J. Dairy Sci.

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

Determination of antioxidant activity of bioactive

peptide fractions obtained from yogurt
+
 DQOGHUH$OROX1 and Z. ner
Sleyman Demirel University, Faculty of Engineering and Architecture, Department of Food Engineering, 32260 Isparta, Turkey

ABSTRACT

In this study, physicochemical and microbiological properties of traditional and commercial yogurt
samples were determined during 4 wk of storage. Proteolytic activity, which occurs during the storage period
of yogurt samples, was also determined. Peptide fractions obtained from yogurts were investigated and the
effect of proteolysis on peptide release during storage
was determined. The antioxidant activities of peptides
released from yogurt water-soluble extracts (WSE) and
from HPLC fractions were determined by 2,2c-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and
2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. The
antioxidant activity of WSE from traditional yogurt was
greater than that of WSE from commercial yogurts. In
analysis by the ABTS method, mean values increased
from 7.697 to 8.739 mM Trolox/g in commercial yogurts, and from 10.115 to 13.182 mM Trolox/g in traditional yogurts during storage. Antioxidant activities
of peptides released from HPLC fractions of selected
yogurt samples increased 10 to 200 times. In all yogurt
samples, the greatest antioxidant activity was shown in
the F2 fraction. After further fractionation of yogurt
samples, the fractions coded as F2.2, F2.3, F4.3, and
F4.4 had the highest antioxidant activity values. Total
antioxidant activity of yogurts was low but after purification of peptides by fractionation in HPLC, peptide
fractions with high antioxidant activity were obtained.
Key words: yogurt, bioactive peptide, antioxidant
activity, proteolysis
INTRODUCTION

Several reactive oxygen species, including the superoxide radical, hydroxyl radical, hydrogen peroxide,
and the peroxide radical, are known to cause oxidative
damage not only to food systems but also to living systems (Liu et al., 2005; Kavas et al., 2007). Antioxidant
peptides present in the food system play a vital role in
Received February 18, 2011.
Accepted July 22, 2011.
1
Corresponding author: haticealoglu@sdu.edu.tr

the maintenance of antioxidant defense systems by preventing the formation of free radicals or by scavenging
free radicals and active oxygen species, which induce
oxidative damage to biomolecules and cause aging, cancer, heart disease, stroke, and arteriosclerosis (Gupta
et al., 2009). Hence, interest is increasing in finding
natural antioxidants from food, because it is believed
that they can protect the human body from the attack
of free radicals and delay the progress of many chronic
diseases, as well as impede lipid oxidative rancidity in
foods (Liu et al., 2005; Kadri et al., 2011).
Natural antioxidants, including rosemarinic acid, catechin, tocopherols, ascorbate, and various phenolic extracts from plants, have been widely used in processed
foods. The search for natural antioxidants has extended
beyond these traditional sources, and several studies
have shown that peptides and protein hydrolysates of
plant and animal origins could possess significant antioxidant activity (Xue et al., 2009). Antioxidants from
natural sources are likely more desirable than those
produced chemically, because some synthetic antioxidants have been reported to be carcinogenic (Liu et al.,
2005).
Today, milk proteins are considered the most important source of bioactive peptides, and an increasing
number of bioactive peptides have been identified in
milk protein hydrolysates and fermented dairy products
(Korhonen and Pihlanto, 2006; Korhonen, 2009; Ong
et al., 2007; Schmelzer et al., 2007; Lpez-Expsito et
al., 2007; Contreras et al., 2009; Srinivas and Prakash,
2010; Kamau et al., 2010; Nagpal et al., 2011). Lactic
acid bacteria used as starters are primarily responsible
for the generation of bioactive peptides during milk
fermentation. Fermented milk products, in addition to
providing energy and nutrients, are an excellent source
of bioactive peptides (Chianese et al., 1997; Smacchi
and Gobbetti, 2000; Gobbetti et al., 2002; Algaron et
al., 2004; Donkor et al., 2007).
Yogurt is a coagulated milk product obtained from
lactic acid fermentation by the action of Lactobacillus bulgaricus and Streptococcus thermophilus (Rasic
and Kurman, 1978; Anonymous, 1999a; Shah, 2003;
Anonymous, 2009). Yogurt is traditionally considered
a healthy food. The functionality of yogurt is further

