Optimization Of Probiotic/ginseng Fermentation Conditions And Its Effect On Rats With Antibiotic-related Diarrhea

In recent years,the phenomenon of antibiotic abuse has received widespread attention.Antibiotic associated diarrhea(AAD)is one of the common side effects in the clinic,which can cause great interference to the intestinal flora and immune system.In clinic,probiotics,prebiotics and other micro-ecological preparations are often used for the prevention and treatment of AAD.Probiotics are active microorganisms that colonize the human body and are beneficial to the host.It can regulate the intestinal flora disorder by inhibiting pathogenic bacteria and improving the intestinal microenvironment.Ginseng is a valuable Chinese medicinal material in China,which has the effection of nourishing vitality and improving immunity.Fermentation of ginseng with probiotics can transform substances in ginseng,play a role in regulating intestinal flora and improving immune function.In order to explore the therapeutic effect of fermented ginseng on AAD,this article first used liquid fermentation to establish a probiotic/ginseng fermentation system and optimized the fermentation conditions;and then used AAD model rats as the research carrier to evaluate the effects of fermented ginseng on AAD rats.The improvement of physiological and pathological conditions,and the evaluation of the therapeutic effect of fermented ginseng on AAD from the aspects of intestinal flora and intestinal inflammation regulation,in order to provide the possibility of AAD treatment and lay the foundation for the development of fermented ginseng.The specific research contents and results of this article are as follows:(1)Establishment of probiotic/ginseng fermentation system18 strains of probiotic bacteria were used for liquid fermentation of ginseng,and the number of viable probiotic bacterias was used as an indicator to optimize the bacterial species to establish a suitable probiotic/ginseng fermentation system.A probiotic strain suitable for growing in ginseng was screened.After 16S rRNA identification,the probiotic strain was Lactobacillus fermentium.(2)Optimization of fennentation conditions and determination of ginsenoside content and antioxidant activityThe response surface method was used to optimize the probiotic/ginseng fermentation system.The optimized fermentation conditions were:fermentation time 42h,ginseng addition amount 8.03%,temperature 28.20?,inoculation amount 2%,the model predicts the number of viable bacteria is 4.372×1013 CFU/mL.After verification,the optimization results are reliable.The content of ginsenoside before and after fermentation was analyzed by HPLC,and the change of SOD enzyme activity before and after fermentation was determined.The results showed that the contents of ginsenosides Rg1,Re,Rb1,Rc,and Rd all increased after fernentation,and the SOD enzyme activity increased by 26%compared with that before fermentation(3)Preliminary study on the therapeutic effect of fermented ginseng on AAD ratsA triple-antibiotic gavage was used to establish an ADD rat model,and different doses of fermented ginseng were used to intervene in AAD rats to investigate their effects on rat body weight and fecal water content;morphological changes of colon tissue cells were observed by H&E staining.The qPCR technology was used to determine changes in intestinal flora.ELISA was used to detect changes in inflammatory factors in intestinal tissue.The results showed that fermented ginseng can improve the symptoms of slow weight gain,abnormal activity,diarrhea and other symptoms caused by antibiotics,and restore the abnormal shape of the colon to a certain extent.Antibiotics can cause disturbance of intestinal bacteria in rats.The qPCR analysis showed that after 5 days of natural recovery,total bacteria,Enterococcus faecalis,Lactobacillus murium,Bifidobacterium,and E.coli could not return to normal levels.After treatment with fermented ginseng,the abundance of total bacteria and the above four bacteria returned to normal levels.PCoA analysis found that the intestinal flora of rats in the fermented ginseng group also tended to recover to the normal group.In addition,antibiotic intervention will result in a significant increase in the expression of TLR4 and NF-?B in the colon of rats,and after treatment with fermented ginseng,its expression drops to normal levels.The above results indicate that fermented ginseng has a certain repairing effect on antibiotic-induced diarrhea,tissue structure variation,intestinal flora and immune damage.For different doses of fermented ginseng,the therapeutic effect also differs,among which FG3(0.5g/kg/d)has the best therapeutic effect.