Kefir Bakteriyosin Bacteriocin

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Kafkas niv Vet Fak Derg 15 (1): 45-50, 2009

Investigation of Bacteriocin Production Capability of Lactic Acid Bacteria Isolated From Foods [1] [2]
[1] This study was summarized from PhD thesis of corresponding author [2] This study was supported by The Scientific and Technologic Research Found of Kafkas University

idem SEZER *

Abamslm GVEN

(Project no: VF-2006-04) * Kafkas University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Kars TURKEY Yayn Kodu (Article Code): 2008/56-A

The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from foods. A total of 12700 LAB were isolated from foods and screened for their bacteriocin production. Among them, 601 isolate showed a clear antimicrobial activity against indicator strains. After neutralization 35 of the cultures retained active. The antimicrobial activities of strains were lost after treatment with proteases. The antimicrobial metabolites were heat stable and were active pH range of 2-12. The bacteriocin which produced by Lactococcus lactis ssp. lactis isolated from kashar samples was partially purified by ammonium sulphate precipitation, dialysis, following ion-exchange chromatography. It was also found that this bacteriocin was cationic. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2.4 kDa, 4 kDa or 15 kDa. In this study antimicrobial substance of LAB from food showed strong antibacterial activity against Gram-negative and Gram-positive indicator microorganisms. Those were also found that these bacteriocins were stable at temperatures used in food manufacturing and storage. Bacteriosinogenic LAB or bacteriocins might be useful as a natural preservative for inhibiting food-borne pathogens in particular Listeria monocytogenes in foods. Keywords: Animals food, Lactic acid bacteria, Bacteriocin, Antimicrobial activity

Summary

Bu almada, gdalardan bakteriyosinojenik laktik asit bakterilerinin (LAB) izolasyonu amalanmtr. Gdalardan izole edilen toplam 12700 LABnin antimikrobiyel etkisi test edilmitir. Bu izolatlardan sadece 601 adedi almada kullanlan indikatr sulara kar antimikrobiyel etki gstermitir. LABnin rettii laktik asitin ve hidrojen peroksitin gl antimikrobiyel etkisi gz nne alndnda bu etkinin ortadan kaldrlmas iin katalaz ilavesiyle birlikte ortam ntrlendiinde antimikrobiyel etkisini srdren sadece 35 izolat olduu belirlenmitir. zolatlarn etkisi proteolitik enzimlerin varlnda kaybolmutur. retilen antimikrobiyel etkili metabolitlerin gda retim ve muhafaza scaklklarnda ve geni pH (pH 2-12) aralnda aktivitelerini srdrdkleri gzlenmitir. Kaar peynirinden izole edilen Lactococcus lactis ssp. lactis suunun rettii bakteriyosin ksmi olarak saflatrlarak tanmlanmaya allmtr. zole edilen bakteriyosinojenik sularn Gram-negatif ve Gram-pozitif indikator mikroorganizmalara kar olduka gl antimikrobiyel etkiye sahip olduu belirlenmitir. Bakteriyosinojenik LAB ve/veya onlarn bakteriyosinleri gda kaynakl patojenlerden zellikle Listeria monocytogenesin inhibisyonu iin doal bir katk maddesi olarak kullanlabilir. Anahtar szckler: Hayvansal gdalar, Laktik asit bakterileri, Bakteriyosin, Antimikrobiyel aktivite letiim (Correspondence)

zet

Gdalardan zole Edilen Laktik Asit Bakterilerinin Bakteriyosin retme Yeteneklerinin Aratrlmas

+90 474 242 68 07/1192 cigdemsezer@hotmail.com

Investigation of Bacteriocin Production...

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INTRODUCTION
Lactic acid bacteria (LAB) can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these compounds 1. Antimicrobial proteinaceous compounds produced by bacteria that are active against other bacteria, despite varying greatly in chemical nature, mode of action and hot specificity, have traditionally been defined as bacteriocins 2 . Bacteriocins and lantibiotics produced by LAB are extremely important in preventing the growth of spoilage and pathogenic bacteria, and have been subject of extensive studies in recent years because of their potential use as novel, natural food preservatives 3. They have an inhibitory effect only on closely related species and on other Gram-positive organisms. Those produced by LAB are of particular interest due to their potential application in the food industry as natural preservatives 3,4. This paper reports the identification of the bacteriocin producing strain isolated from several foods, and physical and chemical properties, and action mode of the bacteriocin.