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enhanced by the release of bioactive peptides during

lactic fermentation (Shah, 2007). These peptides are
encrypted in the milk proteins and released during
fermentation due to the proteolytic activities of the
organisms used (Schanbacher et al., 1997, 1998; Smacchi and Gobbetti, 2000; Korhonen and Pihlanto, 2006;
Roufik et al., 2006; Mller et al., 2008). The results of
some animal and human studies suggest that fermented
milk products do have an antioxidative effect (Pihlanto,
2006).
The objectives of the study were to isolate bioactive peptides fractions from traditional and commercial
yogurts and to investigate the antioxidant activities of
these 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 and
Burdur regions in Turkey. Commercial yogurt samples
were obtained from Sleyman Demirel University, nst Plant, in the first days of the production. All yogurt
samples stored at 4C were analyzed at 1-wk intervals
from d 1 to 4 wk of storage.
For RP-HPLC analysis, HPLC-grade water was used
to prepare the buffers and mobile phases, which were
filtered through 0.45-m filters (Schleicher & Schuell
GmbH, Dassel, Germany) and degassed. All other
chemicals were of analytical grade.
Physicochemical and Microbiological Analysis

Total solids and titratable acidity of yogurt samples

were measured according to the Turkish Standard TS
1330 (Anonymous, 1999a). pH values were determined
electrometrically with a pH meter (WTW, Wielheim,
Germany). Fat and fat in solids were determined by
Gerber method as described in Turkish Standard TS
8189 (Anonymous, 1990).
Yogurt samples were prepared for microbiological
analysis according to the IDF Standard (IDF, 1992).
Samples for counts of lactic acid bacteria were spread
plated on M17 (Merck, Darmstadt, Germany) and de
Man, Rogosa, Sharpe (Merck) agar. Plates were incubated at 37C for 48 to 72 h. Plate count skim milk
agar was used for enumeration of total mesophilic aerobic bacterial (TMAB) at 35C for 24 to 48 h. Eosin
methylene blue (Merck) agar was used to determine
coliform counts at 37C for 24 to 48 h. Yeast and molds
were enumerated by plating on potato dextrose agar
(Merck) acidified with 10% lactic acid at 25C for 4
d (de Man et al., 1960; Terzaghi and Sandine, 1975;
Anonymous, 1999b).
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Determination of Proteolytic Activity

The degree of proteolysis during fermentation of milk

was quantified by determining free NH3 groups using
the o-phthaldialdehyde method with some modifications (Church et al., 1983; Nielsen et al., 2001; Donkor
et al., 2007).
Preparation of Water-Soluble Extract Peptides

Yogurt samples were prepared according to Donkor

et al. (2007). Yogurt samples were centrifuged at 15,000
g (Sigma 3K30, Steinheim, Germany) at 4C for 30
min. The supernatant was filtered separately through
a 0.45-m membrane filter and the filtrate was freezedried (benchtop-SLC freeze dryer, Virtis, Gardiner,
NY). The freeze-dried samples were stored at 25C
for further analysis. Protein content in water-soluble
extracts (WSE) of yogurts were measured by Lowry
method (Lowry et al., 1951).
Radical Scavenging Activity: 2,2c-Azino-Bis
(3-Ethylbenzothiazoline-6-Sulfonic Acid) Method

