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Food Chemistry 150 (2014) 34–40

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Comparison of antioxidant capacity, protein profile and carbohydrate

content of whey protein fractions
Evren Önay-Uçar a, Nazlı Arda a, Murat Pekmez a, Aysße Mine Yılmaz b, Nazlı Böke-Sarıkahya c,
Süheyla Kırmızıgül c, A. Suha Yalçın b,⇑
a
Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, 34134 Vezneciler-Istanbul, Turkey
b
Department of Biochemistry, School of Medicine, Marmara University, 34668 Haydarpasßa-Istanbul, Turkey
c
Section of Organic Chemistry, Department of Chemistry, Faculty of Science, Ege University, 35500 Bornova-Izmir, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Whey is used as an additive in food industry and a dietary supplement in nutrition. Here we report a
Available online 1 November 2013 comparative analysis of antioxidant potential of whey and its fractions. Fractions were obtained by size
exclusion chromatography, before and after enzymatic digestion with pepsin or trypsin. Superoxide rad-
Keywords: ical scavenging, lipid peroxidation inhibition and cupric ion reducing activities of different fractions were
Antioxidant activity checked. Peptides were detected by SDS–PAGE and GC–MS was used to determine carbohydrate content
CUPRAC of the fractions. All samples showed antioxidant activity and the second fraction of the trypsin hydroly-
Whey protein fractions
sate showed the highest superoxide radical scavenging activity. CUPRAC value of this fraction was two-
times higher than that of whey filtrate. The first fraction of the pepsin hydrolysate was the most effective
inhibitor of lipid peroxidation. Each sample exhibited a different polypeptide profile. Different percent-
ages of carbohydrates were identified in whey filtrate and in all second fractions, where galactose was
the major component.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction immunomodulating, opioid and hypocholesterolemic activities of

Different commercial whey products, such as whey powder
(WP), whey protein concentrate (WPC) or whey protein isolate
(WPI), are available in the market. These products are mainly used
as a nutritional ingredient of infant formulas and a protein supple-
ment for body builders and athletes (Bayram, Pekmez, Arda, &
Yalçın, 2008; Hernández-Ledesma, Recio, & Amigo, 2008). Major
components of whey are b-lactoglobulin (b-Lg) and a-lactalbumin
(a-La). Bovine serum albumin (BSA), lactoferrin (LF), immunoglob-
ulins (IgA, IgG, IgM), lactoperoxidase, lysozyme, relaxin, cytokines,
growth factors such as insulin-like growth factor (IGF-1 and -2),
transforming growth factor (TGF-b), platelet-derived growth factor
(PDGF), fibroblast growth factor (FGF), and free amino acids are
also found in whey. In addition to the protein, peptide and pep-
tide-related constituents, whey contains carbohydrates (mainly
lactose), lipids (fatty acids and phospholipids), minerals and vita-
mins (Madureira, Pereira, Gomes, Pintado, & Malcata, 2007).
Biological properties and therapeutic potential of whey proteins
and peptides have been reviewed in detail (Madureira et al., 2007;
Yalçın, 2006). Health-promoting effects of whey are related to anti-
oxidant, acetylcholine esterase (ACE) inhibitory, antimicrobial,
⇑ Corresponding author. Tel.: +90 533 397 87 29.
E-mail address: asyalcin@marmara.edu.tr (A.S. Yalçın).
specific peptides and amino acids, predominantly derived from
b-Lg (Hernández-Ledesma et al., 2008). Since oxidative stress is
associated with several disease states, antioxidant activity of whey
products and hydrolysates has been extensively investigated by
various in vitro techniques (Table 1). Protective effects of dietary
whey have also been examined by in vivo techniques. Its wound
and burn healing properties have been documented (Jahovic
et al., 2005; Velioğlu-Öğünç et al., 2008; Öner, Velioğlu-Öğünç, Cin-
gi, Bozkurt-Uyar, & Yalçın, 2006). Peptides generated from the
digestion of milk and whey proteins are reported to have antioxi-
dative effects (Pihlanto, 2006). Moreover, antioxidant activity is
elevated by fractionation or hydrolysis of whey proteins (Bayram
et al., 2008; Peña-Ramos & Xiong, 2003). Thus, it seems that pep-
tides have priority as functional constituents. Studies on digestion
as well as hydrolysis of whey proteins indicated that some
peptides, which are inactive within the sequence of intact protein
may be activated during gastrointestinal digestion or food
processing.
