Molecular characterization of Enterococcus faecium MTCC 5153 for probiotic properties

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.