Selective enrichment of bioactive propertiesduring ultrafiltration of a tryptic digest ofβ-lactoglobulin

O. Power a,c, A. Fernández b, R. Norris a, F.A. Riera b, R.J. FitzGerald a,c,*a Department of Life Sciences, University of Limerick, Castletroy, Limerick, Irelandb Department of Chemical Engineering and Environmental Technology, University of Oviedo, Oviedo, Spainc Food for Health Ireland, University of Limerick, Castletroy, Limerick, Ireland

A B S T R A C T

Whey proteins are rich sources of bioactive peptides which may play a role in the dietary

management of chronic diseases. Fractionation via ultrafiltration (UF) was investigated for

the enrichment of antioxidant, dipeptidyl peptidase IV (DPP-IV) and angiotensin convert-

ing enzyme (ACE) inhibitory activity in a tryptic hydrolysate of β-lactoglobulin (β-Lg TH). UF

processing selectively enhanced the biofunctional properties of β-Lg TH with the perme-

ate obtained using a 1 kDa polyethersulfone (HFP-1) membrane having highest in vitro mul-

tifunctional bioactivity. Compared to β-Lg TH, the antioxidant activity was 1.7-fold higher

(46,765 ± 2504 vs. 77,251 ± 5124 µmol Trolox equivalent/100 g dw; P < 0.05), DPP-IV half maximal

inhibitory concentration (IC50) decreased threefold (1.6 ± 0.31 vs. 0.53 ± 0.05 mg/mL; P < 0.05)

and there was a twofold reduction in ACE IC50 (0.131 ± 0.005 vs. 0.068 ± 0.018 mg/mL; P < 0.05).

The multifunctional peptide VAGTWY, β-Lg f(15–20) was shown to have potent antioxidant

activity (5.63 µmol TE/µmol peptide). Two new ACE inhibitory peptides, IIAEK and IPAVFK,

having IC50 values of 63.7 ± 7.22 and 144.8 ± 25.3 μM, respectively, were also identified in β-Lg

TH. This study demonstrates the multifunctional bioactive properties of a β-Lg TH and its

associated UF fractions.

© 2014 Elsevier Ltd. All rights reserved.

A R T I C L E I N F O

Article history:

Received 15 October 2013

Received in revised form 31 March

2014

Accepted 2 April 2014

Available online

Keywords:

β-lactoglobulin

Trypsin

Enzyme hydrolysis

Membrane processing

Bioactive peptides

Dipeptidyl peptidase IV

Antioxidant

Angiotensin converting enzyme

1. Introduction

Whey protein is a valuable co-product of cheese manufac-ture, owing to its nutritional and technofunctional proper-ties. The biofunctional properties of whey proteins have beenextensively reviewed (Morris & FitzGerald, 2008; Riera,Fernández, & Muro, 2012). These biofunctional properties areattributed to peptides inactive within the whey protein se-quence, however, once released, by enzymatic hydrolysis, mi-crobial fermentation and physical/chemical processing, they

can have beneficial effects on the cardiovascular, gastrointes-tinal, immune and nervous systems.

β-Lactoglobulin (β-Lg), the predominant whey protein, is as-sociated with much of the bioactivity. β-Lg derived peptideshave been shown to have a wide variety of biofunctionalities(Hernández-Ledesma, Recio, & Amigo, 2008). Therefore, β-Lghydrolysates or peptides could be useful functional ingredi-ents for dietary management of increasingly prevalent dis-eases such as hypertension and type 2 diabetes (T2DM). Inparticular, peptides with multifunctionality could be a noveldisease risk reduction and preventative strategy. This study

* Corresponding author. Tel.: +353 61 202598; fax: +353 61 331490.E-mail address: dick.fitzgerald@ul.ie (R.J. FitzGerald).

http://dx.doi.org/10.1016/j.jff.2014.04.0021756-4646/© 2014 Elsevier Ltd. All rights reserved.

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focused on three complimentary bio-functionalities namely,angiotensin converting enzyme (ACE) inhibition, dipeptidyl pep-tidase IV (DPP-IV) inhibition and antioxidant activity whichcould be beneficial in the dietary management of hyperten-sion and T2DM.

ACE (EC 3.4.15.1) is a key enzyme in a number of blood pres-sure control systems including the renin–angiotensin system(RAS) and the kinin nitric oxide system (KNOS). In the RAS, ACEcatalyzes the formation of angiotensin II, a potent vasocon-strictor and in the KNOS ACE breaks down the potentvasodilatory peptide bradykinin (Murray & FitzGerald, 2007).Therefore, the inhibition of ACE can consequently lead to a de-crease in blood pressure. Food derived ACE inhibitory pep-tides from a range of sources have been reported includingcasein, whey, corn, wheat, fish, algae, ovalbumin, gelatin andsoya (for review see Murray & FitzGerald, 2007; Norris &FitzGerald, 2013 and references therein). ACE inhibitory pep-tides have gained interest as potential preventative agents forhypertension control and as potential active ingredients in func-tional foods. Currently, hypertension affects up to 30% of theworld’s adult population (Kearney et al., 2005). Given the preva-lence of hypertension and its role in the development ofcardiovascular diseases (CVD), identification of novel food-derived ACE inhibitors could provide an alternative to syn-thetic drug inhibitors. Hypertension and T2DM frequentlycoexist and hypertension is more prevalent in the diabetic popu-lation than the general population (U.K. Prospective DiabetesStudy, 1998). The incidence of T2DM is estimated to reach550 million by 2030 (World Health Organisation, 2012). There-fore, dietary strategies to tackle these co-morbidities could bebeneficial.

