proteins. In fact, whereas α-amylase is a
globular protein, PRPs are extended randomly coiled proteins, which offer more
sites to interact with tannins. However, R-amylase seems to bemore specific and
selective than PRPs in the aggregation with samples containing different
amounts of proanthocyanidins (13). α-Amylase from porcine pancreas (hereafter
PPA) is a single polypeptide chain of 496 residues of which 17 are tryptophan
residues, which are responsible for its intrinsic fluorescence (14). Mammalian α-
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amylases show a high degree of homology, so it is considered that in a general
way porcine and human α-amylases are very similar (15, 16).
The presence of polysaccharides in solution is also an important factor that
affects the interaction between tannins and proteins. Several studies have
demonstrated the ability of some neutral and anionic polysaccharides to disrupt
the binding of polyphenols to proteins (17-19). For instance, loss of astringency is
one of the principal changes that occur during the ripening of many edible fruits,
which has been related to the increase of soluble pectins during maturation. Two
mechanisms have been proposed to explain this phenomenon: (I)
polysaccharides form a ternary complex protein/polyphenol/polysaccharide,
which enhances solubility in an aqueous medium; (II) there is a molecular
association in solution between polysaccharides and polyphenols, competing for
protein aggregation (17, 19).
The effect of polysaccharides on polyphenol complexation with proteins has been
studied in solution by techniques such as enzyme activity, NMR, and
nephelometry (17, 19, 20). Whereas NMR gives important data about the
interaction between tannin and protein at a molecular level, nephelometry
measures the formation of high molecular weight structures at a macromolecular
level, taking into account all physical-chemical driving forces involved in the
aggregates’ formation. Over recent years, the fluorescence quenching technique
has been employed, with success, to study the specificity of the protein/tannin
interaction at a molecular level (21-23). Changes on PPA structure resulting from
the interaction with tannins were evaluated by the measurement of intrinsic
fluorescence intensity of protein tryptophan residues. Fluorescence
measurements give information about the molecular environment in the vicinity of
the chromophore molecule. The decrease of protein intrinsic fluorescence
intensity is called quenching, which can occur by different mechanisms, namely,
collisional quenching, when the excited-state fluorophore is deactivated upon
contact with some other molecule in solution (the quencher), or static quenching,
whereby fluorophores form nonfluorescent complexes with quenchers (24).
The aim of this work is to link the fluorescence quenching, nephelometry, and
dynamic light scattering (DLS) techniques to provide insights, at a molecular
level, of the process whereby polysaccharides (arabic gum, β-cyclodextrin, and
pectins with different methoxylation degrees) inhibit α-amylase aggregation with
grape seed procyanidins. α-Amylase/tannin aggregates are thought to influence
food astringency and, on the other hand, inhibit enzyme activity.
166 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
� MATERIALS AND METHODS
Reagents. α-Amylase from porcine pancreas (≥98%) was purchased from
Sigma. Arabic gum was purchased from Aldrich. β-Cyclodextrin (≥99%, HPLC)
was purchased from Fluka. Pectins (from citrus peel and standardized by the
addition of sucrose) with different methoxylation degrees (MD of 27, 58-63, and
72%) were kindly supplied by CPKelco Co.
Grape Seed Tannin Isolation. Condensed tannins were extracted as
described in the literature (25, 26). Briefly, condensed tannins were extracted
from Vitis vinifera grape seeds with an ethanol/water/chloroform solution (1:1:2,
v/v/v), and the chloroform phase, containing chlorophylls and lipids, was rejected.
The resulting hydroalcoholic phase was extracted with ethyl acetate. The organic
solvent was removed using a rotary evaporator (30°C), and the resulting residue,
corresponding to catechin monomers and oligomeric procyanidins, was
fractionated through a TSK Toyopearl gel column TSK Toyopearl HW-40(s) gel
column (100 mm X 10 mm i.d., with 0.8 mL.min-1 methanol as eluent), yielding
four fractions. Fractions I and II were obtained after elution with 99.8% (v/v)
methanol during 1 and 5 h, respectively (cumulative time); fraction III was
obtained after elution with methanol/5%(v/v) acetic acid during the next 14 h; and
fraction IV was obtained after elution with methanol/10% (v/v) acetic acid during
the next 8 h. All of the fractions were mixed with deionized water; the solvent was
eliminated using a rotary evaporator under reduced pressure at 30°C and then
freeze-dried. The composition of procyanidins in each fraction was determined by
direct analysis by ESI-mass spectrometry (Finnigan DECA XP PLUS) as
described in the literature (26). Fraction I contains catechin, galloyl derivatives,
procyanidin dimer, the galloyl derivative, and procyanidin trimer; fraction II
contains procyanidin trimer and tetramer and its galloyl derivatives (mean MW =
950); fraction III contains procyanidin pentamer and the galloyl derivative (mean
MW = 1512); fraction IV contains procyanidin pentamer digalloyl, procyanidin
tetramer tetragalloyl, procyanidin hexamer galloyl, procyanidin heptamer, and the
galloyl derivative (mean MW = 2052). The mean molecular weight was
determined on the basis of the relative abundance of each flavanol in the
fractions.
Fluorescence Quenching and Nephelometry Measuremen ts. The
quenching effect between α-amylase and procyanidin fractions with different
molecular weights was assayed in the presence of increasing concentrations of
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different polysaccharides (arabic gum, β-cyclodextrin, and pectins with different
methoxylation degrees; 27, 58-63, and 72%) present in foods or added during
food processing (27-31). A Perkin-Elmer LS 45 fluorometer was used for
fluorescence quenching and nephelometry measurements. For the fluorescence
quenching assays the excitation wavelength was set to 282 nm and the emission
spectrum was recorded from 300 to 450 nm. Both slits were 10 nm. For
nephelometry analysis, the fluorometer was used as a 90° light scattering
photometer; for that, both excitation and emission wavelengths selected were the
same (400 nm). At this wavelength, protein, tannins, and polysaccharides do not
absorb the incident light (32). Considering the possibility of fluorescence
resonance energy transfer (FRET) between the protein and the procyanidin
fractions, the absorption spectra of both were analyzed (data not shown):
procyanidin fractions have an absorption maximum at 270 nm and decrease
significantly until near 310 nm. The protein’s emission spectrum starts at 320 nm,
and at this λ the polyphenols practically do not absorb. On the other hand,
procyanidin fractions have their emission maximum at 330 nm, whereas the
protein’s absorption spectrumis between 200 and 290 nm. Thus, it does not
seem possible that energy transference between these two molecules may
occur.
The experiments were performed in 100 mM acetate buffer with 12%
ethanol/water (v/v) as solvent (pH 5.0), and stock solutions of α-amylase (100
µM), procyanidin fractions (1 mM), and polysaccharides were prepared in this
buffer. A pH of 5.0 was chosen because it is already known that α-amylase
interactions with polyphenol are high at this pH, and it is close to mouth pH (7.0)
(33).
Because the reactivity of procyanidins toward α-amylase depends on their MW,
the concentration of each procyanidin fraction corresponds to the maximum
aggregation for a fixed concentration of α-amylase (1 µM). Fraction I interacted
weakly with α-amylase and was rejected in this study. The concentrations used
(fraction II = 120 µM; fraction III = 50 µM; fraction IV = 17 µM) were obtained by a
nephelometry curve, corresponding to the aggregation with increasing
concentration of procyanidins (data not shown). The α-amylase concentration
was chosen to be in the linear range of concentration with fluorescence.
In several microtubes, a volume of procyanidin fraction stock solution was mixed
with different volumes (to make a final volume of 250 µL) of acetate buffer. After
this, different volumes of the polysaccharide stock solution were added, and the
168 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
tubes were mixed. The mixture was allowed to stand for 30 min, and the blank
was measured (only for nephelometry) before the addition of 4 µL of the α-
amylase stock solution. After the addition of α-amylase, the mixture was mixed
and allowed to react for 1 h. After this, the microtube was mixed and the emission
spectra and intensity of aggregates were measured in the fluorometer cell.
The effect of polysaccharide on the protein’s fluorescence intensity was
previously analyzed. For this, a mixture of α-amylase (1 µM) with the highest
concentration of each polysaccharide (0.6 g.L-1 for arabic gum, 4.4 g.L-1 for
cyclodextrin, and 80.0 mg.L-1 for all pectins) was prepared, and after 1 h, the
protein’s fluorescence intensity was measured.
Because the procyanidin fractions possess intrinsic fluorescence at λex (282 nm),
their spectrum was measured and subtracted in all fluorescence experiments.
The effect of polysaccharides in the procyanidin fraction emission spectra was
also evaluated; it was observed that polysaccharides did not affect the
procyanidin fractions spectra.
All assays were performed in triplicate.
Dynamic Light Scattering (DLS) Measurements. The size of
aggregates present in solution was determined by DLS. The size of procyanidin
fractions/PPA aggregates and the effect of the carbohydrates were measured
using the Zetasizer Nano ZS Malvern instrument. This technique measures the
time-dependent fluctuations in the intensity of scattered light that occur because
particles undergo Brownian motion. The analysis of these intensity fluctuations
enables the determination of the diffusion coefficients of particles, which are
converted into a size distribution. The sample solution was illuminated by a 633
nm laser, and the intensity of light scattered at an angle of 173 was measured by
an avalanche photodiode. This analysis provides information concerning particle
size (obtained by the parameter intensity) and polydispersity.
These experiments were performed in triplicate.
Statistical Analysis. All assays were performed in n = 3 repetitions. The
mean values and standard deviations were evaluated using analysis of variance
(ANOVA); all statistical data were processed using GraphPad Prism version 5.0
for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).
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� RESULTS AND DISCUSSION
Fluorescence quenching of α-amylase/tannin complexes in the presence of
increasing concentrations of polysaccharides was followed simultaneously with
the measure of α-amylase/tannin aggregates by nephelometry. As the
nephelometry measurements of aggregation are affected by several factors,
mainly by the size of aggregates, this aspect was also evaluated by DLS
technique.
Effect of Arabic Gum on PPA/Procyanidin Fraction Co mplexation.
Fig.4.1 shows the fluorescence emission spectrum (at λex = 282 nm) obtained for
PPA and procyanidin fraction IV (mean MW = 2052) solution in the absence and
increasing concentrations of arabic gum (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 g.L-1). Similar
spectra were obtained with procyanidin fractions II and III (data not shown). As
the concentration of arabic gum rises, the intensity of fluorescence increases.
Fig.4.1 Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM) and procyanidin fraction IV (17 µM; mean MW = 2052) solution, in the absence (full line) and presence of increasing concentrations of Arabic gum (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 g.L–1). The experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).
The effect of different polysaccharides on the protein’s fluorescence intensity was
assayed to determine if the observed variations in fluorescence intensity were
only due to modifications of procyanidin interaction with α-amylase (Fig.4.2A).
300 350 400 4500
20
40
60
80
100
120
λλλλ (nm)
Inte
nsity
arabic gum
concentration
170 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
A.
B. Procyanidins fractions alone Procyanidins fractio ns in presence of polysaccharides
Fig.4.2 A. Fluorescence emission spectrum (at λex = 282 nm) of PPA (1 µM) (solid spectra) and in the presence of the highest polysaccharide concentration [arabic gum 0.6 g.L-1, ; β-cyclodextrin 4.4 g.L-1, - - -; and pectins 80 mg.L-1 (27% MD, ; 58-63% MD, ; and 72% MD, ]. B. Several fluorescence emission spectra of procyanidin fractions alone and in the presence of the highest polysaccharide concentration (arabic gum 0.6 g.L-1, ; β-cyclodextrin 4.4 g L-1, ; and pectin 58-63% MD 80 mg.L-1, ). The experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).
In general, the different polysaccharides studied did not affect significantly the α-
amylase intrinsic fluorescence. On the other hand, procyanidins have been
shown to have much lower fluorescence intensity than the one observed for
proteins at the λex (Fig.4.2B). Nevertheless, procyanidin fraction spectra were
measured and subtracted from the emission spectra of α-amylase. Therefore, the
results obtained in the fluorescence quenching assays are only due to
modifications in the interaction between protein and the quencher (procyanidin).
This means that the quenching effect of procyanidins on PPA fluorescence
decreases in the presence of arabic gum. However, the increase in fluorescence
does not reach the protein’s intrinsic fluorescence itself (∼200 of intensity, in the
same experimental conditions without procyanidins and arabic gum) even for
higher concentrations of polysaccharide (data not shown). The increasing
percentages of intrinsic fluorescence by the presence of arabic gum for all tested
300 340 380 420 4600
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200
250
300
350
λλλλ (nm)
Inte
nsity
300 340 380 420 4600
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Inte
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300 340 380 420 4600
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II
IV
III
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procyanidin fractions are shown in Fig.4.3 (upper data). All procyanidin fractions
followed the same behavior.
Overall, arabic gum was shown to be more efficient in reducing the quenching
effect of PPA for the more highly polymerized fraction (IV).
Fig.4.3 Influence of increasing concentrations of arabic gum in (upper data) variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution and (lower data) percentage of procyanidin/PPA (1 µM) insoluble aggregates measured by nephelometry. Mean MWs of fractions II, III, and IV are 950, 1512, and 2052, respectively. All experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).
For the same solution, the formation of aggregates (macromolecules) between α-
amylase and procyanidins, which are supposed to be in part insoluble, was also
followed by nephelometry (Fig.4.3, lower data). For all of the procyanidin
fractions tested, a decrease in aggregation with the increase in arabic gum
concentration was observed. This decreasing intensity of aggregation seems to
be greater for the highly polymerized fractions. These results are in agreement
with previous works (19, 34).
It is interesting to observe that the aggregation decreases concomitantly with the
increase of fluorescence. The competition between arabic gum and α-amylase
for procyanidins leads to the decrease of the quenching effect and, consequently,
to less aggregation.
Effect of β-Cyclodextrin on PPA/Procyanidin Fraction Complexat ion.
The influence of β-cyclodextrin concentration in procyanidin/PPA intrinsic
fluorescence is similar to that observed with arabic gum (Fig.4.1). The variation of
intrinsic fluorescence and aggregation is shown in Fig.4.4.
0.0 0.1 0.2 0.3 0.4 0.5 0.60
20
40
60
80
100
120
140
160
180
200
Fraction II
Fraction III
Fraction IV
Flu
ores
cenc
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ephe
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Arabic gum (g.L -1)
%
172 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.4.4 Influence of increasing concentrations of β-cyclodextrin in (upper data) variation of the fluorescence of PPA (1 µM) and procyanidin fractions solution and (lower data) percentage of procyanidin/PPA (1 µM) insoluble
aggregates measured by nephelometry. Mean MWs of procyanidin fractions II, III, and IV are 950, 1512, and 2052,
respectively. All experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).
As observed for arabic gum, fluorescence in the presence of β-cyclodextrin
increases concomitantly with the decrease in percentage of aggregation. With
regard to the quenching effect, the lower MW fraction (II) is the most affected,
whereas the higher MW fraction (IV) is the less affected, oppositely to arabic
gum, where fraction IV was the most affected fraction.
For arabic gum and β-cyclodextrin assays, the interaction between procyanidins
(quenchers) with PPA is restrained by the presence of these polysaccharides,
reducing the quenching effect and consequently reducing the aggregation, as
confirmed by nephelometry. Altogether, these results provide strong evidence for
a molecular association in solution between polysaccharides and procyanidins,
competing for protein aggregation and inhibiting the formation of aggregates.
Comparison of these two polysaccharides shows that arabic gum is by far the
most effective in restraining the interaction between PPA and procyanidins,
because the amount required was about 10-fold smaller than that of β-
cyclodextrin. This is probably due to the polysaccharide structures, because β-
cyclodextrin has a cone-shaped structure conferring conformational restrictions
for the interaction with procyanidins. Effectively, β-cyclodextrin is a cyclic
oligosaccharide, composed of seven 1→4-linked α-D-glucopyranoside units. The
interior of the structure is less hydrophilic than the surface, being able to host
hydrophobic molecules in its interior (30, 31). However, some studies (35, 36)
have shown that only small molecules, such as (+)-catechin, can be included
inside the cyclodextrin cavity, which is not the case with procyanidin oligomers.
On the other hand, arabic gum is a heteropolysaccharide composed by a
polysaccharide and hydroxyproline-rich protein moieties able to ensure
hydrophobic interactions with procyanidins (27, 28). This polysaccharide moiety
0.0 1.0 2.0 3.0 4.0 5.00
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Fraction II
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has also a slight acidic character, resulting from the presence of only 20% of
glucuronic acids. Its more acidic character compared to β-cyclodextrin may help
to establish electrostatic and cooperative hydrogen bonds that strengthen the
interaction with procyanidins. This may explain the higher efficiency of arabic
gum in restraining the aggregation between procyanidin fractions and PPA.
These kinds of electrostatic and ionic interactionswith proteins and tannins have
previously been shown with pectins from wines (32, 37). The restraining effect of
both polysaccharides in the aggregation between procyanidin fractions and α-
amylase is lower for fraction II than for the other two fractions. The effect of
polysaccharides in aggregation seems to be affected differently with the increase
in procyanidin MW. This effect was greater for fractions IV and III for arabic gum
and α-cyclodextrin, respectively.
Effect of Pectin with Different MDs on PPA/Procyani din Fraction
Complexation. The variation in intrinsic fluorescence of α-amylase/procyanidin
with different concentrations of pectins is shown in Fig.4.5 (upper data).
A B
C
Fig.4.5 Percentage of procyanidin/PPA (1 µM) insoluble aggregates in the presence of increasing concentrations of different pectins measured by nephelometry. Mean MWs of procyanidin fractions II (a), III (b), and IV (c) are 950, 1512, and 2052, respectively. All experiments were performed in 100 mM acetate buffer (pH 5.0) with 12% ethanol/water (v/v).
0.0 20.0 40.0 60.0 80.00
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Fraction II
Fraction III
Fraction IV
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174 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Oppositely to arabic gum and β-cyclodextrin, the fluorescence remains more or
less constant with the increase of pectin concentration. In these cases, the
quenching effect of α-amylase by procyanidins is not affected by the presence of
pectins: the molecular environment in the vicinity of the chromophore (tryptophan
residues) seems to remain unchanged.
The nephelometry analysis showed a systematic decrease in aggregation with
the increase in pectin concentration, similar to that with arabic gum and β-
cyclodextrin. Once again, this effect was lower for the procyanidin fraction with
lower MW (II).
It is interesting to observe that although the effect of procyanidins in α-amylase
fluorescence is not affected by the presence of pectins, their aggregation ability is
importantly affected. Altogether, these results demonstrate that there is probably
a molecular association between polysaccharides, procyanidin fraction, and
protein in solution, resulting in a ternary complex and thereby forming more
soluble aggregates in aqueous medium. Thus, the fluorescence quenching of
PPA by procyanidin fractions is not reduced, but the formation of less soluble
aggregates is still inhibited.
Pectins were the most effective polysaccharides that inhibited procyanidins/PPA
aggregation, because the amount required was about a 10-fold lower
concentration than that of arabic gum. This is probably due to their more acidic
character and higher flexibility, when compared to the other polysaccharides
tested. In fact, pectins are a very complex mixture of different polysaccharide
molecules with a large range of molecular weights that vary with origin and
extraction conditions. In general, they are composed by partially methylated poly-
R-(1→4)-D-galacturonic acid residues (38). Pectins with high MD have reduced
polarity, whereas pectins with low MD have a few hydrophobic groups.
However, no correlation between the pectins’ MD and the restraining effect on
procyanidin fractions/PPA aggregation was observed. Apparently, differences in
the ratio of hydrophobic/hydrophilic groups between pectins did not seem to be
enough to induce important different behaviors. Overall, among all the studied
polysaccharides, pectins have a higher ionic character capable of establishing
hydrophilic interactions.
Size Measurement of Aggregates. The measurement of the size of
aggregates present in solution resulting from the interaction between the three
procyanidin fractions (II, III, and IV) and PPA in the absence and presence of
carbohydrates was performed by DLS. Table 4.1 shows the average size (Z) of
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aggregates and also the polydispersity of solutions. In general, the polydispersity
of solutions is below 0.5, which means that the Z and polydispersity will give
values that can be used for comparative purposes.
Table 4.1 Average size of aggregates (Z) present in procyanidin fractions and PPA (1 µM) solutions (100 mM acetate buffer with 12% ethanol/water (v/v) pH 5.0) and polydispersity in absence and presence of carbohydrates, measured by dynamic light scattering. Fraction II Fraction III Fraction IV Carbohydrate Z (nm) polydispersity Z (nm) polydispersity Z (nm) polydispersity
none 646 ± 38 0.221 485 ± 49 0.121 465 ± 28 0.346 β-cyclodextrin (4.5 g.L-1) 646 ± 39 0.833 445 ± 26 0.279 416 ± 25 0.021 arabic gum (0.6 g.L-1) 123 ± 7 0.525 156 ± 2 0.246 107 ± 6 0.500 Pectin (80 mg.L-1): MD 27% 249 ± 15 0.495 177 ± 11 0.248 173 ± 10 0.477 MD 58-63% 289 ± 17 0.464 196 ± 12 0.287 164 ± 10 0.425 MD 72% 300 ± 18 0.707 178 ± 11 0.307 219 ± 13 0.477
Despite the decrease of procyanidin fraction concentration relative to
themolecular weight, the aggregates present in solution have approximately the
same size, particularly for procyanidin fractions III and IV (magnitude of 500 nm).
This could be explained by the fact that the concentrations of procyanidin
fractions used correspond to their maximum aggregation with PPA (stoichiometric
concentrations), for which the increase in tannin concentration does not change
aggregation (see Materials and Methods).
In general, the size of aggregates decreases in the presence of carbohydrates.
Exceptionally, the one obtained for fraction II in the presence of β-cyclodextrin did
not change. In that case, a large width of the particle size distribution was also
observed (high polydispersity), indicating a very heterogeneous population of
aggregates.
In the presence of pectin and especially of arabic gum there is a significant
decrease in the size of aggregates, much higher than for β-cyclodextrin. These
carbohydrates have induced a reorganization of the aggregates leading to the
formation of smaller structures that reduce the scattered light, which is in
agreement with the observed decrease of nephelometric measurements.
