| With the increasing aging of the global population,the health problems of the elderly were highly concerned.With the degenerates of function and unbalanced of nutrition,the old people need to take in more protein than the recommended to meet the normal function of body.In addition,high protein diet can make obesity,diabetes and other diseases better.However,excessive protein intake will affect the normal metabolic function of liver,kidney and gastrointestinal tract of the elderly.It’s very important to improve the high protein intake and ensure its fully digestion and absorption without affecting normal health.The research group creatively combines the natural and safe agricultural food of Mulberry and Konjac as raw materials,and invents a kind of solid beverage(hereinafter referred to as "compoud powder").The product can effectively promote the digestion of protein in diet,but the effect and mechanism of the product on animal protein are not clear.In order to explore the above scientific issues,15-month-old BALB/C mice were used as the experimental animals,and the AIN-93 M standard diet was used as the normal control.The animal high protein diet(Based on the AIN-93 M standard diet,animal protein was used to replace part of corn starch,and the protein content was more than 50%.Referred to as "high protein" in this paper)was used as the experimental group,at the same time,different doses of compound powder and Xiaoshi tablets were administered to the stomach respectively,to clarify the mechanism of compound powder on the metabolism of high protein diet,and also to provide theoretical guidance and practical application for protein scientific diet of the elderly.The results are listed below:(1)The results of compound powder on protein metabolism fed with high protein diet in old miceFirstly,body weight,food intake,organ index,water content and pH value of feces and cecal contents were measured,then the protein apparent digestibility and serum biochemical indexes related to protein metabolism were measured by biochemical kit.Then the peptide sequence and 25 kinds of amino acids were measured by proteomics and amino acid high-throughput target metabonomics,to study the effects of different dosages of compound powder on protein digested products.The results showed that there was no significant difference in body weight and organ index of each group,the compound powder increased the water content of cecal contents and feces,decreased the pH value.The protein apparent digestibility in the compound powder intervention groups were increased significantly,BHD group improved from 86.70% to 92.92% or more,which was dose-dependent.FHD group improved from 86.70% to 96.78% or more.The serum index showed that the compound powder increased the GPT and GOT activity and amino acid level,decreased the serum urea nitrogen content,especially the total amino acid content of BHD group was 2084nmol/g,which was no difference between NC group(2049 nmol/g).It indicates that the compound powder increased the amino acids metabolism,and the effect of high fish group was more obvious.Although Xiaoshi tablets can promote the digestion of protein,most of them are excreted in the form of urea nitrogen.The results of fecal peptide identification showed that,in beef protein diet 1326 peptides were identified,the molecular weight of peptides below 3000 Da was 93%,mainly distributed in 1000~1500 Da.The number of specific peptides in each group were 48,154,123,78,70 and 62,respectively.In fish protein diet group,194 peptides were identified,93% of them were below 3000 Da,mainly distributed in 1000~1500 Da.The number of specific peptides in each group were 14,24,15,14,15,17,respectively.This conclusion suggest that the compound powder can promote the digestion of protein,and the digestibility of fish protein is better than that of beef protein.Through polypeptide sequence analysis and function prediction,it was found that the high abundance peptides identified were mainly from actin,myosin,and a few myoglobin and histone.Most of the amino acids of the four proteins were conserved at the end of the sequence,most of the peptides were identified and has a high reliability.The polar amino acids such as lysine and threonine were the main amino acids at the C-terminal,and the nonpolar amino acids such as methionine and alanine and a small amount of polar amino acids such as cysteine and aspartic acid at the N-terminal.It was this sequence structure made the four proteins were not easy to be identified in vivo digestion.The above results showed that the compound powder promoted the protein digestion and metabolism of old mice with high protein diet.(2)Effect of compound powder on the stomach and intestine of old mice fed with high protein dietBased on the structure of the internal environment of the animal intestine,the effect of compound powder on the health of the stomach and the intestine were explored by using the whole intestine pathological section observation technology,including observation the stomach and whole intestine(small intestine: duodenum,jejunum,ileum;large intestine: cecum,colon,rectum),measurement the intestinal texture,the intestinal(duodenum,jejunum,ileum)mucosa tissue(villus height,villus width,crypt depth,muscle thickness),the serum immune factors,LBP,LPS,and SCFAs in cecal contents,to provide scientific basis for the function of compound powder in improving gastrointestinal structure and internal environment health.The results showed that high protein diet is harmful to the intestinal structure of old mice,the compound powder significantly improved the firmness,elasticity,breaking force and breaking distance of the small intestine,it can improve the gastrointestinal morphology and structure of old mice fed with high protein diet,and has more significant effect on the high beef protein group.Xiaoshi tablet had no significant effect on improving the intestinal tract of old mice in high protein diet group.The results of serum immune factors showed that the compound powder reduced the content of pro-inflammatory cytokines(TNF-α,IL-1β),increased the content of anti-inflammatory cytokines(IL-10)of the old mice fed with high protein diet,the LBP,LPS contents were reduced.The content of SCFA in intestinal tract increased significantly with the intervention of compound powder,and the effect was more obvious in the high fish protein diet group.(3)The effect of compound powder on intestinal enzyme activity of old mice fed with high protein diet and its molecular mechanismWith the help of pathway analysis and gene and protein expression level,the specific situation and possible metabolic enzymes and metabolic pathways of metabolism and enzyme activity in old mice were studied.Through the prediction of KEGG pathway of protein metabolism in high beef protein and high fish protein found that the intervention of compound powder can promote the pathway of amino acid metabolism.Furthermore,through the measurement of enzyme activity related to protein metabolism,it was found that the compound powder could improve the pepsin activity fed with high protein diet,increase the trypsin and enteropeptidase activity of old mice fed with high fish protein diet,and reduce the trypsin and enteropeptidase activity of high beef protein diet competitively.RT-qPCR and Western blot were used to investigate the expression of key enzymes in protein metabolism at mRNA and protein levels.It was found that the intervention of compound powder increased the activity of APN,PepT1 and GDH at transcription and translation levels in high protein diet old mice.(4)The effect of compound powder on the intestinal flora of old mice fed with high protein dietThe V3-V4 region of the16 S rRNA in old mice intestinal bacterial was sequenced,Alpha diversity,Beta diversity,community structure and the relationship between intestinal flora and amino acid metabonomics was analysised.The results showed that the high protein diet decreased the Firmicute level,increased the Bacteroides level,while compound powder increased the Firmicute level and decreased Bacteroides level,and the ratio of F/B was increased,it has a dose-response relationship.Further analysis of species composition and abundance at phylum level showed that compound powder increase the Lactobacillus significantly.Finally,the above conclusion was further proved by the correlation analysis of 25 amino acids in intestinal flora and metabolites.It was indicated that the compound powder could improve the composition of intestinal flora in the old mice fed with high protein diet,increase the abundance of Lactobacillus and some non dominant bacteria but beneficial to human health,such as Eubacterium coprostanoligenes、Blautia et al.,reduce the abundance of harmful bacterial competitively,protect intestinal barrier and promote intestinal health,so promote the protein digestion. |
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