For isolation of LAB the serial dilutions of the samples were inoculated (pour plate) onto Chalmers agar 6, M17 agar and MRS agar. Followed by aerobic incubation at 30C for 4872 h to allow the colonies to develop. From MRS agar, M17 agar and modified Chalmers agar, five colonies were randomly taken from each sample. Tests were carried out on each isolate. All isolates were investigated to determine their colony morphology, cell morphology, and Gram staining, catalase, and gas production from glucose. These tests were conducted as a preliminary step in characterization of the selected isolate 7,8. Three methods were used to determine antimicrobial activity. Antimicrobial activity was determined in both colonies of LAB and culture supernatant. Detection of antagonistic activity

Isolation of bacteriocin-producing lactic acid bacteria from samples of food

MATERIAL and METHODS


The bacteria chosen as indicators were Lactobacillus casei, Lb. cellebiosis, Leuconostoc paramesenteroides, Streptococcus lactis, (isolated from kefir samples in our laboratory), Micrococcus luteus, L.monocytogenes, Staphylococcus aureus, Bacillus subtilis, Salmonella enteritidis, Yersinia enterocolitica O: 3 (Refik Saydam Hygiene Center, Turkey). 450 samples of a variety of milk and meat products obtained from different manufacturers in Turkey were analyzed. They included raw milk, yoghurt, butter, cream, tulum cheese, kashar cheese, white cheese and fermented sausage. Samples of kefir were made using with kefir grains in our laboratory 5. Samples Indicators strains

Method 2: Culture supernatant (CS) was obtained by centrifuging (Hettich, Universal 32 R) the culture at 10.000 x g for 10 min at 4C. Mueller Hinton (Oxoid) agar plates were seeded with a bacterial lawn of indicator strains at a 1x105 CFU/ml concentration. After this step, two different processes were applied; Agar disk diffusion methods 10 and Well-infusion methods 11. Method 3: Microtiter plate method was used to determine antimicrobial activity.
Bacteriocin assays

Method 1: One of the methods, briefly, Mueller Hinton (Oxoid) agar plates was seeded with a bacterial lawn of indicator strains at 1x105 CFU/ml concentration. Then, colonies of LAB isolates spotted on the agar plates. The plates were incubated 24h at 30C, antimicrobial activity being detected as translucent halos in the bacterial lawn surrounding the colonies 9.

LAB strains were grown in MRS broth at 30C for 18 h. A cell-free solution was obtained by centrifuging the culture at 10.000 x g for 10 min at 4C. The supernatant fluid was then adjusted to pH 6.5-7 with 5 N NaOH or 5 N HCI, so as to rule out inhibition through the production of organic acids. The supernatant fluid was filtered through a syringe

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filter with a pore size of 0.22 m (Millipore). If antagonism was present, the possible inhibitory action of hydrogen peroxide was eliminated by the addition of a sterile solution of catalase (1 mg/ml-Sigma) at 25C for 2 h. The samples were incubated at in water bath at 60C for 10 min. Any residual antimicrobial activity to treated extract was determined by the agar disk method and well-diffusion method 8,11,12.

If inhibition continued to be present when the extract added the wells had been neutralized and treated with catalase, this was taken to be due to the production of bacteriocin or bacteriocin-like compounds. The proteinaceous nature of the active extract was evaluated by treating this extract with a 1 mg/ml final concentration of proteolytic enzymes [-chymotrypsin (Sigma SIC 4129), Protease (Sigma P4860), Trypsine (Sigma T1426)]. All these enzymes solution were filter-sterilized through a syringe filter with a pore size of 0.22 m (Millipore). An untreated culture extract and enzyme solution (in buffer only) served as controls. The samples and controls were incubated at 37C for 2 h and water bath at 60C for 10 min to inactive the enzymes. Any residual antimicrobial activity to treated extract was determined by the agar disk method and well-diffusion method 12,13. Sensitivity of bacteriocin-like substance to pH and heat