Therefore, as previously mentioned, the disrupting effect of polysaccharides
toward protein/tannin aggregation has been proposed to result from two
mechanisms (Fig.4.6): (i) the ability of polysaccharides to form a ternary complex
protein/polyphenol/polysaccharide, thereby enhancing its solubility in aqueous
medium, resulting in less insoluble aggregates; and (ii) the molecular association
in solution between polysaccharides and polyphenols competing with protein
aggregation.
176 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fig.4.6 Possible mechanisms (i and ii) involved in the inhibition of the aggregation of tannins and proteins by polysaccharides. P, protein; T, tannin; C, polysaccharide/carbohydrate.
From the results obtained herein, it can be proposed that the mechanisms i and
ii, by which polysaccharides restrain the protein/polyphenol aggregation, are
governed by the polysaccharide structure. The results obtained for arabic gum
and β-cyclodextrin strongly suggest that the main mechanism by which these two
compounds inhibit protein/polyphenol aggregation is mechanism ii (Fig.4.6).
Arabic gum and β-cyclodextrin interact with procyanidin fractions, with more or
less strength depending on procyanidin MW and also on the polysaccharide
structure, and inhibit procyanidin interaction with PPA. Effectively, the quenching
effect on α-amylase by procyanidins is decreased by the action of these
polysaccharides promoting an increase in the observed intrinsic fluorescence
intensity of α-amylase. Simultaneously, the aggregation decreases concomitantly
with the increase of polysaccharide concentration.
The interaction between polysaccharides and polyphenols may result from
cooperative hydrogen bonding, between the hydroxyl groups of polysaccharide
and phenolics, and from hydrophobic interactions. The higher effect of arabic
gum, compared to β-cyclodextrin, is probably due to its more acidic character,
resulting from the presence of glucuronic acids able to establish more hydrogen
bonds with procyanidins.
In the case of pectins, the results obtained provide substantial evidence for
demonstrating that the main mechanism by which these compounds inhibit
protein/polyphenol aggregation is mechanism i. Pectins seem to interact with
procyanidin fractions and with PPA to form a ternary complex
protein/polyphenol/polysaccharide, thereby enhancing its solubility in aqueous
medium, resulting in smaller andmore soluble aggregates. Effectively, opposite to
Stechoimetric
concentrations
polyphenol protein
+ Largest network of
protein-tannin
insoluble complexes
+
i)
ii
Protein/tannin/polysaccharide
ternary complex
Soluble complex of
tannin/polysaccharide
polysaccharide
Protein with a few
tannin molecules
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177
arabic gum and β-cyclodextrin, the environment of the tryptophan residues of α-
amylase is not affected by the presence of pectins, so the intrinsic fluorescence
is not changed while solubilization occurs. This could mean that procyanidins still
interact with α-amylase even in the presence of pectins.
Using DLS, it was observed that carbohydrates induce a decrease in aggregates
size. The decrease in nephelometry intensity seems to be due to the formation of
smaller aggregates, which results from the disruption and reorganization of the
aggregates.
Overall, the combination of different analytical techniques – fluorescence
quenching, nephelometry, and DLS – allowed important insights into the two
mechanisms by which different polysaccharides are thought to inhibit
protein/tannin aggregation. This development may help further investigations
regarding the influence of carbohydrates in food taste.
� ABBREVIATIONS USED
PRP, proline-rich protein; PPA, porcine pancreas α-amylase; MD, methoxylation
degree; DLS, dynamic light scattering; FRET, fluorescence resonance energy
transfer.
� ACNOWLEDGMENT
We thank Prof. Graham Jones from the University of Adelaide (Australia) for
scientific advice and grammatical corrections.
� LITERATURE CITED
(1) Khanbabaee, K.; van Ree, T. Tannins: classification and definition. Nat. Prod.
Rep. 2001, 18, 641–649.
(2) Haslam, E. Plant Polyphenols – Vegetable Tannins Revisited – Chemistry
and Pharmacology of Natural Products; Cambridge University Press: Cambridge,
U.K., 1989.
(3) Siebert, K. J.; Troukhanova, N. V.; Lynn, P. Y. Nature of polyphenol-protein
interactions. J. Agric. Food Chem. 1996, 44, 80–85.
(4) Gonçalves, R.; Soares, S.; Mateus, N.; de Freitas, V. Inhibition of trypsin by
condensed tannins and wine. J. Agric. Food Chem. 2007, 55, 7596–7601.
178 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(5) Bennick, A. Interaction of plant polyphenols with salivary proteins. Crit. Rev.
Oral Biol. Med. 2002, 13, 184–196.
(6) Griffiths, D. W. The inhibition of digestive enzymes by polyphenolic
compounds. Adv. Exp. Med. Biol. 1986, 199, 509–516.
(7) Bate-Smith, E. C. Astringency in foods. Food 1954, 23, 124–127.
(8) Haslam, E. The association of proteins with polyphenols. J. Chem. Soc.,
Chem Commun. 1981, 309–311.
(9) Lesschaeve, I.; Noble, A. C. Polyphenols: factors influencing their sensory
properties and their effects on food and beverage preferences. Am. J. Clin. Nutr.
2005, 81 (Suppl.), 330S–335S.
(10) Lu, Y.; Bennick, A. Interaction of tannin with human salivary proline-rich
proteins. Arch. Oral Biol. 1998, 43, 717–728.
(11) Yan, Q.; Bennick, A. Identification of histatins as tannin-binding proteins in
human saliva. Biochem. J. 1995, 311, 341–347.
(12) de Freitas, V.; Mateus, N. Nephelometric study of salivary proteintannin
aggregates. J. Sci. Food Agric. 2001, 82, 113–119.
(13) Mateus, N.; Pinto, R.; Ruão, P.; de Freitas, V. Influence of the addition of
grape seed procyanidins to Port wines in the resulting reactivity with human
salivary proteins. Food Chem. 2004, 84, 195–200.
(14) Elodis, P.; Mora, S.; Krysteva, M. Investigation of the active center of
porcine-pancreatic amylase. Eur. J. Biochem. 1972, 24, 577–582.
(15) Buisson, G.; Duee, E.; Haser, R.; Payan, F. Three dimensional structure of
porcine pancreatic R-amylase at 2.9 Å resolution. Role of calcium in structure
and activity. EMBO J. 1987, 6, 3909–3916.
(16) Pasero, L.; Mazzei-Pierron, Y.; Abadie, B.; Chicheportiche, Y.; Marchis-
Mouren, G. Complete amino acid sequence and location of the 5 disulphide
bridges in porcine pancreatic α-amylase. Biochim. Biophys. Acta 1986, 869, 147–
157.
(17) Ozawa, T.; Lilley, T. H.; Haslam, E. Polyphenol interaction: astringency and
the loss of astringency in ripening fruit. Phytochemistry 1987, 26, 2937–2942.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
179
(18) McManus, J. P.; Davis, K. G.; Beart, J. E.; Gaffney, S. H. Polyphenol
interactions. Part 1. Introduction; some observations on the reversible
complexation of polyphenols with proteins
and polysaccharides. J. Chem. Soc., Perkin Trans. 2 1985, 1429–1438.
(19) de Freitas, V.; Carvalho, E.; Mateus, N. Study of carbohydrate influence on
protein-tannin aggregation by nephelometry. Food Chem. 2003, 81, 503–509.
(20) Carvalho, E.; Póvoas,M. J.; Mateus,N.; de Freitas, V. Application of flow
nephelometry to the analysis of the influence of carbohydrates on protein-tannin
interactions. J. Sci. Food Agric. 2006, 86, 891–896.
(21) Papadopoulou, A.; Green, R. J.; Frazier, R. A. Interaction of flavonoids with
bovine serum albumin: a fluorescence quenching study. J. Agric. Food Chem.
2005, 53, 158–163.
(22) Rawel, H. M.; Frey, S. K.; Meidtner, K.; Kroll, J.; Schweigert, F. J.
Determining the binding affinities of phenolic compounds to proteins by
quenching of the intrinsic tryptophan fluorescence. Mol. Nutr. Food Res. 2006,
50, 705–713.
(23) Soares, S.; Mateus, N.; de Freitas, V. Interaction of different polyphenols
with bovine serum albumin (BSA) and human salivary α-amylase (HSA) by
fluorescence quenching. J. Agric. Food Chem. 2007, 55, 6726–6735.
(24) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer
Academic/Plenum Publishers: New York, 1999.
(25) de Freitas, V. A. P.; Glories, Y.; Laguerre, M. Incidence of molecular
structure in oxidation of grape seed procyanidins. J. Agric. Food Chem. 1998, 46,
376–382.
(26) Gonzalez-Manzano, S.; Mateus, N.; de Freitas, V. A. P.; Santos-Buelga, C.
Influence of the degree of polymerisation in the ability of catechins to act as
anthocyanin copigments. Eur. Food Res. Technol. 2008, 227, 83–92.
(27) Kjoeniksen, A.; Hiorth, M.; Roots, J.; Nyström, B. Shear-induced association
and gelation of aqueous solutions of pectin. J. Phys. Chem. B 2003, 107, 6324–
6328.
180 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(28) Goodruma, L. J.; Patelb,A.; Leykamc, J. F.; Kieliszewski,M. J.Gum arabic
glycoprotein contains glycomodules of both extensin and arabinogalactan-
glycoproteins. Phytochemistry 2000, 54, 99–106.
(29) Sanchez, C.; Renard, D.; Robert, P.; Schmitt, C. Structure and rheological
properties of acacia gum dispersions. Food Hydrocolloids 2002, 16, 257–267.
(30) Biwer, A.; Antranikian, G.; Heinzle, E. Enzymatic production of cyclodextrins.
Appl. Microbiol. Biotechnol. 2002, 59, 609–617.
(31) Saenger, W.; Jacob, J.; Gessler, K.; Steiner, T.; Hoffmann, D.; Sanbe, H.;
Koizumi, K.; Smith, S. M.; Takaha, T. Structures of the common cyclodextrins
and their larger analogues;beyond the doughnut. Chem. Rev. 1998, 98, 1787–
1802.
(32) Carvalho, E.; Mateus, N.; Plet, B.; Pianet, I.; Dufourc, E.; de Freitas, V.
Influence of wine pectic polysaccharides on the interactions between condensed
tannins and salivary proteins. J. Agric. Food Chem. 2006, 54, 8936–8944.
(33) Young, G.; Resca, H. G.; Sullivan, M. T. The yeasts of the normal mouth and
their relation to salivary acidity. J. Dent. Res. 1951, 30, 426–430.
(34) Mateus, N.; Carvalho, E.; Luís, C.; de Freitas, V. Influence of the tannin
structure on the disruption effect of carbohydrates on protein-tannin aggregates.
Anal. Chim. Acta 2004, 513, 135–140.
(35) Jullian, C.; Miranda, S.; Zapata-Torres, G.; Mendizábal, F.; Olea-Azar, C.
Studies of inclusion complexes of natural and modified cyclodextrin with (+)-
catechin by NMR and molecular modeling. Bioorg. Med. Chem. 2007, 15, 3217–
3224.
(36) Kriz, Z.; Koča, J.; Imberty, A.; Charlot, A.; Auzély-Velty, R. Investigation of
the complexation of (+)-catechin by β-cyclodextrin by a combination of NMR,
microcalorimetry and molecular modeling techniques. Org. Biomol. Chem. 2003,
1, 2590–2595.
(37) Vernhet, A.; Pellerin, P.; Prieur, C.; Osmianski, J.; Moutounet, M. Charge
properties of some grape and wine polysaccharide and polyphenolic fractions.
Am. J. Enol. Vitic. 1996, 47, 25–30.
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(38) Pérez, S.; Rodríguez-Carvajal, M. A.; Doco, T. A complex plant cell wall
polysaccharide: rhamnogalacturonan II. A structure in quest of a function.
Biochimie 2003, 85, 109–121.
182 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Capítulo 5
Carbohydrates Inhibit Salivary Proteins Precipitation by Condensed Tannins
Soares, S., Mateus, N., de Freitas, V.
J. Agric. Food Chem. 2012, 60, 3966-3972
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Carbohydrates Inhibit Salivary Proteins Precipitati on by Condensed Tannins
Susana Soares, Nuno Mateus and Victor de Freitas*
Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
Condensed tannins are a group of polyphenols that are associated with the
astringency sensation, as they readily interact and precipitate salivary proteins.
As this interaction is affected by carbohydrates, the aim of this work was to study
the effect of some carbohydrates used in the food industry [arabic gum (AG),
pectin, and poligalacturonic acid (PGA)] on the salivary proteins/grape seed
procyanidins interaction. This was assessed monitoring the salivary proteins that
remain soluble in the presence of condensed tannins with the addition of
carbohydrates (HPLC) and analysis of the respective precipitates (SDS-PAGE).
The results show that pectin was the most efficient in inhibiting protein/tannin
precipitation, followed by AG and PGA. The results suggest that pectin and PGA
exert their effect by formation of a ternary complex
protein/polyphenol/carbohydrate, while AG competes with proteins for tannin
binding (competition mechanism). The results also point out that both hydrophilic
and hydrophobic interactions are important for the carbohydrate effects.
KEYWORDS: proline-rich proteins, grape seed tannins, pectin, arabic gum, polygalacturonic acid
� INTRODUCTION
Condensed tannins are a complex group of polyphenolic polymers of catechin
(polyphenol compounds) that can be found in vegetal foodstuffs, particularly in
fruits, cereal grains, and beverages (red wine, tea, and beer). During foodstuff
consumption, these polyphenols interact with salivary proteins forming insoluble
aggregates that are supposed to be at the origin of the astringency sensation.1-4
Astringency has been defined as a dryness of the oral surface, puckering and
tightening sensations of the oral mucosa typically experienced during the
ingestion of tannin-rich food, in particular red wine.5 It is considered to be a
tactile, diffuse, and poorly localized sensation. In fact, this sensation is often the
186 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
last one to be detected as it can take 15 s or more for the perception to fully
develop.6
Although there are a few theories about the origin of the astringency sensation,
the most widely accepted one relies on the interaction between tannins and
salivary proteins. In general, these tannin.protein interactions are thought to
involve the cross-linking of separate protein molecules by the tannin, which acts
as a polydentate ligand on the protein surface involving hydrophobic and
hydrogen bonds.7-12
Salivary proteins include very structurally diverse proteins such as á-amylase,
albumin, lysozyme, proline-rich proteins (PRPs), histatins, cystatins, and
statherin. The main salivary proteins have been grouped into six structurally
related major classes, namely, histatins, basic proline-rich proteins (bPRPs),
acidic proline-rich proteins (aPRPs), glycosylated proline-rich proteins (gPRPs),
statherin, and cystatins.13,14 These proteins have important biological functions in
saliva associated with calcium binding to enamel, maintenance of ionic calcium
concentration (PRPs and statherin), antimicrobial action (histatins and cystatins),
or protection of oral tissues against degradation by proteolytic activity
(cystatins).15-19
Regarding tannin-protein interactions in vitro, these are reported to be affected by
several factors, in particular ionic strength, pH of the medium, percentage of
ethanol, temperature, and presence of carbohydrates.20-24
The first report of the inhibitory effect of carbohydrates in those interactions
concerned the proposed mechanisms for the astringency loss during fruit
ripening: as the cellular structure softens during fruit ripening, there is an
increase in watersoluble pectin fragments that could prevent the formation of
aggregates between fruit tannins and salivary proteins in the mouth, leading to a
modified astringency response.25-27 Other studies showed that the astringency of
tannins, as well as their interaction with proteins, is reduced by the addition of
carbohydrates.23,28,29
Two mechanisms have been proposed to explain this inhibitory effect of
carbohydrates: (I) carbohydrates form ternary complex protein-polyphenol-
carbohydrates, which enhance solubility in an aqueous medium; (II) there is a
molecular association in solution between carbohydrates and polyphenols hence
competing for protein aggregation.25,27
The effect of carbohydrates in protein-tannin interactions has a great impact in
the perception and choice of foodstuffs. Carbohydrates are frequently used in
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food industry as food colloids (gums) and are also naturally present in several
food products, thereby affecting their astringent features.
Despite the knowledge about the effect of carbohydrates on protein-tannin
interactions, there is still little information when considering this effect on the
interaction of tannins with salivary proteins. In fact, there is a lack of information
in which way the structure of the different salivary proteins and the different
carbohydrates affect their action. Most of the literature on this subject has mainly
reported studies involving other proteins, namely, model proteins such as bovine
serum albumin (BSA) and α-amylase.
Therefore, this work aimed to study the effect of several carbohydrates used in
the food industry as food colloids [Arabic gum (AG), pectin, and polygalacturonic
acid (PGA)] on the interaction between salivary proteins and grape seed fraction
(GSF) (condensed tannins).
� MATERIALS AND METHODS
Reagents. All reagents used were of analytical grade. AG (purity not
provided) was purchased from Aldrich. Pectin (purity 79.5%) and PGA (purity
80%) (both from citrus peel) were purchased from Sigma.
GSF Isolation. Procyanidins were extracted from grape seeds (Vitis
vinifera) with an ethanol/water/chloroform solution (1:1:2, v/v/v). The resulting
solution was centrifuged, and the chloroform phase, containing chlorophylls,
lipids, and other undesirable compounds, was rejected. The hydroalcoholic
phase was then extracted with ethyl acetate, and the organic phase was
evaporated using a rotary evaporator (30°C). The resulting residue
corresponding essentially to oligomeric procyanidins was fractionated through a
TSK Toyopearl HW-40(s) gel column (100 mm × 10 mm i.d., with 0.8 mL.min−1
methanol as eluent), yielding two fractions according to the method described in
the literature.30 The first fraction was obtained after elution with 99.8% (v/v)
methanol during 5 h (240 mL), and the second was eluted with methanol/5% (v/v)
acetic acid during the next 14 h (670 mL). Both fractions were mixed with
deionized water, and the organic solvent was eliminated using a rotary
evaporator under reduced pressure at 30°C and then freeze-dried. The
procyanidin composition of fractions was determined by direct analysis by
electrospray ionization mass spectrometry (ESI-MS) (Finnigan DECA XP PLUS)
as described in the literature.31 The first fraction contains mainly catechins,
188 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
procyanidin dimers, and their galloyl derivatives, and the second fraction contains
essentially procyanidin dimers galloylated, procyanidin trimers and their galloyl
derivatives, and procyanidin tetramers. For the second fraction, the average full
mass spectra was obtained, and the mean degree of polymerization (mDP) was
estimated from the ratio between the sum of the relative abundance of each
compound multiplied by its number of elementary catechin units and the sum of
the relative abundances for all compounds in the fraction. The fraction has a
mean MW of 936 and a polymerization degree average of 3.2. Only the second
fraction named GSF was used herein, as it was shown to be the most reactive
toward proteins.
Saliva Collection. Saliva was collected from six healthy nonsmoking
volunteers, and 2 mL of saliva from each volunteer was used to make a saliva
pool (whole saliva). The collection time was standardized at 2 p.m. to reduce
concentration variability connected to circadian rhythms of secretion.32 The saliva
pool was mixed with 10% trifluoroacetic acid (TFA) (final concentration, 0.1%) to
precipitate several high molecular weight salivary proteins (such as α-amylase,
mucins, carbonic anhydrase, and lactoferrin) and to preserve sample protein
composition, since TFA partially inhibits intrinsic protease activity. After the
centrifugation (8000g for 5 min), the supernatant (acidic saliva, AS) was
separated from the precipitate and used for the following experiments.
Pectin Purification. Pectin was purified by precipitation with ethanol.
Briefly, after dissolution in a small amount of water, ethanol was added to
achieve a 70% (v/v) CH3CH2OH/H2O concentration. The precipitated
carbohydrate was recovered by filtration under vacuum (1 µm filters) and dried.
Pectin was analyzed by colorimetric methods and gas chromatography to
determine sugar composition and esterification degrees.33,34 It was found to be
composed by 85% galacturonic acid, 10% galactose, and 5% other sugars. The
degrees of methylation and acetylation were determined to be 14 and 1%,
respectively.
Protein and Tannin Interaction. The AS sample was analyzed by high-
performance liquid chromatography (HPLC) before and after the interaction with
increasing concentrations of GSF. These experiments were made to obtain the
minimal GSF concentration that precipitates almost totally the salivary proteins.
The control condition was a mixture of AS (150 µL) and water (50 µL) (final
volume, 200 µL). Different volumes of a GSF stock solution (30.0 mM) prepared
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in water were added to AS (150 µL) to obtain the desired final concentrations,
150, 300, or 750 µM. The final volume was adjusted to 200 µL with pure water.
The mixture was shaken and kept for 5 min at room temperature (±20 °C) and
then centrifuged (8000g, 5 min). The supernatant was injected into the HPLC.
After these first experiments, the GSF concentration chosen for the experiments
with the carbohydrates was 300 µM.
Effect of Carbohydrates on Protein/Tannin Interacti on. For the
experiments with carbohydrates, stock solutions of AG (25.0 g.L–1), PGA (30.0
g.L–1), and pectin (10.0 g.L–1) were prepared in water, and different volumes of
these stock solutions were added to GSF solution to obtain different final
concentrations. The final volume (50 µL) was adjusted with water to obtain the
desired carbohydrates final concentration (between 2.4 and 30.0 g.L–1,
depending on carbohydrates). The mixture was shaken and kept at room
temperature (±20 °C) for 30 min. After that, 150 µL of AS was added to the
previous mixture (final volume, 200 µL), which was shaken and kept for 5 min at
room temperature (±20 °C). The mixture was then centrifuged (8000g, 5 min), the
supernatant was injected into the HPLC, and the precipitate was analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH
of the supernatant was measured prior to analysis, and it was 3.1 [which is near
to some beverages pH such as red wine (pH 3.5)].
