Preliminary Study On Imaging Analysis Of Probiotics Based On Fluorescent D-amino Acids Labeling

Objective:According to the needs of D-amino acids for the synthesis of peptidoglycan structure in the process of bacterial growth,this project intends to design and synthesize fluorescent D-amino acids to realize the fluorescent labeling of active bacterial wall,then establish an analysis of oral probiotics in vivo survival rate and division methods,and finally applied them to healthy and ulcerative colitis mice,provide a basis for studies on colonization and survival of gavaged probiotics,and provide guidance for clinical probiotic supplementation.Methods:In this study,fluorescent D-amino acids were synthesized by the coupling of fluorescent groups and D-amino acids.The fluorescent labeling of bacteria was achieved by simply adding them to the bacterial culture medium.After verification by flow cytometry and fluorescence microscopy,it was carried out for intestinal flora labeling;subsequently,a dual labeling method for oral probiotics was established to reveal survival rate and proportion of the flora in mice.Results:1.The live bacteria of Lactobacillus acidophilus,Lactobacillus casei,and Veillonella atypica can be 100%fluorescently labeled in vitro by 3 purified fluorescent D-amino acids while the inactivated bacteria cannot;the labeling process has no obvious effect on the normal growth of bacteria and revealed the way of growth and division.2.The labeling of fluorescent D-amino acid on intestinal flora of mice shows the growth and division of a variety of bacteria in the intestine;the establishment of a double labeling method has successfully investigated survival rates of Lactobacillus acidophilus,Lactobacillus casei and Veillonella atypica were 91.00%,80.03%,and 80.36%after gavage.3.Supplementation of Lactobacillus acidophilus to healthy and ulcerative colitis mice shows that Lactobacillus acidophilus gradually colonizes in healthy mice cause the proportion of the intestinal flora and the survival rate are both increased;Lactobacillus acidophilus supplementation in ulcerative colitis mice successfully alleviated the intestinal dysfunction caused by dextran sodium sulfate and promoted intestinal peristalsis.The proportion of Lactobacillus acidophilus is much lower than the model group and tends to the normal group,and reversed the trend of decreasing survival rate of Lactobacillus acidophilus in ulcerative colitis mice.Conclusion:The bacterial labeling method based on fluorescent D-amino acid labeling can not only reveal the growth and division mode of bacteria in the intestine,but also realize the in vivo tracking of oral probiotics,examine their survival rate under different conditions,and finally provide supplements for clinical probiotics.