Novel strategies to manage T2DM have emerged includingDPP-IV inhibitors. DPP-IV (EC 3.4.14.5) is an ubiquitously ex-pressed aminodipeptidase. DPP-IV principally cleaves dipep-tides containing a penultimate proline or alanine residue fromthe N-terminal of polypeptides including the incretin hor-mones, glucose dependent insulinotropic polypeptide (GIP) andglucagon like peptide-1 (GLP-1) (Mentlein, 1999). Therefore,incretin based therapies that extend or enhance incretin half-life could be a useful strategy in the management of T2DM.DPP-IV inhibitors have been shown to increase circulating activeGLP-1 (Deacon & Holst, 2006), decrease plasma glucose con-centration and improve glucose tolerance in diabetes (Ahrénet al., 2002). Enzymatic hydrolysis has been used to release DPP-IV inhibitory peptides from a variety of food protein sourcesincluding milk proteins (Power, Nongonierma, Jakeman, &FitzGerald, 2013 and references therein) and β-Lg has been high-lighted as an important precursor of DPP-IV inhibitory pep-tides (Lacroix & Li-Chan, 2012b).

The by-products of oxidative metabolism such as reactiveoxygen species and free radicals are part of normal physiol-ogy. However, oxidative stress occurs when there is an excessproduction of free radicals which disturbs redox equilibrium.Oxidative stress has been implicated in the development ofchronic conditions such as CVD and T2DM (Rice-Evans &Diplock, 1993). Therefore, dietary antioxidants are an essen-tial defence to protect the body from oxidative damage. Theantioxidant activity of milk proteins/milk protein-hydrolysateshave been reported and in particular whey protein hydroly-sates have been shown to act as radical scavengers, reducing

agents and inhibitors of lipid peroxidation (Pihlanto, 2006; Power,Jakeman, & FitzGerald, 2013 and references therin). Theantioxidant activity of β-Lg hydrolysates was found to bedependent on the specificity of the enzyme used(Hernandez-Ledesma, Davalos, Bartolome, & Amigo, 2005).

Membrane technology which separates peptides based onsize, charge and hydrophobicity can be used to enrich bioactivepeptides from protein hydrolysates (Muro, Francisco, &Fernández, 2013). Ultrafiltration (UF) and nanofiltration (NF) arewidely used within the dairy industry for concentration andfractionation of milk proteins. UF, in particular, has been usedto increase the bioactivity of whey protein hydrolysates by en-riching for antioxidant (Salami et al., 2010), DPP-IV inhibitory(Lacroix & Li-Chan, 2012a; Nongonierma & FitzGerald, 2013b)and ACE inhibitory peptides (Mullally, Meisel, & FitzGerald,1997a; Pihlanto-Leppälä et al., 2000). However, there do notappear to be any studies on simultaneous enhancement of theantioxidant, DPP-IV and ACE inhibitory activity of β-Lg hydro-lysates using UF. Therefore, the objective of this study was toevaluate the effect of selective membrane fractionation on thebioactive properties of a tryptic hydrolysate of β-Lg (β-Lg TH).Peptide fractions were tested in vitro using ACE and DPP-IV in-hibition, and antioxidant assays to identify β-Lg fractions andassociated peptides with potential multifunctionality.

2. Materials and methods

2.1. Materials

Bovine β-Lg (Davisco Food Co., Minnesota, USA, total proteincontent 97.9%), bovine lung (Gaelic Meats and Livestock Ltd.,Limerick, Ireland), L-1-tosylamide-2-phenylethyl chloromethylketone treated trypsin (TPCK-Trypsin ≥10,000 BAEE units/mgprotein, T1426.1G, Sigma, USA), sodium phosphate monoba-sic (S71505), sodium phosphate dibasic (S7907), trifluoroaceticacid (TFA; T6508), fluorescein sodium salt (F6377), 2,2′-azobis-2-methyl-propanimidamide,dihydrochloride (AAPH, 44091-4), Tris(hydroxymethyl)aminomethane hydrochloride (Trizma®base, T1503), sodium chloride (NaCl; S7653), ethylenediaminetetra acetic acid disodium salt dihydrate (EDTA, E5134), DPP-IV (EC 3.4.14.5, human recombinant; D444943, 8 mU/mL),Trolox™ (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid; 23881-3) were all obtained from Sigma-Aldrich (Dublin,Ireland). 7-Amino-4-methylcoumarin (AMC) standard (Q-1025.0001), H-Gly-Pro-AMC (1-1225-0050) and Diprotin A (H-3825.0050) were obtained from Bachem (Bubendorf, Switzerland).The synthetic peptides (purity: >95%) ALK, IIAEK, ALPMHIR,VAGTWY, IPAVFK, TPEVDDEALEK, GLDIQK were obtained fromGenScript (New Jersey, USA). Sodium hydroxide (NaOH), hy-drochloric acid (HCl), high performance liquid chromatogra-phy (HPLC) grade acetonitrile (ACN) and HPLC grade water wereobtained from VWR (Dublin, Ireland).

2.2. Preparation of a tryptic hydrolysate of β-Lg

A commercial (Davisco) bovine β-Lg substrate was used as asubstrate for tryptic hydrolysate preparation as previously de-scribed by Fernández and Riera (2012) with some modifica-

39j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 3 8 – 4 7

tions. Briefly, a 2.5 L aqueous solution containing 15 g/L of β-Lgwas equilibrated at 37 °C in a batch reaction and adjusted topH 8.0 with 1 M NaOH before the addition of the enzyme. Hy-drolysis was carried out at 37 °C and pH 8.0 at a final enzymeto substrate ratio (E:S) of 1:250 (w/w). The β-Lg TH was ob-tained after 24 h hydrolysis. The degree of hydrolysis (DH) wasdetermined from the quantity of base consumed according tothe pH-stat method (Adler-Nissen, 1986).