HPLC Analysis. Ninety microliters of each solution was injected on a
HPLC Lachrom system (L-7100) (Merck Hitachi) equipped with a Vydac C8
column (Grace Davison Discovery Sciences), with 5 µm particle diameter
(column dimensions 150 mm × 2.1 mm); detection was carried out at 214 nm,
using a UV−vis detector (L-7420). The HPLC solvents were 0.2% aqueous TFA
(eluent A) and 0.2% TFA in acetonitrile (ACN)/water 80/20 (v/v) (eluent B). The
gradient applied was linear from 10 to 40% (eluent B) in 60 min, at a flow rate of
0.30 mL.min–1. After this program, the column was washed with 100% eluent B
for 20 min to elute S type cystatins and other late eluting proteins. After washing,
the column was stabilized with the initial conditions.1,32
SDS-PAGE. The precipitates that resulted from the interaction between
AS and GSF in absence and presence of the highest carbohydrates
concentration, as well as the AS control solution, were analyzed by SDS-PAGE.
The precipitates were resolubilized in 200 µL of electrophoresis sample buffer (50
mM Tris-HCl, pH 6.8, 12% v/v glycerol, 4% SDS, 2.5% v/v β-mercaptoethanol,
190 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
and 0.01% bromophenol blue) to have the same initial volume used in the
previous experiments during precipitation. Eighty microliters of electrophoresis
sample buffer was added to 80 µL of these solutions. The control solution was
composed by 80 µL of the AS solution and 80 µL of a twice-concentrated
electrophoresis sample buffer (100 mM Tris-HCl, pH 6.8, 24% v/v glycerol, 8%
SDS, 5% v/v β-mercaptoethanol, and 0.02% bromophenol blue). The samples
were heated at 60°C for 1 h with shaking and then analyzed by SDS-PAGE in a
tris-tricine buffer system according to the method of Schägger using 16%
acrylamide resolving gel. The stacking gel was 5% acrylamide. The cathode
buffer was 0.1 M Tris, 0.1 M tricine, and 0.1% SDS. The anode buffer was 0.2 M
Tris-HCl, pH 8.9. Electrophoresis was performed on a Bio-Rad MiniProtean Cell
electrophoresis apparatus (Bio-Rad) at constant voltage (150 V). After
electrophoresis, the gels were stained with Imperial Protein Stain (Thermo
Scientific), a Coomassie R-250 dye-based reagent. The staining with Imperial
Protein Stain was done according to the supplier's instructions. The destaining
step was done by washing the gels with water until the bands were visible.
Molecular weights (Sigma) were estimated by comparison with the migration
rates of standard proteins (β-galactosidase from Escherichia coli, 116000 Da;
phosphorylase b from rabbit muscle, 97000 Da; albumin bovine serum, 66000
Da; glutamic dehydrogenase from bovine liver, 55000 Da; ovalbumin from
chicken egg, 45000 Da; glyceraldehyde-3-phosphate dehydrogenase from rabbit
muscle, 36000 Da; carbonic anhydrase from bovine erythrocytes, 29000 Da;
trypsinogen from bovine pancreas, 24000 Da; trypsin inhibitor from soybean,
20000 Da; á-lactalbumin from bovine milk, 14200 Da; and aprotinin from bovine
lung, 6500 Da).
Half Maximal Effective Concentration (EC 50) Calculation and
Statistical Analysis. EC50 values were calculated considering the area variation
of chromatogram peaks of each protein in the presence of carbohydrates. The
software GraphPad Prism 5 (GraphPad Software, Inc.) was used to calculate
EC50 and statistical analysis. Statistical significance of the difference between the
several calculated EC50 was evaluated by one-way analysis of variance, followed
by the Bonferroni test. Differences were considered to be statistically significant
when P<0.05. All of the experiments were performed in n = 3 repetitions.
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� RESULTS
The carbohydrates AG, pectin, and PGA are commonly present in food and are
also used in the food industry as additives. The influence of these three different
ionic carbohydrates on the interaction between salivary proteins and grape seed
condensed tannins was assessed by HPLC analysis and SDS-PAGE. The
disrupting effect of these compounds on protein−tannin aggregates was
assessed by monitoring the increase of the salivary proteins that remain soluble
in the presence of condensed tannins with the addition of each of these
carbohydrates.
Salivary Proteins. The initial acidic treatment of human saliva with TFA is
used to precipitate several high molecular weight salivary proteins (such as α-
amylases, mucins, carbonic anhydrase, and lactoferrin) and to preserve sample
protein composition, since TFA partially inhibits intrinsic protease activity.32
However, peptides and proteins like histatins, basic, acidic, and glycosylated
PRPs, statherin, cystatins, and defensins are soluble in AS solution and may be
directly analyzed by RP-HPLC, as previously described.1,32
The HPLC chromatogram of this AS solution at 214 nm is presented in Fig.5.1.
The top of the figure shows the distribution of the different families of salivary
proteins along the chromatogram that were established previously by proteomic
approaches, namely, ESI-MS and matrix-assisted laser desorption/ionization
time-of-flight/time-of-flight (MALDI-TOF/TOF).1,32
Fig.5.1Typical RP-HPLC profile detected at 214 nm of the AS solution of human saliva in the absence (— ) and presence (- - -) of 300 µM GSF. The vertical dotted lines show the ranges and the main salivary proteins family assigned to each HPLC peptide region: bPRPs, gPRPs, and aPRPs.
The HPLC chromatogram of the AS solution is roughly divided into four salivary
proteins family regions: the first region comprises proteins that belong to the
15 20 25 30 35 40 45 50 55 60 650
2
4
6
bPRPs gPRPs aPRPs statherin
t (min)
Abs
(A
u)
192 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
classes of bPRPs and histatins. The bPRPs identified in this region include IB-8b,
IB-8c, IB-9, IB-4, and P-J, and the histatins include histatins 3, 5, 7, 8, and 9. The
second region comprises mainly one gPRPs, the bPRP3. The next region
corresponds entirely to aPRPs, namely, PRP1 and PRP3, and the last region has
phosphorylated and nonphosphorylated forms of statherin and peptide PB.1
Interaction of Salivary Proteins with Procyanidin Fraction from
Grape Seeds (GSF). The first experiments were made to assess the minimal
procyanidin concentration that leads to a significant precipitation of salivary
proteins and then to study if the carbohydrates were able to inhibit that
precipitation. It was important to establish this concentration since an insufficient
quantity of procyanidins would lead to no significant precipitation of most of the
salivary proteins, and an excess of GSF would lead to free molecules of GSF,
thereby influencing the further effect of carbohydrates. Bearing this, three
increasing GSF concentrations were tested (150, 300, and 750 µM).
From these experiments, the GSF concentration chosen for the experiments with
carbohydrates was 300 µM, since it corresponded to the GSF concentration that
precipitated almost all of the salivary proteins present in the AS (Fig.5.1). In fact,
while for 150 µM GSF only aPRPs and statherin were significantly affected, the
750 µM concentration led to the precipitation of all of the proteins but also to an
excess of free molecules of GSF (data not shown).
It can be seen from Fig.5.1 that the addition of 300 µM GSF reduced significantly
the amount of gPRPs and practically depleted aPRPs and statherin. On the other
hand, bPRPs were not significantly affected. Therefore, the effect of
carbohydrates on the interaction between salivary proteins and tannins was
focused on gPRPs, aPRPs, and statherin proteins.
Effect of Carbohydrates on the Interaction between Salivary Proteins
and GSF. The experimental approach described herein intends to mimic the
phenomenon that occurs in the mouth during food mastication, where tannins
and carbohydrates simultaneously contact with salivary proteins. So, a solution of
tannins−carbohydrates was prepared, to which the AS solution was subsequently
added. The control experimental condition was made only with AS and 300 µM of
GSF. Fig.5.2 shows part of the chromatogram corresponding only to the families
of salivary proteins that were effectively affected by the GSF in the absence or
presence of the different carbohydrates.
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Fig.5.2 Part of the chromatograms of the AS solution after the interaction with GSF (300 µM) in the absence (AS solution + 300 µM GSF) and presence of the several tested carbohydrates (pectin, 5.0 g.L–1; AG, 10.0 g.L–1; and PGA, 20.0 g.L–1).
From the presented results, it can be observed that the carbohydrates in solution
enhance the chromatographic peaks corresponding to salivary proteins, in
especially for aPRPs and statherin. Fig.5.3 shows variation of the
chromatographic peaks area of the several salivary proteins studied with the
increase in the carbohydrate concentration, expressed in percentage of the area
of these proteins relatively to the respective area in control saliva (AS without
tannin).
A B
C
Fig.5.3 Influence of carbohydrate concentration on salivary proteins precipitation by condensed tannins (GSF, 300 µM). (A) AG, (B) PGA, and (C) pectin. These results represent the average of three independent experiments.
30 35 40 45 50 55 60 65 700
2
4
6
8
10
t (min)
Abs
(A
u)AS + GSF
AS + GSF + Pectin
AS + GSF + PGA
AS + GSF + AG
gPRPs aPRPs statherin
0.0 5.0 10.0 15.0 20.0 25.00
20
40
60
80
100
AG (g.L -1)
Are
a (
%)
of c
hrom
ato
gra
phic
pea
ks o
f sa
liva
ry p
rote
ins
0.0 10.0 20.0 30.00
20
40
60
80
100
PGA (g.L -1)
Are
a (
%)
of c
hro
ma
togr
aph
icp
ea
ks o
f sa
liva
ry p
rote
ins
0.0 2.0 4.0 6.0 8.0 10.00
20
40
60
80
100
Pectin (g.L -1)
Are
a (
%)
of c
hro
ma
togr
aph
icp
ea
ks o
f sa
liva
ry p
rote
ins
gPRP
aPRP
statherin
194 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
In general, it is possible to observe that salivary proteins in solution increase
concomitantly with the carbohydrates concentration. The observed changes may
be interpreted as the inhibition of salivary proteins precipitation by tannins in the
presence of increasing concentrations of carbohydrates.
Pectin was by far the most effective carbohydrate in preventing precipitation of
salivary proteins by condensed tannins. While pectin prevents almost total
precipitation of salivary proteins at the concentration of 10.0 g.L−1, AG and PGA
only showed similar effects for concentrations higher than 20.0 g.L−1.
Besides the HPLC analysis of the supernatant, it was also important to analyze
the pellets that resulted from the AS interaction with GSF in the absence and
presence of the different carbohydrates. So, to analyze these pellets, all
precipitates were resolubilized by heating (60°C) in 200 µL of electrophoresis
sample buffer and further analysis by SDS-PAGE. The proteins bands were
subsequently analyzed by densitometry, and the results are presented in Fig.5.4.
Fig.5.4 SDS-PAGE of the pellets that resulted from the interaction between AS and GSF in the absence (C) and presence of the several carbohydrates (pectin, 10.0 g.L−1; AG, 25.0 g.L−1; and PGA, 25.0 g.L−1). The molecular weight markers were substituted by lines, and the molecular mass marked on the left side is expressed in kDa. The gels were stained with Imperial Protein Stain, a Coomassie R-250 dye-based reagent. The table shows the ratio between the densitometry values obtained for total salivary proteins present in AS (control AS) and in each experiment. The values with equal letters are not significantly different (P < 0.05).
The results obtained by SDS-PAGE analysis of the referred pellets, as well as by
the densitometry analysis performed to the gel, clearly demonstrate that the
presence of these carbohydrates, in particular pectin and AG, decreases the
salivary proteins that are precipitated by tannins (Fig.5.4). The maximum of
salivary proteins precipitation results from the interaction between AS and GSF in
the absence of AG and pectin (lane C). The experiment with AS and GSF
resulted in a value of 0.46, while in the presence of pectin or AG, the obtained
value is significantly lower (0.16 and 0.11,respectively). Regarding the PGA
effect, looking at the SDS-PAGE results, it is also possible to observe that it also
Ratio Control AS 1.00 ± 0.07
C (AS + GSF) 0.46 ± 0.00a Pectin 0.16 ± 0.02b
AG 0.11 ± 0.02b PGA 0.43 ± 0.08a
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195
leads to a decrease of salivary proteins precipitation but in a fewer extent than
the other carbohydrates. SDS-PAGE analyses are probably not comparable with
HPLC results because there are other proteins (cystatins and other late eluting
proteins) not analyzed by HPLC that could be precipitated by tannins and so
appear in the SDS-PAGE gel. In this way, SDS-PAGE results only reflect the
efficiency of the carbohydrates in inhibiting tannin−protein interaction.
To compare properly the effectiveness of carbohydrates to inhibit salivary
proteins precipitation by tannins, the half maximal effective concentration (EC50)
was calculated from the HPLC results (Table 5.1).
Table 5.1 Half maximal effective concentration (EC50) in inhibition of tannin/salivary protein precipitation by three carbohydrates: AG, PGA and pectin. The values with equal letters are not significantly different (P<0.05).
EC50 (g.L -1) Carbohydrate gPRP aPRP statherin
AG 4.78 ± 0.39a 10.10 ± 0.16 5.68 ± 0.65a PGA 19.47 ± 0.50 15.12 ± 0.58 f 14.34 ± 1.61f
Pectin 4.75 ± 0.38d 4.83 ± 0.10d 4.77 ± 0.18d
Pectin has been shown to have the lowest EC50 value (around 4−5 g.L−1) being
the most effective in the inhibition of salivary proteins precipitation by tannins,
while PGA was shown to be the less effective carbohydrate in inhibiting
tannin−salivary proteins precipitation with an EC50 between 14 and 19 g.L−1.
Oppositely to AG, the effect of the other carbohydrates is quite similar for the
three groups of salivary proteins, especially for pectin, which indicates that the
influence at this latter is independent of the salivary protein structure. For AG, its
effect is more pronounced for gPRPs and statherin rather than for aPRPs. In fact,
the EC50 obtained for AG and aPRP is nearly twice of the one obtained for the
other salivary proteins. These results seem to suggest that the mechanism by
which AG exerts its effect is probably different and more selective toward salivary
protein structure as compared to pectin and PGA.
It had already been shown that AG inhibits the interaction between a protein (α-
amylase) and condensed tannins by a different mechanism than pectin.23 While
pectin was described to have the ability to form a ternary complex protein-
polyphenol-carbohydrate (Fig.5.5, i), thereby enhancing its solubility in aqueous
medium, resulting in less insoluble aggregates, AG has been described to have
the ability to inhibit protein−tannin interaction by competing with tannins by
protein aggregation (Fig.5.5, ii). Although this previous work was performed with
a different protein (α-amylase),23 a similar behavior could explain the high
196 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
efficiency and lack of selectivity observed herein for pectin and PGA. In the
present case, as tannins complex with both proteins and carbohydrates (pectin or
PGA), there is no competition mechanism and thus less selectivity.
Fig.5.5 Possible mechanism (i and ii) involved in the inhibition of the aggregation of tannins and proteins by carbohydrates. P, protein; T, tannin; and C, carbohydrate.40
PGA and pectin are quite similar polymers of α-(1→4)-linked D-galacturonic acid
units that differ essentially on the degree of methoxylation of their carboxyl
groups.35 This structural similarity probably explains the identical inhibition
mechanism described, leading to the formation of ternary complexes. However,
pectin is more esterified than PGA, which reduces its polarity and consequently
its readiness to hydrophilic interactions. On the other hand, esterification favors
its ability to establish van der Waals interactions. Thus, pectin would be more
prone to hydrophobic interactions than PGA, which favors the interaction with
condensed tannins (that tend to be more hydrophobic molecules). This particular
feature seems to be important to increase the ability of pectin to inhibit salivary
protein−tannin precipitation despite its low selectivity.
Regarding AG, its structure is quite different from the structures of pectin and
PGA, which could explain the different mechanism of inhibition of this
carbohydrate. A competition mechanism is once again proposed herein for AG
(Fig.5.5, ii). The effectiveness of this process is expected to depend on the
relative affinities of tannin toward AG and salivary proteins. If tannins have a
higher affinity to AG than to salivary proteins, AG would be able to prevent
salivary proteins-tannins precipitation. AG is a heteropolysaccharide composed
of a polysaccharide and hydroxyproline-rich protein moieties able to ensure
hydrophobic interactions with procyanidins. The polysaccharide moiety has also
a slightly acidic character, resulting from the presence of 20% glucuronic acids.
The acidic character may help to establish electrostatic and hydrogen bonds that
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197
could help to strengthen the interaction with condensed tannins. On the other
hand, the presence of the protein-proline moiety could also contribute to an
increase in the efficiency of AG to complex strongly with tannins, competing for
the interaction with salivary proteins. This is readily apparent from the obtained
results as AG was found to be the second more efficient carbohydrate in a range
of concentrations generally much lower than the ones for PGA.
Altogether, these results seem to point out that both hydrophilic and hydrophobic
interactions are important for the carbohydrate effects where the hydrophilic
interactions (hydrogen bonds) seem to contribute to the selectivity of the
inhibition, while the hydrophobic ones seem to contribute to the efficiency. This
was especially clear for pectin that was shown to be the most efficient in
inhibiting salivary proteins precipitation by condensed tannins toward the aPRP,
gPRP, and statherin, despite its low selectivity.
Besides the central role of the carbohydrate structure, these results also point to
the importance of the protein structure for the effect of those carbohydrates.
PRPs are classified in different classes (acidic, basic, and glycosylated) based on
small and specific structural differences, in particular richness in acidic or basic
residues and the presence of sugar molecules in their structure.36-38 The acidic
character of aPRPs is confined roughly to the first 30 amino acids, due to the
presence of many aspartic and glutamic acid residues. The remaining part is
basic and, similarly to bPRPs, shows repeated sequences of proline and
glutamine often separated by glycine residues. Regarding the gPRP, there is little
information about the glycosylation pattern of these proteins. Statherin is
abundant in tyrosine residues and is phosphorylated at Ser-2 and Ser-3. This
latter protein also has a highly acidic amino-terminal hexapeptide (first six
residues) that is needed to inhibit calcium phosphate crystal growth. These
structural aspects of salivary proteins are important for their competition with
carbohydrates toward tannins in the competition mechanism previously referred.
In fact, the efficiency of the carbohydrate to inhibit protein-tannin aggregation
depends not only on the carbohydrate structure but also on the affinity between
the salivary proteins and the tannins. This is evident for AG that has been shown
to be less efficient (high EC50) in inhibiting aPRPs-tannin aggregation as
compared to other salivary proteins. In this case, AG has been shown to be more
effective in inhibiting gPRPs-tannins and statherin-tannins precipitation (EC50 =
4.78 and 5.68 g.L–1, respectively) than aPRPs-tannin precipitation (EC50 = 10.10
g.L–1). In fact, aPRPs have been described recently to interact more strongly with
198 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
condensed tannins than the other salivary protein families.1 On the other hand,
gPRPs are known to not interact strongly with tannins as compared to other
salivary proteins because of the presence of the sugar moiety in their structure,39
which probably explains the higher inefficacy of AG to inhibit gPRP-tannin
precipitation.
Overall, the obtained results show that carbohydrates commonly used in the food
industry are able to inhibit the interaction and precipitation of salivary proteins
with tannins and thus influence the perceived astringency of some food products.
The extent and the mechanism by which this inhibition occurs are related with the
carbohydrate structure and in the last instance also to the protein structure.
� AUTHOR INFORMATION
Corresponding Author
*Tel: +351 220402558. Fax: +351 220402659. E-mail: [email protected].
Funding
This work was supported by Fundação para a Ciência e Tecnologia by one Ph.D.
grant (SFRH/BD/41946/2007) and one project grant (PTDC/AGR-
ALI/67579/2006).
Notes
The authors declare no competing financial interest.
� ABBREVIATIONS USED
ACN, acetonitrile; aPRPs, acidic proline-rich proteins; AS, acidic saliva; AG,
arabic gum; bPRPs, basic proline-rich proteins; ESI-MS, electrospray ionization
mass spectrometry; gPRPs, glycosylated proline-rich proteins; GSF, grape seed
fraction; HPLC, high-performance liquid chromatography; MALDI-TOF/TOF,
matrix-assisted laser desorption/ionization time-of-flight/time-of-flight; PGA,
polygalacturonic acid; PRPs, proline-rich proteins; SDS-PAGE, sodium dodecyl
sulfate−polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid
� REFERENCES
(1) Soares, S.; Vitorino, R.; Osório, H.; Fernandes, A.; Venâncio, A.; Mateus, N.;
Amado, F.; De Freitas, V. Reactivity of human salivary proteins families toward
food polyphenols. J. Agric. Food Chem. 2011, 59, 5535−5547.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
199
(2) Bacon, J. R.; Rhodes, M. J. C. Binding affinity of hydrolysable tannins to
parotid saliva and to proline-rich proteins derived from it. J. Agric. Food Chem.
2000, 48, 838−843.
(3) Kallithraka, S.; Bakker, J.; Clifford, M. N. Evidence that salivary proteins are
involved in astringency. J. Sens. Stud. 1998, 13, 29−43.
(4) Lu, Y.; Bennick, A. Interaction of tannin with human salivary proline-rich
proteins. Arch. Oral Biol. 1998, 43, 717−728.
(5) ASTM. Standard Terminology to Sensory Evaluation of Materials and
Products; Annual Book of ASTM Standards; American Society of Testing and
Materials: Philadelphia, PA, 1989; Vol. 15.07.
(6) Bajec, M. R.; Pickering, G. J. Astringency: Mechanisms and perception. Crit.
Rev. Food Sci. Nutr. 2008, 48, 858−875.
(7) Bennick, A. Interaction of plant polyphenols with salivary proteins. Crit. Rev.
Oral Biol. Med. 2002, 13, 184−196.
(8) Cala, O.; Pinaud, N.; Simon, C.; Fouquet, E.; Laguerre, M.; Dufourc, E. J.;
Pianet, I. NMR and molecular modeling of wine tannins binding to saliva proteins:
Revisiting astringency from molecular and colloidal prospects. FASEB J. 2010,
24, 4281−4290.
(9) Canon, F.; Giuliani, A.; Paté, F.; Sarni-Manchado, P. Ability of a salivary
intrinsically unstructured protein to bind different tannin targets revealed by mass
spectrometry. Anal. Bioanal. Chem. 2010, 398, 815−822.
(10) De Freitas, V.; Mateus, N. Structural features of procyanidin interactions with
salivary proteins. J. Agric. Food Chem. 2001, 49, 940−945.
