Determinación de Los Fenoles Totales en Jugos y Superfruits

8/10/2019 Determinacin de Los Fenoles Totales en Jugos y Superfruits

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Determination of the total phenolics in juices and superfruits by a novel chemical method 5

Marjorie B. Medina*

Residue Chemistry and Predictive Microbiology Research Unit, Eastern Regional Research Center, Agricultural Research Service,US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA

A R T I C L E I N F O

Article history:Received 21 December 2010Received in revised form23 February 2011Accepted 28 February 2011Available online 29 March 2011

Keywords:Total phenolsFast Blue BB diazonium dyeFolinCiocalteuSuperfoods and superfruits

Juices

A B S T R A C T

The total phenols in foods and beverages are currently measured with FolinCiocalteu,through its reducing capacity. A novel method was developed to quantify polyphenols orphenolic compounds through direct interactions of polyphenols with Fast Blue BB in analkaline medium and the absorbance was measured at 420 nm. The gallic acid calibrationlines (0500 l g/mL) had a linear correlation ( R) greater than 0.99 in all analysis. The limit of detection was


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contributed by non-phenolic antioxidants and reducing sub-stances (ascorbic acid, glucose, fructose, sulphites) that arecommon food additives or are naturally present in juices,fruits and vegetables. Amino acids (tyrosine, tryptophan)and proteins containing these amino acids also formed a bluecolor with the FolinCiocalteu reagent ( Peterson, 1979, 1983 ).

There is a need for a simple, rapid and direct detection of phenolics or polyphenols in foods, beverages, and agriculturalby-products. The novel total phenolics method utilizing Fast

Blue BB diazonium salt is based on the coupling of phenoliccompounds with the diazonium salt resulting in the forma-tion of azo complexes. Aromatic diazonium ions normallycouple with active substrates such as phenols ( Morrison &Boyd, 1969; Smith & March, 2007). Fast Blue BB salt containsa diazonium group, +N @ N, where the nitrogen is retainedin coupling with the reactive activating group (OH) of thephenolic group. Coupling mostly occurs para to the phenolicactivating group, unless the position is already occupied, then

Fig. 1A Proposed interactions of Fast Blue BB salt with phenolic acids, caffeic and chlorogenic acids in NaOH buffer solution.In this gure, the coupling (N @ N) are shown in the para positions relative to the activating group (OH) of the phenolicmoeity. It is also possible that coupling can occur at the ortho position(s).

80 J O U R N A L O F F U N C T I O N A L F O O D S 3 ( 2 0 1 1 ) 7 9 8 7


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the substitution occurs in the ortho position to the activating group. Phenols are coupled in slightly alkaline solution wherethey can be converted to the more active phenoxide ions. Thedistribution of naturally occurring phenolics, such as avo-noids and the chemical structure of these phenolics were de-scribed by Shahidi and Naczk (2004) . Quercetin, kaempferol,chlorogenic acid, caffeic acid, coumaric acid are found in gojiberies ( Wang, Chang, Stephen Inbaraj, & Chen, 2010 ); cyani-dins, orientin, and taxifolin are present in acai ( Schausset al., 2006). The proposed interaction of gallic acid phenolicgroups with the diazonium moiety of Fast Blue BB indicatedcoupling in the para and ortho OH positions ( Medina,2011). Fig. 1A shows the proposed interactions of Fast BlueBB with phenolic acids (chlorogenic and caffeic acids) wherethese compounds are major constituents of fruits and coffeebeans. The azo complex formation with major phenolic con-stituents (cyanidin, pelargonidin, kaempferol, delphinidin,

orientin, taxifolin) is shown in Fig. 1B. Medina and Nagdy(1993) also showed a proposed interaction between Fast Cor-inth V diazonium group and the phenolic OH of zeranol, aresorcylic compound. The use of other azo-forming dyes fordetection of related phenolic compounds were reported fordetection of veterinary estrogens ( Gunther, 1978; Medina &Schwartz, 1992; Wortberg, Woller, & Chulamorakot, 1978 ); hu-man estrogens ( Weiss, Esterbauar, & Scherr, 1975; Weiss, Win-ter, Scherr, & Bayer, 1976 ); zearalenone mycotoxin ( Scott,Panalaks, Kanhere, & Miles, 1978 ); phenolic constituents of plant cell walls ( Harris, Hartley, & Barton, 1982 ).

