| Lactobacillus is playing an essential role in promoting animal growth,regulating the immune system and balancing the intestinal microecology,and is currently the most widely used probiotics in animal healthcare and disease prevention.However,the development and application of microecological preparations for dogs are relatively few,and most of them are exogenous lactic acid bacteria,and the efficacy of probiotics is species-specific and strainspecific due to the different intestinal microbiota of different animals.Therefore,this study intends to isolate canine-derived lactic acid bacteria from canine feces,screen out excellent lactic acid bacteria strains with good resistance,adhesion,bacterial inhibition,safety and other potential probiotic properties through in vitro tests,and optimize their high-density culture process and lyophilized preparation process to enrich canine-derived strains with probiotic potential,so as to develop new and safe probiotic preparations for canine use and apply them to nutrition or therapy in dogs.In this study,canine feces was used as the object of study.The strains isolated and purified from the samples were identified by morphological observation,16 S r RNA gene sequence determination and construction of phylogenetic tree,and the lactic acid bacteria with excellent resistance were screened according to acid and bile salt tolerance tests and the inhibition activity of supernatant was studied by Oxford cup method.Finally,hemolysis test,drug sensitivity test and mouse gavage test were performed to ensure the safety of the strain for clinical use;the optimal inoculum,p H,carbon and nitrogen sources of Lactobacillus rhamnosus LM1 were screened by single-factor method,and the significant factors and response values were determined by PB test and steepest climb test.The optimization of lyophilization conditions was carried out by screening the optimal centrifugation conditions and the ratio of protective agent,and the optimal lyophilization protective agent and its optimal concentration were screened out by single-factor test,and finally the compound formulation of lyophilization protective agent was optimized by response surface optimization test.The experimental results were as follows:(1)Twelve strains of Lactobacillus were isolated from the feces of five healthy police dogs,of which eight strains were identified as Lactobacillus salivarius,two strains of Lactobacillus animalis,one strain of Lactobacillus rhamnosus,and one strain of Streptococcus paris.Nine isolates showed good acid tolerance and three strains survived 73.72% after 4 h at0.3% bile salt,showed good inhibition of common pathogenic bacteria,and showed high adherence.In vitro biosafety assessment showed that all 3 strains of Lactobacillus were nonhemolytic and sensitive to most antibiotics.In vivo safety tests on Lactobacillus murinus LM1,which had the relatively best overall probiotic profile,showed no adverse effects or mortality in all mice and normal physiological characteristics.The hematological indices were in the normal range and no significant abnormalities were found in the HE staining of the tissue sections of the mice.Therefore,Lactobacillus rhamnosus LM1 can be safely used in production practice.(2)The optimum inoculum of Lactobacillus rhamnosus was 2%,and the optimum p H was7.0-8.0.The medium composition was 30.66 g/L maltodextrin,58.12 g/L yeast extract,2.0 g/L dipotassium hydrogen phosphate,2.0 g/L diammonium hydrogen citrate,5.0 g/L sodium acetate,0.2 g/L magnesium sulfate,0.04 g/L manganese sulfate and 0.99 m L/L Tween 80.The maximum viable bacteria count of 1.15×109 CFU/m L could be achieved when m L/L.The viable bacteria count of the optimized medium was 153.3% higher than that before optimization,and the optimization of Lactobacillus rhamnosus LM1 medium was successfully achieved.(3)The collection rate of Lactobacillus rhamnosus LM1 was relatively high and cost effective under the condition of 8000 r/min for 5 min,and the optimal protectant ratio was 1:8and the optimal protectant compound formula was 16.52% skim milk powder,7.88% sucrose and 14.79% lactose.In this study,a strain of Lactobacillus rhamnosus with excellent and safe probiotic properties was successfully screened from canine feces,and the optimization of the preparation process of lyophilized formulation of Lactobacillus rhamnosus was achieved through optimization tests,in order to provide a basis and reference for the subsequent industrial production. |