(11) Asano, K.; Shinagawa, K.; Hashimoto, N. Characterization of haze-forming
proteins of beer and their roles in chill haze formation. J. Am. Soc. Brew. Chem.
1982, 40, 147−154.
(12) Siebert, K. J.; Troukhanova, N. V.; Lynn, P. Y. Nature of polyphenol-protein
interactions. J. Agric. Food Chem. 1996, 44, 80-85.
(13) Huq, N.; Cross, K.; Ung, M.; Myroforidis, H.; Veith, P.; Chen, D.; Stanton, D.;
He, H.; Ward, B.; Reynolds, E. A review of the salivary proteome and peptidome
200 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
and saliva-derived peptide therapeutics. Int. J. Pept. Res. Ther. 2007, 13, 547-
564.
(14) Humphrey, S. P.; Williamson, R. T. A review of saliva: Normal composition,
flow, and function. J. Prosthet. Dent. 2001, 85, 162-169.
(15) Helmerhorst, E. J.; Oppenheim, F. G. Saliva: A dynamic proteome. J. Dent.
Res. 2007, 86, 680-693.
(16) Schlesinger, D. H.; Hay, D. I.; Levine, M. J. Complete primary structure of
statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva of
the monkey, macaca arctoides. Int. J. Pept. Protein Res. 1989, 34, 374-380.
(17) Oppenheim, F. G.; Xu, T.; Mcmillian, F. M.; Levitz, S. M.; Diamond, R. D.;
Offner, G. D.; Troxler, R. F. Histatins, a novel family of histidine-rich proteins in
human parotid secretion. Isolation, characterization, primary structure, and
fungistatic effects on Candida albicans. J. Biol. Chem. 1988, 263, 7472−7477.
(18) Hay, D. I.; Bennick, A.; Schlesinger, D. H.; Minaguchi, K.; Madapallimattam,
G.; Schluckebier, S. K. The primary structures of six human salivary acidic
proline-rich proteins (prp-1, prp-2, prp-3, prp-4, pif-s and pif-f). Biochem. J. 1988,
255, 15−21.
(19) Kauffman, D. L.; Keller, P. J.; Bennick, A.; Blum, M. Alignment of amino acid
and DNA sequences of human proline-rich proteins. Crit. Rev. Oral Biol. Med.
1993, 4, 287−292.
(20) Carvalho, E.; Mateus, N.; Plet, B.; Pianet, I.; Dufourc, E.; De Freitas, V.
Influence of wine pectic polysaccharides on the interactions between condensed
tannins and salivary proteins. J. Agric. Food Chem. 2006, 54, 8936−8944.
(21) Fontoin, H.; Saucier, C.; Teissedre, P.-L.; Glories, Y. Effect of pH, ethanol
and acidity on astringency and bitterness of grape seed tannin oligomers in
model wine solution. Food Qual. Pref. 2008, 19, 286−291.
(22) Serafini, M.; Maiani, G.; Ferro-Luzzi, A. Effect of ethanol on red wine tannin-
protein (BSA) interactions. J. Agric. Food Chem. 1997, 45, 3148−3151.
(23) Soares, S. I.; Gonçalves, R. M.; Fernandes, I.; Mateus, N.; De Freitas, V.
Mechanistic approach by which polysaccharides inhibit α-amylase/procyanidin
aggregation. J. Agric. Food Chem. 2009, 57, 4352−4358.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
201
(24) Charlton, A. J.; Baxter, N. J.; Khan, M. L.; Moir, A. J. G.; Haslam, E.; Davies,
A. P.; Williamson, M. P. Polyphenol/peptide binding and precipitation. J. Agric.
Food Chem. 2002, 50, 1593−1601.
(25) Luck, G.; Liao, H.; Murray, N. J.; Grimmer, H. R.; Warminski, E. E.;
Williamson, M. P.; Lilley, T. H.; Haslam, E. Polyphenols, astringency and proline-
rich proteins. Phytochemistry 1994, 37, 357−371.
(26) Ozawa, T.; Lilley, T. H.; Haslam, E. Polyphenol interactions: Astringency and
the loss of astringency in ripening fruit. Phytochemistry 1987, 26, 2937−2942.
(27) Haslam, E.; Lilley, T. H.; Warminski, E.; Liao, H.; Cai, Y.; Martin, R.; Gaffney,
S. H.; Goulding, P. N.; Luck, G. Polyphenol complexation – A study in molecular
recognition. In Phenolic Compounds in Food and Their Effects on Health I;
American Chemical Society: Washington, DC, 1992; pp 8−50.
(28) Troszynska, A.; Narolewska, O.; Robredo, S.; Estrella, I.; Hernandez, T.;
Lamparski, G.; Amarowicz, R. The effect of polysaccharides on the astringency
induced by phenolic compounds. Food Qual. Pref. 2010, 21, 463−469.
(29) De Freitas, V.; Carvalho, E.; Mateus, N. Study of carbohydrate influence on
protein-tannin aggregation by nephelometry. Food Chem. 2003, 81, 503−509.
(30) De Freitas, V. a. P.; Glories, Y.; Bourgeois, G.; Vitry, C. Characterisation of
oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion
mass spectrometry. Phytochemistry 1998, 49, 1435−1441.
(31) González-Manzano, S.; Mateus, N.; De Freitas, V.; Santos-Buelga, C.
Influence of the degree of polymerisation in the ability of catechins to act as
anthocyanin copigments. Eur. Food Res. Technol. 2008, 227, 83−92.
(32) Messana, I.; Cabras, T.; Inzitari, R.; Lupi, A.; Zuppi, C.; Olmi, C.; Fadda, M.
B.; Cordaro, M.; Giardina, B.; Castagnola, M. Characterization of the human
salivary basic proline-rich protein complex by a proteomic approach. J. Proteome
Res. 2004, 3, 792−800.
(33) Coimbra, M. A.; Waldron, K. W.; Selvendran, R. R. Isolation and
characterisation of cell wall polymers from olive pulp (Olea europaea L.).
Carbohydr. Res. 1994, 252, 245−262.
202 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(34) Nunes, C.; Rocha, S. M.; Saraiva, J.; Coimbra, M. A. Simple and solvent-
free methodology for simultaneous quantification of methanol and acetic acid
content of plant polysaccharides based on headspace solid phase
microextraction-gas chromatography (HS-SPME-GS-FID). Carbohydr. Polym.
2006, 64, 306−311.
(35) Bemiller, J. N. An introduction to pectins: Structure and properties.
Chemistry and Function of Pectins; American Chemical Society: Washington,
DC, 1986; pp 2-12.
(36) Inzitari, R.; Cabras, T.; Onnis, G.; Olmi, C.; Mastinu, A.; Sanna, M. T.;
Pellegrini, M. G.; Castagnola, M.; Messana, I. Different isoforms and post-
translational modifications of human salivary acidic proline-rich proteins.
Proteomics 2005, 5, 805-815.
(37) Vitorino, R.; Alves, R.; Barros, A.; Caseiro, A.; Ferreira, R.; Lobo, M. C.;
Bastos, A.; Duarte, J.; Carvalho, D.; Santos, L. L.; L. Amado, F. Finding new
posttranslational modifications in salivar proline-rich proteins. Proteomics: Clin.
Appl. 2011, 5, 197-197.
(38) Messana, I.; Inzitari, R.; Fanali, C.; Cabras, T.; Castagnola, M. Facts and
artifacts in proteomics of body fluids. What proteomics of saliva is telling us? J.
Sep. Sci. 2008, 31, 1948-1963.
(39) Sarni-Manchado, P.; Canals-Bosch, J. M.; Mazerolles, G.; Cheynier, V.
Influence of the glycosylation of human salivary proline-rich proteins on their
interactions with condensed tannins. J. Agric. Food Chem. 2008, 56, 9563-9569.
(40) Mateus, N.; Carvalho, E.; Luís, C.; De Freitas, V. Influence of the tannin
structure on the disruption effect of carbohydrates on protein-tannin aggregates.
Anal. Chim. Acta 2004, 513, 135-140.
Capítulo 6
Different Phenolic Compounds Activate Distinct Subsets of Human Bitter Taste Receptors
Soares, S., Kohl, S., Thalmann, S., Mateus, N., Meyerhof, W., de Freitas, V.
Submetido ao J. Agric. Food Chem.
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205
Different Phenolic Compounds Activate Distinct Subs ets of Human Bitter Taste Receptors
Susana Soares, † Susann Khol, ‡ Sophie Thalmann, ‡ Nuno Mateus, † Wolfgang
Meyerhof‡,* and Victor de Freitas†,*
†Chemistry Investigation Center (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
‡ DIFE, German Institute of Human Nutrition, Potsdam-Rehbrücke, 14558 Nuthetal, Germany
Bitterness is a major sensory attribute of several common foods and beverages
rich in polyphenol compounds. These compounds are reported as very important
for health as chemopreventive compounds but they are also known to taste bitter.
So, in this work, we analyzed the activation of the human bitter taste receptors,
TAS2Rs, by six polyphenol compounds. The compounds chosen are present in a
wide range of plant-derived foods and beverages, namely red wine, beer, tea and
chocolate. Pentagalloylglucose (PGG) is a hydrolysable tannin, (-)-epicatechin is
a precursor of condensed tannins, procyanidin dimer B3 and trimer C2 belong to
the condensed tannins, and malvidin-3-glucoside and cyanidin-3-glucoside are
anthocyanins. The results show that the different compounds activate different
subsets of the ~25 TAS2Rs. (-)-Epicatechin activated three receptors, TAS2R4,
TAS2R5 and TAS2R39 whereas only two receptors, TAS2R5 and TAS2R39,
responded to PGG. In contrast, malvidin-3-glucoside and procyanidin trimer
stimulated only one receptor, TAS2R7 and TAS2R5, respectively. Notably,
tannins are the first natural agonists found for TAS2R5 that display high potency
only toward this receptor. The catechol and/or galloyl groups appear to be
important structural determinants that mediate the interaction of these
polyphenolic compounds with TAS2R5. Overall, the EC50 values obtained for the
different compounds vary 100-fold with the lowest values for PGG and malvidin-
3-glucoside compounds suggesting that they could be significant polyphenols
responsible for the bitterness of fruits, vegetables and derived products even if
they are present in very low concentrations.
KEYWORDS: polyphenols, bitterness, tannins
� INTRODUCTION
It has been well-known and common sense that consumption of diets rich in
fruits, vegetables and moderate intake of red wine (i.e., polyphenol-rich foods)
206 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
are inversely correlated to cardiovascular diseases incidence and cancer risk,
probably because the polyphenols modulate several biological reactions that lead
to these disease conditions (1). In fact, during the past years polyphenol
compounds have been suggested as chemoprevention agents.
Polyphenol compounds result from plant secondary metabolism as chemical
defense against predators and are usually divided in non-flavonoids and
flavonoids, the latter being the most relevant ones. Flavonoids include very
diverse compounds such as anthocyanins and flavan-3-ols. Although they are
potentially beneficial to human health in small doses, many of these compounds
are toxic in high doses (2). Several of these compounds are responsible for major
organoleptic properties of vegetables and fruits, namely color and taste. In fact,
some of them are known to have a bitter taste and so, despite their healthy
properties, many people do not like to eat vegetables and other plant-derived
food because of the bitterness associated with polyphenols (3). Debittering of
food has been a longstanding major sensory challenge for the food industry (3),
that requires identification of those compounds making food and beverages taste
bitter. Therefore, studying the sensorial properties of polyphenol compounds is a
prominent subject relevant for people’s food choices, and the chemopreventive
potential of food.
Although bitterness in foods is usually unpleasant, there are some foodstuffs in
which it is a wanted sensory attribute, e.g. red wine and beer. In fact, most of the
sensory analysis data regarding polyphenols bitterness are related to red wine. It
is thought that the bitterness of red wine is mainly induced by polyphenol
compounds (e.g. tannins) (3-5). However, the limited available data regarding
polyphenol’s structure/bitterness relationship are rather inconsistent (5-11). In
general, these works studied the bitterness of polyphenol compounds, such as
polymeric fractions of tannic acid and tannins, as well as flavan-3-ol monomers,
dimers, and trimers, and demonstrated that larger molecules tend to be less bitter
and more astringent (8, 10). Peleg and co-workers (8) found that (-)-epicatechin
was more bitter than the stereoisomer (+)-catechin and that these both were
more bitter than the procyanidin trimers, catechin-(4-8)-catechin-(4-8)-catechin
and catechin-(4-8)-catechin-(4-8)-epicatechin. Robichaud and colleagues (10)
found that tannic acid, a commercial hydrolysable pentagalloylglucose-rich tannin
was more bitter than both, (+)-catechin and a grape seed extract which is rich in
polymeric procyanidins. However, other reports have shown that bitterness of
polyphenols increases with molecular weight (5, 9, 12). Hufnagel and Hofmann
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207
(5) found that procyanidin dimers and a trimer were more bitter than (-)-
epicatechin. These authors also demonstrated by taste reconstruction and
omission experiments, that the bitterness of the red wine could be induced by
subthreshold concentrations of phenolic acid ethyl esters and flavan-3-ols.
Besides these inconsistencies, there is insufficient knowledge about the bitter
taste of polyphenol compounds that do not belong to tannin classes, such as
anthocyanins.
Bitter taste is elicited by a specific subset of taste receptor cells (TRC) localized
in the oral cavity in groups of cells called taste buds, which are embedded in the
epithelium of the gustatory papillae on the tongue and palate. These TRC are
characterized by the expression of members of the TASTE 2 Receptor (TAS2R)
gene family encoding bitter taste receptors (13-17). In humans, this gene family
codes for ~25 taste receptors (TAS2Rs) that are G protein-coupled receptors. So
far, 20 of them have been deorphaned meaning that cognate bitter compounds
have been assigned to them (14, 18-30). In general, TAS2Rs are sensitive to
several or multiple different bitter compounds, a property that has been proposed
to be the basis for recognition of the countless bitter chemicals (19, 31, 32). Due
to the presence of numerous non-synonymous single nucleotide polymorphisms
(SNPs) the TAS2R genes encode functionally distinct receptor variants that form
the basis for variations in bitterness perception in the population (23, 25, 33-35).
In order to understand better structure/bitterness relation, to overcome the
inconsistencies associated with sensorial analysis, and to identify which TAS2Rs
are activated by the various polyphenol compounds an objective, robust, and
reliable method to analyze their bitterness is required. Few recent studies used
heterologous expression experiments to successfully measure TAS2R activation
by purified bitter chemicals that are normally present in food and beverage
sources such as beer hops, cheese, and soy products (18, 26, 29, 30, 36, 37). In
the present report we use this method to examine the activation of the TAS2Rs
by several polyphenol compounds widely spread in food and beverages derived
from plants and commonly present in our daily diet (Fig.6.1).
208 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
β-1,2,3,4,6-Penta-O-galloyl-d-glucopyranose Procyanidin trimer C2
(-)-Epicatechin Malvidin-3-glucoside
Fig.6.1 Chemical structures of the four polyphenol compounds studied.
Based on their extensive prevalence in vegetable, fruits and derived products, we
chose the following six substances that belong to some of the most important
classes of polyphenols (anthocyanins, flavan-3-ols and hydrolysable tannins)
(38): (-)-epicatechin, procyanidin dimer B3 and trimer C2 (flavan-3-ols), malvidin-
3-glucoside and cyanidin-3-glucoside (anthocyanins) and pentagalloylglucose
(PGG) (hydrolysable tannins). (-)-Epicatechin (structural unit of condensed
tannins) and a number of different procyanidin dimers and trimers belong to the
condensed tannins class and are present in a variety of frequently ingested food
including red wine, cocoa, grape seeds, tea, beer and cereals (38, 39). Malvidin-
3-glucoside and cyanidin-3-glucoside belong to the anthocyanin class, being
highly present in red fruits and their derived products, red wine and olives (38,
40). Although PGG is not commonly found in a wide range of foodstuffs, it
belongs to the other class of tannins (hydrolysable tannins) and is present in
pomegranate, green tea and it can also be incorporated in red wine during the
winemaking process (by addition of commercial tannic acid) (41).
CO
O
O CO
OH
OH
OHO
CO
OH
OHOH
O
CO
OH
OH
OH
O
O
CO
OH OH
OH
OH
OH
OH
Galloyl group
O
OH
OH
OH
OH
R1H
R2
O
OH
OH
OH
OH
HH
OH
O
OH
OH
OH
OH
HH
OH
OOH
OH
OH
OH
H
H
OH
Orto-catecholgroup
O
OH
OH
OH
O
OMe
OMe
Glu
+
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209
Our aim was to elucidate if the selected polyphenol compounds are activators of
human bitter taste receptors and to examine if different polyphenols activate
different TAS2Rs.
� MATERIALS AND METHODS
Reagents. All reagents used were of analytical grade. (+)-catechin and (-
)-epicatechin were purchased from Sigma-Aldrich, malvidin aglycon was
purchased from Extrasynthèse, sodium borohydride and tartaric acid were
purchased from Aldrich, tannic acid was purchased from Fluka Biochemica
(Switzerland), taxifolin was purchased from Extrasynthèse (Genay, France) and
Toyopearl HW-40(s) gel was purchased from Tosoh (Tokyo, Japan).
Malvidin-3-glucoside and cyanidin-3-glucoside (anth ocyanins)
isolation. Malvidin-3-glucoside and cyanidin-3-glucoside were isolated as
described in the literature (42). Briefly, these compounds were isolated from a
grape skin extract that was applied to a TSK Toyopearl HW-40(s) gel column.
The elution was made with 10% CH3OH/CH3COOH (v/v) and then the solvent
was evaporated. The resulting residue was applied to a C18 gel column and the
compounds were eluted with 10% methanol acidulated. The solution was again
evaporated and the resulting solution was mixed with distilled water, frozen and
lyophilized. The compounds purity was assessed by HPLC-MS, direct MS and
NMR analysis and was higher than 99%.
Procyanidin dimer (B3) and trimer (C2) (condensed t annins)
synthesis. The synthesis of procyanidin dimer B3 (catechin-(4-8)-catechin) and
trimer C2 (catechin-(4-8)-catechin-(4-8)-catechin) followed the procedure
described in the literature (43). Briefly, taxifolin and (+)-catechin (ratio 1:3) were
dissolved in ethanol and the mixture was treated with sodium borohydride (in
ethanol). The pH was then lowered to 4.5 by addition of CH3COOH/H2O 50%
(v/v), and the mixture stood under argon atmosphere for 30 min. The reaction
mixture was extracted with ethyl acetate. After evaporation of the solvent, water
was added, and the mixture was passed through C18 gel, washed with water,
and recovered with methanol. After evaporation of methanol, the fraction was
passed through a TSK Toyopearl HW-40(s) gel column (300 mm × 10 mm i.d.,
0.8 mL.min-1, methanol as eluent) coupled to a UV-vis detector. Several fractions
were recovered and analyzed by ESI-MS (Finnigan DECA XP PLUS) yielding
210 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
procyanidins dimers (B3 and B6) and trimer (C2). The structure was elucidated
by HPLC-MS and NMR analysis.
β-1,2,3,4,6-Penta-O-galloyl-d-glucopyranose (PGG) (h ydrolysable
tannin) synthesis. PGG was synthesized accordingly to Chen and Hagerman
(44). Briefly, 5.0 g of tannic acid was methanolyzed in 70% methanol in acetate
buffer (0.1 M, pH 5.0) at 65 °C for 15 h. The pH of the reaction mixture was
immediately adjusted to 6.0 with NaOH. Methanol was evaporated under reduced
pressure at <30 °C and water was added to maintain the volume. The solution
was extracted with 3 volumes of diethyl ether and 3 volumes of ethyl acetate. The
ethyl acetate extracts were combined and evaporated, with addition of water to
maintain the volume. The resulting suspension was centrifuged, and the
precipitate was redissolved by heating in 2% methanol solution. PGG precipitated
as the solution cooled to room temperature and was collected by centrifugation.
PGG was washed twice with an ice-cold 2% methanol solution and once with ice-
cold distilled water. The final material was lyophilized to yield a white powder with
an overall mass yield of 23%. The purity of the obtained PGG was assessed by
HPLC analysis (45) and 1H NMR spectroscopy and it was ≤ 99%.
Test compounds preparation. The compounds to be tested were either
dissolved in buffer C1 (130.0 mM NaCl, 5.0 mM KCl, 10.0 mM N-2-
hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 2.0 mM CaCl2, 10.0 mM
glucose [pH 7.4]) or in a mixture of dimethylsulfoxide (DMSO) and buffer C1 not
exceeding a final DMSO concentration of 0.1% (v/v) to avoid toxic effects on the
transfected cells.
Cell transfection and expression of TAS2Rs in heter ologous cells.
Functional expression studies were carried out as described before (18, 30).
Human embryonic kidney (HEK)-293T cells stably expressing the chimeric G
protein subunit Gα16gust44 were seeded into poly-D-lysine coated (10 µg.mL-1)
96-well plates (Greiner Bio-One, Frickenhausen, Germany) under regular cell
culture conditions [Dulbecco’s modified Eagle medium (DMEM)), 10% FCS, 1%
penicillin/streptomycin; 37ºC, 5% CO2, 95% humidity]. After 24-26 h, cells were
transfected transiently with 150 ng expression plasmids based on pEAK10 (Edge
BioSystems) or pcDNA5/FRT (Invitrogen) using 300 ng of Lipofectamine2000
(Invitrogen). In addition to the TAS2R coding sequences the plasmids contained
the first 45 amino acids of rat somatostatin receptor 3 for cell surface localization
followed by the herpes simplex virus (HSV) glycoprotein D epitope for
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211
immunocytochemical detection of the receptor. The TAS2R sequences are
according to Meyerhof et al. (18).
Calcium imaging analysis. Twenty-four to 26 h after transfection, cells
were loaded with the calcium-sensitive dye Fluo-4-acetoxymethylester (2 µM,
Molecular Probes) in serum-free DMEM medium. Probenecid (Sigma-Aldrich
GmbH), an inhibitor of organic anion transport, was added at a concentration of
2.5 mM, to minimize the loss of the calcium indicator dye from cells. One hour
after loading, the wells were washed three times with C1 buffer using a Denley
Cell Washer (Thermo Fisher Scientific, Inc., Waltham, MA). Cells were incubated
in washing buffer in the dark for 30 min between the washing steps.
