determination of antioxidant activity of bioactive peptide fractions obtained from yogurt

7/23/2019 Determination of Antioxidant Activity of Bioactive Peptide Fractions Obtained From Yogurt

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J. Dairy Sci . 94:53055314doi:10.3168/jds.2011-4285 American Dairy Science Association, 2011.

ABSTRACT

In this study, physicochemical and microbiologi-cal properties of traditional and commercial yogurtsamples were determined during 4 wk of storage. Pro-teolytic activity, which occurs during the storage periodof yogurt samples, was also determined. Peptide frac-tions obtained from yogurts were investigated and theeffect of proteolysis on peptide release during storage

was determined. The antioxidant activities of peptidesreleased from yogurt water-soluble extracts (WSE) andfrom HPLC fractions were determined by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Theantioxidant activity of WSE from traditional yogurt wasgreater than that of WSE from commercial yogurts. Inanalysis by the ABTS method, mean values increasedfrom 7.697 to 8.739 mMTrolox/g in commercial yo-gurts, and from 10.115 to 13.182 mMTrolox/g in tra-ditional yogurts during storage. Antioxidant activitiesof peptides released from HPLC fractions of selectedyogurt samples increased 10 to 200 times. In all yogurtsamples, the greatest antioxidant activity was shown inthe F2 fraction. After further fractionation of yogurtsamples, the fractions coded as F2.2, F2.3, F4.3, andF4.4 had the highest antioxidant activity values. Totalantioxidant activity of yogurts was low but after puri-fication of peptides by fractionation in HPLC, peptidefractions with high antioxidant activity were obtained.Key words: yogurt, bioactive peptide, antioxidantactivity, proteolysis

INTRODUCTION

Several reactive oxygen species, including the su-peroxide radical, hydroxyl radical, hydrogen peroxide,and the peroxide radical, are known to cause oxidativedamage not only to food systems but also to living sys-tems (Liu et al., 2005; Kavas et al., 2007). Antioxidantpeptides present in the food system play a vital role in

the maintenance of antioxidant defense systems by pre-venting the formation of free radicals or by scavengingfree radicals and active oxygen species, which induceoxidative damage to biomolecules and cause aging, can-cer, heart disease, stroke, and arteriosclerosis (Guptaet al., 2009). Hence, interest is increasing in findingnatural antioxidants from food, because it is believedthat they can protect the human body from the attackof free radicals and delay the progress of many chronicdiseases, as well as impede lipid oxidative rancidity infoods (Liu et al., 2005; Kadri et al., 2011).

Natural antioxidants, including rosemarinic acid, cat-echin, tocopherols, ascorbate, and various phenolic ex-tracts from plants, have been widely used in processedfoods. The search for natural antioxidants has extendedbeyond these traditional sources, and several studieshave shown that peptides and protein hydrolysates ofplant and animal origins could possess significant anti-oxidant activity (Xue et al., 2009). Antioxidants fromnatural sources are likely more desirable than thoseproduced chemically, because some synthetic antioxi-

dants have been reported to be carcinogenic (Liu et al.2005).Today, milk proteins are considered the most impor-

tant source of bioactive peptides, and an increasingnumber of bioactive peptides have been identified inmilk protein hydrolysates and fermented dairy products(Korhonen and Pihlanto, 2006; Korhonen, 2009; Onget al., 2007; Schmelzer et al., 2007; Lpez-Expsito etal., 2007; Contreras et al., 2009; Srinivas and Prakash2010; Kamau et al., 2010; Nagpal et al., 2011). Lacticacid bacteria used as starters are primarily responsiblefor the generation of bioactive peptides during milkfermentation. Fermented milk products, in addition to

providing energy and nutrients, are an excellent sourceof bioactive peptides (Chianese et al., 1997; Smacchiand Gobbetti, 2000; Gobbetti et al., 2002; Algaron etal., 2004; Donkor et al., 2007).

