HYDROLYSIS OF MAJOR DAIRY PROTEINS BY LACTIC ACID

BACTERIA FROM BULGARIAN YOGURTS

IRENA TZVETKOVA1,4, MICHÈLE DALGALARRONDO1, SVETLA DANOVA2,

ILIA ILIEV3, ISKRA IVANOVA4, JEAN-MARC CHOBERT1,5 and
THOMAS HAERTLÉ1
1
UR 1268 Biopolymères Interactions Assemblages, INRA, Equipe Fonctions et
Interactions des Protéines Laitières
Rue de la Géraudière, B. P. 71627
44316 Nantes Cedex 3, France
2
Institute of Microbiology
Bulgarian Academy of Sciences
Sofia, Bulgaria
3
SIBIO-93 Ltd.
Plovdiv, Bulgaria
4
Department of Microbiology
Sofia University
Sofia, Bulgaria

Accepted for Publication December 1, 2006

ABSTRACT

Twenty-one Lactobacillus strains isolated from three types of Balkan

homemade yogurts were grown on sodium caseinate, b-casein or whey pro-
teins, and the proteolysis was followed by electrophoresis and reversed-phase
high-performance liquid chromatography. The best conditions allowing
obtaining proteolysis without casein precipitation are 0.8% casein in 50-mM
phosphate buffer. The strains tested showed a relatively high proteolytic activ-
ity despite the limited conditions for bacterial growth. Within 72 and 96 h of
incubation, 80–90% of b-casein was consumed. They showed also a pro-
teolytic activity toward a-lactalbumin (ALA), being able to reduce its concen-
tration between 5 and 55%, depending on the strains used. The capacity of the
strains to hydrolyze b-lactoglobulin was lower as compared with hydrolysis of
ALA. Hydrolysis of casein by all strains produced peptides with an antibac-
terial effect against Escherichia coli. Consequently, to obtain a maximal
hydrolysis of the dairy proteins seconded by appearance of antimicrobial
peptides, a combination of strains with different beneficial properties to be
used as starters was proposed.
5
Corresponding author. TEL: +332-40-67-52-30; FAX: +332-40-67-50-84; EMAIL:
chobert@nantes.inra.fr

Journal of Food Biochemistry 31 (2007) 680–702. All Rights Reserved.

680 © 2007, The Author(s)
Journal compilation © 2007, Blackwell Publishing
 MILK PROTEINS PROTEOLYSIS BY LAB 681

PRACTICAL APPLICATIONS

Some of the 21 Lactobacillus strains isolated from three types of Balkan

homemade yogurts may be used to proteolyze milk proteins in order to
produce peptides with an antibacterial effect against Escherichia coli. To
obtain a maximal hydrolysis of the dairy proteins seconded by appearance of
antimicrobial peptides, a combination of strains with different beneficial prop-
erties to be used as starters should be determined. During fermentation
process, milk proteins are acidified by the production of lactic acid and are
hydrolyzed by proteases and peptidases from bacteria. This proteolysis is
followed by a reduction of the number of epitopes and consequently by a
decrease in allergenicity of hydrolyzed proteins. For these reasons, starters as
Lactobacillus strains with beneficial properties able to reduce the allergenicity
of fermented milk products are of great interest for the dairy industry.

INTRODUCTION

Milk is the complete source of nutrients for the neonate, which provides
proteins, lipids, sugars, vitamins and minerals for healthy growth of an infant
(Warner et al. 2001). The newborn human has an immature digestive capacity
in the stomach and small intestine, resulting in a high proportion of intact or
large fragments of food protein molecules passing through the intestinal
mucosal barrier into the immune structures in the intestinal lining (Peyer’s
patches) or into the blood. These intact proteins or large peptides may act as
antigens to the immune system of the young, resulting in allergic hypersensi-
tivity reactions and food protein intolerance. Cow milk proteins belong to the
strongest antigens in human diet (Eigel et al. 1984). Milk proteins are the first
exogenous proteins consumed in large quantities by children (Järvinen and
Suomalainen 2001). Whey proteins (a-lactalbumin [ALA], b-lactoglobulin
[BLG], bovine serum albumin) and caseins are considered as the main cow’s
milk antigens (Räsänen et al. 1992; Nentwich et al. 2004), and they cause an
immunologically mediated adverse reaction (Savilahti et al. 1992).
Nowadays, the cow’s milk allergy ranges from 1.9 to 7.5% of the popu-
lation (Järvinen and Suomalainen 2001). For this reason, the application of
lactic acid bacteria (LAB) in hydrolysis of milk proteins may have important
effects on milk digestibility, on the production of bioactive peptides and may
decrease milk allergenicity and lactose intolerance (Nentwich et al. 2004;
Prioult et al. 2005). Humans have used yogurt and yogurt-like products as the
most popular vehicles for ingestion of probiotic microorganisms. Probiotics
have been the subject of considerable scientific and commercial attention over
the past two decades. Probiotics are live microorganisms that when ingested
682 I. TZVETKOVA ET AL.