Fluorescence changes were recorded at 510 nm following excitation at 488 nm
by a fluorometric imaging plate reader (FLIPR, Molecular Devices) before and
after application of the test compounds in each well. A second application of 100
nM somatostatin-14 (Bachem) activating the endogenous somatostatin receptor
type 2 was used to assess cell vitality. All experiments were performed at least in
duplicates. Mock-transfected cells (cells transfected with empty pcDNA5/FRT
vector used as negative control) were always measured in parallel on the same
microtiter plates using the same compound concentrations used to examine the
cells expressing the various TAS2Rs. All compounds were initially tested at
different concentrations for unspecific calcium responses in untransfected
HEK293T Gα16gust44 cells. Based on this pilot experiment, we used maximal
compound concentrations that were always lower than those concentrations that
generated unspecific responses in the absence of transfected receptor DNAs.
Determination of half maximal effective concentrati ons (EC 50) and
statistical analysis. Having identified responsive TAS2Rs, we examined their
concentration-dependent activation and established half maximal effective
concentrations (EC50) values for their bitter agonists. To calculate the
concentration-response curves, the fluorescence changes of mock-transfected
cells were subtracted from the corresponding values of receptor-expressing cells
by means of the FLIPR384 software (Molecular Devices, Munich, Germany). To
compensate for differences in cell density, signals were normalized to
background fluorescence for each well. Signals were recorded in at least
duplicate wells and the data averaged. Signal amplitudes were then plotted
versus log agonist concentration. EC50 values were calculated using SigmaPlot
9.01 (Systat Software Gmbh, Erkrath, Germany) by nonlinear regression using
212 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
the function (equation 1) where x is the test compound concentration and nH
the Hill coefficient. Statistical significance of the difference between the several
calculated EC50 and of the signal amplitudes was evaluated by one-way analysis
of variance, followed by the Bonferroni test. Differences were considered to be
statistically significant when P < 0.05.
� RESULTS
Identification of TAS2Rs that respond to polypheno l compounds. In
order to identify the TAS2Rs that are sensitive to the selected prototypical
polyphenols, the 25 TAS2Rs, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7,
TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R38,
TAS2R39, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R31, TAS2R45,
TAS2R46, TAS2R30, TAS2R19, TAS2R20, TAS2R50 and TAS2R60 were
expressed individually in HEK293T cells stably expressing the chimeric G protein
Gα16gust44. This construct was designed to couple the activation of TAS2Rs in
heterologous cells to the release of Ca2+ from intracellular stores, which can be
measured using calcium-sensitive fluorescence dyes (46). Receptor-mediated
changes in intracellular calcium concentrations were detected as fluorescence
changes by means of a fluorometric imaging plate reader.
The six polyphenol compounds to be tested (-)-epicatechin, procyanidin dimer
and trimer, malvidin-3-glucoside, cyanidin-3-glucoside and PGG were
administered, at the highest possible concentrations (pre-determined in pilot
experiments, data not shown), by bath application to the 25 different receptor-
expressing cell populations. (-)-Epicatechin was employed up to 16.0 mM
whereas all the other compounds were employed only at µM range [procyanidin
trimer, 100.0 µM; malvidin-3-glucoside, 20.0 µM; PGG, 10.0 µM (Fig.6.2)]. By
tracing cytosolic calcium levels we found that cells expressing TAS2R4, TAS2R5
or TAS2R39 responded to challenge with (-)-epicatechin. Cells transfected with
DNA for TAS2R5 or TAS2R39 were also sensitive to PGG. Elevation of calcium
levels were also seen in cells expressing TAS2R5 that have been exposed to
procyanidin trimer. Notably, above chemicals are the first natural bitter
compounds found for TAS2R5. So far this specialist receptor responded only to
the synthetic compound phenantroline and may therefore be the only TAS2R that
is activated (“specific”) by natural tannins. Finally, we found that malvidin-3-
glucoside elicited signals specifically in cells transfected with DNA for TAS2R7.
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213
(-)-Epicatechin Malvidin-3-glucoside
Procyanidin trimer PGG
Fig.6.2 Fluorescence changes of Fluo4-AM loaded HEK293T-Gα16gust44 cells expressing the TAS2Rs indicated in the graphs following administration of 10.0 mM (-)-epicatechin, 20.0 µM malvidin-3-glucoside, 100.0 µM procyanidin trimer or 10.0 µM PGG. Responses of mock-transfected cells (empty plasmid) are indicated by the thinner solid line. Fluorescence changes of Fluo4-AM loaded HEK293T-Gα16gust44 cells expressing the TAS2Rs indicated in the graphs following administration of 10.0 mM (-)-epicatechin, 20.0 µM malvidin-3-glucoside, 100.0 µM procyanidin trimer or 10.0 µM PGG. Responses of mock-transfected cells (empty plasmid) are indicated by the thinner solid line.
Cells expressing TAS2R1, TAS2R3, TAS2R8, TAS2R9, TAS2R10, TAS2R13,
TAS2R14, TAS2R16, TAS2R38, TAS2R40, TAS2R41, TAS2R42, TAS2R43,
TAS2R31, TAS2R45, TAS2R46, TAS2R30, TAS2R19, TAS2R20, TAS2R50 and
TAS2R60 did not respond to the tested compounds (data not shown). It is also
important to note that procyanidin dimer and cyanidin-3-glucoside did not activate
any receptor, at least in the highest possible concentrations tested (100 µM).
Thus, the data indicate that four of the six polyphenols compounds tested
mediate their bitterness through activation of the 4 human bitter taste receptors,
TAS2R4, TAS2R5, TAS2R7, and TAS2R39.
Functional characterization of activated TAS2Rs. In order to
investigate the activation of the bitter receptors TAS2R4, TAS2R5, TAS2R7 and
TAS2R39 by the polyphenols in greater detail, concentration-response functions
2500
MOCK
hTAS2R5
hTAS2R4
hTAS2R39
5 min
counts 1500
5 min
counts
hTAS2R7 MOCK
5 min
500 counts
hTAS2R5
MOCK
5 min
600 counts
hTAS2R5
hTAS2R39
MOCK
214 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
were recorded and the half maximal effective agonist concentrations (EC50)
established (Fig.6.3). The concentration-response curves followed sigmoid
functions. The calculated EC50 values for the compound-receptor pairs are
presented in Table 6.1 were the values with ≥ are estimates because the dose-
response curves did not saturate and so we were unable to determine an EC50
value.
Collectively, the data demonstrate that the EC50 values for the different
agonist/receptor pairs vary from the µM to the mM range. While the EC50 values
for PGG at both TAS2R5 and TAS2R39 are ≥ 8.5 and ≥ 6.6 µM, respectively,
those for (-)-epicatechin are ~1000fold higher and vary between 3.2 and 3.8 mM.
Like the EC50 values for PGG, those for malvidin-3-glucoside and procyanidin
trimer are also in the micromolar range, being 12.6 µM at TAS2R7 and 35.6 µM
at TAS2R5, respectively.
(a) (b)
(c) (d)
Fig.6.3 Concentration-response curves for HEK293T-Gα16gust44 cells transfected with DNA for the indicated TAS2R following stimulation with the indicated test compound. Error bars represent the confidential interval (P = 0.05). (a) Dose dependent activation was observed for TAS2R4, TAS2R5, and TAS2R39 by (-)-epicatechin (a), for TAS2R5 and TAS2R39 by PGG (b), for TAS2R7 by malvidin-3-glucoside (c), and for TAS2R5 by procyanidin trimer (d).
(-)-Epicatechin [mM]
0.01 0.1 1 10 100
∆F/F
-0.2
0.0
0.2
0.4
0.6
0.8hTAS2R4 hTAS2R39hTAS2R5
PGG [µM]
0.01 0.1 1 10 100
∆ F/F
-0.2
0.0
0.2
0.4
0.6
0.8
hTAS2R39hTAS2R5
Malvidin-3-glucoside [µM]
0.1 1 10 100
∆F/F
-0.2
0.0
0.2
0.4
0.6
0.8
Procyanidin trimer [µM]
0.1 1 10 100 1000
∆ F/F
-0.2
0.0
0.2
0.4
0.6
0.8hTAS2R5
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215
Table 6. 1 EC50 values (µM) for the test compounds and respective receptors. The values with superscript equal letters are not significantly different (P < 0.05). Values with ≥ are estimates because the dose-response curves did not saturate.
EC50 (µM)
TAS2R4 TAS2R5 TAS2R7 TAS2R39
(-)-Epicatechin ≥ 30151.0 3210.0 +/- 42.0a - 3800.0 +/- 200.0b,c
Procyanidin trimer - 35.6 +/- 0.7d - -
PGG - ≥ 8.5 - ≥ 6.6
Malvidin-3-glucoside - - 12.6 +/- 0.7 -
Besides these pronounced differences in the EC50 values, the observed threshold
concentrations, defined as the lowest concentration that resulted in calcium
signals in receptor-transfected cells, are also largely different (Table 6.2).
Whereas PGG induced robust receptor responses already at a concentration of
3.0 µM at both TAS2R5 and TAS2R39, a 300-1000fold higher concentration of (-
)-epicatechin was required to evoke receptor responses at TAS2R4, TAS2R5,
and TAS2R39. Like PGG, threshold values for malvidin-3-glucoside and
procyanidin trimer were in the micromolar range. The lowest concentrations that
induced detectable receptor responses were 6.0 µM malvidin-3-glucoside for
TAS2R7 and 30.0 µM procyanidin trimer for the TAS2R5. Thus the data clearly
demonstrate that PGG, malvidin-3-glucoside and procyanidin trimer act as high
potency agonists on TAS2Rs, whereas (-)-epicatechin is a low potency stimulus.
Table 6.2 Threshold concentrations (µM), defined as the lowest concentration that resulted in calcium signals in receptor-transfected cells, for the test compounds.
Threshold concentrations (µM)
TAS2R4 TAS2R5 TAS2R7 TAS2R39
(-)-Epicatechin 2000.0 1000.0 - 1000.0
Procyanidin trimer - 30.0 - -
PGG - 3.0 - 3.0
Malvidin-3-glucoside - - 6.0 -
Also signal amplitudes, which are related to the efficiency of the receptor
activation, differ across receptors and the polyphenols (Table 6.3). (-)-
Epicatechin, activated TAS2R5 cognate receptor as well as TAS2R4 and
TAS2R39 with high efficacy as revealed by the change in fluorescence, i.e., the
corresponding ∆F/F values. Similarly, PGG was an efficient stimulus for
TAS2R39 and also malvidin-3-glucoside showed high efficiency at its cognate
216 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
receptor. In contrast, the observed efficacy of procyanidin trimer at its cognate
receptor is relatively low.
Table 6.3 Signal amplitudes (given as relative fluorescence changes ∆F/F) for the test compounds.
Signal amplitudes
TAS2R4 TAS2R5 TAS2R7 TAS2R39
(-)-Epicatechin 0.44 +/- 0.04a 0.59 +/- 0.11a - 0.65 +/- 0.04a
Procyanidin trimer - 0.22 +/- 0.04c - -
PGG - 0.36 +/- 0.16d - 0.61 +/- 0.04d
Malvidin-3-glucoside - - 0.54 +/- 0.09 -
� DISCUSSION
It has been well-known and common sense that consumption of diets with high
content of food rich in vitamins, minerals, fibers, and polyphenols, as well as low
levels of saturated fats are directly associated to a low incidence of
cardiovascular and cancer diseases. The well-known French Paradox that
appeared in 1992, it’s a classic example of this association (47). Since that time,
an exponential growing of epidemiological, clinical and experimental data support
the importance of a diet rich in polyphenol-rich foods (1). In fact, these
compounds have been suggested as chemoprevention agents. For instance,
pentagalloylglucose has been shown in vivo to have anti-cancer effect against
prostate, lung, and breast cancers as well as to have anti-diabetes activity (48).
Also cyanidin-3-glucoside has been shown to be able to revert human melanoma
cells from the proliferating to the differentiated state (49). Because of
polyphenols’ beneficial effects on human health, increasing the phytonutrient
content of plant foods is a potent dietary option for preventing diseases and a
challenge for the design of functional food. However, many people do not like to
eat vegetables and/or derived products especially because of their bitter taste
(3). So, the study of the bitterness properties of polyphenol compounds is a
prominent subject that could reflects in people’s food choices, and lastly in
people’s food chemoprevention.
Therefore, in this report we studied the bitterness of 6 polyphenol compounds
commonly present in human diet ((-)-epicatechin, procyanidin dimer B3 and
trimer C2, malvidin-3-glucoside, cyanidin-3-glucoside, and PGG) and showed
that the human bitter taste receptors, TAS2R4, TAS2R5, TAS2R7, and TAS2R39
are sensitive to 4 of the 6 compounds tested. The agonist-receptor pairs display
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217
an interesting activation pattern. Firstly, different compounds activate the same
receptor. For example (-)-epicatechin and PGG activate TAS2R39 whereas (-)-
epicatechin, PGG and procyanidin trimer activate TAS2R5. Secondly, different
receptors are activated by the same compound. This is evidenced by (-)-
epicatechin which activates TAS2R4, TAS2R5 and TAS2R39 and PGG which
stimulates TAS2R5 and TAS2R39. These results confirm well with the
combinatorial activation patterns of TAS2Rs previously reported (18, 20).
Furthermore, by the structural diverse compounds, these data also have
implications for structure-activity relationships. They suggest that the catechol
and/or galloyl group (which has only one more hydroxyl group than catechol) are
critical features (but not essential) for the interaction of polyphenol compounds
with TAS2R5. Firstly, the compounds that activated this receptor, (-)-epicatechin,
procyanidin trimer and PGG have at least one of these groups while the other
tested compounds, malvidin-3-glucoside and cyanidin-3-glucoside, did not.
Secondly, procyanidin trimer and PGG that possess three ortho-catechol groups
and five galloyl groups, respectively, activate TAS2R5 at 100-fold lower
concentrations than does (-)-epicatechin which has only one ortho-catechol
group. However, this structural feature is not required for other compounds that
activate TAS2R5 since the synthetic ligand 1,10-phenanthroline lacks these
groups completely. In fact, it is evident that the presence of these groups is not
sufficient for the responsiveness of this receptor since procyanidin dimer (which
as two ortho-catechol groups) does not activate this receptor. However, it seems
that, when a compound activates TAS2R5, the presence of these groups could
be essential for higher responses/interaction. The importance of the hydroxyl
groups in bitter receptor activation is also referred by Roland and co-workers
(36), who have studied the activation of receptor TAS2R39 by the soy isoflavone
genistein and structurally similar isoflavonoids. These authors concluded that the
presence of three hydroxyl groups in the isoflavonoid ring system which
resembles that of the polyphenols seemed to be more favorable for TAS2R39
activation than the presence of fewer hydroxyl groups. These authors also noted
the importance of glucose residues attached to their test compounds for
TAS2R39 activation. The present report demonstrated that TAS2R39 responded
to PGG which also contains a glucoside moiety. However, malvidin-3-glucoside
and cyanidin-3-glucoside did not activate this receptor. Interestingly, the
glycosylated compounds did not activate the TAS2R16, which has been
suggested to be specific for β-glucopyranosides (19). However, these authors
218 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
(19) also found that hydrophobicity and size of the aglycons are critical for
receptor activation. The great structural differences between the compounds
employed by Bufe and co-workers and those tested here easily explain the
absence of responses of TAS2R16.
A receptor for which the presence of glucose residues seems to be important is
TAS2R7. Whereas malvidin-3-glucoside activated TAS2R7, its aglycon form
(without glucose residue) failed to do so, at least at the tested concentrations.
Our result for malvidin-3-glucoside oppose those of a sensory study that showed
that this anthocyanin does not taste bitter (50). These authors studied the
bitterness of malvidin-3-glucoside in a mixture with cyanidin-3-glucoside made in
a solution saturated with tartaric acid (pH 3.6) and containing ethanol. It is also
important to mention that anthocyanins, co-exist in solution in different forms
because of their well-known pH dependence (51, 52): at low pH (1-2) they are
essentially present in the red cationic form (AH+), but as the pH increases rapid
proton transfer reactions occur leading to the formation of blue quinonoidal bases
(A and A-) and chalcones. Therefore, the two data sets appear not to be
comparable since the use of other compounds and distinct experimental
conditions could influence the perceived bitterness and receptor responses
differently. It is important to point out that we could not test ethanol in the in vitro
assay because it is toxic for cells already at 1%.
Even though the factors that determine bitter intensity are still unknown the
concentration-response functions of the 5 polyphenols and their cognate
receptors predict that PGG appears to be a strong bitter compound in foodstuffs
if present at least in low micromolar concentrations. Firstly, it activates both
receptors, TAS2R5 and TAS2R39. Secondly, it displays high potency evident as
low EC50 values at both cognate receptors. Thirdly, it shows also high efficacy as
revealed by the high signal amplitudes of the activated receptors, in particular for
TAS2R39. Malvidin-3-glycoside and procyanidin trimer are also of high potency,
i.e., they have low EC50 values at their receptors. However, both compounds
activate one receptor with relatively low efficacy, TAS2R7 and TAS2R5,
respectively. This could mean that these polyphenol compounds elicit a weaker
bitter taste than PGG, if they are present in food in similar concentrations as
PGG. (-)-Epicatechin also appears to be a strong bitter chemical. It activates
three receptors, TAS2R4, TAS2R5, and TAS2R39 with high efficacy. The
potencies of this compound at its cognate receptors may be low relative to the
other 4 polyphenols because its EC50 values are in the millimolar range.
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219
However, if (-)-epicatechin is present in food in this concentration range it would
function as a strong activator of bitterness due to its high efficacy. This is
supported by data about β-glucopyranosides which have millimolar potencies at
only one receptor, hTAS2R16, yet elicit strong bitterness and avoidance behavior
(19, 53).
Polyphenol concentrations in vegetables, fruits and derived products depend on
many factors, e.g. culture conditions and degree of ripeness. The tested
polyphenols are present in numerous plant-derived products commonly present
in our diet (e.g. red wine, beer, cocoa, red fruits), even though their
concentrations vary widely (54). For instance, (-)-epicatechin has been identified
in 125 food and beverages and it is known to be present in dark chocolate (mean
content 0.070 mg.g-1), apple (mean content 0.008 mg.g-1) and blackberry (mean
content 0.012 mg.g-1). Malvidin-3-glucoside has been identified in 21 foodstuffs
including black grapes (mean content 0.039 mg.g-1) and beans (mean content
0.0006 mg.g-1). Procyanidin trimer C2 has been identified in beer (0.3 µg.mL-1).
The wide range of concentrations is particular evident in red wine (55-57). (-)-
Epicatechin has been reported to be present in the range of 5.0 to 88.0 mg.L-1 (=
17.0 – 302.0 µM) in over 800 types of red wine (56) and malvidin-3-glucoside is
present in the range of 40.0 to 510.0 mg.L-1 (= 81.0 – 1035.0 µM) in Port wine
(55). Thus, the concentrations of (-)-epicatechin are about 10fold below its EC50
values at all cognate receptors suggesting that this polyphenol may contributes
little to the bitterness of red wine. Malvidin-3-glucoside is present at
concentrations 6-80fold higher than its EC50 value at TAS2R7 suggesting that this
receptor is robustly activated when consuming these types of wine. Regarding
procyanidin trimer and PGG used herein, there are no reported concentrations in
red wine but, as referred previously, this trimer has been reported to be present
in beer at 0.3 µg.mL-1(54). However, procyanidin trimer C1 is known to be
present in red wine at 25.6 mg.L-1 (29.6 µM) (58).
Although the existing results are controversial, some sensory evaluations
perceive smaller polyphenol molecules as bitterer than larger ones (8, 10). Peleg
and co-workers (8) studied the bitterness of several compounds including (-)-
epicatechin, procyanidin dimers and trimers at the concentration of 0.9 g.L-1 in
aqueous ethanol (1% v/v). This concentration corresponds to 3093.0 µM of (-)-
epicatechin and to 1040.0 µM of procyanidin trimer, which in the case of (-)-
epicatechin lies in the concentration range used herein, but for procyanidin trimer
is much higher than the concentration range used herein. At that concentration,
220 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Peleg (8) found that monomers were significantly bitterer than the dimers, which
in turn were significantly bitterer than the trimers.
Hufnagel and Hofmann (5) studied the astringency and bitterness of numerous
compounds from a red wine by sensorial analysis and they demonstrated that the
bitterness of that red wine could be induced by phenolic acid ethyl esters and
flavan-3-ols (including (-)-epicatechin, procyanidin dimers and C1 trimer). But in
contrast to Peleg, they found that the larger molecules were bitterer than smaller
ones. In fact, the bitter taste threshold for (-)-epicatechin was found to be 930.0
µM, and so these data correlate with our observations for the TAS2R39.
However, the bitter taste thresholds they found for procyanidins were between
400 and 500 µM which are much higher than the values found in this work (30.0
µM). This observed correlation between the in vivo and in vitro analysis, with
sensory data showing mostly higher thresholds values in comparison to in vitro
data, is common and has been reported previously (30). On possible explanation
to this fact are the formation of complexes with proline-rich proteins in the saliva
and/or adsorption of these compounds by the oral epithelium which could alter
their effective oral concentrations differently and explain the different
concentration-response functions recorded in vivo and in cell-culture (30). We
can also not exclude the possibility that a particular fully activated TAS2Rs
evokes bitter perception to a lesser degree than another one.
Also, a sensory analysis of fractions of grape seed phenolics showed that
bitterness increases along with molecular size (9). Moreover, Lea and Arnold (12)
showed that bitterness is associated with oligomeric procyanidins reaching a
maximum with the epicatechin tetramer. This is in agreement with our results
since (-)-epicatechin monomer is less bitter that procyanidin trimer.