Although it is clear that major whey constituents have significant
antioxidant potential, active principles and action mechanisms are
not fully understood. In our previous study, b-Lg and a-La could be
easily separated by Sephadex G-200 gel filtration chromatography
supported with proteolytic cleavages, and free radical scavenging
activity of whey and its fractions before and after protease treatment

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.10.119
 Author's personal copy
E. Önay-Uçar et al. / Food Chemistry 150 (2014) 34–40
Table 1
Literature survey of different antioxidant activity tests used for whey proteins and hydrolysates.
Method
Radical scavenging (superoxide)
Radical scavenging (DPPH, superoxide, hydroxyl)
Lipid peroxidation inhibition (TBARS)
Lipid peroxidation inhibition (TBARS)
Lipid peroxidation inhibition (TBARS)
Lipid peroxidation inhibition (TBARS)
Lipid peroxidation inhibition (TBARS), radical (peroxyl)
scavenging, Iron chelating
Lipid peroxidation inhibition (TBARS and conjugated dienes)
Lipid peroxidation inhibition (TBARS)
Lipid peroxidation inhibition
was investigated by diphenylpicrylhydrazyl (DPPH) test (Bayram
et al., 2008). It was observed that the DPPH scavenging activity
was enhanced more than 11-fold with pepsin cleavage.
Here we analysed antioxidant potential of whey and hydrolysed
whey fractions eluted from Sephadex G-50 column and aimed to
find out possible active constituents, focusing on polypeptides
and carbohydrates. We used three test systems for detecting of
antioxidant activity in which superoxide radical scavenging
activity, lipid peroxidation inhibition and cupric ion reducing anti-
oxidant capacity (CUPRAC) of the fractions were evaluated. Poly-
acrylamide gel electrophoresis (SDS–PAGE) was performed to
determine polypeptide profiles. Free carbohydrates were detected
by gas chromatography–mass spectrometry (GC–MS). The results
are expected to contribute to our understanding of which whey
fractions are more active and which bioactive constituents are
found in native and hydrolysed whey samples after food process-
ing or digestion.
2. Materials and methods
2.1. Chemicals and reagents
Whey powder was obtained from Sütasß Milk and Dairy Products
Company (Bursa, Turkey). Pepsin (2500–3500 units/mg), ascorbic
acid, 2-thiobarbituric acid (TBA), neocuproine, nitroblue tetrazo-
lium chloride (NBT), riboflavin, ethlyenediaminetetraacetic acid
(EDTA), L-a-phosphatidylcholine, 6-hydroxy-2,5,7,8-tetrame-
thylchroman-2-carboxylic acid (Trolox) were purchased from Sig-
ma–Aldrich (Germany). Trypsin (approx. 9000 units/mg), ferrous
chloride tetrahydrate and Coomassie brilliant blue R-250 were
purchased from Fluka Riedel-deHaën (Germany). Trichloroacetic
acid, copper chloride, hydrogen peroxide, methanol, ammonium
acetate, sodium carbonate, potassium dihydrogen phosphate,
methionine, barium carbonate, hydrochloric acid, hexane, trimeth-
ylchlorosilan, hexamethyldisilazan, methanol, benzene, chloro-
form, pyridine and sodium hydroxide were purchased from
Merck (Germany). All solvents used in GC–MS analysis were
HPLC-grade. Dipotassium hydrogen phosphate was purchased
from BDH (UK). Sephadex G-50 was purchased from Pharmacia
(Sweden).
2.2. Whey and whey fractions
Whey powder was suspended in hexane (1:16, w/v) and centri-
fuged at 3000g at room temperature for 10 min to remove lipid-
soluble constituents. Hexane washing was repeated three times.