2.3. Permeation experiments

The β-Lg TH was filtered using a Pellicon 2 mini holder(Millipore, USA) equipped with three different UF tangentialflow membrane cassettes: two polyethersulfone (PES) mem-branes having molecular weight cut offs (MWCO) of 1 kDa (Sar-torius; Goettingen, Germany) and 5 kDa (Millipore; MA, USA)and a stabilized cellulose membrane with a MWCO of 2 kDa(Hydrostart, Sartorius). Each membrane has a filtration area of0.1 m2.The fractionation experiments were carried out withoutsalt addition and at pH 8.0. The pH (Fernández, Suárez, Zhu,FitzGerald, & Riera, 2013), ionic strength (Fernández & Riera,2013b) and concentration (Fernández & Riera, 2012) had beenpreviously optimized in order to obtain permeates enriched inbioactive peptides. Transmembrane pressure and the tempera-ture of the hydrolysate were set to 7.5 × 104 Pa and 37 °C, re-spectively. Fractions denoted HFP-1, HFP-5 and HFP-2 correspondto the permeate streams obtained using the 1 kDa PES, 5 kDaPES and 2 kDa Hydrosart membranes, respectively. Finally, thediafiltration retentate (DR) fraction was composed of the pep-tides rejected by the 5 kDa PES membrane after a continuousdiafiltration process. Distilled water was added to the hydro-lysate during the diafiltration process at the same rate as thepermeate was generated until a diafiltration volume of 7.5 Lwas obtained. Samples were freeze dried and stored at −20 °Cuntil further analysis.

2.4. Chromatographic analysis and peptide quantification

All the freeze-dried samples were analyzed by reverse phasehigh performance liquid chromatography (RP-HPLC) and thepeptides present in the chromatographic profile were identi-fied by mass spectrometry as previously described (Fernández& Riera, 2013a).

2.5. Oxygen radical absorbance capacity (ORAC) assay

Antioxidant capacity was evaluated using the fluorescencebased ORAC assay as per the methodology of Harnedy andFitzGerald (2013) with some modifications. The assay was per-formed in a 96 well microplate (Fisher Scientific, Dublin, Ireland).A Trolox standard curve was generated by assaying Trolox stan-dards at concentrations between 10 and 200 μM. Test samples,blank (assay buffer) and Trolox standards were dissolved in75 mM sodium phosphate buffer, pH 7.0 and were added (50 μL)to the appropriate wells and pre-incubated with 50 μL of0.312 µM fluorescein (final concentration) at 37 °C for 10 minin a microplate reader (Biotek Synergy HT,Winooski, USA). Base-line fluorescence was measured at excitation (485 nm) andemission (520 nm) wavelengths after 1 min. The reaction wasinitiated by addition of 25 μL of 44.2 mM AAPH (final concen-

tration) to each well. The microplate was incubated at 37 °Cfor 120 min during which fluorescence was measured every5 min. For each sample, the reaction was deemed to be com-plete if final fluorescence intensity (FIn) was less than 5% ofinitial fluorescence (FI0). Final results were presented as µmolTE per 100 g of dry weight (µmol TE/100 g dw). All data are pre-sented as the mean ± SD of independent triplicate analyses(n = 3).

2.6. DPP-IV inhibition assay

DPP-IV inhibitory activity was evaluated using a fluorescencebased assay as per the methodology of Harnedy and FitzGerald(2013). The assay was performed in a 96 well microplate (FisherScientific, Dublin, Ireland). An AMC standard curve was gen-erated by assaying 100 μL of AMC standards (1–8 μM). DiprotinA was employed as a reference inhibitor (positive control). TheDPP-IV half maximal inhibitory concentration (IC50) value ofDiprotin A was calculated by assaying at concentrationsbetween 1.25 and 100.00 µM.

Test hydrolysates or positive controls were added (10 μL) toeach well and pre-incubated with 30 μL of 20 mM Tris-HCl buffer,pH 8.0, containing 100 mM NaCl and 1 mM EDTA and 50 μL200 μM H-Gly-Pro-AMC at 37 °C for 5 min in a microplate reader(Biotek Synergy HT, Winooski, USA). The reaction was initi-ated by addition of 10 μL of human DPP-IV (8 mU/mL) to thewells. The microplate was incubated at 37 °C for 30 min afterwhich fluorescence was measured at an excitation of 360 nmand an emission of 460 nm. One unit of DPP-IV activity (U) wasdefined as the amount of enzyme which hydrolyses 1 μmoLof H-Gly-Pro-AMC per minute at 37 °C. The DPP-IV IC50 value,the concentration of peptide required to inhibit 50% of theenzyme activity, for each hydrolysate was determined by plot-ting DPP-IV inhibition as a function of hydrolysate concentra-tion. The logarithmic regression equation generated from thisplot was then used to calculate the IC50 value. DPP-IV inhibi-tion (%) and IC50 values for each hydrolysate were expressedas the mean ± SD of independent triplicate analyses.