The antioxidant activity of either WSE or the isolated peptides thereof was assayed according to the
method described by Re et al. (1999). 2,2c-Azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS) was produced by reacting 7 mM
ABTS stock solution with 2.45 mM potassium persulfate (final concentration in 10 mL of water) and keeping the mixture in the dark at room temperature for 12
to 16 h before use.
The solution was diluted in 0.1 M PBS (pH 7.4) to
an absorbance of 0.70 0.02 at 734 nm after equilibration at 30C. Ten microliters of sample or Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) as positive control was added to 1 mL of diluted
ABTS solution and incubated at 30C for 6 min.
Scavenging of the ABTS radical was followed spectrophotometrically (UV-1601, Shimadzu, Kyoto, Japan)
by monitoring the decrease in absorbance at 734 nm. A
reading was taken 1 min after initial mixing and then
periodically up to 6 min. A solvent blank was run in
each assay (negative control). All determinations were
carried out in triplicate, and their average was used as
a datum point. The percentage inhibition of absorbance
at 734 nm was calculated and plotted as a function
of the concentration of antioxidants and of Trolox for
the standard. To calculate the Trolox equivalent antioxidant capacity (TEAC), the gradient of the plot of
the percentage inhibition of absorbance versus sample
concentration was divided by the gradient of the plot
for Trolox to give TEAC at the specific time.

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Radical Scavenging Activity:

2,2-Diphenyl-1-Picrylhydrazyl Method

Water-soluble extract or the isolated peptide fractions thereof were prepared in 0.1 M sodium phosphate
buffer, 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 WSE
or the isolated peptide fractions (sample) or 1.5 mL
of buffer (control) was mixed with 1.5 mL of DPPH
solution, and left in the dark at room temperature for
30 min. Absorbance of the solution was then measured
at 517 nm. The percentage decrease in absorbance of
the sample relative to the control was calculated as the
relative scavenging activity (Aluko and Monu, 2003;
Farzamirad and Aluko, 2008).
RP-HPLC Analysis of WSE

The water-soluble peptides in the traditional and

commercial yogurts were profiled on an RP-HPLC
(LC-20 AT series, Shimadzu) equipped with a Zorbax
300 SB-C3 guard column (7.0 m, 300 , 15 9.4 mm,
Agilent, Waldbronn, Germany) and Zorbax 300 SB-C8
monomeric column (6.5 m, 300 , 250 9.4 mm,
Agilent). The peptides were eluted in a linear gradient
from 100 to 0% solvent A (0.1% trifluoroacetic acid in
deionized water) in solvent B (0.1% trifluoroacetic acid
in 90%, vol/vol, acetonitrile in deionized water) over 80
min. 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 solvents
were filtered through a 0.45-m membrane filter.
The freeze-dried WSE (2.5 g) of yogurt samples were
dissolved in 5-mL aliquots of 0.1% TFA in deionized
water, and centrifuged at 14,000 g for 30 min. The
supernatants were filtered through a 0.45-m membrane filter and then injected (750 L) onto RP-HPLC.
Fractions at 10-min intervals (Figure 1) were collected,
lyophilized, and assayed for antioxidant activity (Donkor et al., 2007).
Statistical Analysis

Statistical analysis was performed using SPSS 17.0

for Windows (SPSS Inc., Chicago, IL). Results were
analyzed by repeated-measures ANOVA. Differences
between the treatment means were compared at the
5% level of significance using Tukeys test. Statistical
comparisons of the results were performed by calculating the correlation values for proteolytic activity, physicochemical variables, and microbiological analysis.
RESULTS AND DISCUSSION
Physicochemical, Microbiological, and Proteolytic
Activity Changes During Storage

The physicochemical and microbiological composition of the yogurt samples is shown in Table 1. Total

Figure 1. The elution profile of water-soluble extract fractions obtained by reversed phase-HPLC.
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4.19 0.75a,A
<10a,B
5.26 0.48a,A
<10a,B
8.60 0.12a,B
9.28 0.28a,A
5.22 0.80a,A
7.14 1.8a,A
5.46 0.85a,A
9.16 1.90a,A
4.17 0.75a,A
<10a,B
5.13 0.50a,A
<10a,B
8.67 0.14a,A
9.05 0.30ab,A
5.53 0.84a,A
7.77 1.88a,A
5.93 0.81a,A
9.42 1.82a,A
ac

A,B

Lactococcus spp.

Lactobacillus spp.

Total mesophilic aerobic bacteria

Molds-yeasts

Different lowercase superscripts depict the statistical difference within a row between time (P < 0.05).
Different uppercase superscripts depict the statistical difference between means for yogurt types (P < 0.05).