The interactions of the Fast Blue BB with gallic acid werepreviously optimized ( Medina, 2011 ). The absorbance mea-sured at 420, 500 and 520 nm yielded an optimum at 420 nmwith a Fast Blue BB concentration of 0.1%. Reaction time at60 min showed a linear response. Effects of the alkali (Na 2 CO3and NaOH) were also compared and results showed that 5%

Fig. 1B Proposed interactions of Fast Blue BB salt with avonoids (cyanidin, pelargonidin, kaempferol, delphinidin, orientin,taxifolin). In this gure, the coupling (N @ N) are shown in the para and ortho positions relative to the activating group (OH)of the phenolic moeity.

J O U R N A L O F F U N C T I O N A L F O O D S 3 ( 2 0 1 1 ) 7 9 8 7 81


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NaOH had a faster completion of reaction compared to 20%Na2 CO3 . These optimum conditions resulted in a three-stepprocedure, transferring the samples to tubes, adding the FastBlue BB reagent and alkali, and the absorbance was read at420 nm. The azo-based assay had highergallic acid equivalent(GAE) values than the standard FolinCiocalteu for totalphenolics in tea and coffee preparations with ratios of 26while the packaged juice and juice drink samples had ratiosfrom


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divided by 10 to directly convert to mg GAE/100 mL beveragesamples.

2.6. Fresh fruits, soft dehydrated fruits and dry powder

Solid soft foods like fruits (2550 g) were cut to small piecesand homogenized in a mini food processor until the samplewas smooth. The homogenates were transferred to plasticstorage bags and kept in 80 C freezer. Samples (2.5 g) wereweighed in 15 mL round bottom tubes, followed by additionof 5 mL 70% ethanol. The samples were homogenized withan ultra-turrax (UT), for 1 min (2 ) processing, mixed with arotary shaker for 60 min and centrifuged at 6000 rpm (2961 g)for 10 min. The supernatant was decanted and transferredto calibrated glass tubes and the volume was measured. Thesamples were extracted again with another 5 mL of 70% eth-anol, homogenized, mixed for 30 min and centrifuged at6000 rpm (2961g) for 10 min. The supernatant was added tothe rst extract and the total volume was measured. Aliquotsof the pooled extracts were diluted with DIH 2 O and analyzedfor total phenols by the Fast Blue BB and FolinCiocalteumethods. Typical dilutions for analysis were 1:5, 1:10 or 1:20(v/v). Highly colored extracts were analyzed at higher dilu-tions. These dilutions can be screened prior to analysis byanalyzing the diluted extracts with the total phenol methodswithout the calibration standards. The excess extracts werestored in 20 C or 80 C for longer storage. Total phenolswere analyzed with FolinCiocalteu (Section 2.2) and Fast BlueBB method (Sections 2.3 and 2.4) The total phenolic content of samples was expressed in GAE values. The calibration curvevalues were in l g GAE/mL of the diluted extract and multi-plied by the dilution factor to obtain l g/mL of the original ex-tract. This value was then divided by a conversion factorconsisting of weight of sample divided by total volume of ex-tract and these resulted to l g GAE/g sample. This GAE value(l g/g) was further divided by 10 to directly convert to mg GAE/100 g samples.

Freeze-dried acai powder (1 g) was extracted utilizing fourextraction procedures. Preparation A: 20 mL 70% ethanol wereadded and homogenized with Ultra-Turrax and centrifuged.After decanting the supernatant, 10 mL of 70% ethanol wereadded to the pellet and mixed in a shaker for 30 min. Themixture was centrifuged and the supernatants were pooledwith the rst extract. Preparation B1: Another 1 g acai aliquotwas extracted with 25 mL of 70% ethanol. Samples weremixed at room temperature in an IKA vibrax rotary shakerfor 1 h, the extract was separated by centrifugation and thesupernatants were analyzed. Preparation B2 (1 + 24 h): Thepellet of the 1 h extract was extracted again with 10 mL of 70% ethanol for 24 h at room temperature in a shaker. Themixture was centrifuged and the supernatant was analyzed.The phenolic values of the 1 h extraction and the pellet ex-tracts were added and reported as 1 h + 24 h GAE values.Preparation B3. A 1 g acai powder was extracted with 20 mLof 70% ethanol for 24 h at room temperature in a shaker.The extracts were diluted to t the gallic acid calibration stan-dard of 0, 10500 ppm. Duplicate samples were analyzed in 3separate trials with the FolinCiocalteu and Fast Blue BBmethods.