2.7. ACE inhibition activity assay and extraction of ACEfrom bovine lung

ACE inhibitory activity was determined using the fluoromet-ric microtiter assay of Sentandreu and Toldra (2006) with somemodifications as described in Norris, Casey, FitzGerald, Shields,and Mooney (2012). Bovine lung ACE was used in place of ACEextracted from rabbit lung acetone powder. It was extractedfrom fresh bovine lung by a modification of the method byMeng, Balcells, Dell’Italia, Durand, and Oparil (1995). Firstly, thelung was minced using a Breville VTP141 meat grinder (Breville,Oldham, UK). Following this, the minced lung (40 g) was ho-mogenized in ACE buffer (200 mL; 100 mM sodium borate buffercontaining 300 mM NaCl, pH 8.3) at 4 °C using an ultraturrax(IKA Labortechnik, Staufen, Germany) at 24,000 rpm for 1 × 30and 2 × 10 s intervals. The homogenate was then centrifuged(Sorvall® RC 5C plus, Fisher Scientific, Dublin, Ireland) at 15,000 gfor 15 min at 4 °C and the pellet, containing the ACE activity,was washed twice with ACE buffer. The pellet was thenrehomogenized with an ultraturrax as earlier in 5 mM aqueousCHAPS solution (100 ml, 40 % w/v) at 4 °C. The solution was

40 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 3 8 – 4 7

left to stir gently overnight at 4 °C. It was then centrifuged at15,000 g for 15 min at 4 °C. The supernatant was retained andlyophilized and the dry sample was stored at −20 °C. For in-hibition studies, an ACE activity of 10 mU/mL was used foreach determination by diluting the lung extract appropri-ately with assay buffer (100 mM sodium borate buffer, 300 mMNaCl, pH 8.3).

Hydrolysates were assayed at final concentrations of 0.001,0.005, 0.010, 0.050, 0.100, 0.500, 1.000 and 10.000 mg/mL. Thesynthetic peptides were assayed at concentrations ranging from0.01, 0.05, 0.10, 0.50, 1.00, 10.00, 50.00, 100.00, 250.00, 500.00 and800.00 μM during determination of ACE IC50 values. IC50 valueswere calculated using GraphPad® Prism 4.0 from sigmoidal doseresponse plots of inhibitor concentration (µM) vs. % inhibi-tion. The values were expressed as the mean IC50 ± SD of in-dividual duplicates, assayed in triplicate.

2.8. Statistical analysis

All analyses were performed in triplicate and presented as themean ± SD. Data were tested for normality (Shapiro–Wilk) andevaluated by analysis of variance (ANOVA; one-way) followedby Tukey’s test and a significance level of P < 0.05 was em-ployed. All analysis was performed using SPSS (SPSS, version19, IBM Inc., Armonk, USA).

3. Results and discussion

3.1. Characterization and peptidic composition of theβ-Lg hydrolysate and fractions

Intact β-Lg was hydrolyzed with TPCK- trypsin for 24 h. TheDH of the β-Lg TH was 8.09% after 24 h of hydrolysis. The theo-retical DH was estimated to be 10%, based on the theoreticalcleavage sites for trypsin within the β-Lg amino acid se-quence. The lower DH achieved may be attributed to the rela-tively low E:S ratio employed herein and the globular natureof whey protein which may have limited enzymatic accessi-bility to peptide bonds (Peng, Xiong, & Kong, 2009).

The chromatographic profiles of the intact β-Lg substrate,β-Lg TH and the associated fractions are shown in Figs. 1a–f.As observed in Figs. 1a and b, a significant degradation of intactβ-Lg, along with peptide formation was observed during hy-drolysis. The peptides present in the hydrolysate were iden-tified by MS as previously reported (Fernández et al., 2013;Rodríguez-Carrio, Fernández, Riera, & Suárez, 2013). The peaknumbers used on Fig. 1b refer to the peptides screened for bio-activity in this work. As it can be observed from the peptideprofiles of the different samples, many of the larger and hy-drophobic peptides (last peaks in the chromatograms, Figs. 1c–e)were rejected by the HFP-1, HFP-2 and HFP-5 membranes whilepeptides having a molecular weight lower than 1 kDa (elutiontime lower than 30 min) were able to permeate these mem-branes (Figs. 1c–e). This was also reflected in the compositionof the DR sample which showed an almost complete absenceof the smaller peptides (peaks 1–3, Fig. 1f).

Processing through different UF membranes resulted in thegeneration of β-Lg fractions containing peptides with differ-

ent physicochemical properties (Fernández & Riera, 2013a;Fernández et al., 2013; Fernández, Zhu, FitzGerald, & Riera, 2013).These properties were dependent on the peptides present inthe hydrolysate, the characteristics of the different mem-branes used and interaction between these parameters. There-fore, it was postulated that these differences in the separationprocess could influence the bioactive properties of the differ-ent β-Lg fractions obtained.

3.2. DPP-IV inhibitory activity

Whey protein derived peptides have been shown to act as DPP-IV inhibitors (Nongonierma & FitzGerald, 2013a; Silveira,Martínez-Maqueda, Recio, & Hernández-Ledesma, 2013;Tulipano, Sibilia, Caroli, & Cocchi, 2011; Uchida, Ohshiba, &Mogami, 2011). In addition, some peptides may also be sub-strates for DPP-IV (Nongonierma & FitzGerald, 2014; Rahfeld,Schierborn, Hartrodt, Neubert, & Heins, 1991). Peptides re-leased during β-Lg hydrolysis could increase the half-life ofactive incretins by acting as true inhibitors or as substrates ofthis enzyme.