Yogurt is a coagulated milk product obtained fromlactic acid fermentation by the action of Lactobacillus bulgaricus and Streptococcus thermophilus (Rasicand Kurman, 1978; Anonymous, 1999a; Shah, 2003Anonymous, 2009). Yogurt is traditionally considereda healthy food. The functionality of yogurt is further

Determination of antioxidant activi ty of bioactivepeptide fractions obtained from yogurt

1and Z. nerSleyman Demirel University, Faculty of Engineering and Architecture, Department of Food Engineering, 32260 Isparta, Turkey

Received February 18, 2011.Accepted July 22, 2011.1Corresponding author: [email protected]


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enhanced by the release of bioactive peptides duringlactic fermentation (Shah, 2007). These peptides areencrypted in the milk proteins and released duringfermentation due to the proteolytic activities of theorganisms used (Schanbacher et al., 1997, 1998; Smac-chi and Gobbetti, 2000; Korhonen and Pihlanto, 2006;

Roufik et al., 2006; Mller et al., 2008). The results ofsome animal and human studies suggest that fermentedmilk products do have an antioxidative effect (Pihlanto,2006).

The objectives of the study were to isolate bioac-tive peptides fractions from traditional and commercialyogurts and to investigate the antioxidant activities ofthese peptide fractions isolated by reverse phase-HPLC(RP-HPLC).

MATERIALS AND METHODS

Fifteen traditional homemade yogurt samples were

collected from different producers in the Isparta andBurdur regions in Turkey. Commercial yogurt sampleswere obtained from Sleyman Demirel University, n-st Plant, in the first days of the production. All yogurtsamples stored at 4C were analyzed at 1-wk intervalsfrom d 1 to 4 wk of storage.

For RP-HPLC analysis, HPLC-grade water was usedto prepare the buffers and mobile phases, which werefiltered through 0.45-m filters (Schleicher & SchuellGmbH, Dassel, Germany) and degassed. All otherchemicals were of analytical grade.

Physicochemical and Microbiological Analysis

Total solids and titratable acidity of yogurt sampleswere measured according to the Turkish Standard TS1330 (Anonymous, 1999a). pH values were determinedelectrometrically with a pH meter (WTW, Wielheim,Germany). Fat and fat in solids were determined byGerber method as described in Turkish Standard TS8189 (Anonymous, 1990).

Yogurt samples were prepared for microbiologicalanalysis according to the IDF Standard (IDF, 1992).Samples for counts of lactic acid bacteria were spreadplated on M17 (Merck, Darmstadt, Germany) and de

Man, Rogosa, Sharpe (Merck) agar. Plates were incu-bated at 37C for 48 to 72 h. Plate count skim milkagar was used for enumeration of total mesophilic aero-bic bacterial (TMAB) at 35C for 24 to 48 h. Eosinmethylene blue (Merck) agar was used to determinecoliform counts at 37C for 24 to 48 h. Yeast and moldswere enumerated by plating on potato dextrose agar(Merck) acidified with 10% lactic acid at 25C for 4d (de Man et al., 1960; Terzaghi and Sandine, 1975;Anonymous, 1999b).

Determination of Proteolytic Act ivity

The degree of proteolysis during fermentation of milkwas quantified by determining free NH3groups usingthe o-phthaldialdehyde method with some modifica-tions (Church et al., 1983; Nielsen et al., 2001; Donkor

et al., 2007).

Preparation of Water-Soluble Extract Peptides

Yogurt samples were prepared according to Donkoret al. (2007). Yogurt samples were centrifuged at 15,000 g(Sigma 3K30, Steinheim, Germany) at 4C for 30min. The supernatant was filtered separately througha 0.45-m membrane filter and the filtrate was freeze-dried (benchtop-SLC freeze dryer, Virtis, Gardiner,NY). The freeze-dried samples were stored at 25Cfor further analysis. Protein content in water-solubleextracts (WSE) of yogurts were measured by Lowry

method (Lowry et al., 1951).

Radical Scavenging Activ ity: 2,2-Azino-Bis

(3-Ethylbenzothiazoline-6-Sulfonic Acid) Method

The antioxidant activity of either WSE or the iso-lated peptides thereof was assayed according to themethod described by Re et al. (1999). 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radi-cal cation (ABTS) was produced by reacting 7 mMABTS stock solution with 2.45 mMpotassium persul-fate (final concentration in 10 mL of water) and keep-ing the mixture in the dark at room temperature for 12to 16 h before use.