Residues were combined and suspended in ultra-pure water at a
35
Sample Reference
Whey protein fractions and hydrolysates Bayram et al. (2008)
Whey protein isolate and hydrolysates Peng et al. (2009)
Whey fractions Tong, Sasaki, McClements, and
Decker (2000a)
Whey protein concentrate (WPC) McCarthy, Kerry, Kerry, Lynch, and
Buckley (2001)
Whey protein isolate (WPI) hydrolysates Peña-Ramos and Xiong (2001)
Whey powder Coronado et al. (2002)
Whey Tong et al. (2000b)
Whey protein isolate (WPI) and hydrolysates Peña-Ramos and Xiong (2003)
Whey protein isolate (WPI), hydrolysed WPI fractions, Peña-Ramos et al. (2004)
commercial WPI hydrolysates
Acid whey (after ultrafiltration, dialysis, heat treatment and Colbert and Decker (1991)
chloroform extraction)
concentration of 6% (w/v) by vortexing and centrifuged at
3000g at room temperature for 10 min. The clear supernatant
filtered through a 0.45 lm membrane filter was directly applied
to a Sephadex G-50 column as whey filtrate (WF), or subjected to
protease (pepsin or trypsin) treatment followed by chromato-
graphic separation. Protein concentration of WF and all fractions
was determined by a modified Lowry method (Waterborg, 2002).
2.3. Protease treatment
Pepsin and trypsin were used for digestion of whey proteins.
The pH of WF was adjusted using 1 M HCl to 1.5 for pepsin diges-
tion or to 9.0 using 1 M NaOH for trypsin digestion. Digestion was
carried out at 37 °C for 30 min with a protein to enzyme ratio of
1:100 (w/w) for pepsin, and 1:50 (w/w) for trypsin. For pepsin
and trypsin digestions, reactions were stopped by adding 1 M
NaOH or 1 M HCl to adjust the pH to 7.8 or 1.5, respectively (Bay-
ram et al., 2008). This was preferred to heating in order to avoid
denaturation of small peptides.
2.4. Chromatographic separations
Whey filtrate and pepsin- or trypsin-hydrolysed WF were sepa-
rated by size exclusion chromatography in a column (1.5  30 cm)
packed with Sephadex G-50. The column was equilibrated and
eluted with 0.02 M phosphate buffer, pH 8.6 at a flow rate of
0.3 mL/min. Fractions of 1–1.5 mL were collected according to their
protein content (A280).
2.5. Protein electrophoresis
Polypeptides were analysed by SDS–PAGE in a Bio-Rad mini gel
system, as described by Walker (2002). A constant current (3 mA/
well for stacking gel and 6 mA/well for running gel) was applied
through the gel, and the gels were stained with silver nitrate
(Ausubel et al., 1989).
2.6. Carbohydrate analysis
Carbohydrate content was investigated by GC–MS (Ye, Yan, Xu,
& Chen, 2006). Briefly, samples were dried under vacuum at 4 °C
overnight, dissolved in MeOH (10 mL) and incubated in an oil bath
at 95 °C for 6 h under reflux by adding 2 N HCl (10 mL) and ben-
zene (10 mL). After cooling, the acidic hydrolysates were extracted
with chloroform and the aqueous phases were neutralised with
BaCO3. Then solid particles were removed, aqueous phase was
evaporated at 40 °C in a rotary evaporator, dried in a vacuum
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36 E. Önay-Uçar et al. / Food Chemistry 150 (2014) 34–40
incubator, solubilised in 1.0 mL of dried pyridine and silylyllated
with trimethylchlorosilan (0.5 mL) and hexamethyldisilazane
(0.5 mL) by refluxing in an oil bath at 65–70 °C for 1 h under CaCl2
tube. Finally, samples were dried with inert nitrogen (N2), dis-
solved in hexane and analysed in a combined GC–MS system (HP
6890–5973) using apolar column (HP-5MS, 5% phenyl methyl
cycloxane, 30 m  0.25 mm  0.25 lm) under defined conditions
(80 °C for 3.5 min; max. temp: 300 °C, inlet temp: 250 °C, pressure:
9.2 psi, split system, 50:1, at a constant flow rate mode with
49.4 mL/min velocity, max. column temp: 325 °C, carrier gas:
He). Final chromatograms of the samples were evaluated by com-
paring the results with standard carbohydrates as well as using a
database (Wiley-Nist) stored in the system.