may have positive effects on human health (Fuller 1991). Beneficial effects
of probiotics on immune-mediated diseases, such as allergy, have been
documented and include stimulating the immune system (Cross et al. 2001;
Ouwehand et al. 2002), synthesizing and increasing the bioavailability
of nutrients (Pessi et al. 1998) and decreasing the prevalence of atopy
(Kalliomäki et al. 2001). Bifidobacteria and lactobacilli are common anaer-
obes in the human intestinal microbiota, and some of them have been reported
to display probiotic properties (Ouwehand et al. 1999). Although LAB are
usually considered to be only weakly proteolytic, some of them cause a
significant degree of proteolysis in multiple fermented dairy products includ-
ing yogurts. Yogurt bacteria are microorganisms with multiple amino acid
auxotrophies (Kok and De Vos 1994). The content of free amino acids and
peptides in milk is insufficient (Zourari et al. 1992; Abu-Tarboush 1996);
therefore, many LAB possess a complex system of proteinases and peptidases,
which enable them to produce essential amino acids during their growth in
milk (Kunji et al. 1996; Chen and Steele 1998).
The proteolytic activities of LAB including yogurt bacteria and probiotic
bacteria have been studied extensively (Booth et al. 1990; Wohlrab and
Bockelmann 1993; Bockelmann et al. 1996; Law and Haandrikman 1997; Ehn
et al. 2005).
Additionally, proteolytic enzymes from LAB produce flavor compounds
and precursors that are essential for cheese flavor development (Mulholland
1997; Chen and Steele 1998). The proteolytic systems of LAB can be func-
tionally divided into three components: (1) cell envelope-associated protein-
ases, which hydrolyze caseins to oligopeptides; (2) peptide transport systems,
of which the oligopeptide transport system is the most important in milk and
cheese; and (3) numerous intracellular peptidases (Kunji et al. 1996; Chen and
Steele 1998). The intracellular peptidases of LAB include both endopeptidases
and aminopeptidases. Endopeptidases, because of their ability to hydrolyze
peptide bonds within a peptide, are of particular interest in targeting peptides
for rapid hydrolysis.
To promote human health, combinations of different bacterial strains
belonging to the genera Lactobacillus, Streptococcus and Bifidobacterium
have been used traditionally in fermented dairy products (Prasad et al. 1998;
Dunne et al. 1999). A variety of LAB isolated from fermented milk products
have been previously reported as displaying beneficial functions for humans,
including antimicrobial (Sandine et al. 1972; Eijsink et al. 1998), antitumor
(Kelkar et al. 1988; Hosono et al. 1990; Adachi 1992) and antimutagenic
activities (Nishioka et al. 1989; Hosono et al. 1990), as well as effects on
modulating the immune system (Perdigon et al. 1988; Fernandes and Shahani
1990), lowering cholesterol levels (Shun et al. 1989) and reducing lactose
intolerance in the host (Alm 1982). Lactobacillus is the most important group
 MILK PROTEINS PROTEOLYSIS BY LAB 683

of starters for the dairy industry. During fermentation process, milk proteins
are acidified by the production of lactic acid and are hydrolyzed by proteases
and peptidases from bacteria. This proteolysis is followed by a reduction of the
number of epitopes and consequently by a decrease in allergenicity of hydro-
lyzed proteins (Cross et al. 2001; Bertrand-Harb et al. 2003; Nentwich et al.
2004). For these reasons, starters as Lactobacillus strains with beneficial
properties able to reduce the allergenicity of fermented milk products are of
great interest for the dairy industry.
In this study, the attention was focused on homemade yogurts from the
Balkan region, as a source of newly potential starters for application in the
food industry. In this context, the aim of this study was to screen the pro-
teolytic activity of newly isolated strains, by determination of the optimal
conditions of proteolysis for reducing milk protein allergenicity.

MATERIALS AND METHODS

Bacterial Strains and Culture Conditions

A total of 21 Lactobacillus strains isolated from three different types of
Balkan homemade yogurts, made from cow, sheep and buffalo milks, were
used. The strains with prefixes “K,” “O” and “B” correspond to strains isolated
from cow, sheep and buffalo yogurts, respectively. The strains with prefix “K”
and prefix “O” were originated from the western part of Bulgaria, in a village
near the mountain “Pirin”; the strains with prefix “B” were originated from the
northern part of Bulgaria, in a village close to the city “Vratza.” The strains
were cultured overnight (16–18 h) on Mann–Rogosa–Sharpe (MRS) broth
(Merck, Darmstast, Germany) at 37C and in limitation of oxygen (tubes or
Petri dishes with the strains were incubated in plastic bags, which limited the
oxygen content).

