ORIGINAL PAPER

Antioxidant properties of whey protein hydrolysates as measuredby three methods

Adriena Dryakova • Anne Pihlanto • Pertti Marnila •

Ladislav Curda • Hannu J. T. Korhonen

Received: 13 October 2009 / Revised: 17 January 2010 / Accepted: 3 February 2010 / Published online: 19 February 2010

� Springer-Verlag 2010

Abstract Four microbial proteases (Alcalase, Flavour-

zyme, Neutrase and Protamex) were used for the prepara-

tion of whey protein hydrolysates. The aim of this research

was to find out whether these hydrolysates can be used as a

source of whey derived antioxidants. Hydrolyzed samples,

including their unhydrolyzed protein solutions were tested

by the ABTS (2,20-azinobis-(3-ethylbenzothiazoline-6-sul-

fonic acid) decolorization assay, by the total radical-trap-

ping potential method and by the assay of liposomes

peroxidation (fluorescence photometry). Antioxidant

properties were enhanced by hydrolysis in most of cases.

Alcalase hydrolysates were found as the most effective

antioxidants as determined by ABTS assay (*50% of

antioxidant activity at 0.1 mg ml-1 of hydrolysate in

reaction) and fluorescence photometry. Liposomes were

oxidized *50% less (1.1 lM of a-tocopherol equivalent)

with Alcalase hydrolysates additive (at 5.85 mg ml-1 of

hydrolysate in reaction). Hydrolysates did not inhibit

the oxidation of liposomes at concentrations below

1.0 mg ml-1 in reaction. On the contrary, results of total

trapping potential method did not agree with findings

observed in other tests. In this assay, Neutrase hydrolysates

showed the best antioxidant properties. Pro-oxidant prop-

erties were observed in solutions containing (prior to the

enzyme Protamex addition only) intact whey protein as

determined by the measurement of the liposome peroxi-

dation. The ABTS assay was optimized for the evaluation

of the antioxidant activity in whey protein hydrolysates.

The reaction time should be prolonged to avoid underes-

timation of the antioxidant activity.

Keywords Whey � Hydrolysates � ABTS � Liposomes �Chemiluminescence

Introduction

The importance of oxidation processes in the body and

foodstuffs has been widely recognized. The oxidative stress

caused by reactive oxygen species plays a significant role

in a number of age-specific diseases and neurodegenerative

disorders [1]. In foods, oxidative reactions cause deterio-

rations in food quality, unacceptable taste or texture, color,

loss of nutritive value at whole and shortening of shelf life.

Compounds which are responsible for destructive and

lethal cellular effects are lipid peroxides and low molecular

weight compounds produced during late stage of the oxi-

dative reactions. The main targets of these reactions are

lipids, proteins, DNA and enzymes. To prevent foods from

undergoing deteriorations and to provide protection against

processes promoting the development of age-specific dis-

eases like cancer, atherosclerosis and diabetes, it is very

important to inhibit the lipid peroxidation occurring in

foodstuffs and the living body [2]. The potential adverse

effects of synthetic antioxidant additives, such as butylated

hydroxyanisole and butylated hydroxytoluene, have stim-

ulated their replacement by natural antioxidants derived

from dietary sources. One practical approach to solve this

problem is the use of antioxidants from natural sources,

mainly non-protein compounds from plants. Some proteins

from certain foods have also been reported to have the

ability to scavenge active oxygen species. For example,

A. Dryakova (&) � L. Curda

Institute of Chemical Technology, Technicka 5,

166 28 Prague 6, Czech Republic

e-mail: adriena89@hotmail.com

A. Pihlanto � P. Marnila � H. J. T. Korhonen

MTT Agrifood Research, 31600 Jokioinen, Finland

123

Eur Food Res Technol (2010) 230:865–874

DOI 10.1007/s00217-010-1231-9

porcine myofibrillar protein [3], soy and gelatin [4], potato

[5], milk proteins of which caseins [6, 7] and finally whey

proteins [8–11] have been shown to have the antioxidant

effect. In addition to proteins, protein hydrolysates have

also been found to exhibit the antioxidant activity. During

protein hydrolysis, protein’s overall antioxidant activity is

enhanced as its tertiary structure is disrupted and the sol-

vent accessibility of released amino acids increases.

