Maria Doneva, Kamelia Loginovska, Svetla Dyankova,

Journal Iliana
of Chemical Technology
Nacheva, and Metallurgy,
Petya Metodieva, Nadya 59, 1, 2024, 15-22
Ninova-Nikolova

APPLICATION OF WHEY PROTEIN HYDROLYSATES

AS A FERMENTATION MEDIUM WITH STRAINS OF LACTIC ACID BACTERIA

Maria Doneva, Kamelia Loginovska, Svetla Dyankova,

Iliana Nacheva, Petya Metodieva, Nadya Ninova-Nikolova

Institute of Cryobiology and Food Technologies

Agricultural academy, 53 Cherni Vrah blvd.
Sofia 1407, Bulgaria
E-mail: maria.doneva@ikht.bg

Received 02 February 2023

Accepted 15 September 2023 DOI: 10.59957/jctm.v59.i1.2024.2

ABSTRACT

The aim of the present study is to obtain enzymatic hydrolysates from whey protein and evaluate their potential
application as a substrate for fermentation with lactic acid bacteria. For hydrolysis of whey proteins, papain is
used in 3 concentrations - 0.1, 0.5 and 1.0 mg mL-1. The degree of hydrolysis (DH) was determined on the obtained
whey protein hydrolysates (WPHs), the highest percentage being achieved with hydrolysate WPH3 using the papain
enzyme at a concentration of 1 mg mL-1. Fermentation of the whey protein hydrolysates was carried out with the
participation of the probiotic strains Lactiplantibacillus plantarum subsp. plantarum NBIMCC 3447 and Lactobacillus
gasseri NBIMCC 2450. The survival of two strains of probiotic lactic acid bacteria was investigated. In the samples
obtained from hydrolyzed whey medium with 0.5 mg mL-1papain, the highest values of viable probiotic bacterial
cells were recorded - for Lactiplantibacillus plantarum NBIMCC 3447 2.5 х 109 CFU mL-1, and for Lactobacillus
gasseri NBIMCC 2450 2.1 х 109 CFU mL-1, respectively. Antioxidant activity is reported on the fermented products.
The WPH2 variant has the highest antioxidant potential - 9.14 mg TE/100 mL.
Keywords: whey protein hydrolysates, papain, fermentation.

