25 

 

Journal homepage: www.fia.usv.ro/fiajournal 

Journal of Faculty of Food Engineering,  

Ştefan cel Mare University of Suceava, Romania  

Volume XIX, Issue 1 - 2020, pag. 25   -  39 

 

 

ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF BOVINE WHEY 

PROTEINS HYDROLYSED WITH SELECTED LACTOBACILLUS STRAINS 

 

*Hayet MESSAOUI
1
, Salima ROUDJ

1
, Nour-Eddine KARAM

1
 

1Laboratory of Microorganisms Biology and Biotechnology, Department of Biotechnology, Faculty of Natural 

Sciences and Life, Oran1 University Ahmed Benbella, B.P. 1524, El M’Naouer, 31000 Oran, Algeria. 

mess_hayet@yahoo.fr  

*Corresponding author 

Received 29th Februaruy 2020, accepted 30th March 2020 

 
Abstract: The present study investigated the antioxidant and antibacterial properties of hydrolysates 
obtained from bovine whey proteins, after hydrolysis with Lactobacillus plantarum strains isolated 
from ewe milk and Lactobacillus brevis isolated from camel milk. The aim here was to valorize whey 

proteins and to constitute local collection of bacteria, with potentially interesting technological and 

biological features. The protein hydrolysis was determined by degree of hydrolysis (DH) and 
electrophoresis SDS–PAGE, and then optimized using temperature, pH, cells/whey proteins ratio and 

time incubation. Antioxidant activity of hydrolysates was evaluated by 2,2-diphenyl-1-picrylhydrazyl 

(DPPH) and nitric oxide (NO) free radicals scavenging methods. The antibacterial activity was 

examined by the agar well diffusion assay. The results revealed that Lb. plantarum LBBS2 and Lb. 
plantarum LBM2 exhibited an optimum of hydrolysis of whey proteins at pH 7, at 30 °C, with (2/1) as 

ratio cells/whey proteins and after 48 h of incubation. Under these conditions, the hydrolysates 

exhibited an antibacterial activity against Gram positive and Gram negative bacteria, including some 
pathogenic and spoilage microorganisms.  Meanwhile, scavenging radical properties by the same 

hydrolysates reached a maximum after 24 h. These results suggest that Lb. plantarum LBBS2 and Lb. 

plantarum LBM2 could be used as new potential adjunct bacteria with interesting proteolytic activity. 
Moreover, milk whey which is the main by-product of food industry could be used for the production 

of molecules with a promising interest as antioxidants and antibacterials. 
 

Keywords: Lactobacillus plantarum, milk whey, hydrolysates, degree of hydrolysis, electrophoresis 
SDS–PAGE. 

 

 

1. Introduction 
 

Lactic Acid Bacteria (LAB) are widely 

utilized in the fermented food industry for 

their technological and health-promoting 

features, such as improving the sensorial 

properties of fermented products, as well 

as providing special characteristics to 

foods particularly the release of 

compounds with health-promoting effects 

[1]. LAB have an efficient proteolytic 

system comprised of a Cell Envelope 

Proteinase (CEP) which initiates protein 

degradation, a transport system and various 

intracellular peptidases. In this regard, 

LAB are able to degrade milk proteins [2, 

3]. Lactobacillus spp. are greatly employed 

in the food industry as starter cultures for 

the manufacture of fermented foods and 

beverages or as probiotics for 

incorporation into functional foods [4]. 

Whey is the principal by-product of the 

food industry. It represents about 85–90% 

of milk volume and retains approximately 

55% of milk nutrients [5]. Previously, 

whey was considered as important 

http://www.fia.usv.ro/fiajournal
mailto:mess_hayet@yahoo.fr


Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

26 

pollutant of the dairy industry, not only 

due to its great organic load, but also 

because of its important volume [6]. In 

fact, continued growth in the production of 

dairy products, especially cheese, has seen 

a concomitant increase in the volume of 

whey worldwide (currently >200 million 

tonnes per year) [7]. Whey is a rich 

mixture of secreted proteins with wide 

variety of functional characteristics for 

biological, nutritional, and food purposes. 

It contains 20% of total milk proteins. 

Nowadays, the perception of whey as a 

pollutant has changed with the discovery 

of its functional and bioactive properties, 

being considered as an additional product 

of cheese manufacture [5]. Among the 

food proteins, most studies have been 

performed on whey proteins due to their 

high nutritional value, inexpensiveness, 

diverse functional properties and 

widespread application in food industry [8, 

6]. 

Oxidative stress, the increased production 

of reactive oxygen species (ROS) and free 

radicals are incriminated in many 

degenerative diseases like cancer, 

atherosclerosis and diabetes [9]. 

Improvement of the body’s antioxidant 

defences through food supplementation 

would appear to a practical initiative to 

reduce the level of oxidative stress. As 

obvious antioxidant feature of whey 

protein hydrolysates was detected in recent 

studies, it was good for their application in 

food industry as functional materials [9, 

10]. 