API 50CHL System Assay

Initial identification of all the strains was performed by API 50CHL
system (BioMerieux, Craponne, France), according to the manufacturer’s
instructions. The fermentation profiles were read after incubation at 37C in
anaerobic conditions, for 3 days.

Proteolytic Activity
Initial screening of the strains for the presence of proteolytic activity was
performed using the well diffusion method (Schillinger and Lucke 1989). A
1.4% skim milk agar was used. Wells were made in the lawn of hardened soft
684 I. TZVETKOVA ET AL.

agars in Petri dishes. Aliquots (70 mL) of supernatant of overnight cultures

(16–18 h) in MRS broth were poured in the wells. The plates were left for 1 h
at room temperature (22C) in sterile conditions before incubating them to the
adequate temperature for the growth of the test organism. A clear zone corre-
sponding to proteolysis was accepted as positive.

Proteolysis of Caseins. To analyze the caseinolytic activity of the

strains, all the strains were cultured on different test media performed to
achieve the optimal conditions for casein degradation. After an overnight
incubation in MRS media, the strains were inoculated (10% inoculum) in the
test media containing different concentrations of sodium caseinate and with
the following composition: 2% glucose and sodium caseinate or b-casein
(concentrations from 0.08 to 2.0%) buffered with sodium phosphate buffer
(25–100 mM) and with or without additives (vitamins, MRS components).
After incubation, 50 mL of the culture was taken at 24, 48, 72 and 96 h
and diluted 1:1 (v/v) with sample buffer containing 9-M urea, 0.5%
2-mercaptoethanol and 300-mg/mL saccharose. The samples were then
analyzed by urea-polyacrylamide gel electrophoresis (urea-PAGE).

UREA-PAGE. Urea-PAGE was performed with 10% polyacrylamide

slab gel with a 4% stacking gel on a Mini Protean II apparatus (Bio-Rad,
Hercules, CA).
The migration buffer contained 50-mM Tris and 0.384-M glycine accord-
ing to Andrews (1983). After running at 10 mA on the stacking gel and 20 mA
on the running gel, staining was performed with Coomassie Brilliant Blue
R-250 followed by a convenient destaining. Gels were scanned and intensity of
the bands was quantified using Quantity One BioRad Software (Bio-Rad,
Marne la Coquette, France).

Proteolysis of Whey Proteins. To analyze the proteolytic activity of the

tested strains toward whey proteins, an MRS broth containing BLG and ALA
was chosen. The strains were cultivated in the presence of 0.05, 0.1 and 0.5%
BLG and ALA. After incubation, the cultures were centrifuged (12,000 ¥ g,
5 min) and 30 mL of the resulting supernatant was diluted with 90-mL sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer containing 4% sodium dodecyl sulfate (SDS), 3% 2-mercaptoethanol,
10% glycerol, 50-mM Tris–HCl, pH 6.8. The samples were heated at 100C for
3 min then analyzed by SDS-PAGE as previously described (Bertrand-Harb
et al. 2003).

SDS-PAGE. The samples were applied to a 15% polyacrylamide slab

gel. The migration buffer contained 50-mM Tris, 0.384-M glycine and 0.1%
 MILK PROTEINS PROTEOLYSIS BY LAB 685

SDS. After running at 10 mA on the stacking gel and 20 mA on the running

gel, staining and destaining were performed as described earlier. The gels were
scanned and intensity of the bands was quantified by Quantity One BioRad
Software.

Reversed-phase High-performance Liquid Chromatography

(RP-HPLC)
RP-HPLC was used to analyze the supernatant of culture of the strains
growing on 0.08% sodium caseinate, 2% glucose in 50-mM phosphate buffer
or on 0.08% b-casein, 2% glucose in 50-mM phosphate buffer. In a first
experiment, after a preculture on MRS broth, the test media was inoculated
with the test culture. For further experiments, before inoculation, the cultures
were washed twice with 0.5% NaCl.
The samples were separated with a Nucleosil C18 column (250 ¥ 0.3 mm
i.d., Macherey-Nagel, Hoerdt, France) using a Waters (Milford, MA) high-
performance liquid chromatography system with the following conditions:
flow-rate, 0.3 mL/min; solvent A, trifluoroacetic acid (TFA) 0.11% (v/v) in
water; solvent B, acetonitrile/water/TFA, 70/30/0.09 (v/v/v). Elution was
performed with a linear gradient of solvent B from 0 to 50% over 10 min, then
50–80% in 20 min; the column was then rinsed with 100% of solvent B.
Detection was performed with a Waters 996 photodiode array detector at
220 nm. The data were analyzed by Millenium32 software (Waters) as
previously described (Bertrand-Harb et al. 2003).