Enzymatic digests of whey proteins and its use as potential

antioxidants have been also demonstrated in several stud-

ies: [12–15].

Most of authors have used for the evaluation of the

antioxidant activity (measurement of lipid oxidation)

methods based on measurement of thiobarbituric acid

reactive substances and assays based on the ability of

scavenging of different type radicals: either stable chro-

mogen 1,1-diphenyl-2-picrylhydrazyl radical (DPPH�) or

short-live radicals, such as fluorescein radical in oxygen

radical absorbance capacity (ORAC). The goal of this

research was to contribute to a number of previous studies

dealing with the antioxidant activity of whey protein

hydrolysates, and to find out whether these hydrolysates,

prepared by commercially available microbial proteases

(Alcalase, Flavourzyme Protamex and Neutrase), can be

used as a potential source of naturally derived antioxidants

and broaden the information about the mechanism of the

antioxidant effect. This effect was investigated in different

phase systems, using different principles: scavenging of the

2,20-azinobis (3-ethylbenzenthiazoline-6-sulfonic acid)

radical, by the assay of liposome peroxidation induced by

ferric ion or by the rate of trapping the luminol radical

induced by 2,2-azo-bis(2-methylpropionamidine) dichlo-

ride thermolysis. These methods are rarely used for the

determination of the antioxidant activity of whey protein

hydrolysates.

Materials and methods

Materials

The powdered whey protein concentrate (WPC), containing

60.8% of protein was supplied by Promil (Czech Republic).

Enzymes used for hydrolysis of WPC: Alcalase 2.4L (ALC);

Flavourzyme 500 MG (FLA); Protamex (PRO) and Neutrase

(NEU) were obtained from Novo Nordisk A/S (Denmark).

2,2-Azo-bis(2-methylpropionamidine) dichloride (ABAP)

was purchased from Acros Organics (Belgium). o-Phthaldi-

aldehyde (OPA) and N-acetylcysteine (NAC) were purchased

from Merck (Germany). Sodium dodecyl sulfate (SDS) was

purchased from Lachema (Czech Republic). (2,20-azinobis

(3-ethylbenzenthiazoline-6-sulfonic acid) diammonium salt

and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid (Trolox; water soluble analogue of vitamin E) and other

chemicals were obtained from Sigma Chemicals (Germany).

Preparation of enzymatic hydrolysates

The WPC was reconstituted in demineralized water to 4%

(w/v) concentration of protein solution and pH was

adjusted by 2 M NaOH or HCl to obtain the optimal value

for enzyme. Solutions were preheated in temperature-

controlled water bath to 45 �C. Protein content in enzymes

was determined as described by Lowry et al. [16]. The

hydrolysis parameters were used as follows: enzyme/sub-

strate ratio, 1/100 (w/w); pH 8 for ALC, pH 7 for FLA and

NEU and pH 6 for PRO, temperature 45 �C. The hydrolysis

was conducted for 3 h with continuous stirring. Samples

were withdrawn at 15; 30; 60; and 180 min from each

proteolytic mixture and were immediately heated at 85 �C

for 10 min to inactivate the enzyme. The unhydrolyzed

sample was taken before the enzyme addition. A small

amount of the freshly prepared hydrolysates was used to

determine the degree of hydrolysis and the rest was freeze-

dried for later use.

Degree of hydrolysis

The degree of hydrolysis (DH) was determined as descri-

bed by Spellman et al. [17] with slight modifications. The

OPA-NAC reagent was prepared by mixing of 1 ml of

0.3 M OPA solution (in methanol); 25 ml of 0.1 M of

Na2B407; 2.5 ml of 20% (w/v) of SDS and 0.05 g of NAC

(diluted in demineralized water). This mixture was diluted

to the final volume of 50 ml with demineralized water. The

reagent was prepared every day fresh. To dissolve the

possible precipitate in hydrolysate, created after inactiva-

tion of enzyme, 1 ml of 1 M NaOH was added to 1 ml of

hydrolysate. Further, hydrolysates were diluted with

demineralized water and then mixed with the OPA reagent

in ratio 1/100 and let to react in dark for 2 min. The

absorbance was measured at 335 nm using spectropho-

tometer (Cary 50, Varian, Australia). DH values were

calculated on the base of totally hydrolyzed WPC that

represented 100% of DH (data not shown) with subtraction

of DH calculated for unhydrolyzed sample (blank). The

average chain length (ACL) was determined as described

by Adler-Nissen [18], where ACL=100/DH. All samples

were measured in duplicates.