Furthermore, the presence of pathogenic 

and spoilage microorganisms in foods, 

combined with new consumer interest 

resulting in restrictions on the use of 

traditional antimicrobial chemical agents, 

puts pressure on food manufacturers to 

develop new preservatives [11]. During the 

past decade, growing attention has been 

focused on the production of antimicrobial 

peptides derived from whey proteins in 

order to reduce the need for antibiotics [8, 

12]. 

Various protein hydrolysates obtained 

through enzymatic catalysis demonstrate 

biological activities, which are usually 

associated with bioactive peptides. Thus, 

production of hydrolysates can be an 

interesting approach to add value to whey. 

In fact, the bioactive peptides are inactive 

while encrypted in the sequence of original 

protein but can be released by: a) 

hydrolysis by digestive enzymes, b) by 

proteolytic microorganisms, and c) by the 

action of plant or microbial proteases [13]. 

In this context, protein-rich by-products 

might be utilized to produce bioactive 

peptides using specific protease producing 

microorganisms. 

To our knowledge, no study has been 

carried out on the antioxidant and 

antibacterial properties of bovine whey 

protein hydrolysates generated by ewe 

milk and camel milk Lactobacillus strains. 

Therefore, the present work was 

undertaken to study and compare, 

antioxidant and antibacterial activities of 

whey protein hydrolysates obtained with 

autochthonous Lactobacillus strains 

isolated from ewe milk and camel milk.  

 

2. Materials and Methods 

 

Bacterial Strains and Growth 

Conditions 

Nine Lactobacillus plantarum strains 

(coded LBM1, LBM2, LBS1, LBSC1, 

LBSC2, LBBS1, LBBS2, LBBG1, 

LBBG2), one strain of Lactobacillus 

paracasei subsp. paracasei coded LBS2 

isolated from ewe milk and one strain of 

Lactobacillus brevis coded CHTD27 

isolated from camel milk, were supplied by 

the Laboratory of Microorganisms Biology 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

27 

and Biotechnology, Oran1 University 

(Algeria). Ewe milk was collected in 

different regions of Algeria, and camel 

milk in Tindouf area (southwest, Algeria). 

The LAB strains were maintained in 

reconstituted skimmed milk (10%) at - 20 

°C.  Standard cultures were prepared by 

inoculation of de Man, Rogosa and Sharpe 

medium (MRS, Fluka, Saint Louis, MO) 

broth at pH 5.7 with the frozen stocks 

followed by incubation at 30 °C for 18 h. 

 

Qualitative Determination of Proteolytic 

Activity  

The proteolytic activity of strains was 

estimated using agar medium with 

reconstituted skimmed milk (10% w/v) at a 

final concentration of 3%. A loopful of 

lactic strain cultures (Optical Density 

OD600nm = 1) was spotted on skimmed milk 

agar and incubated at 30 °C for 48 h. 

Proteolytic activity was detected by the 

occurrence of clear zones around the 

colonies [14]. 

 

Preparation of Whey Proteins 
Whey proteins were prepared from 

collected cow milk as reported by 

Neyestani et al. [15] with some 

modifications. In brief, cream was 

skimmed from fresh cow milk by 

centrifugation (5000 g, 30 min, 4 °C) and 

the top lipid layer removed. The process 

was repeated three times. The Casein was 

precipitated and separated from whey by 

acidification with 1 N HCl at pH 4.6. The 

precipitate was then removed by 

centrifugation (5000 g, 30 min, at 4 °C). 

The clear straw colored whey was obtained 

as a supernatant and filtered through 

Whatman filter paper N°1 (Whatman Ltd., 

Maidstone, UK). Whey globulins were 

eliminated by precipitation with 

ammonium sulfate at 58% saturation (2.3 

M) and at 4 °C in 24 h. The supernatant 

obtained from this step contained α-

lactalbumin (α-Lb), β-lactoglobulin (β-Lg), 

bovine serum albumin (BSA) and 

lactoferrin (Lf). pH was adjusted to 7.0 

using diluted 1 N NaOH solution. The 

whey proteins were dialyzed against 

distilled water, lyophilized, stored at - 20 

°C and referred to as Cow Whey Proteins 

(CWP). The purity of CWP was checked 

using SDS-PAGE. 

 

Preparation of Whey Protein 

Hydrolysates 

Whey protein hydrolysates (WPH) were 

prepared as described by Pescuma et al. 

[16] with slight modifications.  

Lactobacillus strains were inoculated in 

MRS broth medium with 5 mM CaCl2. The 

inoculum was cultured in 300 ml MRS at 

30 °C for 18 h. Cells were collected by 

centrifugation (5000 g, 10 min, 4 °C) at the 

exponential growth phase (OD600nm= 1), 

washed twice with 0.85% (w/v) saline 

solution supplemented with 10 mM CaCl2, 

and suspended in 100 mM sodium 

phosphate (pH 7.0) with 5 mM CaCl2. Cell 

suspensions were kept at 37 °C for 30 min 

for amino acid starvation; non-proliferating 

cells of strains were incubated with 1% 

CWP (w/v) in a cells/cow whey proteins 

ratio (C/CWP) of 2/1 (v/v). CWP were 

previously dissolved in 100 mM sodium 

phosphate (pH 7.0) supplemented with 5 

mM CaCl2 and heated at 80 °C for 30 min 

(to avoid microbial contamination of 

whey). The cells-whey proteins mixture 

was incubated for 24 h at 37 °C; samples 

were withdrawn and then centrifuged 

(5000 g, 10 min, 4 °C). WPH were 

analyzed by estimation of hydrolysis 

degree and by SDS–PAGE. 