The bioactivity data reported herein were expressed per unitweight of freeze dried sample, an approach which is fre-quently employed when dealing with protein hydrolysates andtheir fractions (Senphan & Benjakul, 2014). While it is pos-sible to quantify the nitrogen content of these samples thereis no appropriate Kjeldahl conversion factor for such a complexmixture of proteins, peptides and amino acids. In addition, de-termination of protein content using dye binding based assayssuch as the Lowry assay is sensitive to variations in the aminoacid composition of proteins. In particular, amino acids withoxidizable side chains (tyrosine, tryptophan, cysteine) in-crease color yield, whereas other amino acids such as prolineand hydroxy-proline have been shown to reduce the stabilityof the complex and reduce color formation (Olson & Markwell,2007). Therefore, the presence of these amino acids or pep-tides containing these amino acids at the end terminal mayyield incorrect data and the differences could be accentuatedon fractionation. While the authors acknowledge the limita-tions of the current approach, expression of bioactivity per unitweight of freeze-dried fraction, in this instance, was deemedthe most appropriate format in which to report the results.

The DPP-IV inhibitory activity, tested at a concentration of2 mg/mL, of β-Lg TH and associated fractions (DR, HFP-5, HFP-2, HFP-1) were compared (Table 1). The DR fraction had no DPP-IV inhibitory activity. Compared to β-Lg TH, there was asignificant increase in percentage DPP-IV inhibition for all UFpermeates (HFP-5, HFP-2, HFP-1; Table 1).The DPP-IV IC50 valuesfor the β-Lg TH, HFP-5 and HFP-1 samples ranged from 1.6 ± 0.31to 0.53 ± 0.05 mg/mL (Table 1). UF fractionation gave approxi-mately threefold reduction in DPP-IV IC50 compared to β-Lg TH.However, there was no significant difference, in the DPP-IV in-hibitory potency between the HFP-5 and HFP-1 permeate frac-tions (P > 0.05, Table 1). Interestingly, peak 6 corresponding tothe most potent DPP-IV inhibitory peptide (Table 2), VAGTWY,was the predominant peptide peak on the chromatogram forHFP-1 and HFP-5 (Fig. 1). On a weight basis these fractions wereapproximately 300 times less potent than Diprotin A which hadan IC50 of 0.002 mg/mL.

41j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 3 8 – 4 7

The DPP-IV inhibitory activity of food protein hydroly-sates is largely dependent on the specificity of the enzyme used.Tryptic digestion of β-Lg or a whey protein concentrate en-riched in β-Lg has been shown to release DPP-IV inhibitory pep-tides (Silveira et al., 2013; Uchida et al., 2011). The IC50 values

reported herein for β-Lg TH compare well with previously pub-lished IC50 values (0.075–1.51 mg/mL) for whey protein derivedhydrolysates (Lacroix & Li-Chan, 2012a; Nongonierma &FitzGerald, 2013b) and (0.4–26.4 mg/mL) hydrolysates of otherfood proteins (Aart, Catharina, Zeeland-Wolbers, Maria, & Gilst,

Fig. 1 – Reverse phase high performance liquid chromatographic (RP-HPLC) profiles of the intact β-lactoglobulin (β-lg)substrate (a), β-lg total hydrolysate (β-lg TH, b) and associated fractions obtained after ultrafiltration (UF) experiments (c)polyethersulfone 1 kDa permeate (HFP-1), (d) stabilized cellulose 2 kDa permeate (HFP-2), (e) polyethersulfone 5 kDapermeate (HFP-5), (f) diafiltration retentate (DR). 1b peak number 1 corresponds to peptide ALK f(139–141); peak 2: IIAEKf(71–75); peak 3: GLDIQK f(9–14); peak 4: TPEVDDEALEK f(125–135); peak 5: ALPMHIR f(142–148); peak 6: VAGTWY f(15–20);peak 7: IPAVFK f(79–83).

42 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 3 8 – 4 7

2009; Hatanaka et al., 2012). Size fractionation by UF has pre-viously been shown to enrich the DPPI-IV inhibitory activityof whey protein hydrolysates (Lacroix & Li-Chan, 2012a;Nongonierma & FitzGerald, 2013b). The data reported hereinappear to be the first report showing that UF fractionation wasan appropriate means to enrich the DPP-IV inhibitory activ-ity of a β-Lg TH (Table 1). The decrease in DPP-IV IC50 valuesfor both the HFP-5 and HFP-1 indicates that low molecular masspeptides were primarily responsible for the DPP-IV inhibitoryactivity (Table 1). This is in agreement with other studies re-porting that DPP-IV inhibitory peptides tend to contain two toeight amino acid residues and have a molecular mass less than1 kDa (Lacroix & Li-Chan, 2012b).

3.3. ACE inhibition

The ACE IC50 values of β-Lg TH and the associated UF frac-tions are also shown in Table 1. The ACE IC50 obtained for the

β-Lg TH was 0.131 ± 0.005 mg/mL. There was no difference inthe ACE IC50 values for the DR, HFP-2, HFP-5 fractions com-pared to β-Lg TH (P > 0.05). However, the HFP-1 permeate hada 48% lower (P < 0.05) ACE IC50 than β-Lg TH (0.0682 ± 0.018 vs.0.131 ± 0.005 mg/mL, respectively).Thus, fractionation by UF se-lectively enhanced the ACE inhibitory activity of the β-Lg TH.Many studies have reported an increase in ACE inhibitorypotency of hydrolysates from a range of sources after frac-tionation using UF membranes (Mullally, Meisel, & FitzGerald,1997b). Low molecular weight peptides within protein hydro-lysates are generally responsible for more potent ACE inhibi-tion, regardless of the origin of the protein substrate. Inagreement with this, the β-Lg TH fraction enriched in low mo-lecular mass peptides (HFP-1) had the lowest ACE IC50 (Table 1).Additionally, the chromatogram for HFP-1 (Fig. 1c) shows agreater peak area for peak 5, the most potent ACE inhibitorypeptide (ALPMHIR, Table 2), than is present in the chromato-gram for HFP-5 (Fig. 1e). Mullally et al. (1997a) obtained ACE