Antioxidant activity (ABTS decolorization assay)

The antioxidant activity of hydrolysates (scavenging of the

ABTS radical (ABTS•?) 2,20-azinobis-(3-ethylbenzothia-

zoline-6-sulfonic acid)) was measured using the decolor-

ization assay as described by Re et al. [19] and Virtanen

866 Eur Food Res Technol (2010) 230:865–874

123

et al. [20]. Briefly, 1ml of diluted ABTS•1 solution in

5 mM phosphate-buffered saline (PBS) was added to 10 ll

of sample, or Trolox in ethanol (standard) or PBS (blank),

and an absorbance (734 nm) reading (Lambda 25, Perkin

Elmer, USA) was taken at 30 �C (exactly 1 min after initial

mixing and up to 180 min at 1 min intervals). Experiments

showed that the dependence of the antioxidant activity on

time was linear up to the first 6-min reaction time.

Accordingly, 6-min reaction time was used for all

hydrolysates. All determinations were duplicated. The

antioxidant activity was measured in hydrolysate recon-

stituted in PBS to 5–10 mg ml-1 of initial sample con-

centration (dry matter) that is 0.05–0.1 mg ml-1 in

reaction. The antioxidant activity (%) was calculated with

the following equation: AA (%) = [(A743 blank–A743

sample)/A743 blank] 9 100%. The AA was calculated at

each minute of reaction time and plotted as a function of

the time to calculate the antioxidant activity as a single

quantity, so the antioxidant activity was expressed in terms

of the total contribution of the sample to the antioxidant

activity.

Total radical-trapping potential method (TRAP)

The effect of hydrolysates and the unhydrolyzed material

on total radical trapping was assessed as described by Lissi

et al. [21]. In the assay, 175 ll of sample (5.8–0.4 mg ml-1

of freeze-dried hydrolysate in reaction) or 50 ll Trolox

(20–80 lM) was incubated with 25 ll of luminol (0.02 M

5-amino-2,3-dihydro-1,4-phthal-azinedione) and 50 ll of

buffer (0.01 M sodium phosphate buffer with 0.9% NaCl

pH 7.8) in a micro plate for 10 min at 37 �C. After incu-

bation, 50 ll of ABAP solution (90 mM) was added.

A chemiluminescence reading was taken immediately after

addition and up to 2.5 h using luminometer (Luminoscan

EL1, ThermoScientific, Finland). All determinations were

carried out in duplicate, at each separate concentration of

the sample. Samples and standard were reconstituted in

buffer (0.01 M sodium phosphate buffer with 0.9% NaCl

pH 7.8). The chemiluminescence peak time was plotted as

a function of sample dry matter. Total antioxidant potential

(TRAP)—trapping activity—was expressed as the equiva-

lent of Trolox concentration by regression analysis of

plotted chemiluminescence peak time versus sample dry

matter.

Assay of lipid peroxidation in liposomes

To determine the susceptibility of lipid to peroxidation

with hydrolysates additive, a liposomal system was used.

Lipid peroxidation was tested using the fluorescence plate

reader as previously described by Tirmenstein et al. [22]

with slight modifications. Phosphatidyl choline liposomes

were prepared according to the procedures of Ursini et al.

[23]. a-Tocopherol treated liposomes were used as standard

compounds. Liposomes were stored at 4 �C for at least

1 week prior to use to increase levels of lipid hydroper-

oxides. In assay, 10 ll of liposomes (with standard or not)

and 50 ll of hydrolysates reconstituted in potassium

phosphate–glycine–ascorbate buffer (at concentrations

0.14–5.85 mg ml-1 in reaction; dry matter) were loaded

into the wells of a microplate. One hundred and twenty

microliter of potassium phosphate (50 mM)–glycine

(100 mM)–ascorbate (0.45 mM) buffer (pH 7.4) was

added, and the oxidation was initiated by adding 20 ll of

25 lM ferric chloride/1 mM ADP. The microplates were

incubated at 37 �C, shaken, and fluorescence (EX360nm,

EM460nm) was measured using plate reader (Wallac

Viktor 2, PerkinElmer, Finland). The measurement was

performed at 15-min intervals for total 4 h. Blank (fluo-

rescence360/460 in well containing buffer and ferric

chloride only) was subtracted before calculating the lipo-

some oxidation. At least three independent experiments

were carried out for each sample. The liposome oxidation

(LO) (after 4 h of incubation) was calculated. The lipo-

some oxidation was expressed as the equivalent of Trolox

concentration by regression analysis of plotted LO versus

sample dry matter.