INTRODUCTION into value-added products, such as functional foods,

Whey is a milk serum that is obtained as a waste
product in the production of various types of cheese
and yogurt [1]. Whey is considered the most important
contaminant in the dairy industry, not only because of
its high organic content, but also because of its large
volume [2]. This necessitates limiting its discharge into
sewage systems and more thoroughly investigating the
nutritional and biological properties of whey proteins,
as well as finding new forms for their application.
Whey contains about half of the nutrients that milk
contains, including whey proteins, lactose, minerals
and water-soluble vitamins. Whey proteins possess
important nutritional and functional properties and are a
rich source of amino acids [3]. In the past few decades,
many researches have focused on transforming whey
whey protein hydrolysates, whey permeate, bioethanol,
probiotics, etc. [4, 5].
Hydrolysis technology is of interest as a leading
strategy for converting whey into value-added hydrolyzed
products. Hydrolysis of whey protein is known to release
bioactive peptides. These peptides are inactive in the
parent protein sequence and can be released by enzymes
or by fermentation.
The hydrolysates obtained in this way can exhibit a
number of physiological properties such as antioxidant,
antihypertensive, antimicrobial activity and opioid
activity [6 - 9].
Whey is also successfully transformed into value-
added products through the lactic acid fermentation
process. Under the action of lactic acid microorganisms,
probiotic whey drinks are obtained. By degrading whey
15
 Journal of Chemical Technology and Metallurgy, 59, 1, 2024
proteins, lactic acid bacteria also release low molecular
mass peptides, and the resulting probiotic products can
be used for dietary prophylaxis in diseases related to the
human digestive system [10, 11].
Protein hydrolysates are an essential component in a
wide range of functional food products for infants, children
and the elderly, for people with a compromised immune
system, as nutritional supplements with a beneficial effect
on human health and tone. This determines the increased
interest in their research and extraction.
Hydrolyzed milk protein is a food that is rapidly
absorbed by the human body and has a biostimulating
effect. It is especially useful for people subjected to
prolonged physical or mental stress [12, 13].
The objectives of the present study were to obtain
and to study enzymatic hydrolysates from whey protein
and to evaluate their potential application as a substrate
for fermentation with lactic acid bacteria.
EXPERIMENTAL
Materials
Papain - EC 3.4.22.2 was purchased from
MP Biomedicals LLC. DPPH (1.1-diphenyl-2-
picrylhydrazyl) free radical, Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) and ninhydrin
were supplied by Sigma Aldrich. All chemicals and
reagents used were of analytical grade.
Preparation of whey protein hydrolysate
In this study, commercial Whey Protein Concentrate
(WPC - 80), obtained from Arla Company, Denmark,
were applied as a substrate of hydrolysis reaction. Papain
with activity 10 units/mg protein was used as enzyme.
WPC was mixed with water in the ratio of 1:10 (w/v)
to obtain slurry. For hydrolysis of whey proteins, papain
is used in 3 concentrations - 0.1, 0.5 and 1.0 mg mL-1,
from which 3 variants of hydrolysates: WPH1, WPH2 and
WPH3 are obtained. The hydrolysis process was carried
out in a water bath at a temperature of 45ºС, pH 7.0 for
30 min. The process was terminated by heating to 85ºС
for 5 min, after which the samples were rapidly cooled.
Each hydrolysate was analyzed for degree of hydrolysis
and antioxidant activity.
Degree of hydrolysis
Degree of hydrolysis (DH) was determined by
16
a modified Adler-Nissen method using ninhydrin
reagent [14].
The degree of hydrolysis was determined by
a method based on the reaction of amino acids
with ninhydrin hydrate (C 9Н 6O 4хН 2О) at рН 5.0,
temperature 100°C and a certain period. For this
purpose, the obtained hydrolysates are centrifuged
for 10 minutes at a temperature of 8ºС and 4000 rpm.
0.4 mL of ninhydrin solution is added to 2 mL of the
supernatant. The samples are heated to a temperature
of 100ºС for 10 min after which they are cooled and the
absorption is measured at a wavelength of λ = 570 nm
against a blank sample (Libra S 22, Biochrom UV-Vis
spectrophotometer, USA).
Determination of antioxidant activity
The DPPH radical scavenging activity of whey
protein hydrolysates was evaluated using the method of
Brand-Williams [15]. DPPH radical solution (0.004 %,
w/v) in 95 % ethanol was prepared. A volume of 2 mL of
DPPH solution was added to 2 mL of samples, vortexed
and incubated for 30 min in dark at room temperature
(25°C ± 0.5). Absorbance of each sample was measured
at 517 nm using UV-Vis spectrophotometer (Libra S22,
Biochrom, USA). Ethanol was used as a blank, while
DPPH solution in ethanol was used as a control. Trolox
solutions in ethanol with concentration from 0.045 to
1.5 mmol were used to prepare a standard curve and
quantify the antioxidant activity. The determination of
antioxidant activity was performed in triplicate and the
antioxidant activity was expressed as Trolox equivalent
- mg TE/100 mL.
SDS-polyacrylamide gel electrophoresis
SDS-PAGE was performed by the method of
Laemmli at concentrations of stacking gel - 6 %, running
gel - 12 %, using an OmniPAGE WAVE Еlectrophoresis
System (Cleaver Scientifics) [16].
The samples were diluted with buffer (0.2 М Tris-
HCl, pH 6.80, containing 2 % SDS, 16 % glycerol and 10
mM DDT). Protein Test Mixture for SDS PAGE (SERVA
Electrophoresis), α-lactalbumin, β-lactoglobulin,
α-casein and BSA - bovine serum albumin (Sigma-
Aldrich) were used as standard proteins. Gel was stained
using 0.1 % Coomassie Brilliant Blue (30 - 40 min). The
conditions of electrophoresis were as follows: current
30 mA, initial voltage 110 V, time 3.5 h.
 Maria Doneva, Kamelia Loginovska, Svetla Dyankova,
Iliana Nacheva, Petya Metodieva, Nadya Ninova-Nikolova

