EVALUATION OF TOLERANCE TO GASTRO-INTESTINAL STRESS FACTORS, ANTIMICROBIAL ACTIVITY, ANTIBIOTIC SUSCEPTIBILITY AND CURD FORMING POTENTIAL OF PRESUMPTIVE PROBIOTIC LACTOBACILLUS STRAINS ISOLATED FROM RURAL VICINITY OF PUNJAB, INDIA


Sharma C., Gulati S., Thakur N., Panwar H.
Department of Dairy Microbiology, College of Dairy Science and Technology, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana – 141004, Punjab, INDIA.

Introduction:
Probiotic bacteria are live microorganisms which when administered in adequate amount, confers health benefits to the host. Nowadays, the consumption of probiotic products is growing intensively in developing countries. The milk flora of Punjab (India), a state recognized for its milk production and consumption, remains unexplored. This study was aimed at selecting novel strains of Lactobacillus from rural background of Punjab for their establishment as potential probiotic candidate.

Methods:
Lactobacillus strains were isolated from curd samples, collected from rural background of Punjab; following standard serial dilution method. Gram positive, catalase negative, rods were characterized as lactobacilli using genus specific PCR. Lactobacillus isolates were subjected to gastrointestinal stress i.e. acid and bile conditions. Antibiotic susceptibility of isolates was determined against 26 common antibiotics, following disc diffusion assay. Antimicrobial activity of cell supernatants was evaluated against gram+ve and gram-ve pathogens through agar well diffusion assay. Auto-aggregation potential of LAB isolates was also determined. Antibiotic susceptibility, Antimicrobial activity and auto-aggregation potential were compared to reference probiotic LAB strains. Product viz. Curd forming potential of selected isolates was also evaluated.

Results:
A total of twenty curd isolates were confirmed as Lactobacillus sp. on basis of 250bp PCR product. When subjected to low pH (1.5, 2, 2.5, 4.5) and bile (0.5, 1, 1.5, 2 %) for different time intervals (0 to 4 hrs), varied responses were recorded. Curd isolates, D4 and D6 could resist acid and bile stress most remarkably, as observed with only 2 and 2.5 log (cfu/ml) reduction at pH2.5 and around 2 log (cfu/ml) reduction at 2% bile after 4hr exposure. Among the 26 antibiotics screened, most of the Lactobacillus isolates showed resistance pattern towards cefoxitin (30mcg), vancomycin (30mcg) and teicoplanin (30mcg). In contrary, isolates were intermediate to moderate susceptible towards other antibiotics with highest zone of inhibition (>40mm) recorded with imipenem (10mcg). Culture supernatants from the Lactobacilllus isolates exhibited varying degrees of inhibitory activity pathogens. S. aureus, S. mutans, L. monocytogenes, E. coli and K. pneumoniae were resistant towards all the tested isolates. However, moderate level of antimicrobial activity was exhibited against B. cereus, S. Typhii, P. mirabilis, S. flexneri and P. aeruginosa. None of the isolates exhibited β- haemolytic activity. Highest cell auto-aggregation (6h, %) was observed with standard probiotic, Lactobacillus fermentum (NCDC 214) (50.44% ±0.39) followed by D7 (36.94 ±0.68), D10 (33.38 ±0.43) and D18 (31.40 ±0.40). Isolate D6 exhibit remarkable curd forming potential as observed with good texture and high water holding capacity.

Discussion:
The study highlighted a noticeable heterogeneity in the ability of the strains to withstand harsh growth conditions and some strains meet the criteria for being a potential probiotic candidate. Studies are going on to further establish and validate their probiotic and safety attributes under in vitro and in vivo conditions.

Keywords: Lactobacillus, Probiotic, Acid tolerance, Bile tolerance, Antimicrobial activity, Auto-aggregation

Citation:
Sharma C., Gulati S., Thakur N., Panwar H. (2016). Evaluation of tolerance to gastro-intestinal stress factors, antimicrobial activity, antibiotic susceptibility and curd forming potential of presumptive probiotic Lactobacillus strains isolated from rural vicinity of Punjab, India. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 116.). IPC2016

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