Apart from the molecular size of the bitter polyphenols under study, other factors
could affect the bitterness of the compounds in sensory evaluations, as referred
previously. For example, while ethanol enhances bitterness intensity, varying
wine pH has little or no effect on perceived bitterness (59). Fischer and Noble
(59) prepared eighteen wines, varying in ethanol (8%, 11%, 14% v/v), pH (2.9,
3.2, 3.8) and (+) catechin (100.0 and 1500.0 mg.L-1) using a dealcoholized white
wine concentrate. In a completely randomized design, bitterness intensity was
rated by 20 subjects and it was found that bitterness increased by all three
components. These authors have shown that an increase of 3% v/v ethanol
elevated bitterness more (around 50%) than adding 1400.0 mg.L-1 of catechin to
the same wine (which increased bitterness by 28%). Such increase in catechin is
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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221
not common in the winemaking industry; however differences of 3% v/v ethanol
occur frequently in table wines and will produce a significant increase in
bitterness. Unfortunately, it is impossible to examine this interesting effect of
ethanol by means of the receptor assay, because ethanol is highly toxic for the
cells above 1%.
In summary, the results show that different polyphenol compounds activate
different subsets of the ~25 TAS2Rs. (-)-Epicatechin activated three receptors
TAS2R4, TAS2R5 and TAS2R39 whereas only two receptors, TAS2R5 and
TAS2R39, responded to PGG. In contrast, malvidin-3-glucoside and procyanidin
trimer stimulated one receptor, TAS2R7 and TAS2R5, respectively. Overall, the
EC50 values obtained for the different compounds vary 100-fold with the lowest
values for the compounds that belong to hydrolysable tannins and anthocyanins
classes, suggesting that these polyphenols could be the major compounds
responsible for the bitterness of fruits and derived products, such as red wine.
� AUTHOR INFORMATION
Corresponding Author
*Tel: +351 220402558. Fax: +351 220402659. E-mail: [email protected].
Acknowledgment
This work has been supported by a grant of the German Research Foudation
(DFG) to W.M. (Me 1024/2-3) and also by two grants of the Fundação para a
Ciência e Tecnologia (one PhD grant [SFRH/BD/41946/2007] and one project
grant [PTDC/AGR-ALI/67579/2006]).
� ABREVIATIONS USED DNA, deoxyribonucleic acid, DMSO, dimethylsulfoxide, H1 NMR, nuclear
magnetic resonance, HPLC, high performance liquid chromatography, PGG,
pentagalloylglucose, TRC, taste receptor cells
� REFERENCES
1. Perez-Vizcaino, F.; Duarte, J.; Andriantsitohaina, R. Endothelial function
and cardiovascular disease: Effects of quercetin and wine polyphenols. Free
Radic. Res. 2006. 40, 1054-1065.
222 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
2. Bate-Smith, E. C. Haemanalysis of tannins: The concept of relative
astringency. Phytochemistry 1973. 12, 907-912.
3. Drewnowski, A.; Gomez-Carneros, C. Bitter taste, phytonutrients, and the
consumer: a review. Am. J. Clin. Nutr. 2000. 72, 1424-1435.
4. Delcour, J. A., et al. Flavor thresholds of polyphenolics in water. Am. J.
Enol. Vitic. 1984. 35, 134-136.
5. Hufnagel, J. C.; Hofmann, T. Quantitative reconstruction of the nonvolatile
sensometabolome of a red wine. J. Agric. Food Chem. 2008. 56, 9190-9199.
6. Noble, A. C., Why do wines taste bitter and feel astringent?, in Chemistry
of Wine Flavor. 1998, American Chemical Society. p. 156-165.
7. Kielhorn, S.; Thorngate Iii, J. H. Oral sensations associated with the
flavan-3-ols (+)-catechin and (-)-epicatechin. Food Qual. Prefer. 1999. 10, 109-
116.
8. Peleg, H., et al. Bitterness and astringency of flavan-3-ol monomers,
dimers and trimers. J. Sci. Food Agric. 1999. 79, 1123-1128.
9. Arnold, R. A.; Noble, A. C.; Singleton, V. L. Bitterness and astringency of
phenolic fractions in wine. J. Agric. Food Chem. 1980. 28, 675-678.
10. Robichaud, J. L.; Noble, A. C. Astringency and bitterness of selected
phenolics in wine. J. Sci. Food Agric. 1990. 52, 343-353.
11. Kallithraka, S.; Bakker, J. Evaluation of bitterness and astringency of (+)-
catechin and (-)-epicatechin in red wine and in model solution. J. Sens. Stud.
1997. 12, 25-37.
12. Lea, A. G. H.; Arnold, G. M. The phenolics of ciders: Bitterness and
astringency. J. Sci. Food Agric. 1978. 29, 478-483.
13. Matsunami, H.; Montmayeur, J. P.; Buck, L. B. A family of candidate taste
receptors in human and mouse. Nature 2000. 404, 601-604.
14. Chandrashekar, J., et al. T2Rs function as bitter taste receptors. Cell
2000. 100, 703-711.
15. Adler, E., et al. A novel family of mammalian taste receptors. Cell 2000.
100, 693-702.
16. Behrens, M., et al. The human bitter taste receptor hTAS2R50 is activated
by the two natural bitter terpenoids andrographolide and amarogentin. J. Agric.
Food Chem. 2009. 57, 9860-9866.
17. Shi, P.; Zhang, J., Extraordinary diversity of chemosensory receptor gene
repertoires among vertebrates, in Chemosensory Systems in Mammals, Fishes,
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
223
and Insects, S. Korsching and W. Meyerhof, Editors. 2009, Springer Berlin /
Heidelberg. p. 1-23.
18. Meyerhof, W., et al. The molecular receptive ranges of human TAS2R
bitter taste receptors. Chem. Senses 2009. 35, 157-170.
19. Bufe, B., et al. The human TAS2R16 receptor mediates bitter taste in
response to beta-glucopyranosides. Nat. Genet. 2002. 32, 397-401.
20. Behrens, M., et al. The human taste receptor hTAS2R14 responds to a
variety of different bitter compounds. Biochem. Biophys. Res. Commun. 2004.
319, 479-485.
21. Sainz, E., et al. Functional characterization of human bitter taste
receptors. Biochem. J. 2007. 403, 537-543.
22. Brockhoff, A., et al. Broad tuning of the human bitter taste receptor
hTAS2R46 to various sesquiterpene lactones, clerodane and labdane
diterpenoids, strychnine, and denatonium. J. Agric. Food Chem. 2007. 55, 6236-
6243.
23. Bufe, B., et al. The molecular basis of individual differences in
phenylthiocarbamide and propylthiouracil bitterness perception. Current Biology
2005. 15, 322-327.
24. Kim, U. K., et al. Positional cloning of the human quantitative trait locus
underlying taste sensitivity to phenylthiocarbamide. Science 2003. 299, 1221-
1225.
25. Pronin, A. N., et al. Specific alleles of bitter receptor genes influence
human sensitivity to the bitterness of aloin and saccharin. Current Biology 2007.
17, 1403-1408.
26. Kuhn, C., et al. Bitter taste receptors for saccharin and acesulfame K. J.
Neurosci. 2004. 24, 10260-10265.
27. Pronin, A. N., et al. Identification of ligands for two human bitter T2R
receptors. Chem. Senses 2004. 29, 583-593.
28. Dotson, C. D., et al. Bitter taste receptors influence glucose homeostasis.
PLoS ONE 2008. 3, e3974.
29. Maehashi, K., et al. Bitter peptides activate hTAS2Rs, the human bitter
receptors. Biochem. Biophys. Res. Commun. 2008. 365, 851-855.
30. Intelmann, D., et al. Three TAS2R bitter taste receptors mediate the
psychophysical responses to bitter compounds of hops (Humulus lupulus L.) and
beer. Chemosens. Percept. 2009. 2, 118-132.
224 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
31. Meyerhof, W. Elucidation of mammalian bitter taste. Rev. Physiol.
Biochem. Pharmacol. 2005. 154, 37-72.
32. Meyerhof, W., et al. The Molecular Receptive Ranges of Human TAS2R
Bitter Taste Receptors. Chem. Senses 2010. 35, 157-170.
33. Kim, U., et al. Worldwide haplotype diversity and coding sequence
variation at human bitter taste receptor loci. Hum. Mutat. 2005. 26, 199-204.
34. Roudnitzky, N., et al. Genomic, genetic and functional dissection of bitter
taste responses to artificial sweeteners. Hum. Mol. Genet. 2011. 20, 3437-3449.
35. Drayna, D. Human taste genetic. Annual Review of Genomics and Human
Genetics 2005. 6, 217-235.
36. Roland, W. S. U., et al. Soy isoflavones and other Isoflavonoids activate
the human bitter taste receptors hTAS2R14 and hTAS2R39. J. Agric. Food
Chem. 2011. 59, 11764-11771.
37. Wooding, S., et al. Genetics and bitter taste responses to goitrin, a plant
toxin found in vegetables. Chem. Senses 2010. 35, 685-692.
38. Neveu, V., et al., Phenol-explorer: an online comprehensive database on
polyphenol contents in foods. 2010.
39. Ross, J. A.; Kasum, C. M. Dietary flavonoids: bioavailability, metabolic
effects and safety. Annu. Rev. Nutr. 2002. 22, 19-34.
40. Prior, R. L.; Wu, X. Anthocyanins: Structural characteristics that result in
unique metabolic patterns and biological activities*. Free Radic. Res. 2006. 40,
1014-1028.
41. Ren, Y.; Chen, X. Distribution, bioactivities and therapeutical potentials of
pentagalloylglucopyranose. Curr. Bioact. Compd. 2007. 3, 81-89.
42. Soares, S.; Mateus, N.; De Freitas, V. Interaction of different polyphenols
with bovine serum albumin (BSA) and human salivary a-amylase (HSA) by
fluorescence quenching. J. Agric. Food Chem. 2007. 55, 6726-6735.
43. BráS, N. F., et al. Understanding the Binding of Procyanidins to
Pancreatic Elastase by Experimental and Computational Methods. Biochemistry
2010. 49, 5097-5108.
44. Chen, Y.; Hagerman, A. E. Characterization of soluble non-covalent
complexes between bovine serum albumin and β-1,2,3,4,6-penta-O-galloyl-d-
glucopyranose by MALDI-TOF MS. J. Agric. Food Chem. 2004. 52, 4008-4011.
45. Fernandes, A., et al. Antioxidant and biological properties of bioactive
phenolic compounds from Quercus suber L. J. Agric. Food Chem. 2009. 57,
11154-11160.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
225
46. Ueda, T., et al. Functional interaction between T2R taste receptors and G-
protein α subunits expressed in taste receptor cells. J. Neurosci. 2003. 23, 7376-
7380.
47. Renaud, S.; De Lorgeril, M. Wine, alcohol, platelets, and the French
paradox for coronary heart disease. The Lancet 1992. 339, 1523-1526.
48. Zhang, J., et al. Anti-cancer, anti-diabetic and other pharmacologic and
biological activities of penta-galloyl-glucose. Pharm Res. 2009. 26, 2066.
49. Serafino, A., et al. Differentiation of human melanoma cells induced by
cyanidin-3-O-β-glucopyranoside. The FASEB Journal 2004. 18, 1940-1942.
50. Vidal, S., et al. Taste and mouth-feel properties of different types of
tannin-like polyphenolic compounds and anthocyanins in wine. Anal. Chim. Acta
2004. 513, 57-65.
51. Brouillard, R.; Delaporte, B. Chemistry of anthocyanin pigments. 2. Kinetic
and thermodynamic study of proton transfer, hydration, and tautomeric reactions
of malvidin 3-glucoside. J. Am. Chem. Soc. 1977. 99, 8461-8468.
52. Pina, F., et al. Chemistry and applications of flavylium compounds: a
handful of colours. Chem. Soc. Rev. 2012. 41, 869-908.
53. Mueller, K. L., et al. The receptors and coding logic for bitter taste. Nature
2005. 434, 225-229.
54. Neveu, V., et al., Phenol-Explorer: an online comprehensive database on
polyphenol contents in foods, in Database. 2010.
55. Mateus, N.; Machado, J. M.; De Freitas, V. Development changes of
anthocyanins in Vitis vinifera grapes grown in the Douro Valley and concentration
in respective wines. J. Sci. Food Agric. 2002. 82, 1689-1695.
56. Hollman, P. C. H.; Arts, I. C. W. Flavonols, flavones and flavanols –
nature, occurrence and dietary burden. J. Sci. Food Agric. 2000. 80, 1081-1093.
57. Santos-Buelga, C.; Scalbert, A. Proanthocyanidins and tannin-like
compounds - nature, occurrence, dietary intake and effects on nutrition and
health. J. Sci. Food Agric. 2000. 80, 1094-1117.
58. Carando, S., et al. Levels of flavan-3-ols in french wines. J. Agric. Food
Chem. 1999. 47, 4161-4166.
59. Fischer, U.; Noble, A. C. The effect of ethanol, catechin concentration,
and pH on sourness and bitterness of wine. Am. J. Enol. Vitic. 1994. 45, 6-10.
226 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
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CONCLUSÃO E PERSPETIVAS FUTURAS
Este trabalho centrou-se na compreensão das propriedades sensoriais de
diferentes famílias de polifenóis (amargor e adstringência) e dos mecanismos
envolvidos. Por outro lado, foi estudado de que modo é que diferentes
carboidratos vulgarmente utilizados na indústria alimentar influenciam a
adstringência no contexto do vinho tinto. Para isso, procurou-se determinar: (a),
as principais famílias de proteínas salivares que apresentam mais afinidade para
os taninos; (b), de que forma é afetada a interação taninos/proteínas salivares
quando os taninos estão numa matriz de vinho tinto; (c), como é que o
enriquecimento de um vinho tinto em taninos influência a adstringência; (d),
quais as caraterísticas (solubilidade e tamanho) dos complexos formados pelas
diferentes famílias de proteínas salivares e taninos condensados vs.
hidrolisáveis; (e), como é que os carboidratos (goma arábica, β-ciclodextrina,
pectina e ácido poligalacturónico) inibem a interação taninos/proteínas,
particularmente com as proteínas salivares; (f), quais são os recetores de sabor
amargo ativados por alguns polifenóis frequentemente encontrados em diversos
alimentos.
Para atingir estes objetivos, começou-se por identificar na saliva humana as
respetivas proteínas por uma abordagem proteómica (HPLC-DAD, ESI-MS,
SDS-PAGE e MALDI-TOF). Seguidamente, foi estudada a afinidade entre as
diferentes famílias de proteínas salivares e taninos condensados através da
interação da saliva com procianidinas isoladas de grainhas de uvas. Os
resultados obtidos num ensaio competitivo, i. e., quando todas as proteínas
estão presentes em simultâneo tal como na cavidade oral, demonstram que os
taninos condensados interagem inicialmente com as aPRPs e estaterina e só
depois com as histatinas, gPRPs e bPRPs. Este resultado foi extremamente
importante pois foi observado pela primeira vez que as aPRPs são as PRPs que
apresentam maior afinidade para os taninos, contrariamente ao que era
esperado uma vez que as bPRPs são as mais estudadas e referidas na literatura
como as mais importantes nesta interação. Na verdade, este trabalho é o
primeiro que compara estas duas classes de PRPs.
Foi estudada a interação entre as diferentes proteínas da saliva com os taninos
diretamente no vinho tinto e os resultados foram confrontados com a
adstringência determinada por um painel de provadores. Para isso, foi
suplementado um vinho tinto com diferentes concentrações de taninos
228 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
condensados extraídos de grainhas de uvas. Comparando estes resultados in
vitro com a análise sensorial in vivo verificou-se que para concentrações baixas
de taninos, a complexação/precipitação de aPRPs e estaterina está
correlacionada com a intensidade da adstringência, pois estas proteínas têm
uma taxa muito elevada de complexação/precipitação comparativamente às
outras proteínas salivares. No entanto, para concentrações maiores de taninos,
a adstringência relativa percecionada dos vinhos parece correlacionar-se com
mudanças na composição das PRPs glicosiladas.
Após o estudo inicial da afinidade relativa da interação taninos
condensados/proteínas salivares, pretendeu-se comparar a interação das
proteínas salivares com um tanino hidrolisável. Para isso, foi estudada a
interação de uma procianidina trimérica e da PGG com as proteínas salivares
por HPLC e dynamic light scattering (DLS). Os resultados mostraram que as
proteínas aPRPs e estaterina, interagem em maior extensão com ambos os
taninos, comparativamente com as gPRPs e bPRPs. As bPRPs interagem pouco
com a procianidina trimérica e as gPRPs só complexam com a PGG. Em geral, a
PGG apresentou mais tendência para a formação de complexos insolúveis
enquanto a procianidina trimérica mostrou mais tendência para a formação de
complexos solúveis (exceto com a estaterina). Os resultados indicam que os
diferentes taninos e proteínas salivares estudados apresentam mecanismos
diferentes de interação e consequentemente particularidades diferentes no
desenvolvimento da adstringência.
Após o estudo da interação taninos/proteínas salivares, estudou-se o efeito dos
carboidratos nesta interação usando dois modelos de estudo: taninos
condensados/α-amilase e taninos condensados/proteínas salivares. No primeiro
estudo foram usadas as técnicas de fluorescência, nefelometria e DLS; no
segundo monitorizou-se por HPLC as proteínas salivares que permaneciam
solúveis na presença dos taninos e após a adição dos carboidratos. Os
respetivos precipitados formados foram também analisados por SDS-PAGE.
Globalmente os resultados mostraram que todos os carboidratos reduziram a
formação de agregados insolúveis, e portanto a precipitação, observando-se que
a pectina foi sempre o carboidrato mais eficiente, seguida da goma arábica. No
primeiro estudo, a extinção da fluorescência permitiu ter evidências de qual o
mecanismo mais provável subjacente a essa ação inibidora, sugerindo que a
goma arábica e a β-ciclodextrina inibem a interação tanino/proteína pelo
mecanismo de competição, associando-se aos taninos e impedindo-os de
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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interagir com a proteína. Por outro lado, a pectina parece atuar pela formação de
um complexo ternário tanino/proteína/pectina com solubilidade acrescida em
meio aquoso. Estas observações também foram testemunhadas no estudo com
as proteínas salivares, neste caso para os carboidratos goma arábica e a
pectina.
Na parte final deste trabalho, estudou-se o sabor amargo de alguns polifenóis
pertencentes a diferentes classes [(-)-epicatequina, PGG, procianidinas dimérica
B3 e trimérica C2, malvidina-3-glucósido e cianidina-3-glucósido], identificando
quais dos 25 recetores de sabor amargo dos humanos (hTAS2Rs) são ativados
por estes compostos.
Os resultados obtidos mostraram que diferentes compostos ativam diferentes
hTAS2Rs. (-)-Epicatequina ativou três recetors hTAS2R4, hTAS2R5 e
hTAS2R39, enquanto a PGG só ativou dois recetores, hTAS2R5 e hTAS2R39.
Por outro lado, a malvidina-3-glucósido e o trímero C2 só ativaram um recetor,
hTAS2R7 e hTAS2R5, respetivamente. Um dos resultados mais notáveis é que
os taninos são os primeiros agonistas naturais encontrados para o hTAS2R5,
apresentando grande potência na ativação deste recetor. Para além disso, os
grupos catecol e/ou galhoilo parecem ser estruturalmente importantes na
mediação da interação dos polifenóis com este recetor.
Os valores obtidos de EC50 para os diferentes compostos variam cerca de 100-
vezes, sendo os valores mais baixos os da PGG e malvidina-3-glucósido. Isto
sugere que estes compostos podem ser significativamente importantes no
amargor de frutos, vegetais e produtos derivados, mesmo que estejam presentes
em concentrações muito baixas.
Os resultados deste trabalho contribuíram para uma melhor compreensão do
envolvimento das diferentes famílias de proteínas salivares no desenvolvimento
da adstringência resultante da ingestão de taninos e do papel dos carboidratos
neste fenómeno. Neste trabalho surgiram, pela primeira vez, evidências de que
as diferentes proteínas salivares estão envolvidas em diferentes fases do
desenvolvimento da adstringência. Também pela primeira vez foi demonstrada
uma grande afinidade entre os taninos presentes no vinho e as aPRPs e a
estaterina. Com base na literatura seria de esperar que as bPRPs fossem as
mais eficientes. No entanto, isto não se verificou porque na literatura existem
poucos trabalhos com as aPRPs e nenhum trabalho que englobe o estudo das
diferentes famílias de PRPs em simultâneo.
230 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Relativamente aos carboidratos, pela primeira vez a utilização de várias técnicas
permitiu conhecer melhor os mecanismos responsáveis pela sua ação inibidora
da interação entre os polifenóis e proteínas, nomeadamente com as diferentes
proteínas salivares. O estudo experimental de sistemas ternários envolvendo
três famílias complexas de compostos como os polifenóis, as proteínas e os
carboidratos é um desafio difícil mas, neste caso em particular, a conjugação
das técnicas de fluorescência, DLS e nefelometria permitiu obter resultados
importantes.
No que diz respeito ao sabor amargo, foram identificados pela primeira vez os
hTAS2Rs ativados por alguns polifenóis. Foram obtidas pela primeira vez
evidências de que a malvidina-3-glucósido (uma antocianina) ativa um hTAS2Rs
e portanto pode ter um papel no sabor amargo dos alimentos. E, finalmente, um
dos resultados mais notáveis é que os taninos são os primeiros agonistas
naturais encontrados para o hTAS2R5, apresentando grande potência na
ativação deste recetor.
Apesar da importância dos resultados obtidos, a interação tanino/proteína ainda
é uma área pouco estudada com muitas questões por explorar. Estudos futuros
passam pela compreensão do papel fisiológico das diferentes famílias de
proteínas salivares a nível sensorial e nutricional, considerando a interação com
os polifenóis. Por outro lado, do ponto de vista nutricional, os polifenóis
apresentam propriedades biológicas importantes (prevenção cardiovascular,
anticancerígena, etc), sendo que a interação com as proteínas salivares poderá
afetar a sua biodisponibilidade. Assim, seria importante estabelecer uma relação
estrutura-atividade dos polifenóis (extraídos de frutos ou sintetizados) na
interação com diferentes famílias de proteínas salivares (PRPs básicas, acídicas,
glicosiladas, estaterina e histatinas).
