In Vitro Fermentation Model Was Used To Study Amino Acid Metabolism In Human Intestine

Normal human feces contain 6 kinds of volatile fatty acids.Among them,acetic acid,propionic acid,butyric acid are the products of carbohydrate fermentation.The other two short-chain branched-chain fatty acids,isobutyric acid and isovaleric acid,are formed by the fermentation of Valine and Leucine by intestinal bacteria,but which amino acid is produced by fermentation of valeric acid in short-chain fatty acids There is no literature report yet.The study found that most of the current work in the world is focused on the study of the effects of carbohydrates on the human intestinal microflora,while the effects of amino acids and proteins on the gut microecology are rarely studied.At the same time,with the development of society,bronchial asthma has gradually become an epidemic of children,characterized by chronic respiratory inflammation.The main clinical manifestations include repeated coughing and wheezing,accompanied by difficulty breathing,and usually have long-term repeated bacterial or viral infections.Often manifested as allergies^[1].And most allergies are related to protein.In recent years,Studies have found that in children with asthma,airway inflammation can lead to intestinal flora disorders,followed by a series of gastrointestinal discomfort symptoms such as abdominal pain,bloating,constipation,and diarrhea^[2].Therefore,This study analyzes the correlation between amino acid metabolism of intestinal flora and childhood asthma patients and healthy children,in order to provide new ideas for prevention and improvement of childhood asthma and prognosis.In this study,An in vitro fermentation model was used to study the amino acid metabolism in the human intestine.First,Through an in vitro fermentation model,an in vitro simulation experiment on the amino acid metabolism of intestinal microbes was carried out,aiming to find the amino acids that metabolize to produce isobutyric acid,isovaleric acid and valeric acid;Then the genome before and after the amino acid metabolism was studied by metagenome sequencing Change,and Then use the Caco-2 cell culture method to determine the effect of different types of short-chain fatty acids on the transmembrane resistance?Trans-epithelium electrical resistant,TEER?of Caco-2 cells.Screening and cultivation of aerobic fecal strains through MacConkey media,using in vitro fermentation models to find bacterial strains that metabolize valeric acid,and to explore the differences in amino acid metabolism in the gut flora of asthmatic children and healthy children aged 3-6 years.The research provided guidance.Gas chromatographic results prove that the production of isovaleric acid in the medium containing Leu and Ile is significantly higher than that in the control group NaCl medium,There is a very significant difference,and There is no significant difference in the other 18 amino acids;Tn the medium containing Val,The level of isobutyric acid content increased significantly,compared with the control group NaCl medium,There was a very significant difference,and The other 19 amino acids did not have this significant difference;Tn the medium containing Pro,the yield of valeric acid was Compared with Na Cl medium in the control group,there was a significant increase,and the difference was significant.The other 19 amino acids did not have this significant difference.Metagenome sequencing results have identified the relevant genera of metabolizing amino acids to produce short-chain fatty acids.We found by species-level Kruskal-Wallis rank sum test that compared with the fecal sample flora,the Comamonadaceae,Escherichia,and Holophaga in the Val group,Shigella and other bacteria are significantly higher than stool samples and have very significant differences;In the Ile group,Escherichia,Holophaga,Comamonas and other bacteria are significantly higher than stool samples and have very significant differences;Leu group Among them,Escherichia,Holophaga and other bacteria are significantly higher than fecal samples,and there are very significant differences;Among the fecal fermentation broth of Pro group medium,Escherichia coli,Enterobacteriaceae,Proteobacteria,Klebsiella pneumoniae,Shigella sonnei and other bacteria are significantly higher than the fecal sample flora,there are very significant differences.Cell culture results of short-chain fatty acids found that valeric acid can significantly increase the resistance of Caco-2 cells compared with the control group,but at the same concentration,valeric acid has a weaker ability to strengthen the intestinal barrier;while isovaleric acid can Significantly reduce the resistance of Caco-2 cells.Screening of bacterial strains producing valeric acid through MacConkey's medium,we found that when 3mg proline was added,the content of valeric acid produced by strain E.coli CAU10142 was significantly higher than other strains at the same concentration,but when 7mg or 12mg proline There is no obvious difference in this strain.Gas chromatographic results of asthmatic children and healthy children found that there was no significant difference between the short-chain branched fatty acids produced by the amino acids metabolized by the intestinal flora between asthmatic children and healthy children.But from the point graph of proline metabolism of asthma children and healthy children to produce valeric acid,we can see that most healthy children have significantly lower valeric acid content than asthma children.The results show that isovaleric acid in the colon contents is formed by intestinal microbial metabolism of leucine and isoleucine degradation;isobutyric acid is formed by the metabolism of valine by enterobacteria;produce.Metagenome sequencing results indicate that the bacteria that metabolize proline to produce valeric acid are most likely to be bacteria such as Escherichia coli and Enterobacteriaceae.Cell culture experiments prove that valeric acid can promote the formation of intestinal barrier,and isovaleric acid inhibits the formation of intestinal barrier.The single-strain fermentation experiment speculated that the strain E.coli CAU10142 was involved in the process of metabolizing proline to produce valeric acid.Experiments on the production of valeric acid by the intestinal bacteria of healthy children and asthma children speculate that valeric acid metabolism may be harmful to the human body.