Statistical analysis

Dose dependencies of tested properties were evaluated by

means of regression analysis. Correlation coefficient was

used for the evaluation of the optimization of ABTS assay.

Data were analyzed using the t-test or by multifactor

analysis of variance.

Results and discussion

Degree of hydrolysis

The DH of WPC ranged from 6 to 35%, depending on

protease specificity and substrate affinity (Table 1). Inter-

estingly, ACL of ALC and FLA hydrolysates was quite

similar (9 for ALC; 3 for FLA after 180 min of hydrolysis).

On the contrary, calculated ACL of PRO and NEU

hydrolysates were higher (18 for NEU and 16 for PRO after

180 min hydrolysis) (Table 1). As expected, DH generally

increased and correspondingly to that, the ACL of

hydrolysates decreased, with the course of hydrolysis. The

significant difference (p \ 0.05) of the calculated DH

between FLA hydrolysates and ALC, NEU and PRO

hydrolysates was observed, which is caused by different

nature of enzyme reaction. Flavourzyme (from Aspergillus

oryzae) is a crude enzyme formula probably with both endo

Eur Food Res Technol (2010) 230:865–874 867

123

and exopeptidases activities. The highest DH, i.e. with

highest amount of released amino groups (or peptide with

shortest amino group chain length), was found in Fla-

vourzyme hydrolysates. The specificity of Neutrase (from

Bacillus amyloliquifaciens) and Protamex is not clearly

known. Hydrolysates digested with these enzymes showed

clearly lower DH and contained peptides with longer

amino acid chain length than those prepared by Flavour-

zyme digestion.

Antioxidant activity (ABTS decolorization assay)

The ABTS decolorization assay is applicable to both

lipophilic and hydrophilic compounds and has been widely

used to the assessment of antioxidant activity [24]. All

samples demonstrated antioxidant activity ranking from 7

to 54% (at 6 min reaction time at 0.10 mg ml-1 concen-

tration of hydrolysate in reaction). The dependence of

antioxidant activity on sample concentration (0.05–

0.10 mg ml-1) was found linear (6 min reading) in all

samples (R2 [ 0.9). Before hydrolysis, all samples (unhy-

drolyzed whey protein) showed low antioxidant activity

(7–19.8%) and during 180 min of hydrolysis, the values

increased notably with all tested hydrolysates (40–54.2%).

The development of the antioxidant activity in relation to

hydrolysis time is shown in Fig. 1. Alcalase hydrolysates

were the most effective in scavenging the ABTS•?.

Already, after 15 min of hydrolysis time a 48% antioxidant

activity was found, whereas in Protamex hydrolysates not

even 180 min of hydrolysis produced such activity. Lower

antioxidant activity in Neutrase and Protamex hydrolysates

can be attributed to the specific enzyme cleavage which did

not produce peptides able to scavenge ABTS•?, taking into

the account the relatively lower affinity of enzyme to

substrate (Table 1). It can be also said that peptide mix-

tures of higher chain lengths ([16 of amino groups in

Table 1 Degree of hydrolysis and average chain length calculated in individual whey protein hydrolysates

Time of hydrolysis (min) ALC FLA NEU PRO

DH (%) ACL DH (%) ACL DH (%) ACL DH (%) ACL

0 0 – 0 – 0 – 0 –

15 7.7 13 4.5 22 0.8 125 0.9 111

30 9.0 11 15.2 7 3.8 26 2.5 40

60 10.9 9 20.7 5 3.9 26 4.7 21

180 11.3 6 35.2 3 5.6 18 6.1 16

Time of hydrolysis indicates the time of the hydrolytic reaction when the hydrolysates was taken at in minutes

DH degree of hydrolysis, ACL average chain length, ALC alcalase, FLA flavourzyme, NEU neutrase, PRO protamex

0

10

20

30

40

50

60

Alcalase Flavourzyme Neutrase Protamex

AA

[%

]

