| ObjectiveWeaning refers to the transition of mammalian nutritional status from breastfeeding to solid diet.This process is often accompanied by stress reactions such as metabolic disorders,gastrointestinal dysfunction and mucosal barrier function,which makes the organism face slow growth and reduced immune function.There is evidence that the stress response of the body during weaning is closely related to the intestinal flora,and the overall microbial diversity of the intestine is reduced,but the relationship between intestinal flora changes and metabolic disorders caused by the weaning process remains unclear.Probiotics are microorganisms that regulate the body's gut flora at an appropriate dose to promote host health.Studies have found that probiotics have the effect of stimulating immune enhancement,protecting the intestinal barrier and preventing intestinal infections.At present,most of the research is mainly on the influence of probiotic supplementation on the physiological state of weaning organisms.The relationship between the changes of intestinal flora and the metabolic changes of the body after intervention of probiotics in weaned rats is rarely studied.Therefore,this study investigated the effects of combined probiotics(Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07)on weaned rats and intestinal flora and metabolomics.Try to elucidate the mechanism by which probiotics improve the weaning stress state and promote the growth and development of the body from the perspective of metabolomics and intestinal microecology.Methods1.Effect of compound probiotics on growth and immunity of weaned rats.The compound probiotics solution was prepared in a ratio of 1:1 of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 strain,and the weaned rats were intervened in three doses of low,medium and high doses respectively for 28 days.Urine and fecal samples from day 14 and day 28 of each group were collected,and serum and tissue samples of each group were collected after the intervention.The serum IL-6 content was measured,and the duodenal state,colon tight junction state,and the ratio of CD4 to CD 8 in the immune cells were measured.2.The effect of compound probiotics intervention on urine and fecal metabolomes based on NMR.Urine and fecal samples were detected by NMR technique to obtain the nuclear magnetic resonance spectrum.After the segmentation integration of the spectra,the peaks were assigned to the HMDB database.Multivariate statistical analysis was performed using SIMCA-P+ software to find differential metabolites,and metabatic pathway analysis was performed using MetaboAnalyst to analyze the physiological significance of differential metabolites.3.Intestinal microecological changes after intervention with compound probiotics based on 16S rRNA sequencing.The 16S rRNA sequencing technology was used to sequence the V4-V5 region of the intestinal flora in the fecal samples after the intervention of the probiotics.The ?-analysis and ?-diversity analysis were used to explore the variational intestinal bacteria between the control group and the control group.The differential flora between the groups was screened by LEfSe analysis,and the affected metabolic pathways were predicted by PICRUSt function.Finally,the correlation analysis between the differential flora and the host metabolites of urine samples and feces was analyzed.ResultsAfter intervention with compound probiotics in weaned rats,the intestinal morphological structure related to digestion and absorption function was further developed,and the expression level of IL-6 in serum and the ratio of CD4 to CD8 were increased,and the immune function of the body was enhanced.Through metabolomics analysis,urine samples and fecal samples shared a total of 141 metabolites,of which 53 were metabolites in the feces and 88 were metabolites in the urine samples.Compared with the composite probiotics,the differential metabolite analysis showed that there were 26 differential metabolites in the urine sample and 24 differential metabolites in the fecal sample.Among them,the main up-regulation of urine samples were mainly trimethylamine oxide(TMAO),thioglycolic acid,asparagine,creatine,etc.,and the major down-regulations were mainly isovalerylglycine,?-ketoleucine,glutamine,etc.Significant up-regulation in fecal samples is mainly short-chain fatty acids,including acetic acid,propionic acid,butyric acid and heptanoic acid,and the major down-regulations are mainly amino acids,including proline,alanine,proline and tyrosine.These differential metabolites suggest that complex probiotic intervention affects the metabolism of host energy,amino acids,and short-chain fatty acids.The results of 16S rRNA sequencing showed that the diversity of intestinal flora was significantly increased after the intervention of probiotics,and the difference between the groups was larger,and the high-dose group of probiotics was the most obvious.Compared with the control group,the relative abundances of Moraxella,Bifidobacteria,and Microbacilli were significantly increased in the high-dose intervention group.The enterococci,the genus Zymophyllum,and the micrococcaceae were significantly reduced.It can be seen that probiotic intervention can significantly increase the abundance of various beneficial bacteria in the intestine and reduce the abundance of some harmful bacteria such as enterococci.ConclusionThis experiment combines metabolomics and intestinal microecology analysis techniques to study the effects of compound probiotics on weaned rats and their changes in metabolomics and intestinal microecology.It was found that the compound probiotic intervention not only promoted the growth and development of the weaned rats,but also affected the intestinal micro-ecological structure and host metabolism.The aim of this study was to investigate the relationship between the regulation of metabolic function and intestinal flora by compound probiotics,and to explain the intrinsic mechanism of compound probiotics affecting weaned rats. |
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