 

Determination of the TCA-Soluble 

Proteins  



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

28 

The trichloroacetic acid (TCA)-soluble 

proteins of the WPH were determined by 

the method of Folin-Ciocalteu [17]. An 

aliquot of the hydrolysates was added to an 

equal volume of TCA (12% w/v). The 

mixture was incubated for 30 min at room 

temperature then centrifuged (5000 g, 15 

min, 4 °C). An aliquot (500 μl) of the 

supernatant (WPH) was added to 2500 μl 

of NaOH (0.2 M) and 250 μl of Folin-

Ciocalteu reagent diluted twice with 

distilled water. The reaction mixture is 

stirred and left at room temperature for 10 

minutes. The blue color that develops is 

measured spectrophotometrically (Optizen, 

2120 UV, Mecasys Co., Ltd, Korea) at 750 

nm against blank (mixture without 

hydrolysate). The results were expressed as 

μg of tyrosine per ml referring to a 

standard curve of tyrosine (0–50 µg/ml). 

 

Determination of the Hydrolysis Degree 

(DH) 

The degree of hydrolysis (DH) is defined 

as the percentage of peptide bonds cleaved. 

It was determined by using a modification 

of the method described by Boudrant and 

Cheftel [18] and was defined as follows, 

expressed as per cent:  

 
Where Dt corresponded to the amount of 

TCA soluble peptides after whey protein 

hydrolysis expressed as tyrosine; D0 

corresponded to soluble peptides of 

unhydrolysed whey proteins expressed as 

tyrosine; Dmax corresponded to the 

maximum amount of TCA soluble peptides 

expressed as tyrosine and determined after 

hydrolysis of the whey protein solution by 

HCl (6 N) at 110 °C for 24 h [19]. 

 

Electrophoresis (SDS-PAGE)  

The samples from CWP hydrolysis were 

examined by polyacrylamide-SDS gel 

electrophoresis as described by Laemmli 

[20] using PAGE mini vertical protein 

electrophoresis system (Cleaver Scientific 

Ltd, UK). The polyacrylamide gels 

consisted of 17% resolving gel and 5% 

stacking gel. T0, T24h samples for each 

strain as well as sample of CWP non 

hydrolysed and the molecular weight 

marker (Protein marker standard 11-190 

kDa, New England Biolabs Inc., Ipswich, 

MA, USA) were  loaded separately at a 

total running time of 3 h at 120 V. The gels 

were fixed in a solution of 12% TCA for 

15 min and then stained by using 

Coomassie brilliant bleue R–250 stain 

solution (Merck, Saint Louis, MO) for 5 h 

with 2–3 changes of the destaining 

solution. 

 

Optimization of the Hydrolysis 

Conditions 

Hydrolysis of CWP by the strains was 

conducted as described by Pescuma et al. 

[16] under different conditions of 

temperature, pH, C/CWP ratio and time 

hydrolysis.  

 

Incubation Temperature Effect 
The effect of incubation temperature was 

determined in 10 mM phosphate buffer 

(pH 7.0) in the range of 30 to 45 °C with a 

C/CWP ratio of 2/1 (v/v). Samples were 

withdrawn after 24 h and 48 h of 

incubation and then centrifuged (5000 g, 

10 min, 4 °C). The hydrolysates have been 

analyzed by estimation of hydrolysis 

degree and by SDS–PAGE. 

 

pH Effect  

The effect of pH on the hydrolysis of CWP 

was examined in the pH range of 6 to 8 by 

using different buffers: Acetate buffer 0.2 

M, phosphate buffer 0.2 M, tris 

(hydroxymethyl) aminomethane-HCl 

buffer 0.2 M. The cells-whey protein 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

29 

mixtures were incubated at 30 °C with a 

C/CWP ratio of 2/1(v/v). Samples were 

pulled out after 24 h, 48 h of incubation, 

centrifuged (5000 g, 10 min, 4 °C) and 

analyzed. 

 

C/CWP Ratio Effect  

The enzymatic process was carried out at 

three different C/CWP ratios (1/1, 1/2 and 

2/1). The relationship between the 

hydrolysis of CWP by the strains and the 

cells/whey proteins ratio was studied at 30 

°C and pH 7. Samples were withdrawn 

after 24 h and 48 h of incubation, 

centrifuged (5000 g, 10 min, 4 °C) and 

then analyzed. 