0 min 15 min 30 min 60 min 180 min

Fig. 1 Development of

antioxidant activity (ABTS

decolorization assay) during

hydrolysis; AA, antioxidant

activity (%); time indicates the

time of hydrolysis when the

sample was withdrawn at (min);

sample concentration (dry

matter) = 0.10 mg ml-1;

reading 6 min; data are

displayed as means ± SD

868 Eur Food Res Technol (2010) 230:865–874

123

chain) were not as effective in scavenging the ABTS•? as

hydrolysates with shorter chain lenghts (Table 1). The

activity found in unhydrolyzed samples confirms the gen-

erally known fact that whey protein itself exhibits some

antioxidant properties [25]. The differences observed

among individual unhydrolyzed samples might have been

explained by different tertiary structure of the protein due

to initial pH and heat treatment of the protein solution prior

to hydrolysis.

The antioxidant activity correlated with degree of

hydrolysis of ALC hydrolysates (r = 0.95). In this study,

lower correlation of DH and antioxidant activity was

observed in other hydrolysates (FLA r = 0.90; NEU

r = 0.75; PRO r = 0.70). A strong correlation between

DPPH scavenging activity and DH of ALC hydrolysates of

porcine plasma was observed [26]. Virtanen et al. [20]

found that during fermentation of milk some lactic acid

bacteria produced ABTS radical scavenging activity that

correlated with DH. OPA method determines the content of

primary a and e-amino groups of free amino acids or in

peptides [27]. High degree of hydrolysis thus means high

content of released amino groups and it should presumably

show also good antioxidant activity in FLA hydrolysates.

Nevertheless, the antioxidant activity of hydrolysates was

not found to be directly attributed to DH, suggesting that

the activity is inherent to specific amino acid sequence.

Inconsistent data are reported for antioxidant activity of

whey proteins or their digests. Some authors relate the

antioxidant activity to low molecular fractions of whey,

[14, 28] on the contrary, others report higher antioxidant

activities [15, 25] in relation to high molecular weight

compounds. Pena-Ramos and Xiong [14] detected highest

antioxidant activity in FLA hydrolysate, and Hogan et al.

[29] detected highest antioxidant activity in milk protein

hydrolysates UF fraction (\3 kDa) prepared by microbial

protease (also from Aspergillus oryzae) when compared to

other hydrolysates prepared by alkaline and neutral prote-

ases. Since in this study, hydrolysis by ALC produced

highest antioxidant activity, it should be emphasized that

results of these studies were carried out with different

materials; (e.g. whey protein concentrate/isolate; different

content of non-protein compounds which produce thio-

barbituric acid reactive substances with protein upon

heating) and the methods and evaluation of results often

greatly varies which makes comparison difficult. Finally,

the uses of concrete enzyme/substrate combination and

conditions of hydrolysis will influence the final antioxidant

potential of hydrolysates.

ABTS radical scavenging pattern

As ABTS decolorization assay is based on the production

of stable chromogen, the time dependency of the reaction

and the influence on the ABTS•? radical scavenging ability

was studied. The results showed that hydrolysates had

clearly different pattern to produce chromogen as Trolox

(Fig. 2). The rate of scavenging by Trolox was constant

and it occurred at the beginning of the reaction. Our

samples, on the contrary, mixtures of proteins and peptides,

behaved as a complex. The radicals were scavenged

gradually and this reaction was dose dependent. For

example, the radical scavenging activity of ALC180

hydrolysate (Fig. 2) gradually increased from 33 to 98%

during 60 min of reaction and reached plateau after 3 h

(data not shown) of the reaction (99.2%). The increase in

scavenging activity in time was not so remarkable in less

concentrated sample of the same hydrolysate which con-

firms the proportional relationship between the concentra-

tion of hydrolysates and the scavenging activity. Results of

this measurement indicated the longer time for the reaction

between ABTS•? and whey protein digests would be more

appropriate to avoid the underestimation of the activity.