Table 1 – Oxygen radical absorbance capacity (ORAC) values, dipeptidyl peptidase-IV (DPP-IV) inhibition at aconcentration of 2 mg/mL, DPP-IV half maximal inhibitory concentration (IC50) and angiotensin converting enzyme (ACE)IC50 of a β-lactoglobulin tryptic hydrolysate (β-lg TH) and associated ultrafiltered samples. Diafiltration retentate (DR),polyethersulfone 5 kDa permeate (HFP-5), stabilized cellulose 2 kDa permeate (HFP-2), polyethersulfone 1 kDa permeate(HFP-1). Values represent the mean ± SD, n = 3. Values with different superscript letters indicate significant differences(P < 0.05).

Sample DPP-IV inhibition(%)*

DPP-IV IC50

(mg/mL)ACE IC50

(mg/mL)ORAC (µmol TE/100 g dw)

β-lg TH 52 ± 4.8a 1.6 ± 0.31a 0.131 ± 0.005a 46765 ± 2504a

DR 0 ± 6.2b ND >10 49035 ± 3537a

HFP-5 82 ± 3.5c 0.58 ± 0.03b 0.120 ± 0.026a,b 75976 ± 2817b

HFP-2 76 ± 6.4c ND 0.140 ± 0.027a 55213 ± 507a

HFP-1 83 ± 0.8c 0.53 ± 0.05b 0.068 ± 0.018b 77251 ± 5124b

ND, not determined; TE, Trolox equivalents.* Dipeptidyl peptidase-IV inhibition at a concentration of 2 mg/mL.

Table 2 – Oxygen radical absorbance capacity (ORAC) values, dipeptidyl peptidase-IV (DPP-IV) half maximal inhibitoryconcentration (IC50) and angiotensin-converting-enzyme (ACE) inhibition IC50 of peptides identified in a tryptic digest ofβ-lactoglobulin. Values represent the mean ± SD, n = 3. Within each column values with different letters superscriptindicate significant differences (P < 0.05).

Peptide Peak number1 Sequence ORAC (μmol TE/µmol peptide)

DPP-IV IC50 (μM) ACE inhibitionIC50 (μM)

ALK 1 β-lg f(139–141) 0.010 ± 0.000a >10003 >10003

IIAEK 2 β-lg f(71–75) 0.016 ± 0.000a >10003 63.7 ± 7.2GLDIQK 3 β-lg f(9–14) 0.018 ± 0.001a >10003 5807

TPEVDDEALEK 4 β-lg f(125–135) 0.004 ± 0.001a 578.7 ± 33.0a,5 >10003

ALPMHIR 5 β-lg f(142–148) 0.035 ± 0.006a >10003 42.67

VAGTWY 6 β-lg f(15–20) 5.63 ± 0.947b 74.9 ± 4.4b,4 16828

IPAVFK 7 β-lg f(78–83) 0.002 ± 0.000a 149.1 ± 17.0c,5 144.8 ± 25.3IPI (Diprotin A) κ-CNf(47–49) NA 4.9 ± 0.2d,6 NACaptopril NA NA 9.4 ± 5.9 nMW 3.61 ± 0.0890c,2 NA NA

NA, not applicable.1 Peak number identified on the reverse phase high performance liquid chromatographic (RP-HPLC) profiles in Fig. 1.2 μmol Trolox equivalents (TE)/µmol amino acid.3 No inhibition was observed at highest concentration used in the study 1000 μM.4 IC50 174 μM. Uchida et al. (2011).5 IC50 319, IC50 143 μM. Silveira et al. (2013).6 0.002 ± 0.000 mg/mL.7 Pihlanto-Leppälä et al. (1998).8 Mullally et al. (1997b).

43j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 3 8 – 4 7

IC50 values of 130.0 and 160.4 mg/L after fractionation of a β-Lgtryptic digest with 3 and 1 kDa UF membranes, respectively.ACE IC50 values of 457, 685 and 237 μg/mL for a crude β-Lg trypticdigest, the associated 30 kDa permeate and the associated 1 kDapermeate, respectively, have been previously reported(Pihlanto-Leppälä et al., 2000).The differences in ACE IC50 valuesreported for trypic digests of β-Lg and subsequent fractions invarious studies may be explained by the different experimen-tal conditions employed (such as hydrolysis time, E:S ratio andtemperature).