4.38 0.79a,A
<10a,B
4.94 0.61a,A
<10a,B
8.79 0.10a,A
9.03 0.23ab,A
5.73 0.87a,A
7.13 1.96a,A
6.78 0.72a,A
9.43 1.61a,A
4.28 0.78a,A
<10a,B
4.38 0.75a,A
<10a,B
8.81 0.08a,A
8.83 0.18ab,A
5.83 0.90a,A
6.87 2.02a,A
7.38 0.52a,A
8.91 1.17a,A
3.92 0.74a,A
<10a,B
4.00 0.74a,A
<10a,B
8.74 0.10a,A
8.41 0.21b,A
7.95 0.21a,A
7.46 0.46a,A
7.93 0.21a,A
8.98 0.47a,A
Traditional
Commercial
Traditional
Commercial
Traditional
Commercial
Traditional
Commercial
Traditional
Commercial
Microbiological (log cfu/g)
Coliforms

0.24a,B
0.54a,A
0.07a,A
0.16a,A
0.04b,B
0.08b,A
1.25a,A
2.80a,A

11.09
16.25
1.32
1.20
3.82
4.28
26.79
22.66
0.21a,B
0.48a,A
0.07a,A
0.15a,A
0.05b,B
0.11b,A
1.26a,A
2.81a,A

10.96
16.42
1.30
1.22
3.85
4.23
27.34
22.54
0.22a,B
0.49b,A
0.06a,A
0.14a,A
0.04b,B
0.09b,A
1.24a,A
2.78a,A

11.18
15.66
1.31
1.21
3.86
4.25
25.36
23.87
0.21a,B
0.46a,A
0.07a,A
0.16a,A
0.05c,B
0.11c,A
1.16a,A
2.59a,A

11.28
16.23
1.24
1.16
3.88
4.25
26.12
22.37
0.20a,B
0.44a,A
0.05b,A
0.11b,A
0.04a,B
0.09a,A
1.33a,A
2.97a,A

11.29
16.13
1.12
1.05
3.97
4.41
25.62
22.66

Week 3
Week 2
Week 1
Day 1
Yogurt

Traditional
Commercial
Traditional
Commercial
Traditional
Commercial
Traditional
Commercial
Fat in solids (%)

The main characteristic of an antioxidant is its ability

to trap free radicals. Highly reactive free radicals and

pH

Measurement of ABTS Radical Scavenging Activity

Acidity (%)

Protein contents of WSE of yogurts are shown in

Table 3. The differences in protein content between
traditional and commercial yogurts and the changes
during storage were not significant (P > 0.05).

Physicochemical
Total solids (%)

Protein Content of WSE of Yogurts

Analysis

solids, pH values, coliforms, and yeast-mold counts in

traditional yogurts were significantly different compared
with those of commercial yogurts (P < 0.01). However,
differences in acidity, fat in solids, TMAB (except at wk
4), and lactobacillus and lactococcus counts between
traditional and commercial yogurts were not significant
(P > 0.05). As shown in Table 1, the average coliform
bacteria and yeast-mold counts were much higher in
traditional yogurts than in commercial yogurts.
The changes during storage in total solids, lactic acid
percentage, and fat in solids were not significant (P >
0.05). A decrease in the pH value was observed during
the first 14 d of storage for all yogurts. Subsequently,
the pH value continued to decrease slightly during the
the rest of the storage period (P < 0.01). In terms
of lactic acid percentage, a significant increase was
observed during the first week of storage (P < 0.01).
Both pH and lactic acid percentage measurements are
important because acidification is the key mechanism
during yogurt fermentation.
Proteolysis is the breakdown of large and complex
proteins into smaller, simpler peptides. The average
proteolytic activity of yogurt samples during storage
was estimated by determination of free amino groups,
and results are presented in Table 2. The proteolytic
activity of both yogurt samples was higher compared
with raw skim milk as control. Proteolysis increased
significantly (P < 0.01) during storage. However, proteolytic activity values between traditional and commercial yogurt samples were not significantly different
(P > 0.05). Although the proteolytic activity values
between traditional and commercial yogurt samples did
not differ, as shown in Table 2, coliform bacteria and
yeast-mold counts were higher in traditional yogurts
than in commercial yogurts (Table 1). Proteolytic
activity has a positive correlation (P < 0.01) with coliform bacteria and yeast-mold counts, but a negative
correlation with pH value (P < 0.01).
Similar to results of other researchers, we showed that
differences in the proteolytic activity were dependent
on pH and strains (Shihata and Shah, 2000; Donkor et
al., 2007).