production, growth at pH 49 in 0.1% methylene blue and growth at 0, 10, 15 and 45C, growth in Acetate agar, Voges prouskauer test and the fermentation of carbohydrates test. The phenotypic identification of LAB strains was carried out by means of miniaturized API 50 CH biochemical test (Biomerieux). The result of the identification tests were interpreted using the APILAB PLUS software (Biomerieux) 7,15-17. For purification, the sterilized neutralize supernatant was saturated with 80% (w/v) ammonium sulfate (Merck), and held for 18h with stirring at 4C. The samples were centrifuged at 15.000 x g for 30 min at 4C and pellet was resuspended in 10 ml 10mM sodium phosphate buffer (Merck), pH 6.5. Further purification included dialysis using a Spectra/Por dialysis membrane with a 1 kDa cut-off (Spectra/Por 7 132104a) against the same buffer, overnight at 4C 18,19. For purification of dialyzed was used ionexchange column. The sample was applied to a column (2.5 by 50 cm) of carboxymethyl-cellulose (CM52 Whatman 4037050) prequilibrated with 50mM sodium phosphate buffer (Merck), pH 6.5. After being washed with buffer and 0.3 M NaCI. Antimicrobial activity of filtrate (anionic protein) and precipitate (cationic protein) were tested by the agar disk method and well-diffusion method 20. Protein concentration of the bacteriocin fractions were determined by using the protein assay kit (Sigma BCA1 and B 9643) with bovine serum albumin as the Standard. Determination of molecular weight Partial purification of bacteriocin

The bacteriocin or bacteriocin-like activity of the cell free culture supernatant was analyzed after heat-treatment and at different pH values. The residual antimicrobial activity was determined by the agar disk method and well-diffusion method. The sensitivity of the active substance to different pH was estimated by adjusting the pH of supernatant samples to pH 212 with NaOH or HCI and testing against the indicator strains after 37C 2 min incubated. The pH-treated samples were neutralized to pH 6.57 before measuring the residual activity. The sensitivity of to heat was tested by heating cell free supernatant samples to at 4C for 7 days, at 65C for 30 min, at 121C for 15 min and testing the residual activity by the agar disk method and well-diffusion method 13,14. Isolates were identified as follows: hydrolysis of arginine, growth in 6 and 10% NaCI broth, dextran Identification of LAB

The apparent molecular masses of bacteriocin were estimated by the Tricine-SDS-PAGE method 21. Electrophoresis was performed at 3090 V for 2.5 h using 1016% gradient Tris-Tricine SDS PAGE gels and polypeptide SDS Standard (BioRad 161-0326) as molecular weight standard. Gel was stained with both Coommasie blue stain and silver stain 22 A total of approximately 13500 colonies isolated in each culture medium were examined for their strongest antibacterial activity against one of the

RESULTS

48 Investigation of Bacteriocin Production...

twelve indicator strains. A total of 601 colonies, showing antibacterial activity against at least one of the indicator organisms, were isolated. Results showed that 35/601 LAB cultures retained activity in the cell-free supernatants after neutralization (pH 6.57). All of them showed powerful antilisterial activity, especially against L. monocytogenes, S. aureus, B. subtilis and M. luteus but not against strains of E. coli, Y. enterocolitica and other Gram negative indicator strains. Only three strains showed antimicrobial activity against both Gram negative strains and other strains. Complete inactivation of antimicrobial activity was observed after treatment of bacteriocincontaining cell-free supernatants with -chymotrypsin, protease and trypsine, confirming its proteinaceous nature. Treatment of the bacteriocin with catalase did not result in any changes of antimicrobial activity, indicating that the inhibition recorded was not hydrogen peroxide. While some the bacteriocin-containing cell-free supernatants were stable in wide pH range from 2 to 12 or heat treatment, other was instable (Data not shown). Effects of enzymes, pH and temperature

bands, Tricine-SDS-PAGE offer high resolution, especially for the small protein and peptide range. Samples of bacteriocin on Tricine-SDS-PAGE gel could not be stained with Coomassie blue dye but could be stained with silver (Data not shown). Silver-stained Tricine-SDS-PAGE gel showed several moderately sized protein bands as well as a prominent band of 2.5 kDa. While coomassie blue-stained used methanol and acetic acid for fixation, silver-stained used formaldehyde as strong agent for fixation.

In this study, strains producing bacteriocins were isolated from local foods and antimicrobial compounds produced by those strains were partially purified.