Bacterial strains and cultivation conditions
Probiotic bacteria
Studies were conducted with two probiotic lactic
acid bacteria of the genus Lactobacillus provided by
the National Bank for Industrial Microorganisms and
Cell Cultures. A strain of Lactiplantibacillus plantarum
subsp. plantarum NBIMCC 3447 isolated from
sauerkraut (Denmark) and Lactobacillus gasseri strain
NBIMCC 2450 isolated from faeces (Germany). Both
strains were supplied in freeze-dried form. They were
reconstituted and subcultured 5 times in MRS - broth
(De Man’s medium, Rogosa & Sharpe, Oxoid). They
are cultivated in a thermostat at 37°C for 16 - 18 hours.
were composed of 10 % whey hydrolysate (WPH1,
WPH2 or WPH3), 2 % lactose, 0.5 % yeast extract, 4 %
glucose and distilled water. The control fermentation
media contained 10 % whey, 2 % lactose, 0.5 % yeast
extract, 4 % glucose and distilled water. The pH of
each media was adjusted to 6.8 - 6.9 with 2.0 M NaOH
and autoclaved at 121°C for 6 min. Every medium was
inoculated with 10 % (v/v) inoculum of the respective
strain. The fermentation process was carried out at 37OC
for 16 - 18 hours. The fermentation was performed in
triplicate.
The technological stages of the fermentation process
for each of the two selected strains are presented in Fig. 1.

Fermentation Enumeration of total number of viable probiotic

The fermentation of probiotic strains was carried bacteria (CFU mL-1)
out in 500 mL Erlenmeyer flasks. Overnight culture of The analysis was performed by counting the colony-
each investigated strain, cultivated in MRS broth was forming units per mL on the selective medium - for
prepared as an inoculum. The test fermentation media Lactobacillus - MRS agar. The method of ten-folded

Freeze-dried bacterial
culture L. plantarum
subsp. plantarum
NBIMCC 3447/
Lactobacillus gasseri
NBIMCC 2450
Weighing and Weighing and dissolving the dry
dissolving dry components of the nutrient medium:
substance pH 6.8÷6.9
MRS broth

Sterilization Recovery and Control sample:

of MRS broth activation of the whey, Test Samples:
(in autoclave) bacterial culture 5-fold lactose, Whey hydrolyzate,
121°C for 15 reseeding in MRS glucose, lactose, glucose, yeast extract
min broth yeast extract

Preparation of Sterilization of the nutrient medium: 121°C for 6

inoculum min

Heating the nutrient medium to cultivation

temperature: 37°C

Inoculation with 10 % bacterial culture

Static fermentation (in a thermostat)

37°C, 16 h

Fig. 1. Technological scheme of the fermentation process of Lactiplantibacillus plantarum subsp. plantarum NBIMCC
3447/ Lactobacillus gasseri NBIMCC 2450.

17
 Journal of Chemical Technology and Metallurgy, 59, 1, 2024

dilutions was used. Petri dishes were incubated at 37°C Table 1. Degree of hydrolysis of whey hydrolysates
for 72 hours. The number of lactic acid bacteria was variants.
calculated according to IDF Standard 117B: 1991.
Sample DH (%)
WPH1 15.16 ± 0.54
WPH2 32.02 ± 1.68
ΣС is the sum of all calculated colonies in two
WPH3 54.57 ± 1.78
consecutive dilutions, V is the volume (mL) of the
microbial probe, n1 is the number of petri dishes for the
first dilution, n2 is the number of petri dishes used for
the second dilution, d is the dilution factor.

