Characterization of Lactobacilli Isolated from Indigenous Fermented Foods and Human Gut with Special Reference to Their Probiotic Attributes

Abstract

Lactobacilli are a group of Lactic Acid Bacteria widely used in the dairy industry nowadays. These bacteria are used as natural acidifiers for the inoculation of bulk quantities of milk, vegetables and fruits in order to produce a variety of fermented products and beverages. The market of probiotic products has expanded in the five years and most probiotic effects appear to be strain specific. Thus, there is a need for new and well characterised strains, as well as the elucidation of their mode of functional action. The objective of this study was to isolate, identify and characterise Lactobacillus strains from traditional indegenous fermented food products and children’s gut as potential probiotic candidates in vitro. A model of the stomach-duodenum passage was optimised and used to screen 96 Lactobacillus isolates for their survival under simulated gastrointestinal conditions. Six strains showing good survival were selected, and identified by phenotypic and genotypic methods. All strains possessed bile salt hydrolase, as an indicator of survival to bile and of possible cholesterol utilisation, and some also hydrolysed lactose, which may serve for alleviating lactose intolerance. Furthermore, some strains could inhibit the growth of food borne pathogens by producing organic acids and H2O2, and also coaggregated with pathogens, which may enhance their removal in the gut by blocking their adhesion sites. The strains also adhered well to intestinal cells, indicating that they may at least transiently remain in the gut to exert their probiotic effects. Furthermore several strains of this group produce substances of peptide structure that possesses antimicrobial activity called bacteriocins or Antimicrobial peptides (AMPs). The only commercially available bacteriocin is Nisin produced by Lactococcus lactis. As such, the need of investigating the production of such substances from other strains of LAB to develop a technique for their production on a large scale emerged. Initially six known AMPs producing strain Lactobacillus casei LAM-1 Lactobacillus casei LAM-2 Lactobacillus delbruckeii LKH-2, Lactobacillus delbruckeii LKH-3 and Lactobacillus fermuntum Lamec-29 were selected to its antagonistic activity against seven food borne pathogens namely: Staphylococcus aureus ATCC 9144, Aeromonas hydrophila ATCC 35654, Yersinia enterolitica ATCC 9610, Enterobacter(Chronobacter) sakazakii ATCC 51329, Shigella flexneri 2a, Salmonella typhimurium and Listeria monocytogenes ATCC 19111. The possibility of producing the AMPs of these bacteria in large quantities was investigated through several techniques. Firstly, the bacteria were grown into simple batch cultures without pH control where the physicochemical needs for their optimum growth were determined. Through the determination of the optimum nutritional conditions for the propagation of the Lactobacilli an optimised medium for growth occurred. The selected Lactobacilli were found to produce an amount of bacteriocins ranging between 90 IU/ml top 100 IU/ml equivalent to Nisin. The stability of the potency of the produced AMPs was investigated through numerous techniques. The produced bacteriocins were proven to be bacteriostatic against strains of the same genera although their practical application has to be further investigated. The bacteriocin purified from culture supernatants was found to be approximately of 2.5 kDa and was fairly thermostable (100°C), hydrophobic, resistant to ethanol, its amino acid sequence obtained through LC/MS and MALDI of the purified peptide is as follows: A T R S Y G N G V Y C N N S K C W - N V G E A K E N I A G I V I S G K A S G L. The AMP exhibited antibactericidal activity through rapid loss of ATP and K+ ions by disruption of cell membranes of target pathogens; cell death was evidenced by live dead staining and SEM. Further, the bacteriocin was stable and could inactivate all the above pathogens in an artificially contaminated laboratory prepared food following storage over a pH of 3.5-6. The results of this study indicate a potential possibility of this AMP for application as biopreservative in foods. In conclusion, this study provides a rationale for the further use of four of the selected Lactobacillus strains as probiotics for therapeutic or disease preventive purposes.