| Lactobacillus reuteri was approved by the Ministry of Health as a human health product in 2003.It is also a new type of probiotic lactobacillus recognized internationally and has been widely used in the food fermentation industry.However,current research mainly focuses on the probiotic effects of Lactobacillus reuteri on human beings.The role of Lactobacillus reuteri in regulating intestinal function and treating intestinal diseases has not been studied enough.Therefore,in this study,the vacuum freeze-drying process of Lactobacillus Royi GL005 was optimized,and further applied to the enterotoxigenic Escherichia coli induced rabbit diarrhea disease model to explore its therapeutic effect,providing theoretical basis and data support for the clinical application of Lactobacillus royi in veterinary medicine.The research content of this experiment is mainly divided into four aspects:(1)Vacuum freeze-drying of Lactobacillus reuteri GL005,single factor test and orthogonal test design of protective agent,filler,antioxidant and acid-base regulator,and optimization of composite protective agent.(2)Simulated gastrointestinal tolerance test was conducted on Lactobacillus reuteri GL005 freeze-dried bacteria powder to determine the number of viable bacteria.(3)The storage stability of lyophilized Lactobacillus reuteri GL005 powder was studied,and the number of viable bacteria was tested once a month at 25℃,4℃,-20℃ and-80℃.(4)Based on the rabbit Escherichia coli diarrhea model,Lactobacillus reuteri GL005 freeze-dried bacteria powder was applied to study the therapeutic effect of Lactobacillus reuteri GL005 on the rabbit diarrhea caused by enterotoxigenic Escherichia coli.The test results showed that:(1)The freezing resistance factor was obtained by single factor test screening: Trehalose,sorbitol,sodium thiosulfate and glycine were optimized by orthogonal test.The optimal formula of GL005 protectants was 10%trehalose,4% sorbitol,0.6% glycine and 4% sodium thiosulfate,and the survival rate was 79.61%～88.21% after lyophilization.Compared with related reports,this study improved the lyophilized survival rate of bacteria and reduced the cost of protective agent.(2)The 3 h survival rate of L.reuteri GL005 lyophilized bacteria powder in simulated gastric fluid was 87.9%,and the 4 h survival rate in simulated intestinal fluid was 89.43%.Compared with L.reuteri bacterial fluid,the results showed that the lyophilized bacteria powder of L.reuteri GL005 had strong tolerance to gastric and intestinal fluid.Lactobacillus reuteri GL005 strain of lyophilized bacteria powder has strong gastrointestinal tolerance,which is feasible for development.(3)By exploring under different conditions of 25℃,4℃,-20℃ and-80℃,it is concluded that the stability is the best under -80℃,and the four-month survival rate is 79.52%.(4)Lactobacillus reuteri GL005 freeze-dried bacteria powder has certain therapeutic effect on rabbit diarrhea caused by enterotoxigenic Escherichia coli,and can significantly reduce inflammatory cytokines TNF-α,IL-6,IL-8,IL-12 and IL-1β.Proapoptotic factors caspase-3 and caspase-9,as well as serum DAO and D-lactic acid content,repaired intestinal mucosal damage.1 g/kg Lactobacillus Royi GL005 freezedried bacteria powder could replace 0.04 g/kg Norfloxacin in the treatment of diarrhea caused by Escherichia coli in rabbits.Lactobacillus reuteri GL005 has potential excellent probiotic properties.After lyophilization,its tolerance to gastric and intestinal fluid is improved,and it has good health care effect on rabbit diarrhea disease.This study laid a foundation for the development of rabbit probiotics and provided technical and theoretical support for the production and application of lyophilized Lactobacillus reuteri GL005 powder. |
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