Effects of environmental and physiological stress on the functionality of probiotic microorganisms
The aim of this project was to examine whether exposure to environmental and physiological stress conditions could affect some functional properties for the selection of probiotic microorganisms. The study was focused on two commercial strains of Bifidobacterium animalis ssp. lactis and two non-commercial Bifidobacterium strains, namely B. breve NCTC 11815 and B. longum NCTC 11818. The effects of exposure to acid, bile, osmotic and oxidative stresses on their antimicrobial activity, biofilm formation capacity and antibiotic susceptibility profiles were assessed. The conditions to generate acid stress in the organisms were chosen as pH 3 for one hour, for both B. animalis ssp. lactis strains, and pH 4 for one hour, for B. breve and B. longum. Conditions for bile stress were 1% (w/v) bile for one hour, for both B. animalis ssp. lactis strains and B. breve, and 0.5% (w/v) bile for one hour, for B. longum. Osmotic stress conditions were 3% (w/v) NaCl for one hour, for both B. animalis ssp. lactis strains and B. breve, and 2% (w/v) NaCl for one hour, for B. longum. Oxidative stress was generated for all organisms by shaking at 200 rpm for two hours. The antimicrobial activities of all four bifidobacteria against pathogenic bacteria, namely Escherichia coli NCTC 12900, Salmonella enterica ser. Typhimurium DT124 and S. enterica ser. Enteritidis PT4, were maintained after exposure to each stress, although there appeared to be lower inhibition after exposure to stress. This varied with strain and type of stress. The antibiotic susceptibility profiles of all four bifidobacteria for five antibiotics, namely tetracycline, erythromycin, ampicillin, chloramphenicol and vancomycin, were unchanged after exposure to each stress. The expression of tetracycline resistance gene tet(W) in one of the B. animalis ssp. lactis strains, designated as strain C, was significantly higher (P ≤ 0.05) after exposure to acid, bile and osmotic stresses, although this did not translate to higher resistance of B. animalis ssp. lactis (C) to tetracycline. Effects of each stress on biofilm formation in the four bifidobacteria varied with the strain. In general, more positive effects of exposure to stress were observed in both B. animalis ssp. lactis strains, while more negative effects of exposure to stress were shown by B. breve and B. longum. The expression of exopolysaccharide-synthesis gene gtf01207 in B. animalis ssp. lactis (C) was significantly higher after exposure to osmotic stress, although it also appeared to be higher after exposure to acid and bile stresses. Studying the effects of exposure to stress on in vitro probiotic selection properties could give a better reflection of what applies in vivo, since microorganisms for probiotic use would be inevitably exposed to stresses. This could give a more accurate insight on the potential to provide health benefit. The results of this study may justify the commercial use of the B. animalis ssp. lactis strains.
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