bulgarian yogurts

8/6/2019 Bulgarian Yogurts

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HYDROLYSIS OF MAJOR DAIRY PROTEINS BY LACTIC ACID

BACTERIA FROM BULGARIAN YOGURTS

IRENA TZVETKOVA1,4, MICHLE DALGALARRONDO1, SVETLA DANOVA2,

ILIA ILIEV3

, ISKRA IVANOVA4

, JEAN-MARC CHOBERT1,5

andTHOMAS HAERTL1

1UR 1268 Biopolymres Interactions Assemblages, INRA, Equipe Fonctions etInteractions des Protines LaitiresRue de la Graudire, B. P. 71627

44316 Nantes Cedex 3, France

2Institute of MicrobiologyBulgarian Academy of Sciences

Sofia, Bulgaria

3SIBIO-93 Ltd.Plovdiv, Bulgaria

4Department of MicrobiologySofia UniversitySofia, Bulgaria

Accepted for Publication December 1, 2006

ABSTRACT

Twenty-one Lactobacillus strains isolated from three types of Balkanhomemade 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, 8090% of b-casein was consumed. They showed also a pro-teolytic activity towarda-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 ofALA. Hydrolysis of casein by all strains produced peptides with an antibac-

terial effect against Escherichia coli. Consequently, to obtain a maximalhydrolysis 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:[email protected]

Journal of Food Biochemistry 31 (2007) 680702. All Rights Reserved. 2007, The Author(s)

Journal compilation 2007, Blackwell Publishing

680
mailto:[email protected]:[email protected]


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PRACTICAL APPLICATIONS

Some of the 21 Lactobacillus strains isolated from three types of Balkanhomemade yogurts may be used to proteolyze milk proteins in order toproduce peptides with an antibacterial effect against Escherichia coli. Toobtain a maximal hydrolysis of the dairy proteins seconded by appearance ofantimicrobial peptides, a combination of strains with different beneficial prop-erties to be used as starters should be determined. During fermentationprocess, milk proteins are acidified by the production of lactic acid and arehydrolyzed by proteases and peptidases from bacteria. This proteolysis isfollowed by a reduction of the number of epitopes and consequently by adecrease in allergenicity of hydrolyzed proteins. For these reasons, starters as

Lactobacillus strains with beneficial properties able to reduce the allergenicityof fermented milk products are of great interest for the dairy industry.

INTRODUCTION

Milk is the complete source of nutrients for the neonate, which providesproteins, lipids, sugars, vitamins and minerals for healthy growth of an infant(Warner et al. 2001). The newborn human has an immature digestive capacityin the stomach and small intestine, resulting in a high proportion of intact orlarge fragments of food protein molecules passing through the intestinalmucosal barrier into the immune structures in the intestinal lining (Peyerspatches) or into the blood. These intact proteins or large peptides may act asantigens to the immune system of the young, resulting in allergic hypersensi-tivity reactions and food protein intolerance. Cow milk proteins belong to thestrongest antigens in human diet (Eigel et al. 1984). Milk proteins are the firstexogenous proteins consumed in large quantities by children (Jrvinen andSuomalainen 2001). Whey proteins (a-lactalbumin [ALA], b-lactoglobulin[BLG], bovine serum albumin) and caseins are considered as the main cowsmilk antigens (Rsnen et al. 1992; Nentwich et al. 2004), and they cause animmunologically mediated adverse reaction (Savilahti et al. 1992).

Nowadays, the cows milk allergy ranges from 1.9 to 7.5% of the popu-lation (Jrvinen and Suomalainen 2001). For this reason, the application oflactic acid bacteria (LAB) in hydrolysis of milk proteins may have importanteffects on milk digestibility, on the production of bioactive peptides and maydecrease milk allergenicity and lactose intolerance (Nentwich et al. 2004;Prioult et al. 2005). Humans have used yogurt and yogurt-like products as themost popular vehicles for ingestion of probiotic microorganisms. Probioticshave been the subject of considerable scientific and commercial attention overthe past two decades. Probiotics are live microorganisms that when ingested