The solution was diluted in 0.1 MPBS (pH 7.4) toan absorbance of 0.70 0.02 at 734 nm after equili-bration at 30C. Ten microliters of sample or Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid) as positive control was added to 1 mL of dilutedABTS solution and incubated at 30C for 6 min.Scavenging of the ABTSradical was followed spectro-photometrically (UV-1601, Shimadzu, Kyoto, Japan)by monitoring the decrease in absorbance at 734 nm. Areading was taken 1 min after initial mixing and thenperiodically up to 6 min. A solvent blank was run in

each assay (negative control). All determinations werecarried out in triplicate, and their average was used asa datum point. The percentage inhibition of absorbanceat 734 nm was calculated and plotted as a functionof the concentration of antioxidants and of Trolox forthe standard. To calculate the Trolox equivalent anti-oxidant capacity (TEAC), the gradient of the plot ofthe percentage inhibition of absorbance versus sampleconcentration was divided by the gradient of the plotfor Trolox to give TEAC at the specific time.


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Radical Scavenging Activ ity:

2,2-Diphenyl-1-Picrylhydrazyl Method

Water-soluble extract or the isolated peptide frac-tions thereof were prepared in 0.1 Msodium phosphatebuffer, pH 7.0, containing 1% (wt/vol) Triton X-100,

and 2,2-diphenyl-1-picrylhydrazyl (DPPH; 100 M)was prepared in methanol. An aliquot (1.5 mL) of WSEor the isolated peptide fractions (sample) or 1.5 mLof buffer (control) was mixed with 1.5 mL of DPPHsolution, and left in the dark at room temperature for30 min. Absorbance of the solution was then measuredat 517 nm. The percentage decrease in absorbance ofthe sample relative to the control was calculated as therelative scavenging activity (Aluko and Monu, 2003;Farzamirad and Aluko, 2008).

RP-HPLC Analys is of WSE

The water-soluble peptides in the traditional andcommercial yogurts were profiled on an RP-HPLC(LC-20 AT series, Shimadzu) equipped with a Zorbax300 SB-C3 guard column (7.0 m, 300 , 15 9.4 mm,Agilent, Waldbronn, Germany) and Zorbax 300 SB-C8monomeric column (6.5 m, 300 , 250 9.4 mm,Agilent). The peptides were eluted in a linear gradientfrom 100 to 0% solvent A (0.1% trifluoroacetic acid indeionized water) in solvent B (0.1% trifluoroacetic acidin 90%, vol/vol, acetonitrile in deionized water) over 80min. Separations were conducted at room temperature(20C) at a flow rate of 3 mL/min. The eluted peptides

were detected at 214 nm using a photo diode array detector (SPD-20A, Shimadzu). All samples and solventswere filtered through a 0.45-m membrane filter.

The freeze-dried WSE (2.5 g) of yogurt samples weredissolved in 5-mL aliquots of 0.1% TFA in deionizedwater, and centrifuged at 14,000 gfor 30 min. The

supernatants were filtered through a 0.45-m mem-brane filter and then injected (750 L) onto RP-HPLCFractions at 10-min intervals (Figure 1) were collectedlyophilized, and assayed for antioxidant activity (Don-kor et al., 2007).

Statistical Analysis

Statistical analysis was performed using SPSS 17.0for Windows (SPSS Inc., Chicago, IL). Results wereanalyzed by repeated-measures ANOVA. Differencesbetween the treatment means were compared at the

5% level of significance using Tukeys test. Statisticacomparisons of the results were performed by calculat-ing the correlation values for proteolytic activity, physi-cochemical variables, and microbiological analysis.

RESULTS AND DISCUSSION

Physicochemical, Microbiological, and Proteolytic

Activi ty Changes During Storage

The physicochemical and microbiological composi-tion of the yogurt samples is shown in Table 1. Tota

Figure 1.The elution profile of water-soluble extract fractions obtained by reversed phase-HPLC.