Antimicrobial Assay
Antimicrobial assay was performed as previously described (Bertrand-
Harb et al. 2003) by the well diffusion method by using soft 0.8% agar. After
adjusting the pH at 6.5 by NaOH, the activity of the collected samples (60 mL)
was checked against E. coli American Type Culture Collection 25922 on
Luria–Bertani agar medium (Sigma, St. Louis, MO) and against Listeria
innocua F (Ecole Nationale des Ingénieurs des Techniques des Industries
Agricoles et Alimentaires, Nantes, France) on brain–heart agar medium
(Biokar Diagnostics, Beauvais, France). The plates were incubated overnight
at 37C.
Antimicrobial activity of 24- and 48-h hydrolyzed samples was checked
on the strains cultured on media containing 2% glucose in 100-mM phosphate
buffer added with 0.08, 0.5 and 2.0% sodium caseinate. The neutralized
supernatants (pH 6.5) obtained after 24-h preculture in MRS, and after 4-, 6-
and 24-h culture in milk were also checked for their activity against E. coli.
All experiments were performed in triplicate.
686 I. TZVETKOVA ET AL.

RESULTS AND DISCUSSION

API Tests
API tests showed that all the strains tested are Lactobacillus delbrueckii
ssp. bulgaricus.

Proteolytic Activity
The initial screening of the strains tested on skim milk agar for their
proteolytic activity showed that all the strains have a proteolytic activity and
generate a clear zone even after 4 h of incubation (data not shown).

Proteolysis of Caseinate. The strains tested were incubated in different

conditions in order to determine the best allowing proteolysis without casein
precipitation. After incubation of the strains on test media supplemented with
different concentrations of sodium caseinate in combination with different
concentrations of phosphate buffer and additional MRS components and vita-
mins, it was observed that the growth of the studied strains was inhibited when
concentrations of phosphate buffer higher than 50 mM were used in the incu-
bation media (data not shown). When the test medium contained 0.1, 0.2 or
2.0% casein and lower concentrations of phosphate buffer (25 or 50 mM), the
precipitation of casein occurred rapidly because of a quick acidification of the
media (data not shown). In these conditions, the results obtained by urea-PAGE
were hard to compare because of the formed precipitates. Consequently, a test
medium with 0.08% sodium caseinate and 50-mM phosphate buffer was used as
a model system for all further experiments.
As observed by urea-PAGE (Fig. 1), caseins were degraded by the differ-
ent strains tested after 24, 48, 72 and 96 h of cultivation. After 24 h of incubation
(Fig. 1A), b-casein was degraded by the action of some strains (K3, K6, K15,
K20 and K26). At the same time, new bands with higher mobilities appeared.
After 72 h of incubation (Fig. 1C), almost all b-casein disappeared except when
using the strains O43, O45, B5, B7 and B8. These strains were less active at the
beginning of the incubation, what could be explained by the very limited
conditions used for their growth (the tested media contained only glucose and
protein). Some strains (K3, K6, K15, K20 and K26) adapted well and were then
able to degrade b-casein within the first 24 h. Above 24 h of incubation, all the
other strains started a rapid “consumption” of b-casein, and after 96 h of
incubation (Fig. 1D), near all b-casein content was hydrolyzed by all the strains
tested. The hydrolysis of aS1- and aS2-caseins was smaller. The peptides
observed by polyacrylamide gel electrophoresis after 24 and 48 h of incubation
 MILK PROTEINS PROTEOLYSIS BY LAB 687