681MILK PROTEINS PROTEOLYSIS BY LAB


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may have positive effects on human health (Fuller 1991). Beneficial effectsof probiotics on immune-mediated diseases, such as allergy, have beendocumented and include stimulating the immune system (Cross et al. 2001;Ouwehand et al. 2002), synthesizing and increasing the bioavailabilityof nutrients (Pessi et al. 1998) and decreasing the prevalence of atopy(Kalliomki et al. 2001). Bifidobacteria and lactobacilli are common anaer-obes in the human intestinal microbiota, and some of them have been reportedto display probiotic properties (Ouwehand et al. 1999). Although LAB areusually considered to be only weakly proteolytic, some of them cause asignificant degree of proteolysis in multiple fermented dairy products includ-ing yogurts. Yogurt bacteria are microorganisms with multiple amino acidauxotrophies (Kok and De Vos 1994). The content of free amino acids andpeptides 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 inmilk (Kunji et al. 1996; Chen and Steele 1998).

The proteolytic activities of LAB including yogurt bacteria and probioticbacteria have been studied extensively (Booth et al. 1990; Wohlrab andBockelmann 1993; Bockelmann et al. 1996; Law and Haandrikman 1997; Ehnet al. 2005).

Additionally, proteolytic enzymes from LAB produce flavor compoundsand precursors that are essential for cheese flavor development (Mulholland1997; 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 andcheese; and (3) numerous intracellular peptidases (Kunji et al. 1996; Chen andSteele 1998). The intracellular peptidases of LAB include both endopeptidasesand aminopeptidases. Endopeptidases, because of their ability to hydrolyzepeptide bonds within a peptide, are of particular interest in targeting peptidesfor rapid hydrolysis.

To promote human health, combinations of different bacterial strainsbelonging to the genera Lactobacillus, Streptococcus and Bifidobacteriumhave been used traditionally in fermented dairy products (Prasad et al. 1998;Dunne et al. 1999). A variety of LAB isolated from fermented milk productshave 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 antimutagenicactivities (Nishioka et al. 1989; Hosono et al. 1990), as well as effects onmodulating the immune system (Perdigon et al. 1988; Fernandes and Shahani1990), lowering cholesterol levels (Shun et al. 1989) and reducing lactoseintolerance in the host (Alm 1982). Lactobacillus is the most important group

682 I. TZVETKOVA ET AL.


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of starters for the dairy industry. During fermentation process, milk proteinsare acidified by the production of lactic acid and are hydrolyzed by proteasesand peptidases from bacteria. This proteolysis is followed by a reduction of thenumber 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 beneficialproperties able to reduce the allergenicity of fermented milk products are ofgreat interest for the dairy industry.

In this study, the attention was focused on homemade yogurts from theBalkan region, as a source of newly potential starters for application in thefood industry. In this context, the aim of this study was to screen the pro-teolytic activity of newly isolated strains, by determination of the optimalconditions 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, wereused. The strains with prefixes K, O and B correspond to strains isolatedfrom cow, sheep and buffalo yogurts, respectively. The strains with prefix Kand prefix O were originated from the western part of Bulgaria, in a villagenear the mountain Pirin; the strains with prefix B were originated from thenorthern part of Bulgaria, in a village close to the city Vratza. The strainswere cultured overnight (1618 h) on MannRogosaSharpe (MRS) broth(Merck, Darmstast, Germany) at 37C and in limitation of oxygen (tubes orPetri 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 50CHLsystem (BioMerieux, Craponne, France), according to the manufacturersinstructions. The fermentation profiles were read after incubation at 37C inanaerobic conditions, for 3 days.

Proteolytic Activity

Initial screening of the strains for the presence of proteolytic activity wasperformed using the well diffusion method (Schillinger and Lucke 1989). A1.4% skim milk agar was used. Wells were made in the lawn of hardened soft



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agars in Petri dishes. Aliquots (70 mL) of supernatant of overnight cultures(1618 h) in MRS broth were poured in the wells. The plates were left for 1 hat room temperature (22C) in sterile conditions before incubating them to theadequate 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 thestrains, all the strains were cultured on different test media performed toachieve the optimal conditions for casein degradation. After an overnightincubation in MRS media, the strains were inoculated (10% inoculum) in thetest media containing different concentrations of sodium caseinate and withthe following composition: 2% glucose and sodium caseinate or b-casein(concentrations from 0.08 to 2.0%) buffered with sodium phosphate buffer(25100 mM) and with or without additives (vitamins, MRS components).