 

Antioxidant Activity by DPPH Radical 

Scavenging assay 

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) 

radical scavenging activity of the 

hydrolysates was determined according to 

the method described by Lin et al. [10] 

with minor modifications. At first, 2744 µl 

of methanolic DPPH solution (0.2 mM) 

were added to 56 µl aliquot of hydrolysate 

solution or phosphate buffer (control). The 

mixture was shaken vigorously and kept 

under dark at ambient temperature for 30 

min. Absorbance of each sample and 

control was read at 517 nm against blank 

by using a UV-visible spectrophotometer 

(Optizen, 2120 UV, Mecasys Co., Ltd, 

Korea). The antioxidant activity of 

hydrolysates was estimated by comparison 

with ascorbic acid which is known as 

natural antioxidant. The DPPH radical 

scavenging activity, expressed as the 

percentage of inhibition, was calculated by 

the following equation: 

 
Where AC is the absorbance of the control 

and AS is the absorbance of the sample. 

 

Nitric Oxide Radical Scavenging 

Activity  

The effect of hydrolysates on nitric oxide 

(NO) radical scavenging was investigated 

by using a sodium nitroprusside (SNP) 

generating NO system. Briefly, 100 mM of 

SNP was dissolved in phosphate buffered 

saline (PBS) pH 7.4. The tested 

hydrolysates were added to SNP (10 mM) 

in PBS in a final volume of 2 ml and then 

incubated at 25 °C for 150 min. A control 

experiment without tested hydrolysates 

was conducted in an identical manner. 

After incubation, 1.0 ml of samples was 

diluted with 1.0 ml of Griess reagent (1% 

sulphanilamide and 0.1% 

naphthyletylenediamine dihydrochloride in 

2% H3PO4) [21]. Ascorbic acid was used 

as a positive control. The mixtures were 

kept under dark at room temperature for 30 

min, and then the absorbance measured at 

540 nm against the corresponding blank 

solutions. The percentage of NO inhibition 

was calculated by the following equation: 

 
Where AC is the absorbance of the control 

and AS is the absorbance of the sample. 

 

Assay for Antibacterial Activity 

The antibacterial activity was tested by 

using a well diffusion method in agar [22]. 

The indicator strains used to detect the 

antibacterial activity were  Staphylococcus 

aureus 25923, Pseudomonas aeruginosa 

27853, Escherichia coli 25922 (From 

Pasteur Institute, Algeria), ten (10) 

bacteria: Salmonella typhi, Serratia 

marcescens, Proteus mirabilis, Citrobacter 

freundii, Klebsiella pneumoniae, 

Acinetobacter baumanii, Bacillus subtilis, 

Bacillus cereus, Enterobacter aerogenes 

and Enterococcus faecium (isolated and 

identified at our laboratory). To check the 

antibacterial activity, target strains were 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

30 

grown in nutrient broth overnight at 37 °C. 

The plates were further overlaid with soft 

nutrient agar (0.75% w/v agar) inoculated 

at 1% (v/v) with cultures of indicator 

strains adjusted to OD600nm= 1. Wells of 5 

mm diameter were cut with sterile well 

borer into these agar plates. 50 μl of WPH 

(T0, T24h and T48h of each strain) previously 

filtered through 0.22 μm pore-size filters 

were placed into each well. Plates were 

stored at 4 °C for overnight to permit 

diffusion of samples, and then incubated at 

37 °C for 24 h. The appearance of a clear 

halo around the wells was measured and 

presented as inhibition zones (mm). 

 

Statistical Analyses: 

All experiments were carried out in 

triplicate. The results were expressed as 

mean ± standard error (SE). Mean 

separation and significance were analyzed 

using the IBM SPSS® software (SPSS 

Statistical Software, Inc., Chicago, IL, 

USA). One-way analysis of variance 

(ANOVA) was carried out to discriminate 

among the means. Tukey and Student-

Newman-Keuls procedures were used as 

post-hoc tests. Differences between 

samples were considered as significant at P 

< 0.05. 

 

3. Results and discussion 

 

Proteolytic Activity of Strains  

The proteolytic systems of lactic acid 

bacteria are important means of making 

free amino acids and peptides from milk 

proteins for growth. In this study a total of 

11 Lactobacillus strains were screened for 

proteolysis. The results show that all the 

strains exhibited proteolytic activity 

(Figure 1). Proteolytic activity has been 

reported several times for LAB, including 

Enterococcus, Lactobacillus, Lactococcus, 

and Pediococcus [23]. The Lactobacillus 

genus, produce proteases that can 

hydrolyze α- and β-casein thus, having a 

direct effect on the biochemistry of dairy 

products. Furthermore, the hydrolysis of 

proteins in milk (i.e., casein, β-Lg, and α-

Lb) produces small peptides, which have 

been suggested to present biological 

activities [24]. 

 

However, the strains were different in the 

efficiency proteolysis. The H/C ratio varies 

 from 1.24±0.028 (Lb. paracasei subsp. 

paracasei LBS2) to 2.65±0.028 (Lb.brevis 

CHTD27) and 2.71±0.042 (Lb. plantarum 

LBBS2) which represent the highest values 

(p˂0.05) (Figure 1). In fact, variability in 

proteolysis is commonly reported for 

isolates of lactic acid bacteria from natural 

sources [25]. Each microorganism used in 

food fermentation has different capacities 

to produce enzymes that hydrolyze 

proteins in different conditions [26]. 