2.7. Superoxide radical scavenging activity
Superoxide radical scavenging ability of the fractions was deter-
mined by the method of Martinez, Loureiro, Oliva, and Maestri
(2001) with a slight modification, on the basis of in vitro inhibition
of NBT formation. In this assay system, photochemical reduction of
flavins generate O2-, which reduces NBT, resulting in the formation
of blue formazan. The active principles inhibit the formation of the
blue formazan and % inhibition is proportional to the concentration
of the constituents. Briefly, sample solution was mixed with the as-
say mixture (1:3, v:v), containing 50 mM phosphate buffer, pH 7.8,
13 mM methionine, 2 lM riboflavin, 100 lM EDTA and 75 lM NBT.
Assay mixtures containing ultra-pure water instead of WF or elu-
tion buffer (0.02 M phosphate buffer, pH 8.6) instead of column
fractions were used as blanks. Production of blue formazan was
followed by monitoring the increase in absorbance at 560 nm after
10 min illumination by a fluorescent lamp (15 W). Percentage
inhibition of superoxide anion formation was calculated using
the following formula: % inhibition = [(A0 A1)/A0]  100, where
A0 = Absorbance of the blank; A1 = Absorbance of the sample. Trol-
ox was used as standard and reference antioxidant. All analyses
Fig. 1. Flowchart showing the individual steps followed during sample preparation.
were run in triplicates and the results were expressed as the
mean ± SD of protein concentration (lg/mL) IC50 leading to 50%
inhibition of superoxide formation.
2.8. Lipid peroxidation inhibitory activity
Iron(II)-mediated liposomal peroxidation system was estab-
lished as described earlier (Duh, Duh, & Yen, 1999) with a slight
modification. Small unilamellar vesicles were prepared by sonicat-
ing of 300 mg phosphatidylcholine (soy lecithin) in 30 mL of
10 mM phosphate buffer (pH 7.4) on ice for 2 h. The liposome solu-
tion (250 lL) and sample or blanks (450 lL) was made up to 1.0 mL
of final volume with FeCl2, H2O2 and ascorbic acid, each in a final
concentration of 125 lM. After incubation in a shaker incubator
(CertomatÒ H, Braun) (50 rpm, 30 °C) for 4 h, thiobarbituric acid
reactive substances in the mixture were detected. The mixture
(250 lL) was added to 500 lL of trichloroacetic acid (TCA)–thio-
barbituric acid (TBA)–HCl reagent, containing 15% (w/v) TCA,
0.375% (w/v) TBA and 0.25 M HCl and kept in a boiling water bath
for 15 min. After cooling, the mixture was centrifuged at 1000g
for 5 min and the absorbance of the supernatant was recorded at
532 nm against blank, which contained all reagents except lecithin.
Trolox was used as standard and reference antioxidant. All analy-
ses were run in triplicates and the results were expressed as the
mean ± SD of protein concentration (lg/mL) leading to 50% inhibi-
tion (IC50).
2.9. Cupric ion reducing antioxidant capacity
The cupric reducing antioxidant capacity (CUPRAC) of the
samples was determined using bis(neocuproine)copper(II) chloride
as described by Apak, Güçlü, Özyürek, Karademir, and Erçağ
(2006). Briefly, 1.0 mL of CuCl2 solution (10 2 M), 1.0 mL of freshly
prepared neocuproine alcoholic (96% ethanol) solution
(7.5  10 3 M) and 1.0 mL of ammonium acetate buffer (1 M) were
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E. Önay-Uçar et al. / Food Chemistry 150 (2014) 34–40
mixed with x mL of the sample, and diluted to a final volume of
4.1 mL by adding 1.1-x mL ultra-pure water. Trolox solution
(1  10 3 M) prepared in 96% ethanol was used as standard. The
assay mixture was kept at room temperature for 30 min and absor-
bance at 450 nm against a reagent blank was measured in a micro-
plate reader (BioLab-lQuant). CUPRAC value was calculated as
mmol trolox equivalent per mg protein. In our assay system, the
molar absorptivity of trolox was e = 1.74  104 L/mol/cm.
2.10. Statistical analysis
Data were expressed as mean ± SEM of three independent
experiments, each done in triplicate. Statistical analyses were
performed using one-way ANOVA followed by Tukey’s Multiple
Comparison Test using GraphPad Prism version 5.00 for Windows,
GraphPad Software, San Diego, California, USA (www.graph-
pad.com). The probability values of P < 0.05 were considered as
statistically significant.