After incubation, 50 mL of the culture was taken at 24, 48, 72 and 96 hand diluted 1:1 (v/v) with sample buffer containing 9-M urea, 0.5%2-mercaptoethanol and 300-mg/mL saccharose. The samples were thenanalyzed by urea-polyacrylamide gel electrophoresis (urea-PAGE).

UREA-PAGE. Urea-PAGE was performed with 10% polyacrylamideslab 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 mAon the running gel, staining was performed with Coomassie Brilliant BlueR-250 followed by a convenient destaining. Gels were scanned and intensity ofthe bands was quantified using Quantity One BioRad Software (Bio-Rad,Marne la Coquette, France).

Proteolysis of Whey Proteins. To analyze the proteolytic activity of thetested strains toward whey proteins, an MRS broth containing BLG and ALAwas 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 sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samplebuffer containing 4% sodium dodecyl sulfate (SDS), 3% 2-mercaptoethanol,10% glycerol, 50-mM TrisHCl, pH 6.8. The samples were heated at 100C for3 min then analyzed by SDS-PAGE as previously described (Bertrand-Harbet al. 2003).

SDS-PAGE. The samples were applied to a 15% polyacrylamide slabgel. The migration buffer contained 50-mM Tris, 0.384-M glycine and 0.1%



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SDS. After running at 10 mA on the stacking gel and 20 mA on the runninggel, staining and destaining were performed as described earlier. The gels werescanned and intensity of the bands was quantified by Quantity One BioRadSoftware.

Reversed-phase High-performance Liquid Chromatography

(RP-HPLC)

RP-HPLC was used to analyze the supernatant of culture of the strainsgrowing on 0.08% sodium caseinate, 2% glucose in 50-mM phosphate bufferor on 0.08% b-casein, 2% glucose in 50-mM phosphate buffer. In a firstexperiment, after a preculture on MRS broth, the test media was inoculatedwith the test culture. For further experiments, before inoculation, the cultureswere washed twice with 0.5% NaCl.

The samples were separated with a Nucleosil C18 column (250 0.3 mmi.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) inwater; solvent B, acetonitrile/water/TFA, 70/30/0.09 (v/v/v). Elution wasperformed with a linear gradient of solvent B from 0 to 50% over 10 min, then5080% in 20 min; the column was then rinsed with 100% of solvent B.Detection was performed with a Waters 996 photodiode array detector at220 nm. The data were analyzed by Millenium32 software (Waters) aspreviously 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. Afteradjusting 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 onLuriaBertani agar medium (Sigma, St. Louis, MO) and against Listeriainnocua F (Ecole Nationale des Ingnieurs des Techniques des IndustriesAgricoles et Alimentaires, Nantes, France) on brainheart agar medium(Biokar Diagnostics, Beauvais, France). The plates were incubated overnightat 37C.

Antimicrobial activity of 24- and 48-h hydrolyzed samples was checkedon the strains cultured on media containing 2% glucose in 100-mM phosphatebuffer added with 0.08, 0.5 and 2.0% sodium caseinate. The neutralizedsupernatants (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.



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RESULTS AND DISCUSSION

API Tests

API tests showed that all the strains tested are Lactobacillus delbrueckiissp. bulgaricus.

Proteolytic Activity

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

Proteolysis of Caseinate. The strains tested were incubated in differentconditions in order to determine the best allowing proteolysis without caseinprecipitation. After incubation of the strains on test media supplemented withdifferent concentrations of sodium caseinate in combination with differentconcentrations of phosphate buffer and additional MRS components and vita-mins, it was observed that the growth of the studied strains was inhibited whenconcentrations 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 or2.0% casein and lower concentrations of phosphate buffer (25 or 50 mM), theprecipitation of casein occurred rapidly because of a quick acidification of themedia (data not shown). In these conditions, the results obtained by urea-PAGEwere hard to compare because of the formed precipitates. Consequently, a testmedium with 0.08% sodium caseinate and 50-mM phosphate buffer was used asa model system for all further experiments.

