Study On The Screening Of Clostridium Butyricum And Its Efficacy In Lowering Lipids And Improving Intestinal Flora

Obesity has developed into a disease that threatens human health worldwide,and probiotics have a good preventive/treatment effect on obesity.The therapeutic effects of Clostridium butyricum,an important component of probiotics,on gastrointestinal diseases such as acute and chronic diarrhea and irritable bowel syndrome have been well established,but there is a lack of related research on the prevention/treatment of obesity and its effect on intestinal flora.The aim of this project is to develop Clostridium butyricum with independent intellectual property rights that has the effect of weight loss and lipid reduction and improving intestinal flora.Firstly,the bacteria were isolated and purified from the fecal samples of 33 healthy volunteers,and finally screened one strain of Clostridium butyricum with high butyric acid production named Clostridium butyricum B-3 from the feces of 96-year-old healthy and long-lived elderly people through butyric acid production characteristics,morphological observation and 16S r DNA sequencing.Then,the biological characteristics of C.butyricum B-3 were evaluated using the commercial Clostridium butyricum MIY588 as a positive control.The results showed that its butyric acid production was high,with a maximum amount of about1.2 g/L;the survival rate of simulated gastrointestinal tract was greater than 80%;and it was susceptible to penicillin,ampicillin,norfloxacin,ciprofloxacin,gentamicin and amikacin.It is resistant to erythromycin and cefazolin;it has the ability to produce amylase,cellulase and protease,and the above biological characteristics were better than C.butyricum MIY588.The preliminary investigation on the in vitro antioxidant and lipid-lowering ability of C.butyricum B-3 revealed that its hydroxyl radical scavenging rate,DPPH scavenging rate and reducing power were better than those of the positive control strain;the bile acid salt degradation,pancreatic lipase inhibition and cholesterol scavenging ability of C.butyricum B-3 were comparable to those of C.butyricum MIY588.The above results indicated that C.butyricum B-3 has better potential as a probiotic.The fermentation process of C.butyricum B-3 was optimized,and the types and addition amounts of carbon sources,nitrogen sources and deaerators that had significant effects on the growth of C.butyricum B-3 were screened using a single-factor test,and further response surface optimization tests were conducted on this basis.The optimal medium formulation was obtained as follows:10.0 g/L glucose,16.0 g/L soybean peptone,0.5 g/L L-cysteine hydrochloride,5.0 g/L Na Cl,3.0 g/L sodium acetate trihydrate.Compared with the pre-optimized culture conditions,the biomass of the bacteria was increased by about 18%after optimization.Obesity in mice was induced by high fat diet,which was intervened by gavage intervention of C.butyricum B-3 active bacteria.The physiological indices of body weight,liver histopathology,blood lipid metabolism and intestinal flora were measured to investigate the hypolipidemic and intestinal flora effects of C.butyricum on high-fat diet mice.The results showed that the three different doses of C.butyricum B-3 interventions significantly(P<0.05)slowed down the body weight gain and improved the kidney and liver fat vacuolation lesions in mice fed high-fat diets.The medium dose(5×10~7 CFU/m L)of C.butyricum B-3significantly(P<0.05)decreased serum total cholesterol,total triglyceride,LDL-cholesterol,IL-6,IL-10 and TNF-αcontent,and significantly(P<0.05)increased HDL-cholesterol content in mice,and there was no significant difference between low and high doses in the above indexes.The high dose(5×10~8 CFU/m L)of C.butyricum B-3 significantly(P<0.05)increased the contents of acetic acid,propionic acid and butyric acid short-chain fatty acids in the feces of mice.In addition,C.butyricum B-3 increased the diversity and abundance of intestinal flora in mice fed a high-fat diet.At the phylum level species abundance,C.butyricum B-3 intervention increased the abundance of the Firmicutes and decreased the abundance of the Bacteroidota;Meanwhile,the abundance and proportion of beneficial bacteria,such as the Desulfobacterota,were adjusted upwards and the abundance and proportion of the Verrucomicrobiota were adjusted downwards.The abundance and proportion of beneficial bacteria such as Erysipelotrichaceae,Muribaculum and Lachnospiraceae were up-regulated at the family level species abundance.At the genus level,the abundance and proportion of beneficial bacteria such as Dubosiella were up-regulated,and the abundance of bacteria such as Akkermansiaceae was down-regulated.In summary,C.butyricum B-3 showed effects of weight loss and lipid-lowering and improvement of intestinal flora in high-fat diet mice,which provides theoretical basis and technical support for the development of functional foods related to the treatment/prevention of obesity.