3. Results and discussion
3.1. Whey fractions and polypeptide analysis
Fig. 1 shows the isolation procedure which yielded a total of six
column fractions from whey samples. In our previous study, frac-
tions were obtained from WF after protease (pepsin and trypsin)
treatment with a Sephadex G-200 column (Bayram et al., 2008).
When WF and digested WF was applied to a Sephadex G-50 col-
umn, two fractions were obtained. These fractions were designated
as F-1 and F-2 from WF; FP-1 and FP-2 from pepsin-digested whey
and FT-1 and FT-2 from trypsin-digested whey. Elution profiles are
presented in Fig. 2.
Fig. 3 shows the polypeptide profiles of the fractions. Compari-
son of the bands obtained from F-1 and F-2 fractions of WF showed
that polypeptides between 14.4 and 160 kDa were present in F-1,
whereas no peptide band was observed in F-2. As the concentra-
tions of the samples applied to the gel were equal, peptides and
amino acids found in F-2 fraction presumably eluted out of the
gel during electrophoresis (Peña-Ramos & Xiong, 2001). Electro-
phoretic results observed for fractions obtained from pepsin and
trypsin digestions (FP-2 and FT-2) were similar to F2. In our previ-
ous study (Bayram et al., 2008), it was found that a-lactalbumin
and b-lactoglobulin could be eliminated by treatment with pepsin
and trypsin, and consequently were easily separated from each
Fig. 2. Elution profiles obtained from Sephadex G-200 column chromatography of whey filtrate before and after pepsin/trypsin treatment.
37
other. Here, chromatographic separation with Sephadex G-50 pro-
duced distinct constituents of whey and whey hydrolysates, but
not as effective and in the same manner with regard to peptides
observed in Sephadex G-200 chromatography. However, it seems
that high molecular weight constituents could be easily separated
from low molecular weight ones by Sephadex G-50.
3.2. Superoxide radical scavenging activity
All samples and fractions were able to scavenge superoxide rad-
icals (Table 2). The column fractions as well as digestion products
were more active than WF (P < 0.001, R2: 0.9364). Moreover, F-2,
FP-1, FT-1 and FT-2 had higher activity compared to the reference
antioxidant Trolox. The half maximal inhibitory concentration
(IC50) of Trolox, F-2, FP-1, FT-1, and FT-2 were 0.052 ± 0.002,
0.042 ± 0.005, 0.039 ± 0.010, 0.038 ± 0.005, and 0.029 ± 0.005 mg/
mL, respectively. FT-2 had the highest activity, approximately
1.8-fold more than Trolox. When F-1 and its hydrolysates are com-
pared, FP-1 and FT-1 were significantly more effective than F-1
(P < 0.001). Although FP-2 was less effective compared to F-2
(P < 0.001), FT-2 was not significantly different compared to F-2.
In our previous work with Sephadex G-200 column fractions,
antioxidant activity was found to be increased by protease treat-
ment (Bayram et al., 2008). Similar results were obtained in this
study. To date, several studies on the peptides derived from diges-
tion have been conducted. In general, antioxidant whey proteins or
constituents (mainly, a-La and b-Lg) have been enzymatically
hydrolysed by commercial proteases such as pepsin and trypsin
(Bayram et al., 2008; Hernández-Ledesma, Dávalos, Bartolomé, &
Amigo, 2005; Peña-Ramos & Xiong, 2001), chymotyrpsin (Hernán-
dez-Ledesma et al., 2005; Peña-Ramos & Xiong, 2001) thermolysin
and corolase (Hernández-Ledesma et al., 2005), papain, protease A,
P and F (Peña-Ramos & Xiong, 2001), flavourzyme and protome
(Peña-Ramos & Xiong, 2003; Peña-Ramos, Xiong, & Arteaga,
2004), and alcalase (Colbert & Decker, 1991; Peña-Ramos et al.,
2004). Radical scavenging activity of the hydrolysates resulted
from proteolytic cleavage of a-La and b-Lg have been determined
using oxygen radical absorbance capacity (ORAC) assay by Hernán-
dez-Ledesma et al. (2005). Among the 42 peptide fragments ob-
tained from b-Lg one sequence has been found to possess a very
high radical scavenging activity, even higher than that of BHT
(butylated hydroxy toluene). In another work, alcalase-hydrolysed
whey proteins were fractionated by Sephadex G-10 gel filtration
chromatography and the strongest radical (hydroxyl, superoxide
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38 E. Önay-Uçar et al. / Food Chemistry 150 (2014) 34–40
Fig. 3. Electrophoretic pattern of proteins and polypeptides of whey fractions in
silver nitrate-stained gel.