Accordingly, the reaction time was prolonged to 15 min,

and measurements were repeated with all samples and

compared to earlier results (6 min reading). The differences

were close to zero (lower than AA of blank) when comparing

slopes except for unhydrolyzed material, which can be

attributed to the low ability of the protein itself to scavenge

the radical. This means that reactions run equally fast (AA

per concentration unit). The differences between intercepts

of curves varied between 10 and 20%, which means higher

activities exhibited by concentration unit per time unit that

confirms the proportional relationship between time and the

development of the scavenging activity under this method

arrangement. Similar behavior was observed in the study

from Zulueta et al. [30], where the authors compared the

antioxidant capacity of milk and juice by ABTS assay. They

found that milk had a low inhibition percentage at start time,

gradually increasing and reaching an antioxidant capacity

value similar to that of the 100% juice after 20 min.

Total radical-trapping potential

Trapping activity of hydrolysates was measured by a pro-

cedure based on the measurement of induction times of

chemiluminescence in the dispersion containing luminol,

exposed to a free radical source which is induced by the

thermolysis of ABAP and read on Luminoscan plate reader.

Radical scavenging activities were measured in all samples

including their unhydrolyzed analogues. The trapping

activity in this reaction system was shown to be dose

dependent in range of 1.5–5.8 mg ml-1 of hydrolysates or

unhydrolyzed material (dry matter). Lower concentrations

of tested material (\1.5 mg ml-1) were not able to prevent

the formation of luminol radical. Calculations were done

with respect to the best linear curve fit (R2 [ 0.95).

Eur Food Res Technol (2010) 230:865–874 869

123

Results of this measurement were not parallel with those

obtained by ABTS decolorization assay (Figs. 1, 3).

Trapping radicals by samples in this reaction system was

improved by hydrolysis almost in all cases. Statistical

testing showed significant differences in trapping activity

between hydrolysates and unhydrolyzed protein in all tes-

ted hydrolysates (p \ 0.05). Generally, best trapping

activity was observed in hydrolysates, withdrawn after

180 min of hydrolysis. Best trapping activity among all

samples was found in NEU hydrolysate withdrawn after

180 min of hydrolysis (7.7 mM of Trolox equivalent).

PRO hydrolysate withdrawn after 15 min of hydrolysis

(4.5 mM of Trolox equivalent) had the highest activity

among all PRO hydrolysates. Alcalase hydrolysates were

not effective as Protamex hydrolysates; similar values

(4.5 mM of Trolox equivalent) were found in samples

hydrolyzed by Alcalase for 180 min in comparison with

Protamex hydrolysates where this activity was seen in

sample withdrawn already at 15 min of hydrolysis.

Hydrolysis influenced the trapping activity of FLA

hydrolysates least among all samples. FLA hydrolysates

exhibited the lowest activity in comparison with all

hydrolyzed samples. Released amino groups (shortest

peptide chain lengths—see Table 1) in FLA hydrolysates

were probably either oxidized themselves or they were not

able to prevent the formation of radicals and by that extend

the chemiluminescence peak time.

Samples showed a different pattern from those obtained

by Trolox in this reaction system. Addition of samples

(hydrolyzed or not) did not produce a measurable induction

time (lag phase) but a very moderate decrease in observed

chemiluminescence intensity. This was also observed in the

study from Lissi et al. [21] on human albumin, where the

negative surface charge of albumin was denoted to reduce

the reaction between the reactive center and the luminol-

derived radical-anion. Furthermore, protein solutions prior

to FLA and NEU hydrolysis (equal concentrations) showed

remarkably higher chemiluminescence maxima at peak

induction time than other unhydrolyzed solutions (data not

shown). This might have explained higher chemilumines-

cence peak time observed after the addition of these sam-

ples. Protein treatment prior to hydrolysis may have

resulted in the premature oxidation of some amino acid

residues or alterations in the protein’s tertiary structure that

affected its free radical scavenging activity [12].

Inconsistent results obtained by ABTS assay and TRAP

method may be accounted to the differences and particu-

larly the variation in analytical procedures. Arnao [24]

states, that in the case of natural antioxidants, they may be

multifunctional. The mechanism that is operative or

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

AA

[%]

t [min]

Fig. 2 Time dependency of the antioxidant activity (ABTS decolor-

ization assay) of selected samples; AA = antioxidant activity;

t = time of reading (the activity was measured during first 60 min;

immediately after adding the sample into the reaction); filled squareALC hydrolysate withdrawn after 180 min of hydrolysis; sample

concentration in reaction (ABTS assay) = 0.10 mg ml-1 filled dia-mond ALC hydrolysate withdrawn before the enzyme addition;

sample concentration in reaction (ABTS assay) = 0.10 mg ml-1

open triangle Trolox; concentration in reaction (ABTS

assay) = 2.5 lg ml-1 open circle ALC hydrolysate withdrawn after

180 min of hydrolysis; sample concentration in reaction (ABTS

assay) = 2.5 lg ml-1 closed circle ALC hydrolysate withdrawn

before the enzyme addition; sample concentration in reaction (ABTS

assay) = 2.5 lg ml-1

870 Eur Food Res Technol (2010) 230:865–874

123

dominant in particular situation is dependent on conditions

and yet this will affect the kinetics and hence the antioxi-

dant activity.