As observed by urea-PAGE (Fig. 1), caseins were degraded by the differ-entstrainstestedafter24,48,72and96 hofcultivation.After24 hofincubation(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 whenusing the strains O43, O45, B5, B7 and B8. These strains were less active at thebeginning of the incubation, what could be explained by the very limitedconditions used for their growth (the tested media contained only glucose andprotein). Some strains (K3, K6, K15, K20 and K26) adapted well and were thenable to degrade b-casein within the first 24 h. Above 24 h of incubation, all theother strains started a rapid consumption of b-casein, and after 96 h ofincubation (Fig. 1D), near allb-casein content was hydrolyzed by all the strainstested. The hydrolysis of aS1- and aS2-caseins was smaller. The peptidesobserved by polyacrylamide gel electrophoresis after 24 and 48 h of incubation



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A

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

B

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

C

D

-Casein

S-Casein

-Casein

S-Casein

K24 K26 O42 043 045 O47 B5 B7 B8 C K2 B5 K27CK3 K5 K6 K14 K15 K19 K20 K21K22 C

-Casein

S-Casein

K22 K24 K26K27 O42 043 O45 O47 B5 C B8 B7K2 K3 K5 K6 K14 K15 K19 K20 K21C

-Casein

S-Casein

FIG. 1. UREA-POLYACRYLAMIDE GEL ELECTROPHORESIS OF SAMPLES OBTAINEDAFTER (A) 6-, (B) 24-, (C) 72- AND (D) 96-H CULTIVATION OF THE STRAINS IN THE

PRESENCE OF 0.08% SODIUM CASEINATEThe 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% sodiumcaseinate, 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 hand diluted 1:1 (v/v) with sample buffer containing 9-M urea, 0.5% 2-mercaptoethanol and

300-mg/mL saccharose.



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disappeared after longer incubation. This finding supports the fact that theproteases present in the different strains conserved well their hydrolyticactivities.

The time course of b-casein degradation during incubation period isshown in Fig. 2. The intensity of degradation of b-casein depends on thestrain used. However, after 96 h of incubation, 8090% of b-casein wasdigested by all the strains tested, as estimated by using Quantity One BioRadSoftware.

Proteolysis of Whey Proteins. The ability of the studied LAB strains tohydrolyze the whey proteins ALA and BLG after 24 h of incubation is shownby SDS-PAGE in Figs. 2 and 3, respectively.

The different strains tested display relatively high proteolytic activityagainst ALA. By using Quantity One BioRad Software, it could be estimatedthat the strains tested were able to hydrolyze ALA in the medium; the amountof 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 strainstested is shown in Fig. 3. All the strains tested were able to reduce to someextent the amount of BLG. The yield of BLG degradation quantified byQuantity One BioRad Software is shown in Fig. 4. The strains tested have alower capacity to hydrolyze BLG as compared with ALA. The strains K14 andK27, which were able to reduce to about 50% the amount of ALA, reducedonly 20% of the amount of BLG.

RP-HPLC

Peptide formation after proteolysis by the different strains tested wasfollowed by RP-HPLC. As the presence of some components of MRSmedium heavily impaired a resolution on the chromatograms, a prewashingstep of the cells with 0.5% NaCl before inoculation of the tested media wasperformed.

After cultivation of the strains in the test medium, the samples wereanalyzed by urea-PAGE. The time course of degradation of caseins afterwashing 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 themedium, the strains needed a longer time of adaptation before start of caseindegradation as compared with the results obtained in the absence of a washingstep (Fig. 2). The time course ofb-casein degradation after 6, 24, 72 and 96 hof cultivation was observed by RP-HPLC (data not shown). It could beobserved that even if the strains were less active, a proteolytic activity was stillpresent.



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Time (h)

0 20 40 60 80 100

beta-casein(%)

0

20

40

60

80

100

120

Strain K2

Strain K3

Strain K5

Strain K6

Strain K14

Strain K15

Strain K19

Strain K20

Strain K21

Strain K22

Control

Time (h)

0 20 40 60 80 100

beta-casein(%)

0

20

40

60

80

100

120

Strain K24

Strain K26

Strain K27

Strain O42

Strain O43

Strain O45

Strain O47

Strain B5

Strain B7

Strain B8

Control

A

B

FIG. 2. TIME COURSE OF DEGRADATION OF b-CASEIN BY THE TESTED STRAINSCULTURED 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 themedium 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 wasperformed in triplicate.