3.4. Oxygen radical absorbance capacity assay

The link between oxidative stress and diabetic cardiovascu-lar complications is widely reported (Jay, Hitomi, & Griendling,2006). Therefore, hydrolysates with antioxidant activity in ad-dition to DPP-IV and ACE inhibition may find applications inthe dietary management of T2DM and CVD. In this study, thein vitro antioxidant activity was measured using the ORAC assaywhich measures the ability of an antioxidant to scavenge aperoxyl radical. The ORAC values for β-Lg TH and its associ-ated UF fractions are shown in Table 1. Enzymatic specificityinfluences the antioxidant activity of milk protein hydroly-sates. A variety of different proteinases have been used to gen-erate enzyme hydrolysates of whey protein isolate, β-Lg enrichedwhey protein concentrate and isolated β-Lg with ORAC valuesbetween 70 and 300,000 µmol TE/100 g protein (Adjonu, Doran,Torley, & Agboola, 2013; Hernandez-Ledesma et al., 2005). Theantioxidant activity of β-Lg TH was 46765 ± 2504 µmol TE/100 g dw. This value is lower than that previously reported fora tryptic digest of β-Lg (Hernandez-Ledesma et al., 2005) andthis may be explained by the 12.5-fold lower E:S ratio appliedherein in the generation of β-Lg TH. Peptide size influences theantioxidant activity of protein hydrolysates and size fraction-ation by UF has previously been shown as an effective strat-egy to enrich the antioxidant activity of a milk proteinhydrolysate (Salami et al., 2010). However, to our knowledge,there are no studies that have used UF to enhance the anti-oxidant activity of β-Lg hydrolysates. The data reported hereindemonstrate that size-based fractionation by UF was an ap-propriate means to selectively enhance the antioxidant activ-ity of β-Lg TH. Each UF fraction had antioxidant activity and,in addition, membrane processing modified the magnitude ofthe antioxidant response (Table 1). Compared to β-Lg TH, therewas no change in the antioxidant activity of the DR and HFP-2fractions (P > 0.05, Table 1). However, the HFP-5 and HFP-1 frac-tions had significantly higher antioxidant activity, 75976 ± 2817and 77251 ± 5124 µmol TE/100 g dw, which are 62.5 and 65.2%higher (P < 0.05) than the β-Lg TH, respectively. To our knowl-edge, this is the first study to demonstrate the selective en-hancement of the antioxidant activity of a β-Lg TH followingUF fractionation through membranes having defined MWCOs.

3.5. Bioactivity of selected synthetic β-Lg peptides

LC-MS analysis previously identified 21 peptides in β-Lg TH(Fernández & Riera, 2013a). Three of these tryptic peptides(IPAVFK, VAGTWY, TPEVDDEALEK) were previously reported asDPP-IV inhibitors (Silveira et al., 2013; Uchida et al., 2011), threepeptides (ALPMHIR, GLDIQK, VAGTWY) were ACE inhibitors

(Mullally et al., 1997b; Pihlanto-Leppälä, Rokka, & Korhonen,1998), one peptide (TPEVDDEALEK) was predicted to haveexcellent gastrointestinal stability (Picariello et al., 2010),another peptide (IIAEK) was previously found to havehypocholesterolemic activity (Nagaoka et al., 2001) and ALK,the most hydrophobic tripeptide identified, had a C terminalK residue that was likely to contribute to ACE inhibition. Theseseven peptides contain 3–11 amino acid residues and were se-lected for further investigation in order to characterize thosepeptides potentially responsible for the multifunctional bioactiveproperties of the β-Lg TH (Table 2).

A large number of potent ACE inhibitory peptides have beengenerated by tryptic digestion of milk proteins. Common fea-tures among potent ACE inhibitory peptides suggest they areshort peptides (2–10 amino acids in length) and contain aro-matic or hydrophobic residues at their C-terminus ((López-Fandiño, Otte, & van Camp, 2006). In agreement, the two mostpotent β-Lg derived ACE inhibitory peptides were IPAVFK(144.8 ± 25.3 µM) and IIAEK (63.7 ± 7.22 µM) which contain lessthan six amino acid residues. In contrast, the longer peptideTPEVDDEALEK showed very little ACE inhibitory activity(IC50 => 1000 μM). C-terminal K and R residues are also commonfeatures among many potent ACE inhibitory peptides. Previ-ously reported β-Lg derived ACE inhibitory peptides have IC50

values in the range from 8 to >1000 μM (Tavares & Malcata,2013). Thus, the two newly identified ACE inhibitory peptidesIPAVFK and IIAEK are among the more potent β-Lg derived ACEinhibitory peptides reported to date.

None of the β-Lg derived peptides evaluated have been pre-viously reported as having antioxidant activity and while eachpeptide had antioxidant activity there were large differencesin the magnitude of the ORAC response (Table 2). The mostpotent peptide was β-Lg f(15–20), VAGTWY, having an ORACscore of 5.63 ± 0.9474 μmol TE/µmol peptide.This peptide is morepotent that other β-Lg derived peptides (β-Lg f(19–29), f(145–149), f(42–46) f(58–61), f(95–101)) previously reported to haveORAC scores between 0.3 and 2.5 μmol TE/µmol peptide(Hernandez-Ledesma et al., 2005). Interestingly, when com-pared on a weight per weight basis, this peptide had higherantioxidant activity than the synthetic antioxidant butylatedhydroxylanisole (BHA) i.e., 5.63 µmol Trolox/µmol peptide vs.2.43 µmol Trolox/µmol BHA (Hernandez-Ledesma et al., 2005).The structural characteristics of VAGTWY may contribute tothe antioxidant activity observed. Hydrophobic amino acids con-tribute to the antioxidant activity in particular when they arelocated at the N terminal of a peptide (Li, Li, He, & Qian, 2011).Therefore, the V residue at the N terminal of β-Lg f(15–20) maycontribute to the potency of this peptide.The C terminal aminoacid is another predictor of antioxidant activity (Li et al., 2011)and the amino acids W, E, L, I, M,V and Y are frequently presentat the C terminus of antioxidant peptides. Therefore, the Yresidue at the C terminal may have increased the antioxi-dant activity. Individual amino acids also have antioxidant ac-tivity in the ORAC assay and Trp was found to be the mostpotent (Hernandez-Ledesma et al., 2005). In this study, W wasemployed as a positive control and when evaluated on a weightbasis the hexapeptide VAGTWY was 1.5-fold more potent thanW (Table 2).