and DPPH) quenching effect was reported for short peptides in the
0.1–2.8 kDa range (Peng, Xiong, & Kong, 2009). Thus, it seems that
whey constituents which are able to scavenge superoxide radicals
could be produced by proteolytic cleavage and eluted in different
fractions.
3.3. Lipid peroxidation inhibitory activity
In this study, peroxidation of liposomes was triggered by Fe
(II)–ascorbic acid–H2O2 and the end products of the process were
measured as thiobarbituric acid reactive substances (Table 2). Lipid
peroxidation inhibitory activity of the fractions were rather low
compared to Trolox. FP-1 and FT-1 showed significant activity,
with IC50 values of 0.369 ± 0.031 and 0.420 ± 0.096 mg/mL (R2:
0.885), respectively. When F-1 and its hydrolysates were com-
pared, FP-1 and FT-1 were significantly more effective than F-1
(P < 0.001). FP-2 and FT-2 did not differ significantly compared to
F-2.
Similar results were obtained for the hydrolysates using pure
enzymes (pepsin, papain, trypsin and chymotrypsin) (Peña-Ramos
& Xiong, 2001). These hydrolysates have no, or little preventive
effect on TBARS formation in a liposome model, although WPI
hydrolysed by the commercial crude enzymes, especially protease
F exhibited antioxidant activity. Thus, whey is regarded as an
acceptable ingredient for reducing lipid oxidation in food industry.
The WPI and hydrolysates were also shown to inhibit lipid oxida-
tion in cooked patties (Peña-Ramos & Xiong, 2003). WPI hydroly-
sates obtained from protamex cleavage was more active than
non-hydrolysed and flavorzyme-treated WPI. Antioxidant poten-
tial of whey has been evaluated in raw and cooked pork patties
(McCarthy, Kerry, Kerry, Lynch, & Buckley, 2001) and in wiener
sausages (Coronado, Trout, Dunshea, & Shah, 2002) as well. Antiox-
idant constituents (lipid peroxidation inhibitors) of WPI/WPI
hydrolysates (Peña-Ramos & Xiong, 2001; Peña-Ramos et al.,
2004) and acid whey (Colbert & Decker, 1991) are suggested to
be mostly low molecular weight substances (<10 kDa). The pri-
mary antioxidants in acid whey were proposed to be polar and
heat-stable compounds between 500 and 5000 kDa molecular
weight (Colbert & Decker, 1991). On the other hand, Tong, Sasaki,
McClements, and Decker (2000a, 2000b) found that the high
molecular weight fraction of whey inhibited lipid peroxidation in
a salmon oil emulsion in a dose-dependent manner. It was pro-
posed that lipid peroxidation inhibitory effect could be related to
the amino acid profile as well as to peptide fractions that mainly
contained hydrophobic (Ile, Leu, Val, Thr, Ser, Ala) and positively
charged (Lys, Arg, His) amino acids (Peña-Ramos et al., 2004).
Sinha, Radha, Prakash, and Kaul (2007) suggested that high content
of sulphur-containing amino acids in whey proteins supported
antioxidant function. In our study, the most active fractions (FP-1
and FT-2) were found to exhibit distinctly different peptide pro-
files. Thus, our findings confirm that enzymatic hydrolysis of WF
by pepsin and tyrpsin produces more active fragments and these
fragments may exist in different fractions depending on chroma-
tography procedure.