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The three strains K5, K19 and K27 showed similar results. They weregrowing on medium containing either sodium caseinate or b-casein. The timecourse ofb-casein degradation obtained by urea-PAGE (Fig. 7, insert) showsthat after 6 h of incubation, a new band with a higher electrophoretic mobilityappeared. 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 selectedstrains cultivated either on b-casein or on sodium caseinate showed the sameresults.

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

After 24 h incubation

ALA(%)

0

20

40

60

80

100

K2

K3

K5

K6

K14

K15

K19

K20

K21

K22

K24

K26

K27

O42

O43

O45O47

B5

B7

B8

Control

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 OF0.1% ALA

The strains with prefixes K, O and B correspond to strains isolated from cow, sheep andbuffalo yogurts, respectively. Thirty microliters of the culture supernatant was diluted with 90-mLSDS-PAGE sample buffer containing 4% sodium dodecyl sulfate, 3% 2-mercaptoethanol, 10%

glycerol, 50-mM TrisHCl, pH 6.8.M, milk; C, control (a medium with ALA, without bacteria).



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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

After 24 h incubation

BLG(%)

0

20

40

60

80

100

K2K3

K5

K6

K14

K15

K19

K20

K21

K22

K24

K26

K27O42O43

O45O47

B5

B7

B8

Control

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 PRESENCEOF 0.1% BLG

The strains with prefixes K, O and B correspond to strains isolated from cow, sheep andbuffalo 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 TrisHCl, pH 6.8.

M, milk; C, control (a medium with BLG, without bacteria).



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A

B

C

D


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


b-casein

aS-casein


b-casein

aS-casein

FIG. 5. UREA-POLYACRYLAMIDE GEL ELECTROPHORESIS OF SAMPLES OBTAINEDAFTER (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 WITH0.5% NaCl

The strains with prefix K, O and B correspond to strains isolated from cow, sheep and buffaloyogurts, 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 themedium 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 wastaken 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.



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Time (h)

0 20 40 60 80 100

beta-casein(%)

0

20

40

60

80

100

Strain K2

Strain K3

Strain K5

Strain K6

Strain K14

Strain K15

Strain K19

Strain K20

Strain K21

Strain K22

Control

Time (h)

0 20 40 60 80 100

beta-casein(%)

0

20

40

60

80

100

Strain K24

Strain K26

Strain K27

Strain O42

Strain O43

Strain O45

Strain O47

Strain B5

Strain B7

Strain B8

Control

A

B

FIG. 6. TIME COURSE OF DEGRADATION OF b-CASEIN BY THE TESTED STRAINSCULTURED ON 0.08% SODIUM CASEINATE AFTER WASHING OF CULTURES WITH

0.5% NaClStrains 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. Onlythe bacterial cultures were washed twice with 0.5% sodium chloride before inoculation and then

incubated in the medium. Each experiment was performed in triplicate.



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Time (min)

0 10 20 30 40

AU

0.0

0.1

0.2

0.3

0.4

K5

K19

K27

Control

Time (min)

0 10 20 30 40

AU

0.0

0.1

0.2

0.3

0.4

6 h 24 h

Time (min)

0 10 20 30 40

AU

0.0

0.1

0.2

0.3

0.4

Time (min)

0 10 20 30 40

AU

0.0

0.1

0.2

0.3

0.4

Time (min)

0 10 20 30 40

AU

0.0

0.1

0.2

0.3

0.4

48 h 72 h

96 h1 2 3 4 5 6 7 8 9 1011 1213141516 17 1819 20

6 h 24 h 72 h48 h96 h

FIG. 7. TIME COURSE OF DEGRADATION OF b-CASEIN DURING THE CULTIVATION OFSTRAINS 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% glucosein 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 beforeinoculation and then incubated in the medium.

AU, absorbance at 220 nm.



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By RP-HPLC (Fig. 7), it could be observed that within the first 6 h ofincubation, the peak ofb-casein disappeared almost completely and a greatnumber of more hydrophilic peaks appeared. The area of the peaks and theirretention times were almost identical for the three strains analyzed, implyingthat these strains harbor similar proteolytic systems.