Based on this screening, the strains Lb. 

plantarum LBM2, Lb. plantarum LBBS2 

from ewe milk, and Lb.brevis CHTD27 

from camel milk which have displayed the 

greatest proteolytic activity among the 

strains tested (p˂0.05), were further 

subjected to whey protein hydrolysis and 

whether potentially healthful bioactive 

peptides could be produced. 

Ability of Strains to Hydrolyse Whey 

Proteins 

The hydrolysis reaction was carried out on 

sodium phosphate at 37 °C, pH 7.0, with 

ratio C/CWP of 2/1 for 24 h. 

To compare the extent of hydrolysis, whey 

protein hydrolysis in terms of degree of 

hydrolysis was determined by measuring 

amount of amino acids and peptides 

liberated, using the Folin-Ciocalteu 

reagent. The results showed that CWP 

were degraded by all the strains studied; 

however, the efficiency of hydrolysis was 

strain dependant. 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

31 

 
Fig. 1. Proteolytic activity of whole cells on Milk-Agar (3%) medium (A); Halo/Colony values (B) obtained at 
30 °C after 48 h of incubation. Bars represent the standard error from triplicate determinations. Values with 

different letters (a-g) indicate results significantly different between the strains (P < 0.05). 

 

As shown in Figure 2, the DH value in 

WPH generated by Lb. plantarum LBBS2 

(24.92%±0.61) was significantly higher 

than Lb. plantarum LBM2 (16.51%±0.39) 

and Lb. brevis CHTD27 (10.46%±0.48) 

which showed the lowest rate of hydrolysis 

(P<0.05).  

 

 
Fig 2. DH values of the hydrolysates obtained by 

the strains at 37 °C, pH 7 with ratio C/CWP (2/1) 

after 24 h of incubation. Bars represent the standard 

error from triplicate determinations. Values with 

different letters (a-c) indicate significant differences 

between hydrolysates (P<0.05). 

 

Previous research specified that the 

activity of proteinases and peptidases 

varied among lactic acid bacteria species 

[27, 28]. Nevertheless, in previous 

research, the strain Lb. brevis CHTD27 

from camel milk which is weakly 

proteolytic on whey proteins, was able to 

cleave to a greater extent caseins. This 

better performance on caseins could be due 

to protease specificity of this bacterium 

[29]. 

Electrophoresis SDS-PAGE revealed 

different peptide profiles from CWP 

hydrolysis according to the strain tested. 

The electrophoresis diagram (Figure 3) 

results of CWP (lane 2) showed four bands 

i.e. Lf (88,000 kDa), BSA (69,000 kDa), β-

Lg (18,400 kDa) and α-Lb (14,300 kDa). 

Hydrolysis revealed a decrease in protein 

intensity at T24h compared to T0 for the 

strains Lb. plantarum LBM2 (lanes 5, 6) 

and Lb. plantarum LBBS2 (lanes 7, 8) but 

no changes were observed in the intensity 

of the proteins for Lb. brevis CHTD27 

(lanes 3, 4). These data were consistent 

with earlier results of proteolytic activity 

by assessment of DH. Several studies 

revealed that an increase in DH of the 

proteins decreased their molecular size by 

creating small peptides [9, 24]. 

 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

32 

 
Fig 3. SDS-PAGE analysis of CWP hydrolysed by the strains at 37 °C, pH 7 with ratio C/CWP (2/1) after 24 h 

of incubation. (1) Molecular weight marker ; (2) Non hydrolysed CWP ; (3) and (4) Hydrolysates obtained with 

the strain CHTD27 at T0, T24h respectively ; (5) and (6) Hydrolysates obtained with the strain LBM2 at T0,  T24h 

respectively ; (7) and (8) Hydrolysates obtained with the strain LBBS2 at T0, T24h respectively. 

Optimization of Hydrolysis of Whey 

Proteins by the Strains 

In order to enrich hydrolysates with 

potentially bioactive peptides, CWP 

hydrolysing ability of the strains was 

optimized under different conditions. 

Results proved that hydrolysis 

temperature, pH, C/CWP ratio and 

incubation time had a great effect on the 

DH of whey proteins. The strains exhibited 

activity at temperatures ranging from 30 

°C to 40 °C with an optimum at 30 °C 

(P<0.05) (Table 1). Guo et al. [28] also 

reported an optimum temperature of 30 °C 

for hydrolysis of whey proteins by Lb. 

helveticus LB13. This could be explained 

by the fact that an overly high temperature 

might have caused proteinase denaturation 

and lowered enzyme activity. The effect of 

pH on the hydrolysis was examined for the 

pH range 6 to 8. Activity was maximal at 

pH 7 (P<0.05). Pan et al. [30] also 

optimized the proteolytic conditions of Lb. 

helveticus JCM1004 at pH 6.5–7.0. The 

ratio C/CWP of 2/1 revealed optimum 

hydrolysis of whey proteins by the strains 

(P<0.05). The increase of C/CWP ratio 

resulted in an increase in DH of whey 

proteins. The results could be due to 

greater hydrolysis of the proteins when 

more proteases were added [31]. The 

incubation time also influenced the 

degradation extent of whey proteins which 

was maximal after 48 h (P<0.05). Similar 

results were observed for the strains Lb. 

acidophilus CRL 636, Lb. delbrueckii 

subsp. bulgaricus CRL 656 and 

Streptococcus thermophilus CRL 804 

when the incubation period on whey 

protein concentrate was extended [32]. 

