| Xinjiang natural fermented yogurt is one of the traditional specialties of ethnic minorities in Xinjiang.In the long-term natural fermentation process,affected by factors such as climate environment and geographical location,the unique microbial community in yogurt is domesticated and cultivated into a large number of usable excellent biological resources.However,with the development of industrial science and technology,traditional techniques have been continuously replaced by new technologies,and the valuable lactic acid bacteria resources in naturally fermented yogurt have gradually disappeared.Therefore,this research took the self-made natural fermented yogurt in the homes of ethnic minority herders in Kashgar and Korla regions of Xinjiang as the research object.The lactic acid bacteria in the yogurt were separated,purified and identified,and the collected lactic acid bacteria were tested for in vitro resistance,and a strain with the best comprehensive resistance was screened for constipation and obesity animal model experiments to evaluate its preventive effect on constipation and obesity,which would provide experimental basis for the development and utilization of excellent lactic acid bacteria resources in natural fermented yogurt in Xinjiang.The main research methods and results are as follows:(1)Isolation and identification of lactic acid bacteria in naturally fermented yogurt and evaluation of resistance in vitro.The lactic acid bacteria were isolated and purified based on traditional separation and purification technology,and the suspected lactic acid bacteria were initially screened out based on the morphological characteristics of the colony and the results of Gram staining.Then 16 S r DNA sequence homology was used to analyze the species relationship of lactic acid bacteria.Finally,the p H 3.0 artificial gastric juice,0.3% bile salt and surface hydrophobicity of the lactic acid bacteria were determined,and the best in vitro resistance strain was selected for follow-up research.The results showed that 15 strains of lactic acid bacteria for patent preservation were isolated from 10 yogurt samples,all of which were Lactobacillus,including 11 Lactobacillus plantarum,1 Lactobacillus casei,1 Lactobacillus pentosus and 2 Lactobacillus delbrueckii subsp.bulgaricus.The results of the in vitro resistance test showed that13 strains of lactic acid bacteria grew in artificial gastric juice at p H 3.0,and the survival rate of 9 strains was >50%,accounting for 60% of all strains resistant to artificial gastric juice testing.The growth rate of all strains in the medium containing0.3% bile salt,and the growth efficiency of 6 strains in 0.3% bile salt is >10%.Among them,Lactobacillus plantarum KFY02 has a strong tolerance in 0.3% bile salt,the growth efficiency is 24.67%.The surface hydrophobicity of 15 strains of lactic acid bacteria was tested,and the hydrophobicity of 6 strains reached more than10%,accounting for 40% of the tested strains.Among them,3 strains belong to medium hydrophobicity,and their hydrophobicity rates are 28.17%,28.01%,and26.81%.The comprehensive resistance results found that the strain numbered KFY02 had the best resistance.Its survival rate in p H3.0 artificial gastric juice was 93.46%,its growth efficiency in 0.3% bile salts was 24.67%,and its surface hydrophobicity was 28.17%.It was selected as the subsequent experimental strain,the patented strain was deposited as Lactobacillus plantarum KFY02,and the deposit number was CGMCC 15638.(2)Regulating effect of Lactobacillus plantarum KFY02 on constipationMontmorillonite powder was used to induce a mouse constipation model.The improvement effect of Lactobacillus plantarum KFY02 on the symptoms of constipation in mice was evaluated by through the weight change of the constipated mice,the fecal water content,the propulsion rate of activated carbon in the small intestine,the time of first black stool discharge,the observation of small intestinal pathology sections,serum biochemical indicators,the level of gastrointestinal hormones in the serum,the expression level of small intestine-related mRNA,and the abundance of the microbial community in the feces of mice.The results show that compared with the model group,Lactobacillus plantarum KFY02 can significantly increase the fecal water content of constipated mice,shorten the discharge time of the first black stool in constipated mice,increase the intestinal advancement rate of constipated mice,and relieve the damage of small intestine villi caused by montmorillonite powder modeling.By significantly increasing the activity of T-SOD,CAT and GSH-Px enzymes in mouse serum,reducing MDA content,thereby reducing the degree of oxidative damage in mice with constipation.It can significantly increase the levels of VIP,MTL,Gastrin,Ach E and SP cytokines in mouse serum,reduce the levels of SS and ET-1,stimulate intestinal peristalsis by regulating gastrointestinal hormones,shorten the transit time of food in the small intestine,and promote defecation,improve the symptoms of constipation.In addition,Lactobacillus plantarum KFY02 can significantly up-regulate the mRNA expression of c-Kit,SCF,GDNF,e NOS and n NOS in the small intestine,and reduce the mRNA expression of TRPV-1 and i NOS.At the same time,Lactobacillus plantarum KFY02 can increase the abundance of beneficial bacteria Bacteroides,reduce the abundance of harmful bacteria Firmicutes,and increase the abundance and diversity of the intestinal microbial community of constipated mice.The abundance of Akkermansia improves the intestinal environment,thereby inhibiting the occurrence and development of constipation and alleviating constipation.(4)Lactobacillus plantarum KFY02 regulates lipid metabolism in obese mice induced by high-fat diet.High-fat diet was used to induce obesity model in mice,mouse body weight,organ index,histopathological section,liver function related indicators,blood lipid level,serum inflammatory factor level,liver and adipose tissue mRNA and protein expression levels were used to evaluate the regulation effect of lactic acid bacteria on lipid in obese mice.The results showed that the body weight and organ index of the model group were significantly higher than those of the normal group.The liver tissue sections showed that there were a large number of fatty vesicles in hepatocytes,and the fatty vesicles of epididymal fat were significantly larger than those of the normal group,indicating that the obesity model was successfully modeled,Lactobacillus plantarum KFY02 can significantly reduce the activity of liver function-related enzymes in serum and liver tissues,reduce lipid content,and slow down liver cell damage and fat accumulation caused by high-fat diet.Reduce the level of pro-inflammatory cytokines in the serum,increase the concentration of anti-inflammatory cytokines,and relieve the inhibition of chronic tissue inflammation,Lactobacillus plantarum KFY02 treatment can increase the mRNA and protein expression levels of obesity-related genes LPL,PPAR-α,CYP7A1 and CPT1 A in mouse liver and epididymal adipose tissue,and promote lipid metabolism,reduce FAS,PPAR-γ and C/EBP-α mRNA and protein expression levels,alleviate fat accumulation.In summary,Xinjiang natural fermented yogurt has rich lactic acid bacteria resources.Through in vitro resistance screening and in vivo animal model evaluation,it is found that Lactobacillus plantarum KFY02 is effective in constipation mice induced by montmorillonite powder and obese model mice induced by high-fat diet.It has a significant preventive effect and can be further developed and utilized as a probiotic with the functions of relieving constipation and reducing fat. |
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