Study On The Evaluation Of Bacillus Probiotics And Its Specific Germination Response

Bacillus probiotics are ingested in the form of spores.The germination and proliferation in vivo are the key factors exerting efficient effect on hosts.There was no systematic study on the germination characteristic of spores associated with the probiotic properties in vivo.In this dessertation,6 strains of Bacillus species from 6commercial probiotics were systematically studied.On the basis of comparative analysis of its probiotic properties and safety in vitro,the germination characteristics of different Bacillus species,the corresponding molecular mechanisms and the intestinal protective effects in vivo were mainly discussed,so as to provide a research basis for the evaluation of Bacillus’s activity and germination breeding.The research contents and related results are as follows:Six strains of Bacillus probiotics were isolated from 6 medicinal probiotics.The probiotic characteristics in vitro were compared including enzyme producing capacity,antibacterial and antioxidant activity,surface hydrophobicity and acid resistant stability of the vegetative cells.The results showed that all of the six strains of Bacillus had certain capacity to produce amylase,cellulase and protease,and all with certain surface hydrophobicity,and the vegetative cells of the strains were fully resistant to acid,except that two strains of Bacillus licheniformis L-1 and Bacillus licheniformis L-2 with the fermentation supernatants showed certain antibacterial activity.However,based on the comprehensive evaluation of the beneficial effects of the six strains in vitro,the probiotic differences were not significant.The safety of the Bacillus strains was future studied systematically.Their safety in vitro was evaluated including antibiotic resistance analyses,antibiotic resistance plasmid detection and virulence genes detection.It was found that all of the six strains had no risk of dissemination of bacterial resistance.However,one or more enterotoxin genes were specifically detected in each strain.Therefore,the safety risk of the use of probiotics Bacillus should be paid more attention in isolates’screening.The probiotic activity of Bacillus related with the germination characteristics was emphasized.Firstly,the spores of the six Bacillus strains were evaluated for their ability to respond to nutrient germination agents.The spores of six strains were specific to the germination response with different nutrient germination agents,but L-alanine could promote the germination of all the six strains.Among them,the response of Bacillus subtilis S-2 to L-Ala was the most sensitive.Meanwhile,the addition of L-alanine significantly promoted the growth of B.subtilis S-2 in a restricted medium in vitro.Based on a laboratory preserved strain Bacillus subtilis312 almost without response to L-Ala as a control,the differentially expressed genes of the two strains responding to L-Ala germination were studied base on the comparative transcriptome analyses,eight genes was significantly up-regulated compared with B.subtilis 312,including ger D,ger KC,ger KB,ger AA,ger AB,ger AC,lgt and spo0A.The effect of B.subtilis S-2 induced by L-Ala on the elimination of intestinal pathogens was studied based on the SD(Sprague-Dawley)rat challenge model based on the labelled strain of Escherichia coli K99-m Cherry.The experiment was divided into four treatments with 6 replicates including the positive treatment(challenge group),negative control(not challenge group),B.subtilis S-2 spore treatment and B.subtilis S-2 spore plus L-Ala treatment.The feeding time lasted for 12 days.After two days of adaptation,the rats were ingested 10~7CFU B.subtilis S-2 spores by suspending in drinking water.On the seven day,the rats were challenged intragastrically by 1 m L of 10~9 CFU/m L enterotoxic E.coli K99-m Cherry in all treatments except the negative control.Six rats in each experimental group were randomly selected and killed for the collection of the intestine samples at 12 h after challenge.The fecal samples were collected at 12 h,24 h,48 h and 72 h after challenge.The growth performance,the amount of E.coli K99-m Cherry shedded in feces,and the number of E.coli K99-m Cherry in each intestinal sections were measured.The results showed that the daily weight gain and feed intake in the B.subtilis S-2 spore plus L-Ala treatment were significantly higher than those of the positive treatment,and were of no significant difference from those of the negative control(not challenge group).At 24 h,the number of E.coli K99-m Cherry colonies in the B.subtilis S-2 spore plus L-Ala treatment was lower than that in the positive control group and the B.subtilis S-2 treatment.The B.subtilis S-2 spore plus L-Ala treatment had the lowest bacterial concentration in each intestinal segment.The results showed that the B.subtilis S-2 spore induced by L-Ala could help protect rats from pathogen clonoinzation in the gastrointestinal tract.In conclusion,based on a systematic evaluation of the probiotic characteristics and safety of six commercial probiotic Bacillus strains,this experiment explored the germination characteristics of spores in vitro and in vivo.It is found that the nutritional germination agent L-Ala can significantly promote the probiotic efficiency of sporeformers in vivo.The study suggests that the Bacillus probiotics can be screened based on their germination potential.