3. Results and discussion

3.1. Fast Blue BB method

Fig. 2 shows that linearity and the slope of the calibrationlines improved with 90 min reaction time compared to60 min. The results were higher at concentrations >100 ppmwhen analyzed at 60 min vs 90 min. Therefore, the Fast BlueBB procedure in this study utilized the 90 min reaction timefor measuring the phenolics in the beverage and fruit sam-ples. The typical interactions with gallic acid showedR = 0.9985 (0.0007 SD) at 90 min vs R = 0.9909 (0.0047 SD) at60 min in six calibration lines. The mean calibrationlines were Y = 0.133395 + 0.0048102x (90) min and Y =0.1832466 + 0.00227735x (60 min) which showed slightchanges in slopes and y-intercepts. The limits of detection(LOD) was


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3.3. Analysis of superfruit juices and juice mix

The United States Food and Drug Administration (FDA) guide-lines on fruit juices indicate that beverages that purport tocontain juice (fruit or vegetable juice) must declare the % juice. This includes non-carbonated beverages, full strength(100%) juices, concentrated juices, diluted juices, and bever-ages that purport to contain juice but contain no juice ( Codeof Federal Regulations, 2009a ). Beverages that are 100% juicemay be called juice. However, beverages that are diluted toless than 100% juice must have the word juice qualied witha term such as beverage, drink, or cocktail ( Code of Fed-

eral Regulations, 2009b ). However, the Food and Drug Admin-istration has no guidelines on superfoods but instead theagency has issued guidelines on antioxidants ( Code of FederalRegulations, 2009c ). A superfood is perceived to have highnutrient density with preventive or healing properties of many chronic diseases. A superfood is dened as a naturalfood regarded as especially benecial because of its nutrientprole or its health-protecting qualities ( Oxford Dictionary,2009). Sloan (2008) predicted that superfoods was second inthe Top 10 Food Trends. Sloan reported that this was a resultof recent scientic validation of the health benets of super-foods convincing the consumers the key benets of a newtrend in whole food nutrition.

Our study showed that some fruits and juices show hightotal phenolic values and may be considered as superfoods.The juices analyzed in this study were diluted with DIH 2 Oprior to analysis. Analysis of juices from superfruits areshown in Table 2 with GAE values of up to 1800 mg/100 mL(600 mg per serving) in the Miracle Fruits juice mix by theFast Blue BB method compared to about 1400 mg GAE/100 mL by the FolinCiocalteu method. The acai commercialdrinks, mixes and smoothies had GAE values ranging from294 to 1256 mg/100 mL. The Fast Blue BB:FolinCiocalteuGAE ratios ranged from 1.9 to 4.0. Juices and juice mixes withdeep colors had GAE ratios >2 such as in most acai prepara-tions. Fresh squeezed blood orange 295 mg/100 mL GAE value

and a ratio of 2.28. Juices with higher Fast Blue BB:FolinCio-

calteu values had Fast Blue BB:FolinCiocalteu ratio of around1 or less suggest the presence of high concentration of non-phenolic compounds. Pomegranate is perceived to be a super-fruit. Analysis of the fresh pomegranate seeds extracted bymashing in a mortar and pestle resulted in mean GAE valuesof 182 and 152 mg/100 g by Fast Blue BB and FolinCiocalteumethods with GAE ratio of 1.2.

3.4. Fresh fruits, soft dehydrated fruits and dry powder

Fresh fruits were stored in 80 C prior to sample preparationor homogenization. After extraction, the extracts were also

stored at 20 or 80 C for repeat analysis. The results fromanalysis of nine fruit samples are shown in Table 3 . Blueber-ries had the highest GAE values of 1291 mg GAE/100 g indomestic fruits. The black grapes and black plums had thenext highest with 572 and 441 GAE/100 g, respectively. Thepeach and pear samples were analyzed with and withoutskins, and results showed higher GAE values in fruits withskin when analyzed with Fast Blue BB and FolinCiocalteumethods. Again, the inter-assay variability (RSD) was lessthan 10%. Blueberries and black plum had the highest phen-olics ratio of 4.1 and 3.4, respectively. The number ( n) of FastBlue BB analysis were at times lower than the number of anal-ysis with FolinCiocalteu method. Initial analysis of samples

containing high phenolics with Fast Blue BB resulted in highGAE values (>500 l g/mL) where the optical density could notbe measured and this replicate analysis was disregarded. Atvarying sample dilutions, the GAE values fell outside the reli-able GAE concentration (>50 and