Active acidity
The active acidity (pH value) was measured before,
during and after fermentation using a 920-UK pH-meter
(Bante, China). The active acidity or pH was measured
prior to fermentation and immediately after completion
of the fermentation with the appropriate culture.

Statistical analysis
The results were processed using MS Office Excel
2016. All data were presented as mean values with their
standard deviations (mean ± SD). The validity of the
Fig. 2. SDS-PAGE analysis of whey hydrolysates: 1 -
differences between the studied samples was established WPC, 2 - WPH1, 3 - WPH2, 4 - WPH3, 5 - Protein marker
by the Student’s t-test. The results are considered to be SigmaMarker low range, 6 - protein mixture - BSA (serum
significant when P < 0.01. albumin), α-casein, β-lactoglobulin and α-lactoalbumin.

RESULTS AND DISCUSSION

To evaluate the course of enzymatic hydrolysis of
whey proteins, the concept of the degree of hydrolysis
(DH) is used. DH was defined as the percentage of
hydrolyzed peptide bonds. The DH values for the
three variant hydrolysates are presented in Table 1.
The highest percentage degree of hydrolysis was
achieved with hydrolysate WPH3 using the enzyme
papain at a concentration of 1 mg mL-1.
The change in the state of the protein substances in
the variants and the control serum sample is established
by their electrophoretic separation is presented in Fig. 2.
Changes in the state of the protein substances in
the three variants hydrolysates, compared to the control
sample, were observed after their electrophoretic
separation (Fig. 2). In SDS-PAGE analysis, all proteins
were compared with purified protein markers. The
electrophoregrams of the WPH2 and WPH3 show changes
in the bands of the α-lactoalbumin and β-lactoglobulin
18
fractions and the appearance of new low molecular mass
bands. In these variants of hydrolysates, treated with
0.5 and 1 mg mL-1 papain, there was also a significant
change in the type and number of protein bands relative
to the original whey protein. The type and intensity of
WPH1 bands was altered to a lesser extent compared
with WPH2 and WPH3.
Biofermentation experiments were carried out
with two selected probiotic strains of lactic acid
bacteria with proven health-promoting properties,
Lactiplantibacillus plantarum subsp. plantarum
NBIMCC 3447 and Lactobacillus gasseri NBIMCC
2450 [17, 18]. Fermentation of whey hydrolysates is a
stage in the process of obtaining probiotic products with
a combination of physiological effects.
Many of the probiotic bacteria of human origin
possess highly pronounced antioxidant and antimicrobial
potential. Lactobacillus strains with pronounced
 Maria Doneva, Kamelia Loginovska, Svetla Dyankova,
Iliana Nacheva, Petya Metodieva, Nadya Ninova-Nikolova

high antioxidant activity tolerate well and survive
oxidative stress from free radicals [19]. In 2020,
Rastogi et al. investigated and confirmed that L. gasseri
strains of human origin exhibit natural antioxidants
and eliminate free radicals [20]. In 2006 and 2015,
Kachouri et al. demonstrated the antioxidant potential
of Lactiplantibacillus plantarum strains isolated from
fermented plant juices and emphasized that the strength
of probiotic properties depends on the concentration of
live cells [21, 22]. They use strains of L. plantarum that
ferment plant residues from olives, thereby increasing
antioxidant activity.
When carrying out fermentation with the participation
of the probiotic strains Lactiplantibacillus plantarum
subsp. plantarum NBIMCC 3447 and Lactobacillus
gasseri NBIMCC 2450, the technological parameter
active acidity of the medium at the beginning and at the
end of the fermentation process was monitored.
Active acidity is expressed by the concentration of
hydrogen ions contained in the fermentation medium.
It is inversely dependent on the number of viable cells
accumulated in the medium during the fermentation
process, i.e. with a high number of viable bacterial
cells, the active acidity of the medium is lower. The
results obtained for the active acidity of the fermentation
medium with the participation of Lactiplantibacillus
plantarum subsp. plantarum NBIMCC 3447 and
Lactobacillus gasseri NBIMCC 2450 in whey culture
and hydrolysates are presented in Figs. 3 and 4.
The presented results show that the active acidity
in the fermentation process gradually decreases until
reaching the maximum permissible value at the end of
the fermentation process.
Total number of active probiotic microorganisms
The number of viable cells of Lactiplantibacillus
plantarum subsp. plantarum NBIMCC 3447 and
Lactobacillus gasseri NBIMCC 2450 determined in the
cultivation process of protein hydrolysates are listed in
Tables 2 and 3.
The obtained results show that the cultivation of both
probiotic bacterial strains Lactiplantibacillus plantarum
subsp. plantarum NBIMCC 3447 and Lactobacillus
gasseri NBIMCC 2450 was successfully carried out in
whey hydrolysate media. The results for the number of
viable cells in the experimental variants have values
higher than the controls, which proves the correct choice
of conditions for carrying out fermentation with specific
substrates, such as the hydrolyzed ones.
The highest values of viable probiotic bacterial cells
(for Lactiplantibacillus plantarum NBIMCC 3447 - 2.5
х 109, and for Lactobacillus gasseri NBIMCC 2450 -