DISCUSSION

According to result of test, when LAB to classify, 15 of this 35 isolates were Lb. plantarum, 12 of isolates Lc. lactis ssp. lactis, four of isolates Lb. brevis, three of isolates Lb. paracasei ssp. paracasei, one of isolates Leuc. mesenteroides ssp. mesenteroides were as identified. Thus far a great number of LAB isolates were worked however bacteriocins were identified very hard. Because of difficultly of partial purification of bacteriocin only one bacteriocin which has a strong antilisterial activity was partially purified from a total of 35 isolates. The bacteriocin produced Lc. lactis ssp. lactis was partially purified from the supernatant fraction of cultures by 80% ammonium sulfate precipitation. For partial purification, ion-exchange column was used. Bacteriocin was determined cationic. The apparent molecular masses of bacteriocin were estimated by the Tricine-SDSPAGE method. SDS-PAGE method was unsuccessful in samples containing small sized protein. Resolution of the small peptides was difficult because they appeared as faint, diffuse Partial purification of bacteriocin

Although 31500 colonies were isolated from foods, bacteriocin productions among the LAB isolated were detected in thirty-five strains. Various factors may have influence, such as culture medium, condition of incubation, target microorganisms, variability and sensitivity of methods used to for determine antimicrobial activity. Bacteriosinogenic strains showed strong antimicrobial activity against Gram positive indicator microorganism but only three of 35 showed activity against Gram-negative bacteria. Nisin exhibits antimicrobial activity towards a wide range of Gram-positive bacteria, and is particularly effective against spores. It shows little or no activity against Gram-negative bacteria, yeast and moulds 23. However, Vignolo et al.11 reported bacteriocins of LAB against Gram-negative bacteria, contrary to other researchers 24,25. Total 12 Lc. lactis ssp. lactis strain produced substrate show that one from the other different data against tolerant test, activity test. The production

There are many methods for determine antimicrobial activity of LAB. In this study, using broth culture of LAB to determine antibacterial activity gave better results than colonies of them on solid medium. In this study, results showed that incubation period must not 12-24 h. Antimicrobial activity of isolates was not determined after 12-24 h. Antimicrobial activity may be lost at the end of six hours period possibly due to purity of supernatant.

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Our study result compared to the results of other researches differed. This distinction being greatly influenced by several factors such as origin of isolates, different antimicrobial substance could be produced by bacteria, variable methods for determination of antimicrobial affect and sensitivity of methods, bacteriocin dose and degree of purification, physiological state of the indicator cells (e.g. growth phase) and experimental conditions (e.g. temperature, pH, presence of agents disrupting cell wall integrity and other antimicrobial compounds, etc.), variable methods for purification of bacteriocin and sensitivity of methods.

Lc. lactis ssp. lactis isolated from kefir showed strong antimicrobial activity against E. coli . In addition most of those isolates continued to demonstrate antimicrobial activity indicator microorganisms at wide pH range from 2 to 12 and heat treatment (121C for 15 min). Despite the fact that LAB-bacteriocins shows little or no activity against Gram negative bacteria, this isolate is important which showed strong antimicrobial activity against E. coli. Antimicrobial substance of Lb. plantarum species in identified this research showed variable sensitivity at heat and pH treatment. Lb. plantarum was identified in almost all analyzed sample foods. Leuc. mesenteroides ssp. mesenteroides isolated from butter and Lb. brevis isolated from butter, cream and kefir showed strong antimicrobial activity against L. monocytogenes and S. aureus but not against strains of E. coli, Y. enterocolitica and other Gram negative indicator strains. The sensitivity to temperature, pH and enzymes and inhibitory effects against indicator microorganisms of antimicrobial compounds produced by species is varied in strains.

of more than one bacteriocin by a single LAB strain has been reported previously. Several bacteriocins were produced by Lc. lactis ssp. lactis. For example lactococcin, lacticin, nisin 26-28.

well in an in-vitro situation, it should be applied to a food system. Several studies show that different results were obtained while bacteriosinogenicLAB used as food preservative in food system or in vitro. The production of a certain bacteriocin in laboratory media does not imply its effectiveness in a food system. This subject was needed overmuch research. The study of bacteriocins as preservatives in foods can be misleading and confusing unless they have been fully characterized.
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