A abordagem experimental passaria pela determinação das constantes de
associação e estequiometria dos agregados, na ausência e na presença de
carbohidratos existentes nos alimentos ou usados na indústria alimentar
(pectina, goma arábica, etc). Este estudo envolveria diferentes técnicas,
nomeadamente HPLC, ESI-MS e RMN. Na interpretação dos resultados seria
importante recorrer à modelação molecular de modo a determinar os principais
locais moleculares da interação tanto na proteína como no polifenol.
A nível nutricional seria importante determinar a estabilidade dos complexos
taninos/proteínas salivares no estômago e intestino e a eventual
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
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231
biodisponibilidade dos taninos, recriando um ambiente de digestão simulado in
vitro.
232 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
REFERÊNCIAS BIBLIOGRÁFICAS
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
235
Adler, E., Hoon, M. A., Mueller, K. L., Chandrashekar, J., Ryba, N. J. P. e Zuker,
C. S. (2000). "A Novel Family of Mammalian Taste Receptors." Cell 100(6): 693-
702.
Akiyama, H., Sakushima, J.-I., Taniuchi, S., Kanda, T., Yanagida, A., Kojima, T.,
Teshima, R., Kobayashi, Y., Goda, Y. e Toyoda, M. (2000). "Antiallergic Effect of
Apple Polyphenols on the Allergic Model Mouse." Biological & Pharmaceutical
Bulletin 23: 1370-1373.
Ames, B. N., Profet, M. e Gold, L. S. (1990). "Dietary Pesticides (99.99% All
Natural)." Proceedings of the National Academy of Sciences 87(19): 7777-7781.
Arnold, R. A., Noble, A. C. e Singleton, V. L. (1980). "Bitterness and Astringency
of Phenolic Fractions in Wine." Journal of Agricultural and Food Chemistry 28(3):
675-678.
Asano, K., Shinagawa, K. e Hashimoto, N. (1982). "Characterization of Haze-
Forming Proteins of Beer and Their Roles in Chill Haze Formation." Journal of
the American Society of Brewing Chemists 40(4): 147-154.
Azevedo, J., Fernandes, I., Faria, A., Oliveira, J., Fernandes, A., De Freitas, V. e
Mateus, N. (2010). "Antioxidant Properties of Anthocyanidins, Anthocyanidin-3-
Glucosides and Respective Portisins." Food Chemistry 119(2): 518-523.
Bacon, J. R. e Rhodes, M. J. C. (1998). "Development of a Competition Assay for
the Evaluation of the Binding of Human Parotid Salivary Proteins to Dietary
Complex Phenols and Tannins Using a Peroxidase-Labeled Tannin." Journal of
Agricultural and Food Chemistry 46(12): 5083-5088.
Bacon, J. R. e Rhodes, M. J. C. (2000). "Binding Affinity of Hydrolyzable Tannins
to Parotid Saliva and to Proline-Rich Proteins Derived from It." Journal of
Agricultural and Food Chemistry 48(3): 838-843.
Bastianetto, S., Yao, Z. X., Papadopoulos, V. e Quirion, R. (2006).
"Neuroprotective Effects of Green and Black Teas and Their Catechin Gallate
Esters against Beta-Amyloid-Induced Toxicity." European Journal of
Neuroscience 23: 55–64.
236 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Bate-Smith, E. C. (1954). "Astringency in Foods." Food 23: 124.
Baxter, N. J., Lilley, T. H., Haslam, E. e Williamson, M. P. (1997). "Multiple
Interactions between Polyphenols and a Salivary Proline-Rich Protein Repeat
Result in Complexation and Precipitation." Biochemistry 36(18): 5566-5577.
Behrens, M. e Meyerhof, W. (2006). "Signaling in the Chemosensory Systems."
Cellular and Molecular Life Sciences 63(13): 1501-1509.
Bennick, A. (1975). "Chemical and Physical Characteristics of a Phosphoprotein
from Human Parotid Saliva." Biochemistry Journal 145(3): 557-567.
Bennick, A. (2002). "Interaction of Plant Polyphenols with Salivary Proteins."
Critical Reviews in Oral & Biology Medicine 13(2): 184-196.
Bors, W., Heller, W., Michel, C. e Saran, M. (1990). "Radical Chemistry of
Flavonoid Antioxidants." Advances in Experimental Medicine and Biology 264:
165-70.
Boze, H., Marlin, T., Durand, D., Perez, J., Vernhet, A., Canon, F., Sarni-
Manchado, P., Cheynier, V. e Cabane, B. (2010). "Proline-Rich Salivary Proteins
Have Extended Conformations." Biophysical Journal 99(2): 656-665.
Brouillard, R. (1983). "The in Vivo Expression of Anthocyanin Colour in Plants."
Phytochemistry 22(6): 1311-1323.
Bufe, B., Hofmann, T., Krautwurst, D., Raguse, J. D. e Meyerhof, W. (2002). "The
Human Tas2r16 Receptor Mediates Bitter Taste in Response to Beta-
Glucopyranosides." Nature Genetics 32(3): 397-401.
Cala, O., Pinaud, N., Simon, C., Fouquet, E., Laguerre, M., Dufourc, E. J. e
Pianet, I. (2010). "NMR and Molecular Modeling of Wine Tannins Binding to
Saliva Proteins: Revisiting Astringency from Molecular and Colloidal Prospects."
The FASEB Journal 24(11): 4281-4290.
Canon, F., Giuliani, A., Paté, F. e Sarni-Manchado, P. (2010). "Ability of a
Salivary Intrinsically Unstructured Protein to Bind Different Tannin Targets
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
237
Revealed by Mass Spectrometry." Analytical and Bioanalytical Chemistry 398(2):
815-822.
Carpenter, G. H. e Proctor, G. B. (1999). "O-Linked Glycosylation Occurs on
Basic Parotid Salivary Proline-Rich Proteins." Oral Microbiology and Immunology
14(5): 309-315.
Carvalho, A. R. F., Oliveira, J., De Freitas, V., Mateus, N. e Melo, A. (2010). "A
Theoretical Interpretation of the Color of Two Classes of Pyranoanthocyanins."
Journal of Molecular Structure-Theochem 948(1-3): 61-64.
Carvalho, E., Mateus, N., Plet, B., Pianet, I., Dufourc, E. e De Freitas, V. (2006).
"Influence of Wine Pectic Polysaccharides on the Interactions between
Condensed Tannins and Salivary Proteins." Journal of Agricultural and Food
Chemistry 54(23): 8936-8944.
Carvalho, E., Póvoas, M. J., Mateus, N. e De Freitas, V. (2006). "Application of
Flow Nephelometry to the Analysis of the Influence of Carbohydrates on Protein-
Tannin Interactions." Journal of the Science of Food and Agriculture 86(6): 891-
896.
Castagnola, M., Inzitari, R., Rossetti, D. V., Olmi, C., Cabras, T., Piras, V.,
Nicolussi, P., Sanna, M. T., Pellegrini, M., Giardina, B. e Messana, I. (2004). "A
Cascade of 24 Histatins (Histatin 3 Fragments) in Human Saliva." Journal of
Biological Chemistry 279(40): 41436-41443.
Chandrashekar, J., Mueller, K. L., Hoon, M. A., Adler, E., Feng, L., Guo, W.,
Zuker, C. S. e Ryba, N. J. P. (2000). "T2Rs Function as Bitter Taste Receptors."
Cell 100(6): 703-711.
Charlton, A. J., Baxter, N. J., Lilley, T. H., Haslam, E., Mcdonald, C. J. e
Williamson, M. P. (1996). "Tannin Interactions with a Full-Length Human Salivary
Proline-Rich Protein Display a Stronger Affinity Than with Single Proline-Rich
Repeats." FEBS Letters 382(3): 289-292.
Charlton, A. J., Haslam, E. e Williamson, M. P. (2002). "Multiple Conformations of
the Proline-Rich Protein/Epigallocatechin Gallate Complex Determined by Time-
238 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Averaged Nuclear Overhauser Effects." Journal of the American Chemical
Society 124(33): 9899-9905.
Charlton, A. J., Haslam, E. e Williamson, M. P. (2002). "Multiple Conformations of
the Proline-Rich Protein/Epigallocatechin Gallate Complex Determined by Time-
Averaged Nuclear Overhauser Effects." Journal of the American Chemical
Society 124(33): 9899-9905.
Day, A. J., Cañada, F. J., Dı́Az, J. C., Kroon, P. A., Mclauchlan, R., Faulds, C. B.,
Plumb, G. W., Morgan, M. R. A. e Williamson, G. (2000). "Dietary Flavonoid and
Isoflavone Glycosides Are Hydrolysed by the Lactase Site of Lactase Phlorizin
Hydrolase." FEBS Letters 468(2): 166-170.
De Freitas, V., Carvalho, E. e Mateus, N. (2003). "Study of Carbohydrate
Influence on Protein-Tannin Aggregation by Nephelometry." Food Chemistry
81(4): 503-509.
De Freitas, V. e Mateus, N. (2001). "Structural Features of Procyanidin
Interactions with Salivary Proteins." Journal of Agricultural and Food Chemistry
49(2): 940-945.
De Freitas, V. e Mateus, N. (2012). "Protein/Polyphenol Interactions: Past and
Present Contributions. Mechanisms of Astringency Perception." Current Organic
Chemistry 16(6): 724-746.
De Freitas, V. a. P., Glories, Y. e Laguerre, M. (1998). "Incidence of Molecular
Structure in Oxidation of Grape Seed Procyanidins." Journal of Agricultural and
Food Chemistry 46(2): 376-382.
De La Iglesia, R., Milagro, F. I., Campión, J., Boqué, N. e Martínez, J. A. (2010).
"Healthy Properties of Proanthocyanidins." BioFactors 36(3): 159-168.
Delcour, J. A., Vandenberghe, M. M., Corten, P. F. e Dondeyne, P. (1984).
"Flavor Thresholds of Polyphenolics in Water." American Journal of Enology and
Viticulture 35(3): 134-136.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
239
Déprez, S., Brezillon, C., Rabot, S., Philippe, C., Mila, I., Lapierre, C. e Scalbert,
A. (2000). "Polymeric Proanthocyanidins Are Catabolized by Human Colonic
Microflora into Low-Molecular-Weight Phenolic Acids." The Journal of Nutrition
130(11): 2733-2738.
Deprez, S., Mila, I., Huneau, J.-F., Tome, D. e Scalbert, A. (2004). "Transport of
Proanthocyanidin Dimer, Trimer, and Polymer across Monolayers of Human
Intestinal Epithelial Caco-2 Cells." Antioxidants & Redox Signaling 3(6): 957-967.
Douglas, W. H., Reeh, E. S., Ramasubbu, N., Raj, P. A., Bhandary, K. K. e
Levine, M. J. (1991). "Statherin: A Major Boundary Lubricant of Human Saliva."
Biochemical and Biophysical Research Communications 180(1): 91-97.
Drewnowski, A. e Gomez-Carneros, C. (2000). "Bitter Taste, Phytonutrients, and
the Consumer: A Review." The American Journal of Clinical Nutrition 72(6):
1424-1435.
Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R.,
Engemann, S., Pastore, A. e Wanker, E. E. (2008). "EGCG Redirects
Amyloidogenic Polypeptides into Unstructured, Off-Pathway Oligomers." Nature
Structural & Molecular Biology 15(6): 558-566.
Faria, A., Calhau, C., De Freitas, V. e Mateus, N. (2006). "Procyanidins as
Antioxidants and Tumor Cell Growth Modulators." Journal of Agricultural and
Food Chemistry 54(6): 2392-2397.
Faria, A., Pestana, D., Teixeira, D., De Freitas, V., Mateus, N. e Calhau, C.
(2010). "Blueberry Anthocyanins and Pyruvic Acid Adducts: Anticancer
Properties in Breast Cancer Cell Lines." Phytotherapy Research 24(12): 1862-
1869.
Fernandes, A., Fernandes, I., Cruz, L. S., Mateus, N., Cabral, M. e De Freitas, V.
(2009). "Antioxidant and Biological Properties of Bioactive Phenolic Compounds
from Quercus Suber L." Journal of Agricultural and Food Chemistry 57(23):
11154-11160.
240 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Fulcrand, H., Benabdeljalil, C., Rigaud, J., Cheynier, V. e Moutounet, M. (1998).
"A New Class of Wine Pigments Generated by Reaction between Pyruvic Acid
and Grape Anthocyanins." Phytochemistry 47: 1401-1407.
Funatogawa, K., Hayashi, S., Shimomura, H., Yoshida, T., Hatano, T., Ito, H. e
Hirai, Y. (2004). "Antibacterial Activity of Hydrolyzable Tannins Derived from
Medicinal Plants against Helicobacter Pylori." Microbiology and Immunology
48(4): 251-261.
Gee, J. M., Dupont, M. S., Day, A. J., Plumb, G. W., Williamson, G. e Johnson, I.
T. (2000). "Intestinal Transport of Quercetin Glycosides in Rats Involves Both
Deglycosylation and Interaction with the Hexose Transport Pathway." The
Journal of Nutrition 130(11): 2765-2771.
Gibbons, R. J. e Hay, D. I. (1988). "Human Salivary Acidic Proline-Rich Proteins
and Statherin Promote the Attachment of Actinomyces Viscosus Ly7 to Apatitic
Surfaces." Infect Immunology 56(2): 439–445.
Glendinning, J. I. (1994). "Is the Bitter Rejection Response Always Adaptive?"
Physiology and Behavior 56(6): 1217-1227.
Goldstein, J. L. e Swain, T. (1963). "Changes in Tannins in Ripening Fruits."
Phytochemistry 2(4): 371-383.
Goncalves, R., Soares, S., Mateus, N. e De Freitas, V. (2007). "Inhibition of
Trypsin by Condensed Tannins and Wine." Journal of Agricultural and Food
Chemistry 55(18): 7596-7601.
Green, M. S. e Jucha, E. (1986). "Association of Serum-Lipids with Coffee, Tea
and Egg Consumption in Free-Living Subjects." Journal of Epidemiology and
Community Health 40(4): 324-329.
Griffiths, D. W. (1986). "The Inhibition of Digestive Enzymes by Polyphenolic
Compounds." Advances in Experimental Medicine and Biology 199: 509-516.
Gu, L., Kelm, M. A., Hammerstone, J. F., Beecher, G., Holden, J., Haytowitz, D.,
Gebhardt, S. e Prior, R. L. (2004). "Concentrations of Proanthocyanidins in
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
241
Common Foods and Estimations of Normal Consumption." Journal of Nutrition
134(3): 613-617.
Guo, Q., Zhao, B., Li, M., Shen, S. e Xin, W. (1996). "Studies on Protective
Mechanisms of Four Components of Green Tea Polyphenols against Lipid
Peroxidation in Synaptosomes." Biochimica et Biophysica Acta (BBA) - Lipids
and Lipid Metabolism 1304(3): 210-222.
Gusman, H., Travis, J., Helmerhorst, E. J., Potempa, J., Troxler, R. F. e
Oppenheim, F. G. (2001). "Salivary Histatin 5 Is an Inhibitor of Both Host and
Bacterial Enzymes Implicated in Periodontal Disease." Infection and Immunity
69(3): 1402–1408.
Guyot, S., Doco, T., Souquet, J.-M., Moutounet, M. e Drilleau, J.-F. (1997).
"Characterization of Highly Polymerized Procyanidins in Cider Apple (Malus
Sylvestris Var. Kermerrien) Skin and Pulp." Phytochemistry 44(2): 351-357.
Gyémánt, G., Zajácz, Á., Bécsi, B., Ragunath, C., Ramasubbu, N., Erdodi, F.,
Batta, G. e Kandra, L. (2009). "Evidence for Pentagalloyl Glucose Binding to
Human Salivary a-Amylase through Aromatic Amino Acid Residues." Biochimica
et Biophysica Acta (BBA) - Proteins & Proteomics 1794(2): 291-296.
Hagerman, A. E. e Butler, L. G. (1980). "Determination of Protein in Tannin-
Protein Precipitates." Journal of Agricultural and Food Chemistry 28(5): 944-947.
Hagerman, A. E. e Butler, L. G. (1981). "The Specificity of Proanthocyanidin-
Protein Interactions." Journal of Biological Chemistry 256(9): 4494-4497.
Hagerman, A. E. e Klucher, K. M. (1986). "Tannin-Protein Interactions." Progress
in Clinical Biological Research 213: 67-76.
Hagerman, A. E., Rice, M. E. e Ritchard, N. T. (1998). "Mechanisms of Protein
Precipitation for Two Tannins, Pentagalloyl Glucose and Epicatechin16 (4-8)
Catechin (Procyanidin)." Journal of Agricultural and Food Chemistry 46(7): 2590-
2595.
242 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Hagerman, A. E. e Robbins, C. T. (1987). "Implications of Soluble Tannin-Protein
Complexes for Tannin Analysis and Plant Defense Mechanisms." Journal of
Chemical Ecology 13(5): 1243-1259.
Hanlin, R. L., Hrmova, M., Harbertson, J. F. e Downey, M. O. (2010). "Review:
Condensed Tannin and Grape Cell Wall Interactions and Their Impact on Tannin
Extractability into Wine." Australian Journal of Grape and Wine Research 16(1):
173-188.
Haslam, E. (1996). "Natural Polyphenols (Vegetable Tannins) as Drugs: Possible
Modes of Action." Journal of Natural Products 59(2): 205-215.
Haslam, E., Lilley Terence, H., Warminski, E., Liao, H., Cai, Y., Martin, R.,
Gaffney Simon, H., Goulding Paul, N. e Luck, G. (1992). Polyphenol
Complexation - a Study in Molecular Recognition. Phenolic Compounds in Food
and Their Effects on Health I, American Chemical Society. 506: 8-50.
Haslam, E. C. (1998). Polyphenols - Structure and Biosynthesis. Pratical
Polyphenolics: From Structure to Molecular Recognition and Physiological
Action, Cambridge University Press.
Hatano, T. e Hemingway, R. W. (1996). "Association of (+)-Catechin and
Catechin-(4a®8)-Catechin with Oligopeptides." Chemical Communications(22):
2537-2538.
Hatton, M. N., Loomis, R. E., Levine, M. J. e Tabak, L. A. (1985). "Masticatory
Lubrication. The Role of Carbohydrate in the Lubricating Property of a Salivary
Glycoprotein-Albumin Complex." Biochemical Journal 230(3): 817-820.
Hay, D. I., Smith, D. J., Schluckebier, S. K. e Moreno, E. C. (1984). "Basic
Biological Sciences Relationship between Concentration of Human Salivary
Statherin and Inhibition of Calcium Phosphate Precipitation in Stimulated Human
Parotid Saliva." Journal of Dental Research 63(6): 857-863.
Hayashi, N., Ujihara, T. e Kohata, K. (2005). "Reduction of Catechin Astringency
by the Complexation of Gallate-Type Catechins with Pectin." Bioscience,
Biotechnology, and Biochemistry 69(7): 1306-1310.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
243
Hertog, M. G. L., Feskens, E. J. M., Hollman, P. C. H., Katan, M. B. e Kromhout,
D. (1993). "Dietary Antioxidant Flavonoids and Risk of Coronary Heart Disease:
The Zutphen Elderly Study." Lancet 342(8878): 1007-1011.
Hladik, C. M. e Simmen, B. (1996). "Taste Perception and Feeding Behavior in
Nonhuman Primates and Human Populations." Evolutionary Antropology 5(2):
58-71.
Hollman, P. C. H. e Katan, M. B. (1999). "Dietary Flavonoids: Intake, Health
Effects and Bioavailability." Food and Chemical Toxicology 37(9-10): 937-942.
Holt, R. R., Lazarus, S. A., Sullards, M. C., Zhu, Q. Y., Schramm, D. D.,
Hammerstone, J. F., Fraga, C. G., Schmitz, H. H. e Keen, C. L. (2002).
"Procyanidin Dimer B2 [Epicatechin-(4β-8)-Epicatechin] in Human Plasma after
the Consumption of a Flavanol-Rich Cocoa." The American Journal of Clinical
Nutrition 76(4): 798-804.
Howitz, K. T., Bitterman, K. J., Cohen, H. Y., Lamming, D. W., Lavu, S., Wood, J.
G., Zipkin, R. E., Chung, P., Kisielewski, A., Zhang, L.-L., Scherer, B. e Sinclair,
D. A. (2003). "Small Molecule Activators of Sirtuins Extend Saccharomyces
Cerevisiae Lifespan." Nature 425(6954): 191-196.
Hu, Y., Guo, D.-H., Liu, P., Cao, J.-J., Wang, Y.-P., Yin, J., Zhu, Y. e Rahman, K.
(2011). "Bioactive Components from the Tea Polyphenols Influence on
Endogenous Antioxidant Defense System and Modulate Inflammatory Cytokines
after Total-Body Irradiation in Mice." Phytomedicine 18(11): 970-975.
Hufnagel, J. C. e Hofmann, T. (2008). "Orosensory-Directed Identification of
Astringent Mouthfeel and Bitter-Tasting Compounds in Red Wine." Journal of
Agricultural and Food Chemistry 56(4): 1376-1386.
Hufnagel, J. C. e Hofmann, T. (2008). "Quantitative Reconstruction of the
Nonvolatile Sensometabolome of a Red Wine." Journal of Agricultural and Food
Chemistry 56(19): 9190-9199.
Intelmann, D., Batram, C., Kuhn, C., Haseleu, G., Meyerhof, W. e Hofmann, T.
(2009). "Three Tas2r Bitter Taste Receptors Mediate the Psychophysical
244 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Responses to Bitter Compounds of Hops (Humulus Lupulus L.) and Beer."
Chemosensory Perception 2(3): 118-132.
Jin, Y.-R., Im, J.-H., Park, E.-S., Cho, M.-R., Han, X.-H., Lee, J.-J., Lim, Y., Kim,
T.-J. e Yun, Y.-P. (2008). "Antiplatelet Activity of Epigallocatechin Gallate Is
Mediated by the Inhibition of Plc[Gamma]2 Phosphorylation, Elevation of Pgd2
Production, and Maintaining Calcium-ATPase Activity." Journal of Cardiovascular
Pharmacology 51(1): 45-54.
