| Abstract | Probiotics are beneficial microorganisms, which provide several health benefits when consumed in adequate amount, lactic acid bacteria constitute major group of probiotic commonly used as starter cultures, biological control agents and pharmaceuticals. Besides several health benefits the lactic acid bacteria also have antagonistic activity towards foodborne pathogens. The lactic acid bacteria and their antimicrobial compounds have the potential to be used as biopreservative agents. The main aim of this study was to investigate the presence of lactic acid bacteria with probiotic characteristics from raw buffalo (Bubalus bubalis) milk including bacteriocin or bacteriocin like inhibitory substance producing strains and determine their biopreservation potential.
Bacterial colonies (n = 160) were isolated from 40 raw milk samples. Based on the Grams staining, cell morphology, catalase, motility and cytochrome oxidase test, 21 isolates were identified as lactobacilli and evaluated for evaluated for tolerance to pH (2, 3, 4.5, 6.5), bile (0.5, 1% w/v) and lysozyme (50 and 100 μg/mL). Six out of 21 lactobacilli isolates showing highest resistance to low acid, high bile and lysozyme were selected for further probiotic characterization. The survival in gastrointestinal fluids, auto aggregation, hydrophobicity, adhesion to Caco-2 cell lines, antibiotic susceptibility, antibacterial activity through agar well assay, β-galactosidase activity, exopolysaccharide production and heat resistance were evaluated. The viability of six potential probiotic isolates in simulated gastric and intestinal fluid ranged from 6.60-7.03 Log CFU/mL and 8.36-8.74 Log CFU/mL, respectively. The isolates showed auto aggregation (14-89%), hydrophobicity (11-81%) and adhesion to Caco2 cell lines (21-69%). The lactobacilli isolates were positive for β-galactosidase activity, exopolysaccharide production, heat resistant and negative for hemolysis. Isolates were susceptible to most of the tested antibiotics and showed antibacterial activity. Isolate 11 showed the potential of bacteriocin or bacteriocin like inhibitory substance production. All six lactobacilli isolates (11, 19, 60, 93, 112) were identified through molecular method (16S rDNA sequencing) as L. paraplatarum (isolate 11), L. plantarum (isolate 19), L. pentosus (isolate 42), L. fermentum (isolate 60), L. pentosus (isolate 93) and L. reuteri (isolate 112). All probiotic strains produced probiotic-based yogurt and survived (8.21 - 8.61 Log CFU/mL) in refrigerated storage temperature (4°C) after 28 days.
Furthermore, the potential bacteriocin like inhibitory substance producing strain and its antimicrobial compound was characterized. Lactobacillus paraplantarum 11 was further evaluated in kinetic assay for tolerance to pH, bile, gastrointestinal fluids and auto aggregation activity at different time intervals. The bacteriocin like inhibitory substance was partially purified through ammonium sulphate purification and antibacterial activity was evaluated against foodborne pathogens, the antimicrobial compound was characterized for stability in chemicals, enzymes, heat and pH. Minimum inhibitory and minimum bactericidal concentration, time kill kinetics and biopreservation potential was evaluated in meat. Lactobacillus paraplantarum survived in low pH, high bile concentration and gastrointestinal fluids at different time intervals and showed auto aggregation potential during 24 h incubation. The partially purified bacteriocin-like inhibitory substance showed antibacterial activity against indicator bacteria including multi drug resistant pathogens. It was susceptible towards proteolytic enzymes, thermostable, active in chemicals and pH 2 - 7. Minimum inhibitory and minimum bactericidal concentration ranged from 31.25 to 62.5 μg/mL and from 62.5 to 125 μg/mL, respectively. Time-kill kinetic revealed the reduction in growth of pathogens in time-dependent manner. Bacteriocin-like inhibitory substance
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showed the biopreservative effect in meat artificially contaminated with Listeria monocytogenes.
Furthermore, antimicrobial and biopreservation potential of six (n= 6) isolated probiotic strains was determined. Live culture probiotic cell were evaluated for antagonistic activity, co-culture, co-aggregation activities with pathogens. The effect of cell free supernatant on release of nucleic acid and growth of pathogens at different time intervals was determined. Biopreservation potential of all six probiotic strains was evaluated in raw milk. The live culture of probiotic strains inhibited the growth of foodborne pathogens with inhibition zones ranging from 10 - 21 mm, in co - culture assay, pathogens population was reduced after incubation at 37 °C for 24 h. The probiotic strains showed co-aggregation capabilities through aggregation with pathogens. The cell free supernatants of probiotic strains caused the release of 260 nm absorbing material (DNA and RNA), which indicated the antagonistic activity on foodborne pathogens. In biopreservation assay the live culture, probiotic strains Lactobacillus paraplantarum 11, Lactobacillus plantarum 19, Lactobacillus pentosus 42, Lactobacillus fermentum 60, Lactobacillus pentosus 93, Lactobacillus reuteri 112 were effective in reducing the Listeria monocytogenes population in raw buffalo milk with complete inhibition after 6 - 8 days. The results suggests that lactic acid bacteria isolated from buffalo milk have probiotic potential including antagonistic activity against foodborne pathogens and can be used in variety of food applications. |