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916710,003 mg/100 g with Fast Blue BB but the mean FolinCiocalteu GAE values were 23802984 with Fast Blue BB:Fo-linCiocalteu ratios of 3.33.4. Higher phenolic values wereobtained in samples extracted (2x) with ultra-turrax homoge-nization (preparation A) followed with a 30 min shakerextrac-

tion of the pellet compared to a 24 h extraction on a rotaryshaker (preparation B). That is, the homogenization extrac-tion yielded higher results than the 24 h shaking method.With the shaker method, the 1 h extraction had higher GAEvalues than the 24 h extraction. There was also a color fading

Table 3 Total phenolics (mg GAE/100 g) in select fresh and dried fruits by Fast Blue BB (FBBB) and FolinCiocalteumethods.

Samples FBBB methodmg GAE/100 g

FBBB method FolinCiocalteumg

GAE/100 g

FolinCiocalteu FBBB:Folin Ciocalteu

Mean (n) RSD (%) Mean (n) RSD (%) RatioFresh fruitsBlueberry 1291 (4) 7 315 (4) 7 4.1Cherry, sweet bing 205 (6) 5.2 80 (6)

81 (6)4.04.7

2.62.5

Grape, black 572 (3) 5 372 (3) 5 1.5Peach, white with skin 160 (3) 3 98 (3) 4 1.6Peach, white peeled 91 (3) 5 46 (3) 0 2Plum, red 161 (3) 1 69 (3) 5 2.3Plum, Black 441 (6) 5.4 131 (6) 9.2 3.4Raspberry 393 (4) 2 163 (4) 2 2.4Strawberry, organic 282 (3) 2 159 (3) 13 1.8

Dry fruitsa Goji-WF (dry) 562 (6) 1.3 1036 (3) 1.2 0.54Goji-B (dry) 530 (6) 10.3 895 (3) 10.2 1.70b Jujube (dry) 595 (3) 17 944 (3) 3 0.63Prune 337 (3) 4 399 (3) 1 0.84

Polyphenols measured as gallic acid equivalents (GAE) per 100 mL. Each value is the mean of multiple analyses ( n). Each data represents GAEvalues derived from ( n) inter-assay trials.

a Goji berry is the common name for wolfberry or Lycium barbarum .b Jujube or, red date or chinese date, is a species of Ziziphus in the buckthorn family Rhamnacae . Samples were extracted with a rotary shaker.

Each data represents GAE values derived from ( n) inter-assay trials.

Table 2 Total phenolics (mg GAE/100 mL) in juices of superfruits by Fast Blue BB (FBBB) and FolinCiocalteu methods.

Super fruit juices/mixes FBBB methodmg GAE/100 g (n)

RSD (%) FolinCiocalteumg GAE/100 g (n)

RSD (%) RatioFolin Ciocalteu:FBBB

Acai + Lime 1227 (3)1256 (3)

50

338 (3)310 (3)

53

3.64.0

Acai-IStored ( 80)

858 (2)685 (3)

62

315 (3)291 (3)

10.81

2.72.35

Acai-M 495 (3) 4 276 (3) 3 1.79Acai-Z

Stored ( 80)395 (2)284 (2)

5.14.5

114 (3)99 (2)

1.83.0

3.462.87

Berry boost 172 (3) 1 283 (3) 2 0.61Black currant 261 (3) 5 115 (3) 3 2.27Black currant nectar, organic 120 (3) 2 70 (3) 3 1.7Blood orange, fresh squeeze 295 (3) 5 129 (3) 3 2.28Miracle fruit juice mix 1 1795 (2)

1868 (4)1750 (3)

243

1390 (2)na1463

4

2

1.07

1.20Miracle fruit juice mix 2 1101 (3) 4 816 (3) 11 1.35Prune juice 243 (3)

296 (3)113

184 (3)179 (3

11

1.321.65

Pomegranate juice, fresh extract of seed coat 193 (3)168 (3)

48

161 (3)142 (3)

16

1.201.18

Purple corn drinkChicha

Stored ( 80)526 (2)501 (2)

1.13.6

142 (4)144 (2)

52.1

3.73.5

Polyphenols measured as gallic acid equivalents (GAE) per 100 mL. Each data represents GAE values derived from ( n) inter-assay trials.



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with the 24 h method. However, the double extraction(1 h + 24 h extraction of the pellet) yielded higher results thanthe 1 h alone and the homogenization method. The phenolicresults from all three trials had


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Determinación de Los Fenoles Totales en Jugos y Superfruits

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