The DPP-IV IC50 of Diprotin A and the synthetic peptides arealso shown in Table 2. The IC50 value obtained for Diprotin A

44 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 3 8 – 4 7

was 4.9 ± 0.2 μM and this is within the range previously re-ported in the literature, i.e., 3.5–210.0 μM (Hatanaka et al., 2012;Tulipano et al., 2011). Peptide amino acid sequence (Hatanakaet al., 2012) and structure (Nongonierma & FitzGerald, 2013a)have all been shown to contribute to DPP-IV inhibitory activ-ity of a peptide. Of the seven peptides analyzed, three pep-tides (VAGTWY, IPAVFK, TPEVDDEALEK) had potent DPP-IV IC50

values between 74.9 and 578.9 μM and these were in line withthe IC50 values previously reported in the literature for thesepeptides (Silveira et al., 2013; Uchida et al., 2011).The other fourpeptides (GLDIQK, IIAEK, ALK, ALPMHIR) evaluated had DPP-IV IC50 values greater than 1000 μM and only ALPMHIR has beenpreviously evaluated for DPP-IV inhibition. Trypsin is anendoproteinase with specificity for cleavage after R and K resi-dues. Five peptides studied had a C terminal K residue and threehad no inhibitory activity (Table 2). It has been previously sug-gested that the presence of a K residue at the C terminal mayinfluence DPP-IV inhibition by reducing the hydrophobic prop-erties of the peptide. The hexapeptide, IPAVFK, had an IC50 of149 μM (Table 2). However, the truncated peptides, IPA and IPAVFhave IC50 values of 49 and 44 μM, respectively (Silveira et al.,2013; Tulipano et al., 2011). The addition of the K residue atthe C terminal resulted in a threefold increase in the DPP-IVIC50. Similarly, the dipeptide AL has been shown to have an IC50

of 880 μM (Nongonierma & FitzGerald, 2013a). However, the tri-peptide ALK had an IC50 > 1000 μM. The most potent DPP-IV in-hibitory peptide evaluated herein was VAGTWY. Notably thispeptide contains a C-terminal Y residue which highlights thenon-specific cleavage by the trypsin preparation. It has beenpreviously suggested that trypsin can have secondary speci-ficity. This activity is modulated by five or six residues sur-rounding the cleavage site. Aliphatic, aromatic and basicresidues are preferred at the carboxy side of the peptides whichmay explain the presence of the Y residue in this peptide. In-terestingly, this peptide has previously been identified as theactive peptide in a β-Lg tryptic hydrolysate which had a gluco-regulatory effect in mice (Uchida et al., 2011).

This analysis of synthetic peptides demonstrates that thebioactivities reported here are peptide specific, dependent onthe amino acid sequence and structure of the peptide. Therelationship between the bioactivity of the peptides studiedhere (Table 2) and the relative abundance of these peptideswithin each fraction (Fig. 1) was evaluated. However, no clearrelationship was established between peptide composition ofthe permeate fractions and the bioactive properties reportedhere (data not shown). This suggests that the bioactive prop-erties of β-Lg TH reported here may be due to the combinedaction of multiple peptides. VAGTWY was the only peptidefound to have ACE inhibition, DPP-IV inhibition and antioxi-dant activities (Table 2). This is the first report of the potentantioxidant activity for this peptide and the previously re-ported DPP-IV and ACE inhibitory activity has been con-firmed. Interestingly, an antimicrobial activity has also beenreported for this peptide (Pellegrini, Dettling, Thomas, &Hunziker, 2001). In addition, our analysis has shown that IIAEKis an ACE inhibitor (Table 2) and this peptide has previouslybeen shown to have hypocholestrolemic activity (Nagaoka et al.,2001).

Understanding more about the structure activity relation-ship of bioactive peptides may assist in identifying novel β-Lg

derived multifunctional peptides in the future. The in vivo sta-bility and bioavailability of the β-Lg derived peptides presentin these fractions remain to be established. While some studieshave previously shown bioactive effects of β-Lg derived pep-tides in vivo, in mice, further studies are required to demon-strate the efficacy of these β-Lg fractions in humans.

4. Conclusions

This is the first report of the multifunctional (DPP-IV, ACE andantioxidant) potential of a β-Lg TH and the selective enhance-ment of these bioactivities using UF.The hexapeptide,VAGTWY,was found to have all three bioactivities and this is, to ourknowledge, the first report of the potent antioxidant activityof this peptide. Two new potent ACE inhibitors, IPAVFK, IIAEKhave also been identified in β-Lg TH. Further work is requiredto confirm if these in vitro bioactivities of the β-Lg fractionstranslate in vivo. Ultimately, β-Lg derived peptides may formpart of a functional food based dietary strategy for the man-agement of CVD and T2DM.

Acknowledgments

Work described herein was supported by Enterprise Irelandunder grant number CC20080001 by the Irish Research Council(IRC) in the form of a studentship to author R. Norris. A. Fer-nandez acknowledges the award of a PhD fellowship from theSevero Ochoa Programme (Principado de Asturias Govern-ment). R.J. FitzGerald acknowledges the financial support of theHigher Education Authority under the Programme for Re-search in Third Level Institutions (cycle 4) as part of the Na-tional Development Plan 2007–2013.

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