β-Casein

αS-Casein

K2 K3 K5 K6 K14 K15 K19 K20 K21 C K22 K24 K26 K27 O42 O43 O45 O47 B5 C B7 B8 C

β-Casein
αS-Casein

K2 K3 K5 K6 K14 K15 K19 C K20 K21K22 K24 K26 K27 O42 043 C O45 O47 B5 B7 B8

β-Casein

αS-Casein

K3 K5 K6 K14 K15 K19 K20 K21K22 C K24 K26 O42 043 045 O47 B5 B7 B8 C K2 B5 K27C

β-Casein

αS-Casein

K2 K3 K5 K6 K14 K15 K19 K20 K21C K22 K24 K26K27 O42 043 O45 O47 B5 C B8 B7

FIG. 1. UREA-POLYACRYLAMIDE GEL ELECTROPHORESIS OF SAMPLES OBTAINED

AFTER (A) 6-, (B) 24-, (C) 72- AND (D) 96-H CULTIVATION OF THE STRAINS IN THE
PRESENCE OF 0.08% SODIUM CASEINATE
The strains with prefixes “K,” “O” and “B” correspond to strains isolated from cow, sheep and
buffalo yogurts, respectively. C denotes the control (the control is a medium made of 0.08% sodium
caseinate, 2% glucose in 50-mM phosphate buffer, incubated under the same conditions as the
medium inoculated by bacteria). Fifty microliters of the culture was taken at 24, 48, 72 and 96 h
and diluted 1:1 (v/v) with sample buffer containing 9-M urea, 0.5% 2-mercaptoethanol and
300-mg/mL saccharose.
688 I. TZVETKOVA ET AL.

disappeared after longer incubation. This finding supports the fact that the
proteases present in the different strains conserved well their hydrolytic
activities.
The time course of b-casein degradation during incubation period is
shown in Fig. 2. The intensity of degradation of b-casein depends on the
strain used. However, after 96 h of incubation, 80–90% of b-casein was
digested by all the strains tested, as estimated by using Quantity One BioRad
Software.

Proteolysis of Whey Proteins. The ability of the studied LAB strains to

hydrolyze the whey proteins ALA and BLG after 24 h of incubation is shown
by SDS-PAGE in Figs. 2 and 3, respectively.
The different strains tested display relatively high proteolytic activity
against ALA. By using Quantity One BioRad Software, it could be estimated
that the strains tested were able to hydrolyze ALA in the medium; the amount
of unhydrolyzed ALA was between 5 and 55%, depending on the strain used
(Fig. 3). The strains K14, K15, K20, K27 and B7 showed the highest pro-
teolytic activity against ALA.
Proteolysis of 0.1% BLG after 24-h cultivation of the different strains
tested is shown in Fig. 3. All the strains tested were able to reduce to some
extent the amount of BLG. The yield of BLG degradation quantified by
Quantity One BioRad Software is shown in Fig. 4. The strains tested have a
lower capacity to hydrolyze BLG as compared with ALA. The strains K14 and
K27, which were able to reduce to about 50% the amount of ALA, reduced
only 20% of the amount of BLG.
RP-HPLC
Peptide formation after proteolysis by the different strains tested was
followed by RP-HPLC. As the presence of some components of MRS
medium heavily impaired a resolution on the chromatograms, a prewashing
step of the cells with 0.5% NaCl before inoculation of the tested media was
performed.
After cultivation of the strains in the test medium, the samples were
analyzed by urea-PAGE. The time course of degradation of caseins after
washing of the cells with 0.5% NaCl is shown in Figs. 5 and 6.
When the cells were washed with 0.5% NaCl before inoculation of the
medium, the strains needed a longer time of adaptation before start of casein
degradation as compared with the results obtained in the absence of a washing
step (Fig. 2). The time course of b-casein degradation after 6, 24, 72 and 96 h
of cultivation was observed by RP-HPLC (data not shown). It could be
observed that even if the strains were less active, a proteolytic activity was still
present.
 MILK PROTEINS PROTEOLYSIS BY LAB 689

120

100
beta-casein(%)

80 Strain K2
Strain K3
Strain K5
60 Strain K6
Strain K14
Strain K15
40 Strain K19
Strain K20
Strain K21
20 Strain K22
Control
A
0
0 20 40 60 80 100

Time (h)

120

100

Strain K24
beta-casein (%)

80 Strain K26
Strain K27
Strain O42
60 Strain O43
Strain O45
Strain O47
40 Strain B5
Strain B7
Strain B8
20 Control

B
0
0 20 40 60 80 100

Time (h)

FIG. 2. TIME COURSE OF DEGRADATION OF b-CASEIN BY THE TESTED STRAINS

CULTURED ON 0.08% CASEINATE AND 50-mM PHOSPHATE BUFFER
Strains “K,” “O” and “B” are issued from yogurts made with cow, sheep and buffalo milk,
respectively. Control is pure medium (0.08% sodium caseinate in 50-mM phosphate buffer, 2%
glucose); control, not inoculated with bacteria, was incubated under the same conditions as the
medium inoculated by the tested bacteria. The samples from the control were taken at the same
times as the samples from the medium inoculated by the tested strains. Each experiment was
performed in triplicate.
690 I. TZVETKOVA ET AL.