Fig. 3. Change of the active acidity of the fermentation medium with the participation of Lactiplantibacillus plantarum
subsp. plantarum NBIMCC 3447 when cultured in whey and whey hydrolysates with papain enzyme concentrations of
0.1; 0.5; 1.0 mg mL-1.

19
 Journal of Chemical Technology and Metallurgy, 59, 1, 2024

Fig. 4. Change in the active acidity of the fermentation medium with the participation of Lactobacillus gasseri NBIMCC
2450 in the cultivation of whey and whey hydrolysates with papain enzyme concentrations of 01; 0.5; 1.0 mg mL-1.

Table 2. Number of viable cells Lactiplantibacillus plantarum subsp. plantarum NBIMCC 3447 when cultured in whey
and whey hydrolysates.
Survival after fermentation
Sample Lactiplantibacillus plantarum subsp. plantarum NBIMCC 3447
CFU mL-1 log CFU mL-1
WPC 1.4х 109 9.14
WPH1 1.6 х 109 9.20
WPH2 2.5 х 10 9
9.40
WPH3 3.1 х 10 8
8.49

Table 3. Number of viable cells of Lactobacillus gasseri NBIMCC 2450 in culture of whey and whey hydrolysates.
Survival after fermentation Lactobacillus gasseri NBIMCC 2450
Sample
CFU mL-1 log CFU mL-1
WPC 1.1 х 109 9.04
WPH1 1.3 х 109 9.11
WPH2 2.1 х 10 9
9.32
WPH3 2.3 х 10 8
8.36