3.4. Cupric ion reducing antioxidant capacity
In order to determine cupric ion reducing antioxidant capacity
of the samples, a simple method abbreviated as the CUPRAC meth-
od, utilising the copper(II)-neocuproine reagent as the chromo-
genic oxidising agent was used. CUPRAC method has been
utilised for the first time as an antioxidant activity test for whey
fractions (Table 2). The highest CUPRAC value was observed in
FT-2 (0.350 ± 0.044 mmol TR/mg protein) compared to WF
(P < 0.001, R2:0.929). CUPRAC values of FT-1 and FP-1 fractions
were also notably high (0.309 ± 0.054 and 0.260 ± 0.059 mmol
TR/mg protein, respectively). Protease treatments increased the
copper reducing capacity of whey. Especially trypsin treatment en-
hanced the CUPRAC value. The results were compared to data on
some herbal teas (Apak et al., 2006) as there is no previous data
on CUPRAC values of whey and whey hydrolysates. All fractions
were active as much as some of the herbal teas and teabags such
as common buckthorn (Rhamnus cathartica), common mallow
(Malva sylvestris), stinging nettle (Urtica dioica) and lime (Tilia euro-
paea). These results correlated with a previous report (Tong et al.,
2000b) and suggested that whey and whey hydrolysates could
have metal ion reducing/chelating efficacy.
3.5. Carbohydrates
Carbohydrates and polyols were not detected in the first
column fractions (F-1, FP-1 and FT-1), probably because these mol-
ecules were not free in these fractions and/or the volume of the
eluent was inadequate. Alpha-D-galactose, a-D-mannose, b-D-ta-
lose, b-D-gulose, a-D-talose, a-D-glucose, b-D-glucose, b-D-galact-
ose, a-L-altrose, b-D-mannose and myo-inositol were found in WF
as well as in F-2, FP-2 and FT-2, in a mean ratio of 22.43%,
18.37%, 11.19%, 10.28%, 7.45%, 7.36%, 7.09%, 5.53%, 3.61%, 3.24%
and 0.19%, respectively (Table 3). The percentage of total sugar
and sugar-like components of the fractions were 97.25, 97.16,
98.99 and 93.46 for WF, F-2, FP-2 and FT-2, respectively.
These results showed that galactose was the most abundant su-
gar (27.96%) in examined fractions. High amount of galactose may
have resulted from the culture used in cheese production or impro-
per storage (>4 °C) of whey powder (Rao, Wendorff, & Smith,
2004). Only one polyol, myo-inositol was detected, although its
percentage was rather low (0.14–0.23%). It is well known that
inositol and inositol-like compounds have considerable roles in
regulating vital cellular functions such as signal transduction, cell
proliferation and differentiation. Recently, they have received
much attention for their roles in antioxidant defence mechanisms
as well as cancer prevention and therapy (Vucenik & Shamsuddin,
2006). Thus, myo-inositol can be suggested as an active constituent
in whey fractions.
It is known that sugars are capable of enhancing antioxidant
defence mechanism in plants. Mannose treatment to the leaves
of etiolated wheat seedlings can modulate the expression of the
enzymatic antioxidant defence system in wheat leaves (Hameed,
Iqbal, & Malik, 2009). On the other hand, glycated proteins have
been found to show antioxidant activity (Sun et al., 2006). Sugar–
ovalbumin complexes, such as altrose/allose–ovalbumin (OVAs),
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E. Önay-Uçar et al. / Food Chemistry 150 (2014) 34–40 39

Table 2
Superoxide radical scavenging, lipid peroxidation inhibitory and cupric ion reducing activity of whey fractions.e

Sample Superoxide radical scavenging Lipid peroxidation inhibitory Cupric ion reducing activityc
activitya (IC50, mg/ml) activityb (IC50, mg/ml) (mmol Troloxd/mg protein)
Trolox 0.052 ± 0.002 0.084 ± 0.001 –
Whey filtrate (WF) 0.134 ± 0.006 1.253 ± 0.300 0.174 ± 0.006
Whey fraction 1 (F-1) 0.066 ± 0.001 0.942 ± 0.225 0.211 ± 0.054
Whey fraction 2 (F-2) 0.042 ± 0.005 0.675 ± 0.092 0.104 ± 0.007⁄
Pepsin fraction 1 (FP-1) 0.039 ± 0.010⁄⁄ 0.369 ± 0.031⁄⁄ 0.260 ± 0.059⁄⁄,d
Pepsin fraction 2 (FP-2) 0.075 ± 0.019dd 0.865 ± 0.156 0.188 ± 0.003  
Trypsin fraction 1 (FT-1) 0.038 ± 0.005⁄⁄ 0.420 ± 0.096⁄ 0.309 ± 0.054⁄⁄⁄,dd
Trypsin fraction 2 (FT-2) 0.029 ± 0.004 0.488 ± 0.075 0.350 ± 0.044⁄⁄⁄,   
a ⁄⁄
: P < 0.01 in comparison with F-1; dd: P < 0.01 in comparison with F-2, R2:0.9286.