(4)The effect of fermented ginseng-probiotics mixture on intestinal flora and intestinal inflammationIllumina MiSeq sequencing and Luminex detection technology were used to analyze the effects of fermented ginseng on the expression of inflammatory factors in the intestinal flora and colon of AAD rats.The effect of the process on the efficacy of ginseng.It was found that the intervention of antibiotics will lead to a decrease in the abundance and diversity of intestinal flora species.Fermented ginseng and ginseng-probiotic mixture can increase species abundance and diversity.Fermented ginseng is superior to the intervention treatment of ginseng-probiotic mixture.From the analysis of species differences at the order level,the intestinal flora of AAD rats after treatment with fermented ginseng can restore the relative abundance of the probiotic Lactobacillales,reduce the relative abundance of the conditionally pathogenic bacteria Erysipelotrichales,and restore the Bacteroidales order,Clostridiales To normal levels.And the therapeutic effect of fermented ginseng is better than the ginseng-probiotic mixture.After the intervention of fermented ginseng,compared with the natural recovery group,the strains whose relative abundance increased significantly were Lactobacillus sp.And Candidatus Stoquefichus sp.;those that fell were Bacteroides sp.,Clostridioides sp.And Fournierella sp.After treatment with the ginseng-probiotic mixture,the only strain whose relative abundance was significantly reduced was Bacteroides sp.Among them,Lactobacillus is a common probiotic in intestinal flora,and Clostridioides is a common pathogenic bacteria in intestinal flora,which can induce intestinal epithelial cell necrosis and cause inflammation.It shows that at the genus level,fermented ginseng can restore the abundance of beneficial bacteria and reduce the abundance of harmful bacteria;the recovery of the ginseng-probiotic mixture is not significant.In addition,the Heapmap and PCoA analysis of the genus level shows that the intestinal bacteria have a tendency to return to normal after fermented ginseng treatment.The ginseng-probiotic mixture treatment group is more discrete and has a certain similarity with the natural recovery group.It shows that the ginseng-probiotic mixture is not as good as fermented ginseng in the overall recovery of intestinal flora.Analysis of the expression of inflammatory factors IL-1?,IL-6,IL-10,and TNF-? in colon tissue found that fermented ginseng can partially restore the abnormal expression of inflammatory factors in colon tissue caused by antibiotics,and the effect is superior to ginseng-probiotics Bacterial mixtureAnalysis of the correlation between the intestinal flora and the expression of inflammatory factors found that certain strains of intestinal bacteria showed a certain correlation with the expression of inflammatory factors,including:Lachnospiraceae sp.,Ruminococcaceae sp.,Prevotellaceae sp.Lactobacillus sp.Genus,Clostridioides sp.,Parasutterella sp.Combined with the change of intestinal flora at the genus level,it is speculated that the recovery of intestinal inflammation by fermented ginseng may be related to the abundance changes of Lactobacillus sp.,Bacteroides sp.,And Clostridioides sp.,which can be reduced by increasing Lactobacillus sp.Bacteroides sp.,Clostridioides sp.Abundance to reduce the expression of intestinal inflammatory factorsBased on the above results,the liquid fermentation was used to establish a probiotic/ginseng fermentation system.The fermentation conditions were optimized with the number of viable probiotic bacteria as indicators,and the changes of ginsenoside content and SOD enzyme activity before and after fermentation were measured.In order to study the carrier,the therapeutic effect of fermented ginseng on antibiotic-associated diarrhea was evaluated from two aspects of intestinal flora structure and intestinal inflammation regulation,and on the basis of this,the relationship between intestinal flora structure and intestinal inflammation was further explored.Through the research in this paper,I hope to provide research materials for the treatment of antibiotic-related diarrhea with fermented ginseng,and provide a basis for enriching the application:fields of ginseng.