Week 4

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Table 1. Physicochemical and microbiological composition (mean SD) of the yogurt samples

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Table 2. The proteolytic activity (mean SD) of yogurt samples

Proteolytic activity (absorbance at 340 nm)
Yogurt
Traditional
Commercial
ac

Day 1

Week 1
c,A

Week 2
b,A

0.37 0.05
0.37 0.12c,A

Week 3
ab,A

0.58 0.07
0.49 0.15b,A

0.60 0.08
0.54 0.17ab,A

Week 4
ab,A

0.69 0.08
0.58 0.17ab,A

0.71 0.08a,A
0.62 0.17a,A

Different lowercase superscripts depict the statistical difference within a row between time (P < 0.05).
Different uppercase superscripts depict the statistical difference between means for yogurt types (P < 0.05).

AB

oxygen species are present in biological systems from a

wide variety of sources. These free radicals may oxidize
nucleic acids, proteins, lipids, or DNA and can initiate
degenerative disease (Zainoldin and Baba, 2010). The
TEAC (free radical and superoxide anion scavenging
activity) values of WSE for all types of yogurts are
shown in Table 4. In terms of antioxidant activity, a
significant difference was observed during the first week
of storage in the traditional yogurt WSE (P < 0.01).
Apart from that, changes in antioxidant activity in
both traditional and commercial yogurts during storage
were not significant (P > 0.05; Table 4). These mean
TEAC values ranged from 7.697 to 8.739 mM Trolox/g
in commercial yogurts, and from 10.115 to 13.182 mM
Trolox/g in traditional yogurts.
A few studies related to the production of antioxidant peptides in milk fermented with lactic acid bacteria indicate that the development of radical scavenging
activity is a strain-specific characteristic, and radical
scavengers are related to proteolysis (Kudoh et al.,
2001; Ryhnen et al., 2001; Hernndez-Ledesma et al.,
2005; Virtanen et al., 2007; Gupta et al., 2009).
Large chemical and microbiological differences were
observed between the traditional and commercial yogurt
samples (Table 1). These differences are believed to be
the cause of the differences between the TEAC values
because the antioxidant activity values of yogurt WSE
were found to be positively correlated with proteolytic
activity and negatively correlated with pH values (P
< 0.01). Gupta et al. (2009) concluded that the antioxidant activity (TEAC) of cheese is dependent on the
degree of proteolysis but only to a certain extent and is
dependent on the type of the starter culture used.
Virtanen et al. (2007) investigated the production of
antioxidant activity during fermentation of milk with
commonly used lactic starter cultures and the connec-

tion to proteolysis and bacterial growth. In their study,

molecular distribution profiles showed that fermentates
with high scavenging activity also possessed a greater
proportion of peptides in the molecular mass range of 4
to 20 kDa, whereas those with low scavenging activity
had mostly large polypeptides and compounds <4 kDa.
Measurement of DPPH Radical Scavenging Activity