b ⁄
: P < 0.05, ⁄⁄ P < 0.01 in comparison with F-1, R2:0.8849.
c ⁄
: P < 0.05, ⁄⁄: P < 0.01, ⁄⁄⁄: P < 0.001 in comparison with WF; d: P < 0.05, dd: P < 0.01 in comparison with F-1;   : P < 0.01,    : P < 0.001 in comparison with F-2, R2:0.9286.
d
Molar extinction coefficient of trolox (e) = 1.74  104 L/mol/cm.
e
Results are given as the mean of three replicates ± SD.

Table 3
Carbohydrate and polyol content of whey filtrate (WF) and second fractions (F-2), pepsin hydrolysate (FP-2) and trypsin hydrolysate (FT-2). RT = retention time.

Carbohydrates and polyols WF F-2 FP-2 FT-2

RT % RT % RT % RT %
a-L-Altrose 20.19 3.96 20.19 3.41 20.24 3.79 20.19 3.28
a-D-Mannose 22.28 18.60 22.29 18.47 22.41 17.71 22.28 18.69
b-D-Mannose 22.47 3.24 22.48 2.91 22.59 3.44 22.48 3.35
b-D-Gulose 24.36 10.38 24.36 10.26 24.48 10.28 24.36 10.19
b-D-Galactose 24.96 5.02 24.96 5.52 25.08 6.12 24.96 5.45
a-D-Galactose 26.39 22.35 26.40 24.20 26.59 21.82 26.36 21.33
b-D-Glucose 27.46 7.00 27.46 6.88 27.62 7.62 27.46 6.84
b-D-Talose 28.11 11.93 28.10 11.15 28.31 11.41 28.09 10.28
a-D-Talose 28.73 7.15 28.72 6.96 28.93 8.94 28.72 6.74
a-D-Glucose 32.06 7.44 32.06 7.18 32.10 7.63 32.06 7.17
Myo-inositol 34.68 0.18 34.68 0.22 34.68 0.23 34.68 0.14

talose/galactose–OVAs, gulose–OVA and mannose/glucose–OVAs _

are effective antioxidants. According to the results of our study,
at least some fractions of whey hydrolysates can be regarded as
a source of carbohydrates, which are able to exhibit or support
antioxidant activity both in free form or attached to the peptides.
4. Conclusion
This study dealed with the polypeptide and carbohydrate
content of antioxidant whey fractions obtained from chromato-
graphic separation of native and hydrolysed whey solutions. These
fractions were able to act as chelators of prooxidative metal ions
and scavengers of superoxide radical ions rather than directly
inhibiting lipid peroxidation. Active fragments were produced as
a result of proteolytic cleavage of whey proteins, especially with
trypsin. The second fraction of trypsin hydrolysate containing
small peptides and several carbohydrates, was the most effective
antioxidant fraction. Our data confirms that whey should be re-
garded as a valuable by-product with antioxidant potential. Anti-
oxidant constituents may be produced by proteolytic degradation
during gastrointestinal digestion as well as food processing. De-
tailed chemical and structural analyses of peptides, peptic and
tryptic peptides and glycated polypeptides of bioactive fractions
using sensitive methods of proteomics, as well as in vivo trials on
protective effects of whey fractions against oxidative DNA and
protein damage are in progress.
Acknowledgements
This work was supported by Turkish Scientific and Technologi-
cal Research Council (SBAG-2972/104S507), Marmara University
Research Fund (SAG-YLS-290506-0092 and SAG-YYP-290906-
0208) and Istanbul University Research Fund (UDP-3724/
05052009). We also thank Izmir Area Management Office of the
Institute of Hygiene, for the GC–MS analyses.
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