2.1 х 109) were recorded in the samples obtained from
hydrolyzed whey medium with 0.5 mg mL-1 papain.
Cell viability decreased in whey hydrolysate samples
obtained after treatment with a high concentration of
papain - 1.0 mg mL-1 papain. This trend was observed
with both probiotic strains. The difference in the number
of viable cells in these samples is due to the higher degree
of hydrolysis of the whey and the insufficient amount
of protein components necessary for the cultivation of
20
the probiotic cultures.
A comparative analysis of the antioxidant effect of
the control whey samples, starting protein hydrolysates
and the obtained fermented products was performed.
The antioxidant effect was measured by the DPPH
method, with a Trolox standard, which is one of the
most commonly used methods for screening antioxidant
activity of foods.
The results are presented in Table 4 as Trolox
 Maria Doneva, Kamelia Loginovska, Svetla Dyankova,
Iliana Nacheva, Petya Metodieva, Nadya Ninova-Nikolova
Table 4. Antioxidant activity of whey hydrolysates and fermented products.
Sample
Unfermented samples
(hydrolyzed with papain)
plantarum NBIMCC 3447
WPC - control (without
1.67 ± 0.02a
enzyme pre-treatment)
WPH1 4.07 ± 0.05b
WPH2 6.83 ± 0.21c
WPH3 5.15 ± 0.18d
Mean values with different superscript letters within a column were significantly different (p < 0.01)
equivalents antioxidant capacity (TE) mg/100 mL
product.
After hydrolysis with papain, the whey samples
showed a significant (p < 0.01) increase in antioxidant
activity, which was highest in WPH2. The fermentation
carried out with the studied strains of lactobacilli further
increased the antioxidant activity of the final products.
The highest values were obtained in the WPH2 samples,
respectively: 9.14 mg TE/100 mL (after fermentation
with Lb. plantarum subsp. plantarum NBIMCC 3447)
and 8.76 mgTE/100 mL (after fermentation with Lb.
gasseri NBIMCC 2450).
The observed differences can be explained by the
fact that the bioactive properties of protein hydrolysates
depend on the concentration of the enzyme and the strain
involved in the hydrolysis process [11, 23].
According to the obtained results, antioxidant
activity increases during the fermentation process,
and the use of Lactiplantibacillus plantarum and
Lactobacillus gasseri improves the nutritional value of
the obtained fermented products.
CONCLUSIONS
The development of new, functional bioproducts
based on enzymatically obtained whey hydrolysates
fermented with selected strains of lactic acid bacteria
is promising due to their wide potential for application
in food technology. Biofermentation experiments
were performed with selected probiotic strains of
lactic acid bacteria with proven health-promoting
Antioxidant activity
(mg ТЕ/100 mL)
Hydrolyzed and fermented samples
Lb. plantarum subsp.
Lb. gasseri NBIMCC 2450
2.65 ± 0.18a 2.31 ± 0.03a
6.07 ± 0.07b 5.61 ± 0.09b
9.14 ± 0.03c 8.76 ± 0.26c
6.14 ± 0.08d 5.90 ± 0.12d
properties Lactiplantibacillus plantarum subsp.
plantarum NBIMCC 3447 and Lactobacillus gasseri
NBIMCC 2450. The obtained results show that the
cultivation process of both probiotic bacterial strains,
Lactiplantibacillus plantarum subsp. plantarum
NBIMCC 3447 and Lactobacillus gasseri NBIMCC
2450, was successfully carried out in whey hydrolysate
media. The results for the number of viable cells in the
experimental variants were higher than the controls,
which proves the potential for the application of whey
hydrolysates as a substrate for fermentation with lactic
acid bacteria. Increased antioxidant activity of the
fermented products compared to WPC was observed.
Therefore, the resulting whey hydrolysates can be
applied to produce probiotic products with a combination
of physiological effects.
Acknowledgements
The authors acknowledge the financial support of
the Bulgarian National Science Fund under contract
КП-06-Н66/1.
REFERENCES
1. L. Tratnik, Mlijeko-tehnologija, biokemija i
mikrobiologija, Zagreb, Hrvatska Mljekarska
Udruga, 1998.
2. R. Walzem, C. Dillard, B. German, Whey components:
Millennia of evolution create functionalities for
mammalian nutrition: What we know and what we
21
 Journal of Chemical Technology and Metallurgy, 59, 1, 2024
may be overlooking, Crit. Rev. Food Sci. Nutr., 42,
2002, 353-375.
3. P. Jelen, H. Roginski, J.F. Fuquay, P.F. Fox, Whey
Processing, Encyclopedia of Diary Sciences, URED:
Academic Press- An Imprint of Elsevier, 4, 2003,
p. 2740.
4. J. Yadav, S. Yan, S. Pilli, L. Kumar, R. Tyagi, R.
Surampalli, Cheese whey: A potential resource to
transform into bioprotein, functional/nutritional
proteins and bioactive peptides, Biotechnol. Adv.,
33, 6, 2015, 756-774.
5. G. Regester, G. McIntosh, V. Lee, G. Smithers,
Whey proteins as nutritional and functional food
ingredients, Food Aust., 48, 1996, 123-127.
6. A. Brandelli, D.J. Daroit, A.P.F. Corrêa, Whey as
a source of peptides with remarkable biological
activities, Food Res. Int., 73, 2015, 149-161.
7. H. Korhonen, Milk-derived bioactive peptides: from
science to applications, J. Funct. Foods, 1, 2009,
177-187.
8. J. Choi, L. Sabikhi, A. Hassan, S. Anand, Bioactive
peptides in dairy products, Int. J. Funct. Foods, 65,
2012, 1-12.
9. A. Clemente, Enzymatic protein hydrolysates in
human nutrition, Trends Food Sci. Technol., 11, 7,
2000, 254-262.
10. A. Pihlanto, Lactic fermentation and bioactive
peptides, in Lactic Acid Bacteria - R & D for Food,
Health, Livestock Purposes, M. Kongo, Ed., Novi
Sad, InTech Prepress, 2013, 310-331.
11. T. Virtanen, A. Pihlanto, S. Akkanen, H. Korhonen,
Development of antioxidant activity in milk whey
during fermentation with lactic acid bacteria, J. Appl.
Microbiol., 102, 1, 2007, 106-115.
12. R. Castro, M. Domingues, A. Ohara, P. Okuro,
J. Santos, R. Brexó, H. Sato, Whey protein as a
key component in food systems: Physicochemical
properties, production technologies and applications,
Food Struct., 14, 2017, 17-29.
13. V. Beresteijn, E.C. Peeters, R.A. Kaper, J. Meijer, R.
Robben, D. Schmidt, Molecular Mass Distribution,
Immunological Properties and Nutritive Value of
Whey Protein Hydrolysates, J. Food Prot., 57, 1994,
22
619-625.
14. M. Navarrete del Toro, F. García‐Carreño, Evaluation
of the progress of protein hydrolysis, Current
Protocols in Food Analytical Chemistry, 10, 1, 2003,
B2.2.1-B2.2.14.
15. W. Brand-Williams, M.E. Cuvelier, C.L.W.T. Berset,
Use of a free radical method to evaluate antioxidant
activity, LWT - Food Sci. Technol., 28, 1, 1995,
25-30.
16. U. Laemmli, Cleavage of structural proteins during
the assembly of the head of bacteriophage T4, Nature,
227, 5259, 1970, 680-5.
17. X. Zhao, X. Zhong, X. Liu, X. Wang, X. Gao,
Therapeutic and Improving Function of Lactobacilli
in the Prevention and Treatment of Cardiovascular-
Related Diseases: A Novel Perspective From Gut
Microbiota, Front. Nutr., 8, 2021, p. 299.
18. C. Raveschot, B. Cudennec, F. Coutte, C. Flahaut,
M. Fremont, D. Drider, P. Dhulster, Production of
bioactive peptides by Lactobacillus species: from
gene to application, Front. Microbiol., 2018, p. 2354.
19. A. Chooruk, S. Piwat, R. Teanpaisan, Antioxidant
activity of various oral Lactobacillus strains, J. Appl.
Microbiol., 123, 2017, 271-279.
20. S. Rastogi, V. Mittal, A. Singh, In vitro Assessment
of Antioxidant and Antimicrobial Potential of
Lactobacillus gasseri Strains Isolated from Human
Milk and Infant Faeces, J. Pure Appl. Microbiol., 14,
2, 2020, 1305-1315.
21. F. Kachouri, M. Hamdi, Use Lactiplantibacillus
plantarum in olive oil process and improvement
of phenolic compounds content, J. Food Eng., 77,
2016, 746-752.
22. F. Kachouri, H. Ksontini, M. Kraiem, K. Setti, M.
Mechmeche, M. Hamdi, Involvement of antioxidant
activity of Lactiplantibacillus plantarum on functional
properties of olive phenolic compounds, J. Food
Technol. -Mysore, 52, 12, 2015, 7924-7930.
23. A. Corrêa, D. Daroit, J. S. M. Coelho, F. Lopes,
J. Segalin, P. Risso, A. Brandelli, Antioxidant,
antihypertensive and antimicrobial properties of
ovine milk caseinate hydrolyzed with a microbial
protease, J. Sci. Food Agric., 91, 12, 2011, 2247-54.