2,2-Diphenyl-1-picrylhydrazyl is a stable free radical

that shows maximum absorbance at 517 nm (Szabo et
al., 2007; Park et al., 2008; Zhang et al., 2008). The
absorbance decreases because of a color change from
purple to yellow as the radical is scavenged by antioxidants. The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen-donating
ability (Bandonien and Murkovic, 2002).
In the present study, antioxidant activity was tested
using different methods (ABTS and DPPH). The difference between the radical scavenging capacity for DPPH
and ABTS could be due, in part, to the difference in
the solubility of the radicals and their diffusivity in
the reaction medium. Although DPPH scavenging is a
widely used method for the assessment of free radical
scavenging activity of natural products, it has a notable
limitation when used to imitate the role of hydrophilic
antioxidants. This is because DPPH can be dissolved
only in organic media (especially in alcoholic media)
and not in aqueous solution. A further disadvantage
is that DPPH serves both as oxidizing substrate and
as the reaction indicator molecule; therefore, the assay
may easily lead to the problem of spectral interference. In contrast, ABTS can be solubilized in aqueous
and organic media; thus, radical scavenging activities
of both hydrophilic and lipophilic compounds can be
measured (Tang et al., 2010).

Table 3. Protein contents (mean SD) of water-soluble extracts (WSE)

Protein content of WSE (mg/mL)
Yogurt
Traditional
Commercial
a

Day 1

Week 1
a,A

0.35 0.01
0.31 0.03a,A

Week 2
a,A

0.37 0.01
0.33 0.03a,A

Week 3
a,A

0.37 0.01
0.35 0.04a,A

Week 4
a,A

0.39 0.02
0.32 0.04a,A

0.41 0.02a,A
0.33 0.05a,A

Different lowercase superscripts depict the statistical difference within a row between time (P < 0.05).
Different uppercase superscripts depict the statistical difference between means for yogurt types (P < 0.05).

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Table 4. Radical scavenging activity (mean SD) of water-soluble extracts by ABTS [2,2c-azino-bis(3ethylbenzothiazoline-6-sulfonic acid)] method
Trolox equivalent antioxidant capacity (mM Trolox/g of lyophilized sample)
Yogurt
Traditional
Commercial
a,b

Day 1

Week 1
b,A

10.12 0.41
7.70 0.93a,B

Week 2
a,A

12.34 0.56
7.97 1.25a,B

Week 3
a,A

12.82 0.60
7.71 1.35a,B

Week 4
a,A

13.18 0.64
7.96 1.43a,B

13.15 0.78a,A
8.74 1.74a,B

Different lowercase superscripts depict the statistical difference within a row between time (P < 0.05).
Different uppercase superscripts depict the statistical difference between means for yogurt types (P < 0.05).

A,B

Our results, which are in agreement with these findings, suggest that the ABTS method was more sensitive
than the DPPH assay for the measurement of antioxidant activity of water-soluble proteins and peptides in
an aqueous solution. Inhibition values of WSE during
storage are shown in Table 5. As shown in Table 5, in
the DPPH method, a fluctuation was observed in the
percentage inhibition values during storage period. This
fluctuation was most likely the result of blur arising due
to dissolved peptides during the DPPH assay. Because
of this, only the ABTS method was used during peptide
analysis, and the DPPH method was considered unsuitable.
Chen et al. (2003) found that the ABTS method was
suitable and sensitive to determine the antioxidant capacity in milk and milk fractions. Nishino et al. (2000)
reported that heat treatment enhanced the DPPH
radical scavenging activity of skim milk, with the activity being further increased by fermentation with Lactobacillus casei Shirota. These authors also suggested
that casein hydrolysate present in the fermented milk
might enhance radical scavenging activity. Kudoh et al.
(2001) found DPPH radical scavenging activity in peptides isolated from milk fermented with Lactobacillus
delbrueckii ssp. bulgaricus. The amino acid sequence of
the peptide was determined to be Ala-Arg-His-Pro-HisPro-His-Leu-Ser-Phe-Met, which corresponded with
the amino acid sequence of -casein from 96 to 106.
Liu et al. (2005) investigated the effect of fermentation by kefir grains on the scavenging activity of DPPH
radicals of milk and soymilk. Both milk kefir and soymilk kefir displayed significantly greater scavenging
activities than milk and soymilk, indicating that some