K5 K3 K14 C ALA M K21 O45 O42 K2 K26 K6 C ALA M K22 K15 K19 B5 K20 K24 B7 K27 C M

Control
100 O43
O47
K5 O42 O45 B8
K2 K21
80 B5
K26
K19
K3 K6 K22 B7
ALA (%)

K15 K24
60 K14 K20

K27

40

20

After 24 h incubation

FIG. 3. SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS

(SDS-PAGE) AND RELATIVE QUANTIFICATION OF a-LACTALBUMIN (ALA) IN SAMPLES
OBTAINED AFTER 24-H CULTIVATION OF THE STRAINS IN THE PRESENCE OF
0.1% ALA
The strains with prefixes “K,” “O” and “B” correspond to strains isolated from cow, sheep and
buffalo yogurts, respectively. Thirty microliters of the culture supernatant was diluted with 90-mL
SDS-PAGE sample buffer containing 4% sodium dodecyl sulfate, 3% 2-mercaptoethanol, 10%
glycerol, 50-mM Tris–HCl, pH 6.8.
M, milk; C, control (a medium with ALA, without bacteria).

The three strains K5, K19 and K27 showed similar results. They were
growing on medium containing either sodium caseinate or b-casein. The time
course of b-casein degradation obtained by urea-PAGE (Fig. 7, insert) shows
that after 6 h of incubation, a new band with a higher electrophoretic mobility
appeared. b-Casein band disappeared almost completely after 48 h of incuba-
tion in the case of the strain K27. After 96-h incubation, the three selected
strains cultivated either on b-casein or on sodium caseinate showed the same
results.
 MILK PROTEINS PROTEOLYSIS BY LAB 691

K5 K3 K14 C BLG M K21 O45 O42 M K27 C

K2 K26 K6 C BLG M K22 K15 K19 B5 M C K20 K24 B7

Control
100
K6 K20 K26
O45
K3 K14 K21 O42O43 O47
K2 K5 K19 K24 K27
B8
80 K15 B7
K22 B5
BLG(%)

60

40

20

After 24 h incubation
FIG. 4. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) AND RELATIVE QUANTIFICATION OF b-LACTOGLOBULIN (BLG) IN
SAMPLES OBTAINED AFTER 24-H CULTIVATION OF THE STRAINS IN THE PRESENCE
OF 0.1% BLG
The strains with prefixes “K,” “O” and “B” correspond to strains isolated from cow, sheep and
buffalo yogurts, respectively. Thirty microliters of the culture supernatant was diluted with 90-mL
SDS-PAGE sample buffer containing 4% sodium dodecyl sulfate, 3% 2-mercaptoethanol, 10%
glycerol, 50-mM Tris–HCl, pH 6.8.
M, milk; C, control (a medium with BLG, without bacteria).
692 I. TZVETKOVA ET AL.

b-casein
aS-casein

K2 K3 K5 K6 K14 K15 K19 K20 K21 C K22 K24 K26 K27 O42 O43 O45 O47 B5 C B7 B8 C

b-casein
aS-casein

K2 K3 K5 K6 K14 K15 K19 K20 K21 C K22 K24 K26 K27 O42 O43 O45 O47 B5 C B7 B8 B8 C

b-casein
aS-casein

K2 K3 K5 K6 K14 K15 K19 K20 K21 C K22 K24 K26 K27 O42 O43 O45 O47 B5 C B7 B8 C

b-casein
aS-casein

K2 K3 K5 K6 K14 K15 K19 K20 K21 C K22 K24 K26 K27 O42 O43 O45 O47 B5 C B7 B8 C

FIG. 5. UREA-POLYACRYLAMIDE GEL ELECTROPHORESIS OF SAMPLES OBTAINED

AFTER (A) 6, (B) 24, (C) 72 AND (D) 96 H CULTIVATION OF THE STRAINS IN THE
PRESENCE OF 0.08% SODIUM CASEINATE AFTER WASHING OF CULTURES WITH
0.5% NaCl
The strains with prefix “K,” “O” and “B” correspond to strains isolated from cow, sheep and buffalo
yogurts, respectively. C denotes the control (the control is a medium made of 0.08% sodium
caseinate, 2% glucose in 50-mM phosphate buffer, incubated under the same conditions as the
medium inoculated by bacteria. Only the bacterial cultures were washed twice with 0.5% sodium
chloride before inoculation and then incubated in the medium). Fifty microliters of the culture was
taken at 24, 48, 72 and 96 h and diluted 1:1 (v/v) with sample buffer containing 9-M urea, 0.5%
2-mercaptoethanol and 300-mg/mL saccharose.
 MILK PROTEINS PROTEOLYSIS BY LAB 693

100

80
Strain K2
beta-casein (%)

Strain K3
Strain K5
60 Strain K6
Strain K14
Strain K15
40 Strain K19
Strain K20
Strain K21
Strain K22
20 Control

A
0
0 20 40 60 80 100

Time (h)

