| A large number of bacteria inhabit the intestinal tract of a healthy human body.The dominant flora and the secondary flora maintain a dynamic balance to maintain the normal nutrient absorption,material transformation and physiological and biochemical functions of the intestinal tract.The intestinal flora,as a system,collaborates with other systems of the body to maintain the health of the body.A large number of studies have shown that Clostridium butyricum is one of the important probiotics in the human intestinal tract,which can inhibit the proliferation and growth of harmful and corrupt bacteria in the intestinal tract,and play an important role in the prevention and treatment of intestinal diseases and the regulation of intestinal microecological balance.The purpose of this study was to isolate Clostridium butyricum from intestinal tract of healthy people,optimize its culture process,and finally investigate the effects of Clostridium butyricum and Akkermansia muciniphila on diabetic mice respectively.First of all,in the feces provided by healthy people,through the PCR identification of specific primers and the specificity of Clostridium butyricum.Clostridium butyricum proliferation medium was used to screen Clostridium butyricum through the strict anaerobic characteristics,carbon source utilization characteristics and morphological characteristics of Clostridium butyricum.Finally,10 strains(named GYJK-2021D1-D10)with more than 99%similarity to Clostridium butyricum were identified by 16SrRNA.Firstly,the yield of butyric acid and total short chain fatty acid(SCFAs)in the fermentation broth was used as indexes to investigate the culture effect of the above 10 strains.Finally,one strain GYJK-2021D4 was found to have excellent acid-producing characteristics.Taking Clostridium butyricum(named strain YX)from the drug "Taikangning" as the control,the culture process of strain GYJK-2021D4 was optimized.Based on Clostridium proliferation medium(RCM),the single factor investigation method was used to determine the best type and concentration of carbon source and nitrogen source,respectively:Glucose 0.3%,maltodextrin 0.3%,yeast extract 1.2%,soybean peptone 1.2%.The results showed that the optimal culture temperature of Clostridium butyricum GYJK-2021D4 was 37℃,the optimal initial pH of bacterial growth was 6.5,and the optimal inoculation amount was 10%.Subsequently,the fermentation process of Clostridium butyricum GYJK-2021D4 and Akkermansia muciniphila GYJK-2021A1(AKK for short)were investigated using optimized Clostridium butyricum medium and AKK optimized synthesis medium,respectively.Secondly,a type I diabetic mouse model was established,and fresh Clostridium butyricum and AKK bacteria were ingavaged to investigate their health improvement effects on diabetic mice.The factors included fasting blood glucose,lipid metabolism and intestinal microflora structure.In this study,ICR mice were used as model animals to replicate type Ⅰ diabetes.Streptozotocin was intraperitoneally injected to mice at a modeling dose of 0.04mg/10g for 5 consecutive days.The mice were divided into three groups(model group/AKK group/Clostridium butyricum group)with 10 mice in each group.AKK group was given strain A1 solution(OD6001.0-1.3),Clostridium butyricum group was given strain D4 solution(OD600 1.3-1.6),0.4mL per animal,twice a day,continuous gavage for 14 days.After intragastric administration,fasting blood glucose and lipid indexes of each group were detected.The intestinal flora of mice were analyzed by Illumina high-throughput sequencing technology.The results showed that the probiotics AKK and Clostridium butyricum could improve the fasting blood glucose and blood lipids of type Ⅰ diabetic mice and form a new intestinal microflora structure.This study laid a foundation for the development of living bacteria preparation for the treatment of Diabetes Mellitus. |
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