Under optimal conditions, the DH values 

in whey protein hydrolysates obtained with 

Lb. plantarum LBBS2 and Lb. plantarum 

LBM2 were significantly higher than the 

strain Lb. brevis CHTD27 which showed 

the lowest rate of hydrolysis (P<0.05). 

Analysis by SDS-PAGE revealed different 

profiles (data not shown) according to the 

strains and parameters studied. The 

peptides pattern produced from CWP 

hydrolysis differed among the 

Lactobacillus strains used, suggesting 

different protease specificities. 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

33 

Table 1.  

Effect of temperature, pH, ratio cells (C)/cow whey proteins (CWP) and time incubation on hydrolysis of CWP by 

the strains CHTD27, LBM2 and LBBS2, expressed by measurement of DH (%) (mean ± standard error, n=3). 

  CHTD27 LBM2 LBBS2 

  24 h 48 h 24 h 48 h 24 h 48 h 

30 °C 18.01±0.47b 28.66±1.42a 38.33±0.56d 64.71±0.75a 39.80±1.04cd 82.24±0.47a 

37 °C 10.80±0.85c 19.49±0.85b 17.19±1.41g 38.98±0.75d 34.88±0.75e 51.27±0.56b 

40 °C 06.37±0.47d 12.93±1.04c 11.95±0.56i 16.21±0.47g 22.93±1.13i 38.16±0.47d 

45 °C 04.08±0.47d 06.71±0.37d 10.80±0.75i 15.06±0.94gh 14.89±0.66k 30.62±0.47f 

pH 6 00.00±0.00e 00.00±0.00e 00.00±0.00j 00.00±0.00j 00.00±0.00l 00.00±0.00l 

pH 7 18.83±1.70b 31.12±1.89a 38.00±0.66d 58.32±0.37c 35.21±0.85e 80.28±0.94a 

pH 7.5 00.00±0.00e 00.00±0.00e 21.12±0.38f 33.58±1.41e 27.51±0.28h 41.33±0.66c 

pH 8 00.00±0.00e 00.00±0.00e 00.00±0.00j 00.00±0.00j 19.32±0.65j 35.38±0.94e 

C/CWP 00.00±0.00e 00.00±0.00e 00.00±0.00j 00.00±0.00j 0.00±0.00l 00.00±0.00l 

C/2CWP 00.00±0.00e 06.38±0.85d 13.42±0.85hi 20.79±1.23f 16.53±0.37k 28.98±0.85fg 

2C/CWP 16.21±2.08b 28.33±0.8a 39.15±0.37d 60.77±0.56b 36.03±0.28e 81.09±0.66a 

Different letters (a, b…) in hydrolysates of the same strain indicate significant differences at different conditions 

of hydrolysis (P < 0.05). 

 

Results were related to the degree of 

hydrolysis of CWP upon hydrolysis by the 

strains. As depicted in Figure 4, at 30 °C, 

pH 7, ratio C/CWP (2/1) and at 48 h of 

hydrolysis, the strain Lb. plantarum 

LBBS2 exhibited the most important 

activity. The protein bands became lighter 

at T48h resulting in higher hydrolysis. 

Furthermore, small fragments appeared 

with molecular weights lower than Lf and 

BSA. This is due to the production of some 

peptides obtained after CWP hydrolysis. 

The strain Lb. plantarum LBM2 showed 

less pronounced hydrolysis than Lb. 

plantarum LBBS2, whereas the strain 

Lb.brevis CHTD27 revealed thicker 

protein bands which are indicatives of 

lower hydrolysis. These results point to a 

different proteolytic behavior for the 

strains.  

Antioxidant Properties of Whey Protein 

Hydrolysates (WPH) 

The results indicated that all WPH reduced 

the DPPH radical to a yellow-coloured 

compound, manifestly because of the 

DPPH radical accepting an electron or 

hydrogen to become a stable molecular. 

However, antioxidant capacity was strain-

specific and reached to maximum at 24 h 

of hydrolysis compared with intact whey 

proteins (T0) for all the strains. As shown 

in Figure 5, WPH derived from Lb. 

plantarum LBBS2 resulted in significantly 

slightly higher antioxidant capacity 

(65.98%±0.11) compared to antioxidant 

activity of ascorbic acid (62.16%±2.69), 

followed by Lb. plantarum LBM2 

(55.59%±1.30), whereas WPH from 

Lb.brevis CHTD27 exhibited the lowest 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

34 

(33.99%±2.42) antioxidant capacity (P< 0.05).  