components of the antioxidants present in the kefir

grains were transferred to milk and soymilk during fermentation. McCue and Shetty (2005) produced yogurt
in a similar way from soymilk using kefir grains and
they related the radical scavenging activity of DPPH
to the mobilization of phenolic compounds (Smet et
al., 2008).
Gmez-Ruiz et al. (2008) determined the antioxidant activity of ovine milk casein fractions before and
after their hydrolysis with gastrointestinal enzymes.
The results show that enzymatic hydrolysis enhanced
the antioxidant activity in all fractions but especially
in the -casein fraction. Zainoldin and Baba (2010)
enriched yogurt with different fruits and investigated
the antioxidant activity using the DPPH radical scavenging method. It was shown that all fruit-enriched
yogurts showed an increment in percentage of inhibition compared with plain yogurt (19.16 to 45.74%)
and it was suggested that addition of dragon fruit
into yogurt might change or enhance the percentage
of inhibition.
Separation of Peptide Fractions by RP-HPLC

To monitor the changes in peptide profiles during

storage of WSE of yogurts, WSE from d 1 and 28 of
storage were analyzed by RP-HPLC. Figure 2 shows
the RP-HPLC profiles obtained from d 1 and 28 of yogurt WSE. The chromatograms of yogurt samples were
similar: during storage, the peaks increased in height
but no significant changes in the chromatographic profile occurred. Therefore, in further purification trials, d
28 samples were used.

Table 5. Radical scavenging activity (mean SD) of water-soluble extracts (WSE) by DPPH (2,2-diphenyl1-picrylhydrazyl) method
Inhibition values (%) of WSE during storage
Yogurt
Traditional
Commercial

Day 1

Week 1
a,A

17.36 2.41
18.67 5.40a,A

a,b

a,A

17.14 5.29
24.42 11.83ab,A

Week 2
a,A

16.78 3.58
16.54 8.01a,A

Week 3
a,A

15.96 2.30
17.29 5.16a,A

Week 4
17.70 4.86a,A
31.22 10.87b,B

Different lowercase superscripts depict the statistical difference within a row between time (P < 0.05).
Different uppercase superscripts depict the statistical difference between means for yogurt types (P < 0.05).

A,B

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Figure 2. Reversed phase-HPLC peptide profile of yogurt sample at d 1 and 28. Color version available in the online PDF.

Papadimitriou et al. (2007) investigated the proteolysis of traditional sheep milk yogurt and the effect
thereon of a probiotic strain (Lactobacillus paracasei
ssp. paracasei DC412) when used as an adjunct to the
normal yogurt culture; they also identified the major
peptides produced during storage. Both products had
similar physicochemical properties and proteolysis patterns throughout storage. The peptide profiles obtained
were similar and only some quantitative differences
were observed, concerning the area of some peaks. The
chromatograms of WSE of probiotic yogurt showed
that most of the peaks were formed by d 2 of storage.
Further Fractionation of Selected Yogurt Samples

Water-soluble extracts of yogurt samples were subjected to fractionation by RP-HPLC as described in

the Materials and Methods section. In the first step, 6

fractions were collected, concentrated under vacuum,
and lyophilized, and then antioxidant activities were
measured. Figure 3 shows TEAC values of RP-HPLC
peptide fractions (F)1 to F6.
The highest antioxidant activity was found in F2;
fractions F3, F4, and F5 had relatively high antioxidant activity. Fractions with low antioxidant activity
were not fractionated any further. The 4 fractions with
high antioxidant activity (F2, F3, F4, F5) were further
separated at 1.33-min intervals, collected, lyophilized,
and assayed for antioxidant activity.
The highest antioxidant activity was found in subfractions F2.2, F2.3, F4.3, and F4.4, as shown in Table
6. The total antioxidant activity of yogurts was low
but after purification by fractionation in HPLC, high
antioxidant activity peptide fractions were obtained.