Hydrolysis of sodium caseinate by the different strains tested is shown byurea-PAGE (Fig. 8, insert) and by RP-HPLC (Fig. 8).Theobtained results showthat there are differences in the extent of proteolytic activity observed with thedifferent 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 or2.0% sodium caseinate in 100-mM phosphate buffer against E. coli and L. in-nocua has been studied (Fig. 9A and B, respectively). The resulting peptidesobtained after hydrolysis of sodium caseinate have an antibacterial effect (zoneof 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 wasalso 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 ahigh antibacterial activity (zone of inhibition) against E. coli only after 24-hgrowth.

When the strains tested were cultivated on MRS for 24 h, only theneutralized supernatants from the strains K2 and K22 have an antibacterialactivity against E. coli, demonstrating the production of antibacterial sub-stances by these strains. The zone of inhibition of the strain K22 was largerthan that obtained in the presence of the strain K2.

CONCLUSIONS

The experiments were performed to analyze the proteolytic activity ofyogurt bacteria. Sodium caseinate and b-casein were used as markers of thisactivity. The best conditions allowing casein hydrolysis without precipitationare 0.8% casein in 50-mM phosphate buffer. Under these conditions, thestrains tested showed a relatively high proteolytic activity despite the limitedconditions for bacterial growth. Even if the degradation of b-casein is astrain-specific process, within 72 and 96 h of incubation, 8090% ofb-casein



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Time (min)

0 10 20 30 40

AU

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0.2

0.4

0.6

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0 10 20 30 40

AU

0.0

0.2

0.4

0.6

Time (min)

0 10 20 30 40

AU

0.0

0.2

0.4

0.6

Time (min)

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Time (min)

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K5

K19

K27

Control

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

6 h 24 h

h27h84

96 h

6 h 24 h 48 h 72 h 96 h

FIG. 8. TIME COURSE OF DEGRADATION OF SODIUM CASEINATE DURING THECULTIVATION 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% sodiumcaseinate, 2% glucose in 50-mM phosphate buffer. The control is a medium made of 0.08% sodiumcaseinate, 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% sodiumchloride before inoculation and then incubated in the medium.

AU, absorbance at 220 nm.



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A

B

FIG. 9. ANTIMICROBIAL ACTIVITY AGAINST (A) ESCHERICHIA COLIAND (B) LISTERIAINNOCUA OF STRAINS CULTIVATED FOR 48 H ON 2% SODIUM CASEINATE, 2%

GLUCOSE IN 100-mM PHOSPHATE BUFFER



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A

B

FIG. 10. ANTIMICROBIAL ACTIVITY AGAINST ESCHERICHIA COLIOF STRAINSCULTIVATED FOR 24 H (A) IN MILK OR (B) ON MANNROGOSASHARPE



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was consumed by the strains. The strain K27 analyzed by RP-HPLC andurea-PAGE showed the highest proteolytic activity.

The strains tested showed proteolytic activity toward ALA. They wereable 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 highestproteolytic 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 withALA. Even if ALA was more easily degradable by the bacteria, the strain K26was able to reduce by 30% the level of BLG in the medium.

The beginning of casein degradation was delayed when the cells werewashed before inoculation of the test medium. RP-HPLC analysis confirmedthat, as a result of proteolysis by the tested strains, b-casein was cleaved intomore hydrophilic protein, which suggests that the strains tested possess also anexopeptidase activity.

The hydrolysis of casein by all the strains tested produced peptides with anantibacterial effect againstE. coli. The neutralized supernatants from the strainsK2andK22grownonMRSalsohadanactivityagainstE. coli that indicates thepresence of antibacterial substances of bacterial origin (bacteriocins).

Consequently, to obtain the most beneficial effect, a combination ofstrains with different properties could be used as starters.

ACKNOWLEDGMENTS

This work was supported by the Socrates-Erasmus exchange program forthe Ph.D. fellowship awarded to Irena Tzvetkova and in part by the NATOScience for Peace project CBP.EAP.SFPP 982164 Study of antimicrobial andhypoallergenic products of lactic acid bacteria.

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