Biological Characteristics And Safety Assessment Of Probiotic Enterococcal Strains

Probiotic bacteria are defined as living micro-organisms‘and could exerthealth benefits beyond inherent basic nutrition by ingestion in certain numbers.Enterococci, belonging to the group of microorganisms known as LAB (lactic acidbacteria), are Gram-positive, non-spore-forming, catalase-negative, oxidase-negative,facultative anaerobic cocci that occur singly, in pairs or in chains. As LAB,enterococci are known to normally reside in human and animal GI (gastrointestinaltract) and can be isolated from foods of milk, meat, cheese and fermented vegetables.Currently, probiotic pharmaceuticals containing enterococcal species are widely usedin the treatment of diarrhea, dysbacteriosis and intestinal inflammation, and certainproducts have been performed to demonstrate their good therapeutic effects.Although enterococci have been successfully applied in food and probiotic products,in contrast to other LAB, these microorganisms are not generally recognized as safe.In view of the value and potential risk of the enterococcus application, it is necessaryto evaluate their safety. In this study, the probiotic enterococcal strains from probioticproducts were examined to provide more information of the properties and safetyaspects for their application.1. Five isolates from thirty probiotic preparations were identified as enterococci by16S rDNA sequencing and biochemical tests. Strains LS15, PF03, BF10and SL13were Enterococcus faecalis, and strain MM08was Enterococcus faecium.2. Biological characteristics were analyzed of the isolates, which include acidtolerance, pepsin and trypsin tolerance, bile salts tolerance and organic acidsproduction. The five isolates could maintain viability (>104cfu ml-1) at pH3.0for2h. All the bacteria could survive (>104cfu ml-1) at high bile salt concentration (at1.0、2.0、3.0and4.0g l-1) for a long time (24h). The viable numbers could maintainat a high level (at least106cfu ml-1) during exposure to pepsin or trypsin (at2.0,4.0,6.0and8.0g l-1) for all strains. Compared with the Ent. faecalis isolates, the Ent.faecium MM08showed a better ability to survive in the adverse condition. The fivestrains mainly produce lactic acid, formic acid, acetic acid and citric acid (fermentedtime at4h,12h,24h and48h). The formic acid exhibited minimum content (<0.42 mg ml-1), and the lactic acid (10~11mg ml-1) constituted a large proportion of theorganic acids with a significant difference (P <0.05).3. The virulence genes detection show that Ent. faecalis strains LS15, PF03, BF10and SL13expressed a highly similar set of virulence factors (with the ace, gelE andefaA genes found). For Ent. faecium strain MM08, no virulence genes were detected.There were no hemolytic reactions found for all the five strains. The enterococcusmainly were resistant or intermediate resistant to erythromycin, ciprofloxacin andgentamicin.4. The expression levels of the enterococcal virulence genes are usually influencedby specific environmental factors. For safety considering, real-time PCR was appliedto detect the enterococcal virulence genes expression in simulated intestinal medium.The efaA of LS15and ace of BF10were both up-regulated (6-8folds), and the othersmaintained at the normal expression level; For Ent. faecalis SL13, the gene ace, gleEand efaA expression profiles at different NaCL concentration, pH gradient and thepresence of antibiotics were also analysed. The expression of the three virulencegenes increased to25-40folds in environment of high concentration of NaCL. Whenthe pH reached9.0, expression levels of the three genes were signifcant up-regulated(for ace,72-fold increasing; gelE,10-fold increasing and efaA,4-fold increasing).The expression levels of the three genes were different for the stress treatment ofgentamycin sulfate (for ace,2-fold increasing; gelE, close to blank and efaA,0.3-folddecreasing)5. The effects of ingestion of probiotic strain E. faecalis SL13on body weight inmice were investigated, and the expression levels of genes ace, gelE and efaA ofSL13in intestinal tract of mice were also studied compared with that in vitro. At20thday, average weight gains were8.55and4.90g for mice respectively fed E. faecalisSL13preparation and normal saline. Expression levels of genes ace and gelE werenot significant difference, but that of efaA could increase by5.88times. The aboveresults showed E. faecalis SL13could improve mice growth and its virulence gene efaA was affected by the factors in the intestine.