Inhibition of lipid peroxidation

The development of lipid peroxidation inhibition was

studied in all samples including unhydrolyzed material

using Fe–ADP system by measuring the inhibition of

phosphatidylcholine liposome peroxidation induced by

ferric ion. Results were expressed as a-tocopherol equiva-

lent. Samples did not inhibit the oxidation of liposomes at

concentrations below 1.0 mg ml-1 in reaction. Inhibition

of liposome oxidation was observed at concentrations of

5.85 mg ml-1 of the hydrolysate in reaction. Hydrolysis

influenced antioxidant properties of tested hydrolysates

depending on the enzyme used (Fig. 4). The decrease in

liposome oxidation in comparison with blank (liposomes

with no additives) was observed in all tested samples

(p \ 0.0003), except the unhydrolyzed sample withdrawn

before hydrolysis by PRO (–0.5 lM of a-tocopherol).

Liposomes containing this sample were oxidized 20%

more than blank (liposomes with no additives). Significant

effect of hydrolysis was most markedly seen in PRO

hydrolysates (p \ 0.05); however, liposomes with these

additives showed highest rates of liposome oxidation

against blank (0.4–0.7 lM of a-tocopherol). On the con-

trary, the lowest rates of liposome oxidation were found in

samples containing ALC hydrolysates; the lipid oxidation

was inhibited by approximately 50% (corresponds to

*1 lM of a-tocopherol) in comparison with blank, as

observed in the sample containing ALC15 and remained

stable in relation to the time of hydrolysis. However, the

influence of hydrolysis on improving ALC hydrolysates

antioxidant properties was not found significant (p \ 0.05).

Low rates of liposome oxidation treated with ALC

hydrolysates can be attributed to the ALC cleavage speci-

ficity for aromatic residues (Phe, Trp, Tyr) which are

potential radical scavengers [31]. Significant difference

between the influence of the unhydrolyzed protein and

NEU and FLA hydrolysates addition on the liposome

oxidation was found (p \ 0.01). Hydrolysis did not induce

the antioxidant activity in NEU hydrolysates: liposomes

were oxidized even more with NEU hydrolysates additives,

in comparison with the liposomes containing their unhy-

drolyzed analogues. Rival et al. [7] suggested a theory,

supported by results from measurements of the antioxidant

activity of caseins and casein peptides, that casein and their

digests might be a preferred target over fatty acid free

radical intermediate(s) and can either initiate further

unwanted radical-mediated side reactions or terminate

chain reactions depending on the stability of the resulting

protein/peptide radical. Furthermore, proteins/peptides can

be oxidized during the process, according to a site or

sequence-specific mechanism. NEU hydrolysates, in this

reaction system, were less effective to slow down the

development of lipid peroxidation in liposomes. Hydroly-

sate, withdrawn after 3 h of hydrolysis by FLA was the

most effective among all FLA hydrolysates (1.0 of lM of

a-tocopherol). Other FLA hydrolysates were less effective,

and 15 min of hydrolysis by FLA even led to the forma-

tion of prooxidant properties (0.2 of lM of a-tocopherol).

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Alcalase Flavourzyme Neutrase Protamex

Tro

lox

[mM

]

0 min 15 min 30 min 60 min 180 min

Fig. 3 Development of the

trapping activity (Total radical-

trapping potential method)

during hydrolysis; trapping

activity was expressed as the

Trolox equivalent (mM), on

equal concentration of samples

4 mg ml-1 in reaction—dry

matter; time indicates the time

of hydrolysis when the sample

was withdrawn at (min)

Eur Food Res Technol (2010) 230:865–874 871

123

Pena-Ramos and Xiong [14] investigated the antioxidant

activity of whey hydrolysates measured by TBARS in Fe-

ascorbate liposomal system. Whey protein isolate hydro-

lyzed by commercial crude enzyme protease F (exo and

enprotease from Aspergillus oryzae) was found to be most

effective. Liposomes containing these hydrolysates were

shown to have lowest amount of TBARS among tested

hydrolysates.