100

80
Strain K24
beta-casein (%)

Strain K26
Strain K27
60 Strain O42
Strain O43
Strain O45
40 Strain O47
Strain B5
Strain B7
Strain B8
20 Control

B
0
0 20 40 60 80 100

Time (h)

FIG. 6. TIME COURSE OF DEGRADATION OF b-CASEIN BY THE TESTED STRAINS

CULTURED ON 0.08% SODIUM CASEINATE AFTER WASHING OF CULTURES WITH
0.5% NaCl
Strains “K,” “O” and “B” are issued from yogurts made with cow, sheep and buffalo milk,
respectively. The control is a medium made of 0.08% sodium caseinate, 2% glucose in 50-mM
phosphate buffer, incubated under the same conditions as the medium inoculated by bacteria. Only
the bacterial cultures were washed twice with 0.5% sodium chloride before inoculation and then
incubated in the medium. Each experiment was performed in triplicate.
694 I. TZVETKOVA ET AL.

0.4 0.4
6h 24 h

0.3 0.3
AU

AU
0.2 0.2

0.1 0.1

0.0 0.0
0 10 20 30 40 0 10 20 30 40

Time (min) Time (min)

0.4 0.4
48 h 72 h

0.3 0.3
AU

AU

0.2 0.2

0.1 0.1

0.0 0.0
0 10 20 30 40 0 10 20 30 40

Time (min) Time (min)

0.4
96 h 1 2 3 4 5 6 7 8 9 1011 1213141516 17 1819 20

0.3
AU

0.2 6h 24 h 48 h 72 h
96 h

K5
0.1 K19
K27
Control
0.0
0 10 20 30 40

Time (min)

FIG. 7. TIME COURSE OF DEGRADATION OF b-CASEIN DURING THE CULTIVATION OF

STRAINS K5, K19 AND K27
1: K5 – 6 h; 2: K19 – 6 h; 3: K27 – 6 h; 4: control – 6 h; 5: K5 – 24 h; 6: K19 – 24 h; 7: K27 –
24 h; 8: control – 24 h; 9: K5 – 48 h; 10: K19 – 48 h; 11: K27 – 48 h; 12: control – 48 h; 13:
K5 – 72 h; 14: K19 – 72 h; 15: K27 – 72 h; 16: control – 72 h; 17: K5 – 96 h; 18: K19 – 96 h; 19:
K27 – 96 h; 20: control – 96 h. Reversed-phase high-performance liquid chromatography analysis
of supernatant of culture of strains K5, K19 and K27 growing until 96 h on 0.08% b-casein, 2%
glucose in 50-mM phosphate buffer. The control is a medium made of 0.08% b-casein, 2% glucose
in 50-mM phosphate buffer, incubated under the same conditions as the medium inoculated by
bacteria. Only the bacterial cultures were washed twice with 0.5% sodium chloride before
inoculation and then incubated in the medium.
AU, absorbance at 220 nm.
 MILK PROTEINS PROTEOLYSIS BY LAB 695

By RP-HPLC (Fig. 7), it could be observed that within the first 6 h of

incubation, the peak of b-casein disappeared almost completely and a great
number of more hydrophilic peaks appeared. The area of the peaks and their
retention times were almost identical for the three strains analyzed, implying
that these strains harbor similar proteolytic systems.
Hydrolysis of sodium caseinate by the different strains tested is shown by
urea-PAGE (Fig. 8, insert) and by RP-HPLC (Fig. 8). The obtained results show
that there are differences in the extent of proteolytic activity observed with the
different strains used. The strain K27 showed the highest proteolytic activity.

Antibacterial Activity Against E. coli and L. innocua

Antibacterial Activity after Growing on the Test Media. The antibac-

terial activity of the tested strains after growing on medium containing 0.5 or
2.0% sodium caseinate in 100-mM phosphate buffer against E. coli and L. in-
nocua has been studied (Fig. 9A and B, respectively). The resulting peptides
obtained after hydrolysis of sodium caseinate have an antibacterial effect (zone
of inhibition) against E. coli but not against L. innocua.

Antibacterial Activity of the Strains Tested after Growing in Milk or

on MRS. The antibacterial activity of the tested strains against E. coli was
also checked after 4-, 6- and 24-h culture in milk or after a 24-h culture in MRS
(Fig. 10).
The resulting peptides obtained after hydrolysis of milk proteins have a
high antibacterial activity (zone of inhibition) against E. coli only after 24-h
growth.
When the strains tested were cultivated on MRS for 24 h, only the
neutralized supernatants from the strains K2 and K22 have an antibacterial
activity against E. coli, demonstrating the production of antibacterial sub-
stances by these strains. The zone of inhibition of the strain K22 was larger
than that obtained in the presence of the strain K2.