Figure 3. Trolox equivalent antioxidant capacity (TEAC) values of reversed phase-HPLC peptide fractions F1 to F6.
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Antioxidant activity of peptides released from HPLC

fractions of selected yogurt samples were increased 10
to 200 times.
In different studies, it was demonstrated lactic acid
bacteria scavenge reactive oxygen species and show
antioxidative activity (Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus, and
Bifidobacterium longum). Furthermore, Strep. thermophilus and Lb. delbrueckii ssp. bulgaricus produced
an antioxidative effect on the inhibition of linoleic acid
peroxidation and the inhibition of low density lipoprotein oxidation in vivo (Lin and Yen, 1999; Jimnez et
al., 2008).
Tsai et al. (2008) fermented milk for up to 5 h at
43C with Strep. thermophilus and Lb. bulgaricus. A
protease, flavorzyme, was added at the beginning of fermentation. During the 5 h of fermentation, the soluble
protein content increased from 4.9 to 57.4 mg/g and
the peptide content increased from 2.1 to 32.8 mg/g.
Hernndez-Ledesma et al. (2007) determined
oxygen radical absorbance capacity (ORAC) of the
-lactoglobulinderived peptides. The radical scavenging activity values ranged from 4.45 to 7.67 mol

Table 6. Trolox equivalent antioxidant capacity values (mean SD)

in yogurt water-soluble extract sub-fractions
Sub-fraction

(mM Trolox/g)

F2.1
F2.2
F2.3
F2.4
F2.5
F2.6
F2.7
F2.8

349.78
2,812.99
2,463.89
666.03
1,615.90
838.18
463.84
273.46

23.12
12.35
24.21
23.57
20.54
12.45
19.43
16.23

F3.1
F3.2
F3.3
F3.4
F3.5
F3.6
F3.7
F3.8

980.10
667.65
1,063.39
1,501.64
1,477.63
1,251.69
1,783.26
1,763.30

12.46
11.10
12.13
32.67
16.43
17.54
23.54
22.23

F4.1
F4.2
F4.3
F4.4
F4.5
F4.6
F4.7

419.40
561.90
2,157.35
1,986.68
1,388.01
1,473.18
705.62

23.11
14.43
32.22
23.65
22.22
23.25
10.15

F5.1
F5.2
F5.3
F5.4
F5.5
F5.6
F5.7

1,045.59
457.60
710.95
368.74
514.84
719.42
219.27

12.34
22.56
22.32
15.32
24.45
32.12
11.34

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(Trolox equivalents/mol of peptide). The presence

and position of amino acids Trp, Tyr, and Met were
thought to be responsible for the antioxidant activity.
Songisepp et al. (2004) reported the total antioxidant
activity of probiotic cheese and it was found to increase
with an increase in ripening period, but they reported
the antioxidant activity of the probiotic culture isolated
from cheese. Ripening of cheese leads to formation of
numerous peptides that originate mainly from casein
as a result of proteolysis. Addition of adjunct cultures
changes the proteolytic rate and its pattern, resulting
in the release of more peptides in the cheese.
CONCLUSIONS

The antioxidant activity was higher in traditional

yogurt than in commercial yogurts, most likely reflecting differences in chemical and microbiological values.
Traditionally produced yogurts are widely consumed
but their physicochemical and microbiological properties did not meet the standards (Anonymous, 1999a;
Anonymous, 2009). In particular, the yeast-mold and
coliform bacteria counts were found to be above the
permissible limits. Commercial yogurts were found appropriate to the standards cited above. In this study,
the ABTS and DPPH methods were used to measure
the total antioxidative capacity of yogurt WSE. Of
these 2 methods, the ABTS method was the more sensitive. The antioxidant activity in WSE was greater in
traditional yogurts than in commercial yogurts. Antioxidant activity of peptides released from HPLC fractions of selected yogurt samples was increased 10 to 200
times. Total antioxidant activity in yogurts was low but
after purification of peptides by fractionation in HPLC,
fractions with high antioxidant activity were obtained.
The antioxidant activities are characteristics of yogurt
peptides and such activity will expand their applications as functional food ingredients. Currently, further
research studies are being carried out to determine the
mechanism of action as well as the in vivo antioxidant
properties of these yogurt-derived peptides.
ACKNOWLEDGMENTS

This work was funded by Sleyman Demirel University, Unit of Scientific Research Projects, Turkey (project no.1580-D-07). The authors thank Ender Olcayto
(Scotland) for his help in editing the language of the
manuscript.
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