Noticeably different behavior of PRO unhydrolyzed

sample (also observed when measured by ABTS decolor-

ization assay) remains unclear. The solution (containing

intact whey protein) prior to adding PRO enzyme was

adjusted to the pH 6 in comparison with other enzymes

where the hydrolysis run at relatively neutral pH (or

alkaline). In contrast to this observation, Shon and Haque

[32] studied the antioxidant ability of native and thermized

sour whey in oxidation-catalyzed systems. The antioxidant

ability increased with decreasing pH (pH 3–7) in terms of

decreased TBARS and peroxide values. Lower lipid oxi-

dation rates at pH values below the pI of the whey powder

in the presence of iron are seem to be due to the electro-

static repulsion between iron and droplet surfaces [33].

However, the treatment prior to hydrolysis might have

negatively affected the tertiary structure of the protein and

influence its ability to slow down the development of lipid

oxidation. Elias et al. [34] studied the influence of native

and denatured (50–95 �C) b-lactoglobulin on oxidative

stability of stabilized menhaden oil-in water emulsions at

pH 7. Conformational changes were seen in heated b-lac-

toglobulin as detected by amount of exposed tryptophan

residues. Also, solubility of major whey protein, b-lacto-

globulin, was observed to decrease upon heating [35]. The

combination of lower pH and heating may lead to forma-

tion of insoluble protein aggregates [36], which in conse-

quence may lead to the worse access of active compounds

to the surface of liposomes.

Conclusions

Antioxidant activity as measured by three different meth-

ods (ABTS, TRAP and inhibition of lipid peroxidation in

liposomal system) was detected in all whey protein

hydrolysates. Antioxidant properties were enhanced by

hydrolysis in most of cases. The best antioxidant properties

were found in ALC hydrolysates as determined by ABTS

decolorization assay and by the measurement of the lipid

peroxidation inhibition. ALC hydrolysates scavenged the

ABTS radical most effectively. FLA, NEU and PRO did

not exhibit so good antioxidant activity in this reaction

system. The highest total trapping potential was observed

in NEU and PRO hydrolysates among all tested hydrolyzed

samples. ALC hydrolysates inhibited most effectively the

lipid oxidation development in liposomes. Hydrolysis of

whey protein by NEU did not improve the ability of

samples to inhibit the development of lipid oxidation in

liposomal system.

The differences among tested samples may be appointed

to the different reaction system used. Used enzymes pro-

duced digests that exhibited different scavenging charac-

teristics which shows that combination of enzyme/substrate

is the key parameter in searching for compounds having

antioxidant properties in protein hydrolysates. Protein

treatment (heating, pH adjustment) prior to hydrolysis

-0.5

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

Alcalase Flavourzyme Neutrase Protamex

α-T

ocop

hero

l [M

]µ

0 min 15 min 30 min 60 min 180 min

Fig. 4 Development of the

liposome peroxidation (Assay

of lipid peroxidation in

liposomes) during hydrolysis;

LO was expressed as the

a-tocopherol equivalent (lM),

on equal concentration of

sample 5.85 mg ml-1 in

reaction—dry matter; time

indicates the time of hydrolysis

when the sample was withdrawn

at (min)

872 Eur Food Res Technol (2010) 230:865–874

123

should be also considered as this influence the protein

folding which can lead to the worsening of the antioxidant

properties. Unhydrolyzed protein solutions adjusted to pH

6 prior to hydrolysis were not effective in scavenging of

ABTS and even promoted a lipid oxidation in liposomal

system. The special care should be taken when optimizing

hydrolysis, especially when related to the pH. The rela-

tionship between pH and measured antioxidant activities

remained to be unclear. ALC whey protein hydrolysates

were shown to have a certain antioxidant potential and can

be used as a source of naturally derived antioxidants e.g. in

food; pharmaceutical industry or cosmetics.

Acknowledgments This work was supported by the Ministry of

Education, Youth and Sports of the Czech Republic (research project

MSM 6046137305) and by the Socrates—Erasmus program.

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