CONCLUSIONS

The experiments were performed to analyze the proteolytic activity of

yogurt bacteria. Sodium caseinate and b-casein were used as markers of this
activity. The best conditions allowing casein hydrolysis without precipitation
are 0.8% casein in 50-mM phosphate buffer. Under these conditions, the
strains tested showed a relatively high proteolytic activity despite the limited
conditions for bacterial growth. Even if the degradation of b-casein is a
strain-specific process, within 72 and 96 h of incubation, 80–90% of b-casein
696 I. TZVETKOVA ET AL.

6h 24 h
0.6 0.6

0.4 0.4
AU

AU
0.2 0.2

0.0 0.0
0 10 20 30 40 0 10 20 30 40

Time (min) Time (min)

48 h 72 h
0.6 0.6

0.4 0.4
AU

AU

0.2 0.2

0.0 0.0
0 10 20 30 40 0 10 20 30 40

Time (min) Time (min)

96 h
0.6 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 1920

0.4
AU

6h 24 h 48 h 72 h 96 h
0.2
K5
K19
K27
0.0 Control
0 10 20 30 40

Time (min)

FIG. 8. TIME COURSE OF DEGRADATION OF SODIUM CASEINATE DURING THE

CULTIVATION OF STRAINS K5, K19 AND K27
1: K5 – 6 h; 2: K19 – 6 h; 3: K27 – 6 h; 4: control – 6 h; 5: K5 – 24 h; 6: K19 – 24 h; 7: K27 –
24 h; 8: control – 24 h; 9: K5 – 48 h; 10: K19 – 48 h; 11: K27 – 48 h; 12: control – 48 h; 13:
K5 – 72 h; 14: K19 – 72 h; 15: K27 – 72 h; 16: control – 72 h; 17: K5 – 96 h; 18: K19 – 96 h; 19:
K27 – 96 h; 20: control – 96 h. Reversed-phase high-performance liquid chromatography analysis
of supernatant of culture of strains K5, K19 and K27 growing until 96 h on 0.08% sodium
caseinate, 2% glucose in 50-mM phosphate buffer. The control is a medium made of 0.08% sodium
caseinate, 2% glucose in 50-mM phosphate buffer, incubated under the same conditions as the
medium inoculated by bacteria. Only the bacterial cultures were washed twice with 0.5% sodium
chloride before inoculation and then incubated in the medium.
AU, absorbance at 220 nm.
 MILK PROTEINS PROTEOLYSIS BY LAB 697

FIG. 9. ANTIMICROBIAL ACTIVITY AGAINST (A) ESCHERICHIA COLI AND (B) LISTERIA
INNOCUA OF STRAINS CULTIVATED FOR 48 H ON 2% SODIUM CASEINATE, 2%
GLUCOSE IN 100-mM PHOSPHATE BUFFER
698 I. TZVETKOVA ET AL.

FIG. 10. ANTIMICROBIAL ACTIVITY AGAINST ESCHERICHIA COLI OF STRAINS

CULTIVATED FOR 24 H (A) IN MILK OR (B) ON MANN–ROGOSA–SHARPE
 MILK PROTEINS PROTEOLYSIS BY LAB 699

was consumed by the strains. The strain K27 analyzed by RP-HPLC and
urea-PAGE showed the highest proteolytic activity.
The strains tested showed proteolytic activity toward ALA. They were
able to reduce the concentration of ALA in the medium between 5 and 55%,
depending on the strains used. The strains K14 and K27 showed the highest
proteolytic activity being able to reduce the concentration of ALA approxi-
mately by 50%.
The capacity of the strains to hydrolyze BLG was lower as compared with
ALA. Even if ALA was more easily degradable by the bacteria, the strain K26
was able to reduce by 30% the level of BLG in the medium.
The beginning of casein degradation was delayed when the cells were
washed before inoculation of the test medium. RP-HPLC analysis confirmed
that, as a result of proteolysis by the tested strains, b-casein was cleaved into
more hydrophilic protein, which suggests that the strains tested possess also an
exopeptidase activity.
The hydrolysis of casein by all the strains tested produced peptides with an
antibacterial effect against E. coli. The neutralized supernatants from the strains
K2 and K22 grown on MRS also had an activity against E. coli that indicates the
presence of antibacterial substances of bacterial origin (bacteriocins).
Consequently, to obtain the most beneficial effect, a combination of
strains with different properties could be used as starters.

ACKNOWLEDGMENTS

This work was supported by the Socrates-Erasmus exchange program for

the Ph.D. fellowship awarded to Irena Tzvetkova and in part by the NATO
Science for Peace project CBP.EAP.SFPP 982164 Study of antimicrobial and
hypoallergenic products of lactic acid bacteria.

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