Molecular characterization of Enterococcus faecium MTCC 5153 for probiotic properties

Badrinath, V. (2010) Molecular characterization of Enterococcus faecium MTCC 5153 for probiotic properties. PhD thesis, University of Mysore.

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Abstract

Probiotics fall into the group of organisms known as lactic acidproducing bacteria and are normally consumed in the form of yogurt, fermented milks or other fermented foods. In the search for new probiotics, Enterococcus spp and each of Lactobacillus plantarum and Pediococcus pentosaceus species isolated from fermented Idli batter and fermented milk, have been characterized. Total 12 isolates were selected based on bacteriocin production ability and studied for its probiotic properties such as acid and bile tolerance, adherence property, cholesterol assimilation, bile salt hydrolase activity, prebiotic utilization. The results were compared with L. rhamnosus GG and two of enterocin-A producing starter cultures. Based on the survivability of the cultures for 2 hrs at pH 2.5, four isolates, showing survivability in the range of 91±5 to 41±7 %, were selected for further study. Strains tested for bile tolerence, L. plantarum S and Ent. faecium IB6 were resistant (delay in growth: 10 min) and P. pentosaceus Cu5 and Ent. faecium RJ4 were tolerant (delay in growth 35 and 45 min) to 0.3 % Ox-gall concentration, and found to assimilate cholesterol (42±5 to 5±0.1 g/mg of dry wt.) as well as possessed bile salt hydrolase activity. Strains tested for -galactosidase showed high (750±50 miller units) and moderate (300±10 miller units) and low (100±5 miller units) activity. And all the selected isolates showed inhibition against several food borne pathogens (25±2 to 5±0.2 mm), and has the ability to utilize the raffinose and mannitol. Based on the above probiotic properties, Ent. faecium MTCC 5153 was selected due to its potentiality of bacteriocin production and evaluated further for non pathogenicity by PCR, RFLP and profiling of 16S rRNA gene product. The Ent. faceium MTCC 5153 has devoid of cytolycin genes conformed by gene specific PCR. Non haemolytic activity was conformed on blood agar and sensitive to many antibiotics. Ent. faecium MTCC 5153 was further studied for enzyme -galactosidase. Conditions were optimized for maximum production of enzyme using different nitrogen, carbon sources and further analysed for the effect of different concentration of these sources. -galactosidase ( -gal) was purified from Ent. faecium MTCC 5153 by conventional chromatographic techniques. The purified enzyme had a specific activity of 24.06 U/mg of protein with Km and Vmax values of 2 mM and 18.2 mM/min/mg of protein, respectively. The yield of purified -gal was 10.65% and it has homodimers with a molecular weight of ~90 kDa consisting of two 43 kDa subunits. The enzyme was found to be stable in pH range of 8.0-9.0 with an optimum pH of 8 and the optimum temperature was 40 C. The enzyme was activated in presence of metal ions, such as Mg+2, Mn+2, Ca+2, K+ and Na+ and was inhibited by Zn+2, Co+2 and Cu+2. Chemical modifiers (N-bromosuccinamide and Diethylpyro carbonate) inactivated the enzyme indicating the role of tryptophan and histidine moieties for activity. The purified -gal was able to synthesize oligosaccharides from lactose by transgalactosylation activity and about ~14% of oligosaccharides formation was observed. Possible application of Ent. faecium MTCC 5153 in skim-milk was studied. Enterocin A was concentrated from Ent. faecium MTCC 5153 and studied for its efficacy in controlling Listeria monocytogenes in skim-milk. Enterocin-A was concentrated from culture supernatants and molecular weight was estimated and found to be of ~4.8 kDa. Enterocin-A from Ent. faecium MTCC 5153 was stable in the pH range of 4-8,. Based on the SEM observation, bactericidal action of bacteriocin was evident by the pore formation in cell wall of Listeria monocytogenes. The culture was tested in skim milk and examined for bacteriocin production during incubation for 24 h at 37 C. About 800 AU/ml of antimicrobial activity was observed at 12 h of incubation and also results in the reduction of viable counts of listeria from 134 X 104 CFU/ml to less than detection level (101 dilution) at the end of fermentation (24 h at 37 C). The enterocin A was adsorbed on 3 types of packaging material. The antimicrobial activity was observed by formation of inhibition zone around the packing material during 7 days period. The work reported in this thesis resulted in exploring the suitability of enterocin-A producing Ent. faecium MTCC5153 for probiotic properties. Several probiotic properties were studied, which includes, ability to survive at low pH conditions (~2.5 pH for 2 h) and bile tolerance, ability to show hypocholesterolemic activity by cholesterol assimilation, by producing bile salt hydrolase activity, antagonistic against various food-borne pathogens, ability to produce -galactosidase. This strain produces novel -galactosidase of low molecular weight (~90 kDa) and enzyme has transgalactosylation activity. Ent. faecium MTCC 5153 has the ability to develop the antioxidant activity, able to produce bacteriocins and had effectively inhibited the growth of Listeria monocytogenes Scott-A. Partially purified enterocin-A can be easily coated on to the packing material and had the stability over 7 days under refrigerated conditions.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Probiotics, lactic acid bacteria, Enterococcus spp,
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology
Divisions:	Food Microbiology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	14 May 2012 09:47
Last Modified:	14 May 2012 09:47
URI:	http://ir.cftri.res.in/id/eprint/10739

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