 

 
Fig. 4. SDS-PAGE analysis of CWP hydrolysed by the strains at optimal conditions: 30 °C, pH 7, with ratio 
C/CWP (2/1) after 48 h of incubation. (1) Non hydrolysed CWP; (2) and (3) Hydrolysates obtained with the 

strain CHTD27 at T0, T48h respectively; (4) and (5) Hydrolysates obtained with the strain LBM2 at T0, T48h 

respectively; (6) and (7) Hydrolysates obtained with the strain LBBS2 at T0, T48h respectively. 

 

The results of NO radical scavenging assay 

were similar to those of DPPH radical 

scavenging activity. At 24 h of hydrolysis, 

WPH from Lb. plantarum LBBS2 showed 

significantly the highest antioxidant 

activity (57.41%±1.52) similar to ascorbic 

acid (61.71%±0.98), followed by Lb. 

plantarum LBM2 (48.29%±0.60), while 

WPH from Lb.brevis CHTD27 displayed 

the lowest radical scavenging properties 

(37.61%±1.10) (P<0.05) (Figure 5).  

It was demonstrated that Lb. plantarum 

strains screened from conventional 

fermented food possess several functional 

features, including antioxidant properties 

[33]. The results observed in our 

experiments are in accordance with those 

reported by Lin et al. [10] who found that 

the antioxidant activity of whey protein 

concentrate solution was improved by 

enzymatic hydrolysis. This was in 

agreement with observations of other 

results that indicated a higher DPPH 

radical scavenging activity for an 

enzymatically-prepared whey protein 

hydrolysates than whey protein isolates 

[34]. The increased antioxidant activity of 

samples could be attributed to the 

hydrolysis of proteins/peptides by the 

proteases of Lactobacillus strains. 

Non hydrolysed whey proteins showed a 

relatively low antioxidant activity. These 

results relating to the antioxidant activity 

of whey proteins are consistent with 

previous studies which reported that whey 

is essentially consisting of polar 

antioxidant compounds [35]. 

However, the radical scavenging activity 

of hydrolysates decreased for all strains 

upon further hydrolysis and was not 

directly connected to DH after 24 h (Figure 

5). This suggests that high DH may result 

in further degradation of antioxidant 

peptides into short peptides with less 

activity or none [24]. These results were in 

agreement with the findings of Virtanen et 

al. [9] on 25 strains of LAB which showed 

that the radical scavenging activity is more 

dependent on specific proteolytic enzymes 

of bacterial strains than on high proteolytic 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

35 

state of strains. The ability of protein 

hydrolysates to inhibit damaging changes 

caused by lipid oxidation seems to be 

related to the nature and the composition 

of the various peptide fractions produced, 

depending on the protease specificity [36]. 

Antioxidative properties of the peptides are 

related to their composition, structure, and 

hydrophobicity [6]. According to the 

results obtained in our work, the 

development of antioxidative activity was 

strain dependent and generally enhanced 

during hydrolysis but was not directly 

correlated with hydrolysis time [9]. 

 

 

 
Fig. 5. Antioxidant activities of CWP hydrolysates expressed as percentage inhibition of DPPH (A), and NO (B) 
radicals. Error bars represent the standard error of the mean of triplicate experiments. Values with different 

letters (a-f)  indicate results significantly different (P < 0.05). 

 

Antibacterial Activity  

As depicted in Table 2, no inhibitory 

activity was observed nor for non-

hydrolysed whey proteins (T0) nor for the 

hydrolysates at T24h for all the strains. 

Nonethless, hydrolysates obtained after 

48h from the strains Lb. plantarum LBM2 

and LBBS2 exhibited growth inhibition 

against the target strains tested except 

Bacillus subtilis that was not affected. 

Salami et al. [8] noted that hydrolysis of 

cow whey proteins showed a tendency for 

increased antimicrobial potential when 

compared to non-hydrolysed whey 

proteins. Furthermore, the substrate 

specificities of CEPs determine the pools 

of generated peptides, which are 

responsible for the functional activities 

exerted [24]. WPH derived from the strain 

Lb. plantarum LBM2 showed antagonistic 

effects against 7 tested strains (2 Gram 

positive and 5 Gram negative), WPH from 

Lb. plantarum LBBS2 were  able to inhibit 

6 target microorganisms (1 Gram positive 

and 5 Gram negative), whereas 

hydrolysates obtained with Lb. brevis 

CHTD27 were unable to inhibit the growth 

of the thirteen target bacteria assayed.  

Furthermore, the highest inhibition halo 

zones were obtained with WPH from Lb. 

plantarum LBBS2. This could be related to 

its proteolytic activity which was 

significantly the most important (P < 0.05) 

(Table 2). 

Molecular distribution profiles obtained at 

optimal conditions showed that 

hydrolysates obtained by the strains Lb. 

plantarum LBBS2 and LBM2 which 

exhibited antibacterial inhibition also 

possessed a higher proportion of peptides, 

in contrast with the strain Lb. brevis 

CHTD27 (Figure 4). 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

36 

 

Table 2.  