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solids, pH values, coliforms, and yeast-mold counts intraditional yogurts were significantly different comparedwith those of commercial yogurts (P< 0.01). However,differences in acidity, fat in solids, TMAB (except at wk4), and lactobacillus and lactococcus counts betweentraditional and commercial yogurts were not significant

(P> 0.05). As shown in Table 1, the average coliformbacteria and yeast-mold counts were much higher intraditional yogurts than in commercial yogurts.

The changes during storage in total solids, lactic acidpercentage, and fat in solids were not significant (P>0.05). A decrease in the pH value was observed duringthe first 14 d of storage for all yogurts. Subsequently,the pH value continued to decrease slightly during thethe rest of the storage period (P < 0.01). In termsof lactic acid percentage, a significant increase wasobserved during the first week of storage (P< 0.01).Both pH and lactic acid percentage measurements areimportant because acidification is the key mechanismduring yogurt fermentation.

Proteolysis is the breakdown of large and complexproteins into smaller, simpler peptides. The averageproteolytic activity of yogurt samples during storagewas estimated by determination of free amino groups,and results are presented in Table 2. The proteolyticactivity of both yogurt samples was higher comparedwith raw skim milk as control. Proteolysis increasedsignificantly (P< 0.01) during storage. However, pro-teolytic activity values between traditional and com-mercial yogurt samples were not significantly different(P> 0.05). Although the proteolytic activity values

between traditional and commercial yogurt samples didnot differ, as shown in Table 2, coliform bacteria andyeast-mold counts were higher in traditional yogurtsthan in commercial yogurts (Table 1). Proteolyticactivity has a positive correlation (P< 0.01) with co-liform bacteria and yeast-mold counts, but a negativecorrelation with pH value (P< 0.01).

Similar to results of other researchers, we showed thatdifferences in the proteolytic activity were dependenton pH and strains (Shihata and Shah, 2000; Donkor etal., 2007).

Protein Content of WSE of Yogurts

Protein contents of WSE of yogurts are shown inTable 3. The differences in protein content betweentraditional and commercial yogurts and the changesduring storage were not significant (P> 0.05).

Measurement of ABTS Radical Scavenging Activity

The main characteristic of an antioxidant is its abilityto trap free radicals. Highly reactive free radicals and Ta

ble1.

Physicochemicaland

microbiologicalcomposition(meanSD)

oftheyogurtsamples

Analysis

Yogurt

Day

1

Week1

Week2

Week3

Week4

Physicochemical

Totalsolids(%)

Traditional

11.2

9

0.20a,B

11.2

80.21a,B

11.1

80.22a,B

10.9

60.21a,B

11.0

90.24a,B

Commercial

16.1

3

0.44a,A

16.2

30.46a,A

15.6

60.49b,A

16.4

20.48a,A

16.2

50.54a,A

Acidity(%)

Traditional

1.12

0.05b,A

1.240.07a,A

1.310.06a,A

1.300.07a,A

1.320.07a,A

Commercial

1.05

0.11b,A

1.160.16a,A

1.210.14a,A

1.220.15a,A

1.200.16a,A

pH

Traditional

3.97

0.04a,B

3.880.05c,B

3.860.04b,B

3.850.05b,B

3.820.04b,B

Commercial

4.41

0.09a,A

4.250.11c,A

4.250.09b,A

4.230.11b,A

4.280.08b,A

Fatinsolids(%)

Traditional

25.6

2

1.33a,A

26.1

21.16a,A

25.3

61.24a,A

27.3

41.26a,A

26.7

91.25a,A

Commercial

22.6

6

2.97a,A

22.3

72.59a,A

23.8

72.78a,A

22.5

42.81a,A

22.6

62.80a,A

Microbiological(logcfu/g)

Coliforms

Traditional

3.92

0.74a,A

4.280.78a,A

4.380.79a,A

4.170.75a,A

4.190.75a,A

Commercial

determination of antioxidant activity of bioactive peptide fractions obtained from yogurt

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