Jöbstl, E., Howse, J. R., Fairclough, J. P. A. e Williamson, M. P. (2006).
"Noncovalent Cross-Linking of Casein by Epigallocatechin Gallate Characterized
by Single Molecule Force Microscopy." Journal of Agricultural and Food
Chemistry 54(12): 4077-4081.
Jobstl, E., O'connell, J., Fairclough, J. P. A. e Williamson, M. P. (2004).
"Molecular Model for Astringency Produced by Polyphenol/Protein Interactions."
Biomacromolecules 5(3): 942-949.
Joslyn, M. A. e Goldstein, J. L. (1964). "Astringency of Fruits and Fruit Products
in Relation to Phenolic Content." Advances in Food Research 13: 179-217.
Kallithraka, S. e Bakker, J. (1997). "Evaluation of Bitterness and Astringency of
(+)-Catechin and (-)-Epicatechin in Red Wine and in Model Solution." Journal of
Sensory Studies 12: 25-37.
Kaur, C. e Kapoor, H. C. (2001). "Antioxidants in Fruits and Vegetables – the
Millennium’s Health." International Journal of Food Science & Technology 36(7):
703-725.
Kawamoto, H., Nakatsubo, F. e Murakami, K. (1995). "Quantitative Determination
of Tannin and Protein in the Precipitates by High-Performance Liquid-
Chromatography." Phytochemistry 40(5): 1503-1505.
Khafif, A., Schantz, S. P., Chou, T. C., Edelstein, D. e Sacks, P. G. (1998).
"Quantitation of Chemopreventive Synergism between (-)-Epigallocatechin- 3-
Gallate and Curcumin in Normal, Premalignant and Malignant Human Oral
Epithelial Cells." Carcinogenesis 19(3): 419-424.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
245
Kielhorn, S. e Thorngate Iii, J. H. (1999). "Oral Sensations Associated with the
Flavan-3-ols (+)-Catechin and (-)-Epicatechin." Food Quality and Preference
10(2): 109-116.
Kim, U., Wooding, S., Ricci, D., Jorde, L. B. e Drayna, D. (2005). "Worldwide
Haplotype Diversity and Coding Sequence Variation at Human Bitter Taste
Receptor Loci." Human Mutation 26(3): 199-204.
Levine, M. e Keller, P. J. (1977). "The Isolation of Some Basic Proline-Rich
Proteins from Human Parotid Saliva." Archives of Oral Biology 22(1): 37-41.
Luck, G., Liao, H., Murray, N. J., Grimmer, H. R., Warminski, E. E., Williamson,
M. P., Lilley, T. H. e Haslam, E. (1994). "Polyphenols, Astringency and Proline-
Rich Proteins." Phytochemistry 37(2): 357-371.
Maehashi, K., Matano, M., Wang, H., Vo, L. A., Yamamoto, Y. e Huang, L.
(2008). "Bitter Peptides Activate hTAS2Rs, the Human Bitter Receptors."
Biochemical and Biophysical Research Communications 365(4): 851-855.
Manach, C., Scalbert, A., Morand, C., Remesy, C. e Jimenez, L. (2004).
"Polyphenols: Food Sources and Bioavailability." American Journal of Clinical
Nutrition 79(5): 727-747.
Margolskee, R. F. (2002). "Molecular Mechanisms of Bitter and Sweet Taste
Transduction." Journal of Biological Chemistry 277(1): 1-4.
Mateus, N., Carvalho, E., Luís, C. e De Freitas, V. (2004). "Influence of the
Tannin Structure on the Disruption Effect of Carbohydrates on Protein-Tannin
Aggregates." Analitycal Chimical Acta 513(1): 135-140.
Mateus, N., Marques, S., Goncalves, A. C., Machado, J. M. e De Freitas, V.
(2001). "Proanthocyanidin Composition of Red Vitis Vinifera Varieties from the
Douro Valley During Ripening: Influence of Cultivation Altitude." American
Journal of Enology and Viticulture 52(2): 115-121.
246 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Mateus, N., Silva, A. M. S., Rivas-Gonzalo, J. C., Santos-Buelga, C. e De Freitas,
V. (2003). "A New Class of Blue Anthocyanin-Derived Pigments Isolated from
Red Wines." Journal of Agricultural and Food Chemistry 51(7): 1919-1923.
Matsunami, H., Montmayeur, J. P. e Buck, L. B. (2000). "A Family of Candidate
Taste Receptors in Human and Mouse." Nature 404(6778): 601-604.
Mcarthur, C., Sanson, G. D. e Beal, A. M. (1995). "Salivary Proline-Rich Proteins
in Mammals: Roles in Oral Homeostasis and Counteracting Dietary Tannin."
Journal of Chemical Ecology 21(6): 663-691.
Mcmanus, J. P., Davis, K. G., Beart, J. E., Gaffney, S. H., Lilley, T. H. e Haslam,
E. (1985). "Polyphenol Interactions. Part 1. Introduction; Some Observations on
the Reversible Complexation of Polyphenols with Proteins and Polysaccharides."
Journal of Chemical Society, Perkin Transaction 2: 1429-1438.
Mcrae, J. M. e Kennedy, J. A. (2011). "Wine and Grape Tannin Interactions with
Salivary Proteins and Their Impact on Astringency: A Review of Current
Research." Molecules 16(3): 2348-2364.
Mehansho, H., Butler, L. G. e Carlson, D. M. (1987). "Dietary Tannins and
Salivary Proline-Rich Proteins: Interactions, Induction, and Defense
Mechanisms." Annual Review of Nutrition 7(1): 423-440.
Messana, I., Inzitari, R., Fanali, C., Cabras, T. e Castagnola, M. (2008). "Facts
and Artifacts in Proteomics of Body Fluids. What Proteomics of Saliva Is Telling
Us?" Journal of Separation Science 31(11): 1948-1963.
Meyerhof, W. (2005). "Elucidation of Mammalian Bitter Taste." Reviews of
Physiology, Biochemistry and Pharmacology 154: 37-72.
Meyerhof, W., Batram, C., Kuhn, C., Brockhoff, A., Chudoba, E., Bufe, B.,
Appendino, G. e Behrens, M. (2009). "The Molecular Receptive Ranges of
Human TAS2r Bitter Taste Receptors." Chemical Senses 35(2): 157-170.
Morley, N., Clifford, T., Salter, L., Campbell, S., Gould, D. e Curnow, A. (2005).
"The Green Tea Polyphenol (−)-Epigallocatechin Gallate and Green Tea Can
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
247
Protect Human Cellular DNA from Ultraviolet and Visible Radiation-Induced
Damage." Photodermatology, Photoimmunology & Photomedicine 21(1): 15-22.
Murray, N. J. e Williamson, M. P. (1994). "Conformational Study of a Salivary
Proline-Rich Protein Repeat Sequence." European Journal of Biochemistry
219(3): 915-921.
Naczk, M., Oickle, D., Pink, D. e Shahidi, F. (1996). "Protein Precipitating
Capacity of Crude Canola Tannins: Effect of pH, Tannin, and Protein
Concentrations." Journal of Agricultural and Food Chemistry 44(8): 2144-2148.
Naczk, M. e Shahidi, F. (2006). "Phenolics in Cereals, Fruits and Vegetables:
Occurrence, Extraction and Analysis." Journal of Pharmaceutical and Biomedical
Analysis 41(5): 1523-1542.
Noble, A. C. (1998). Why Do Wines Taste Bitter and Feel Astringent? Chemistry
of Wine Flavor, American Chemical Society. 714: 156-165.
Obreque-Slier, E., López-Solís, R., Peña-Neira, Á. e Zamora-Marín, F. (2010).
"Tannin–Protein Interaction Is More Closely Associated with Astringency Than
Tannin–Protein Precipitation: Experience with Two Oenological Tannins and a
Gelatin." International Journal of Food Science & Technology 45(12): 2629-2636.
Oh, H. I., Hoff, J. E., Armstrong, G. S. e Haff, L. A. (1980). "Hydrophobic
Interaction in Tannin-Protein Complexes." Journal of Agricultural and Food
Chemistry 28(2): 394-398.
Oizumi, Y., Mohri, Y., Hirota, M. e Makabe, H. (2010). "Synthesis of Procyanidin
B3 and Its Anti-Inflammatory Activity. The Effect of 4-Alkoxy Group of Catechin
Electrophile in the Yb(Otf)3-Catalyzed Condensation with Catechin Nucleophile."
The Journal of Organic Chemistry 75(14): 4884-4886.
Okuda, T., Mori, K. e Hatano, T. (1985). "Relationship of the Structures of
Tannins to the Binding Activities with Hemoglobin and Methylene Blue." Chemical
and Pharmaceutical Bulletin 33: 1424-1433.
248 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Oliveira, J., Fernandes, V., Miranda, C., Santos-Buelga, C., Silva, A., De Freitas,
V. e Mateus, N. (2006). "Color Properties of Four Cyanidin-Pyruvic Acid
Adducts." Journal of Agricultural and Food Chemistry 54(18): 6894-6903.
Oppenheim, F. G., Offner, G. D. e Troxler, R. F. (1985). "Amino Acid Sequence
of a Proline-Rich Phosphoglycoprotein from Parotid Secretion of the Subhuman
Primate Macaca Fascicularis." Journal of Biological Chemistry 260(19): 10671-
10679.
Oppenheim, F. G., Xu, T., Mcmillian, F. M., Levitz, S. M., Diamond, R. D., Offner,
G. D. e Troxler, R. F. (1988). "Histatins, a Novel Family of Histidine-Rich Proteins
in Human Parotid Secretion. Isolation, Characterization, Primary Structure, and
Fungistatic Effects on Candida Albicans." Journal of Biological Chemistry
263(16): 7472-7477.
Ozawa, T., Lilley, T. H. e Haslam, E. (1987). "Polyphenol Interactions:
Astringency and the Loss of Astringency in Ripening Fruit." Phytochemistry
26(11): 2937-2942.
Peleg, H., Gacon, K., Schlich, P. e Noble, A. C. (1999). "Bitterness and
Astringency of Flavan-3-ol Monomers, Dimers and Trimers." Journal of the
Science of Food and Agriculture 79(8): 1123-1128.
Porto, P. A. L. D. S., Laranjinha, J. A. N. e De Freitas, V. A. P. (2003).
"Antioxidant Protection of Low Density Lipoprotein by Procyanidins:
Structure/Activity Relationships." Biochemical Pharmacology 66(6): 947-954.
Prasanna, V., Prabha, T. N. e Tharanathan, R. N. (2007). "Fruit Ripening
Phenomena – an Overview." Critical Reviews in Food Science and Nutrition
47(1): 1-19.
Prieur, C., Rigaud, J., Cheynier, V. e Moutounet, M. (1994). "Oligomeric and
Polymeric Procyanidins from Grape Seeds." Phytochemistry 36(3): 781-784.
Pronin, A. N., Xu, H., Tang, H., Zhang, L., Li, Q. e Li, X. (2007). "Specific Alleles
of Bitter Receptor Genes Influence Human Sensitivity to the Bitterness of Aloin
and Saccharin." Current Biology 17(16): 1403-1408.
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
249
Raj, P. A., Johnsson, M., Levine, M. J. e Nancollas, G. H. (1992). "Salivary
Statherin. Dependence on Sequence, Charge, Hydrogen Bonding Potency, and
Helical Conformation for Adsorption to Hydroxyapatite and Inhibition of
Mineralization." Journal of Biological Chemistry 267(9): 5968-76.
Ramassamy, C. (2006). "Emerging Role of Polyphenolic Compounds in the
Treatment of Neurodegenerative Diseases: A Review of Their Intracellular
Targets." European Journal of Pharmacology 545(1): 51-64.
Rasmussen, S. E., Frederiksen, H., Struntze Krogholm, K. e Poulsen, L. (2005).
"Dietary Proanthocyanidins: Occurrence, Dietary Intake, Bioavailability, and
Protection against Cardiovascular Disease." Molecular Nutrition and Food
Research 49(2): 159-174.
Rawel, H. M., Meidtner, K. e Kroll, J. (2005). "Binding of Selected Phenolic
Compounds to Proteins." Journal of Agricultural and Food Chemistry. 53(10):
4228-4235.
Renaud, S. e De Lorgeril, M. (1992). "Wine, Alcohol, Platelets, and the French
Paradox for Coronary Heart Disease." The Lancet 339(8808): 1523-1526.
Ricardo-Da-Silva, J. M., Cheynier, V., Souquet, J.-M., Moutounet, M., Cabanis,
J.-C. e Bourzeix, M. (1991). "Interaction of Grape Seed Procyanidins with Various
Proteins in Relation to Wine Fining." Journal of the Science of Food and
Agriculture 57(1): 111-125.
Richard, T., Vitrac, X., Merillon, J. M. e Monti, J. P. (2005). "Role of Peptide
Primary Sequence in Polyphenol-Protein Recognition: An Example with
Neurotensin." Biochimica et Biophysica Acta (BBA) - General Subjects 1726(3):
238-243.
Rio, D. D., Borges, G. e Crozier, A. (2010). "Berry Flavonoids and Phenolics:
Bioavailability and Evidence of Protective Effects." British Journal of Nutrition
104: S67-S90.
Robichaud, J. L. e Noble, A. C. (1990). "Astringency and Bitterness of Selected
Phenolics in Wine." Journal of the Science of Food and Agriculture 52: 343-353.
250 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Robinovitch, M. R., Ashley, R. L., Iversen, J. M., Vigoren, E. M., Oppenheim, F.
G. e Lamkin, M. (2001). "Parotid Salivary Basic Proline-Rich Proteins Inhibit HIV-
1 Infectivity." Oral Diseases 7(2): 86-93.
Roland, W. S. U., Vincken, J.-P., Gouka, R. J., Van Buren, L., Gruppen, H. e
Smit, G. (2011). "Soy Isoflavones and Other Isoflavonoids Activate the Human
Bitter Taste Receptors hTAS2R14 and hTAS2R39." Journal of Agricultural and
Food Chemistry 59(21): 11764-11771.
Rudnitskaya, A., Nieuwoudt, H., Muller, N., Legin, A., Du Toit, M. e Bauer, F.
(2010). "Instrumental Measurement of Bitter Taste in Red Wine Using an
Electronic Tongue." Analytical and Bioanalytical Chemistry 397(7): 3051-3060.
Sabatini, L. M., Carlock, L. R., Johnson, G. W. e Azen, E. A. (1987). "cDNA
Cloning and Chromosomal Localization (4q11-13) of a Gene for Statherin, a
Regulator of Calcium in Saliva." American Journal of Human Genetics 41(6):
1048–1060.
Santos-Buelga, C. e Scalbert, A. (2000). "Proanthocyanidins and Tannin-Like
Compounds - Nature, Occurrence, Dietary Intake and Effects on Nutrition and
Health." Journal of the Science of Food and Agriculture 80(7): 1094-1117.
Sarni-Manchado, P., Cheynier, V. e Moutounet, M. (1999). "Interactions of Grape
Seed Tannins with Salivary Proteins." Journal of Agricultural and Food Chemistry
47(1): 42-47.
Scalbert, A. e Williamson, G. (2000). "Dietary Intake and Bioavailability of
Polyphenols." Journal of Nutrition 130(8): 2073S-2085S.
Schlesinger, D. H., Hay, D. I. e Levine, M. J. (1989). "Complete Primary Structure
of Statherin, a Potent Inhibitor of Calcium Phosphate Precipitation, from the
Saliva of the Monkey, Macaca Arctoides." International Journal of Peptide and
Protein Research 34(5): 374-380.
Shi, P. e Zhang, J. (2009). Extraordinary Diversity of Chemosensory Receptor
Gene Repertoires among Vertebrates. Chemosensory Systems in Mammals,
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
251
Fishes, and Insects. S. Korsching and W. Meyerhof, Springer Berlin / Heidelberg.
47: 1-23.
Shim, J. S., Kang, M. H., Kim, Y. H., Roh, J. K., Roberts, C. e Lee, I. P. (1995).
"Chemopreventive Effect of Green Tea (Camellia Sinensis) among Cigarette
Smokers." Cancer Epidemiology Biomarkers & Prevention 4(4): 387-391.
Siebert, K. J., Troukhanova, N. V. e Lynn, P. Y. (1996). "Nature of Polyphenol-
Protein Interactions." Journal of Agricultural and Food Chemistry 44(1): 80-85.
Simon, C., Barathieu, K., Laguerre, M., Schmitter, J. M., Fouquet, E., Pianet, I. e
Dufourc, E. J. (2003). "Three-Dimensional Structure and Dynamics of Wine
Tannin-Saliva Protein Complexes. A Multitechnique Approach." Biochemistry
42(35): 10385-10395.
Singh, A., Holvoet, S. e Mercenier, A. (2011). "Dietary Polyphenols in the
Prevention and Treatment of Allergic Diseases." Clinical & Experimental Allergy
41(10): 1346-1359.
Soares, S., Mateus, N. e De Freitas, V. (2007). "Interaction of Different
Polyphenols with Bovine Serum Albumin (BSA) and Human Salivary a-Amylase
(HSA) by Fluorescence Quenching." Journal of Agricultural and Food Chemistry
55(16): 6726-6735.
Souquet, J. M., Cheynier, V., Brossaud, F. e Moutounet, M. (1996). "Polymeric
Proanthocyanidins from Grape Skins." Phytochemistry 43: 509-512.
Taguri, T., Tanaka, T. e Kouno, I. (2004). "Antimicrobial Activity of 10 Different
Plant Polyphenols against Bacteria Causing Food-Borne Disease." Biological &
Pharmaceutical Bulletin 27(12): 1965-1969.
Tang, H. R., Covington, A. D. e Hancock, R. A. (2003). "Structure-Activity
Relationships in the Hydrophobic Interactions of Polyphenols with Cellulose and
Collagen." Biopolymers 70(3): 403-413.
Ueda, T., Ugawa, S., Yamamura, H., Imaizumi, Y. e Shimada, S. (2003).
"Functional Interaction between T2R Taste Receptors and G-Protein Α Subunits
252 FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a capacidade de interação com proteínas da saliva e recetores do sabor
Expressed in Taste Receptor Cells." The Journal of Neuroscience 23(19): 7376-
738.
Vanderspek, J. C., Wyandt, H. E., Skare, J. C., Milunsky, A., Oppenheim, F. G. e
Troxler, R. F. (1989). "Localization of the Genes for Histatins to Human
Chromosome 4q13 and Tissue Distribution of the mRNAs." American Journal of
Human Genetics 45(3): 381–387.
Vergé, S., Richard, T., Moreau, S., Nurich, A., Merillon, J.-M., Vercauteren, J. e
Monti, J.-P. (2002). "First Observation of Solution Structures of Bradykinin-Penta-
O-Galloyl--Glucopyranose Complexes as Determined by Nmr and Simulated
Annealing." Biochimica et Biophysica Acta (BBA) - General Subjects 1571(2): 89-
101.
Vidal, S., Francis, L., Noble, A., Kwiatkowski, M., Cheynier, V. e Waters, E.
(2004). "Taste and Mouth-Feel Properties of Different Types of Tannin-Like
Polyphenolic Compounds and Anthocyanins in Wine." Analytical Chimical Acta
513(1): 57-65.
Visioli, F., De La Lastra, C. A., Andres-Lacueva, C., Aviram, M., Calhau, C.,
Cassano, A., D'archivio, M., Faria, A., Favé, G., Fogliano, V., Llorach, R.,
Vitaglione, P., Zoratti, M. e Edeas, M. (2011). "Polyphenols and Human Health: A
Prospectus." Critical Reviews in Food Science and Nutrition 51(6): 524-546.
Vitorino, R., Alves, R., Barros, A., Caseiro, A., Ferreira, R., Lobo, M. C., Bastos,
A., Duarte, J., Carvalho, D., Santos, L. L. e Amado, F. L. (2011). "Finding New
Posttranslational Modifications in Salivary Proline-Rich Proteins." PROTEOMICS
– Clinical Applications 5(3-4): 197-197.
Wakabayashi, K. (2000). "Changes in Cell Wall Polysaccharides During Fruit
Ripening." Journal of Plant Research 113(3): 231-237.
Wang, J., Ferruzzi, M. G., Ho, L., Blount, J., Janle, E. M., Gong, B., Pan, Y.,
Nagana Gowda, G. A., Raftery, D., Arrieta-Cruz, I., Sharma, V., Cooper, B.,
Lobo, J., Simon, J. E., Zhang, C., Cheng, A., Qian, X., Ono, K., Teplow, D. B.,
Pavlides, C., Dixon, R. A. e Pasinetti, G. M. (2012). "Brain-Targeted
FCUP Influência dos compostos polifenólicos no sabor dos alimentos: Relação entre a sua estrutura e a
capacidade de interação com proteínas da saliva e recetores do sabor
253
Proanthocyanidin Metabolites for Alzheimer's Disease Treatment." Journal of
Neuroscience 32(15): 5144-5150.
Williamson, M. P. (1994). "The Structure and Function of Proline-Rich Regions in
Proteins." Biochemical Journal 297: 249-260.
Williamson, M. P., Trevitt, C. e Noble, J. M. (1995). "NMR Studies of Dextran
Oligomer Interactions with Model Polyphenols." Carbohydrate Research 266(2):
229-235.
Wolfe, K. L., Kang, X., He, X., Dong, M., Zhang, Q. e Liu, R. H. (2008). "Cellular
Antioxidant Activity of Common Fruits." Journal of Agricultural and Food
Chemistry 56(18): 8418-8426.
Wroblewski, K., Muhandiram, R., Chakrabartty, A. e Bennick, A. (2001). "The
Molecular Interaction of Human Salivary Histatins with Polyphenolic
Compounds." European Journal of Biochemistry 268(16): 4384-4397.
Yan, Q. e Bennick, A. (1995). "Identification of Histatins as Tannin-Binding
Proteins in Human Saliva." Biochemical Journal 311: 341-347.