Antibacterial activity of hydrolysates obtained from the strains at t0, t24h and t48h against Gram positive 

and Gram negative bacteria. 

  Samples tested 

Microorganisms 

  

CHTD27 LBM2 LBBS2 

T0 T24h T48h T0 T24h T48h T0 T24h T48h 

Gram
+
                   

Staphylococcus aureus 

25923 
na na na na na na na na 12.95±0.086a 

Bacillus subtilis na na na na na na na na na 

Bacillus cereus na na na na na 07.60±0.17d na na na 

Enterococcus faecium na na na na na 08.94±0.20c na na na 

Gram
-
                   

Pseudomonas aeruginosa 

27853 
na na na na na 07.8±0.11d na na na 

Acinetobacter baumanii na na na na na na na na 09.36±0.069c 

Escherichia coli 25922 na na na na na 07.86±0.054d na na na 

Salmonella typhi na na na na na 06.85±0.17e na na na 

Proteus mirabilis na na na na na na na na 09.15±0.11c 

Citrobacter freundii na na na na na 07.96±0.16d na na na 

Klebsiella pneumoniae na na na na na na na na 10.71±0.23b 

Serratia marcescens na na na na na 09.55±0.11c na na 12.35±0.13a 

Enterobacter aerogenes na na na na na na na na 12.84±0.12a 

Results are expressed as the mean of the diameter of the inhibition zone in mm±standard error, n=3. Different 

letters (a-e) indicate significant differences between hydrolysates (P<0.05). na: no antibacterial activity 

 

Thus, the pattern of antibacterial activity 

observed for Lb. plantarum LBBS2 and 

Lb. plantarum LBM2 strains could be 

associated with their proteolytic activity on 

whey proteins, since most antibacterial 

capacity accords with the greatest 

proteolytic activity and the increase of 

peptides production. 

Pellegrini et al. [37] reported that 

proteolytic digestion of bovine β-Lg by 

trypsin generates four peptide fragments 

with bactericidal activity. Nevertheless, the 

most studied antimicrobial peptides are the 

lactoferricins derived from Lf. The 

antimicrobial activity of lactoferricin 

seems to be related with the net positive 

charge of the peptides. These cationic 

peptides kill sensitive microorganisms by 

increasing cell membrane permeability [6]. 

Indeed, the difference in amino acid 

sequence, charge distribution, net charge, 

size, and amphipathicity as well as 

secondary structure could be responsible 

for the differential behavior of antibacterial 

peptides against bacteria [38].  

 

4. Conclusion 

In this study, whey proteins extracted from 

cow milk were used in order to analyze 

their degradation by strains of 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
– 39 

 

 

 

37 

Lactobacillus isolated from ewe milk and 

camel milk. Weak hydrolysis of whey 

proteins was noted for the camel milk 

strain Lb. brevis CHTD27. However, 

interesting rate of hydrolysis was observed 

for two ewe milk strains Lb. plantarum 

LBBS2 and Lb. plantarum LBM2, 

suggesting the potential interest of these 

strains. Optimum of hydrolysis of bovine 

whey proteins was obtained at pH 7, 30 °C, 

with ratio cells/whey proteins of (2/1) and 

after 48 h of incubation. Hydrolysates 

derived from Lb. plantarum LBBS2 and 

Lb. plantarum LBM2 exhibited antioxidant 

and antibacterial activities suggesting that 

proteases of these strains have the ability 

to release bioactive peptides from cow 

whey proteins. At optimal conditions, 

hydrolysates from the strains Lb. 

plantarum LBBS2 and Lb. plantarum 

LBM2 have displayed a large activity 

spectrum towards different Gram positive 

and Gram negative bacteria. However, the 

highest antioxidant activity was obtained 

from these same hydrolysates at 24 h. 

Based on these interesting properties, whey 

proteins could be used for the production 

of molecules with a promising interest as 

antibacterials and antioxidants. The strains 

Lb. plantarum LBBS2 and Lb. plantarum 

LBM2 could represent promising 

candidates to be used as potential food 

biopreservatives. Meanwhile, additional 

studies are needed to prove the safety of 

these strains, to identify and characterize 

peptides produced and nutraceutical 

properties.   

 

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Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
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Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 

Volume XIX, Issue 1 – 2020 

 

 
Hayet MESSAOUI, Salima ROUDJ, Nour-Eddine KARAM, Antioxidant and antibacterial activities of bovine whey 
proteins hydrolysed with selected lactobacillus strains, Food and Environment Safety, Volume XIX, Issue 1 – 2020, pag. 25 
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	1. Introduction
	4. Conclusion
	In this study, whey proteins extracted from cow milk were used in order to analyze their degradation by strains of Lactobacillus isolated from ewe milk and camel milk. Weak hydrolysis of whey proteins was noted for the camel milk strain Lb. brevis CHT...
	[1]. DAS D. & GOYAL, A., Lactic acid bacteria in food industry.  Microorganisms in sustainable Agriculture and Biotechnology, Springer, pp. 757-772, (2012).