Regulation Of Short-term Intake Of Different Dietary Proteins On Gut Microbiota And Gut Barrier

Protein is a major part of human nutritional diets.Meat proteins are of high biological value,which are rich in essential amino acids,while there are limited amino acids in plant proteins,for example,the grains are deficient in lysine and the beans are dificinet in methionine.In recent years,studies have shown that dietary nutrients can regulate the composition and metabolism of gut microbiota.The human microbiome,known as the "second genome" of human beings,are also an "invisible" organ in the host,which has an important impact on the body health.Due to the differences in amino acid composition,structure and the digestibility,proteins from varied sources could provide different substrates for microorganisms in gut and finally result in differential microbial community and metabolic characteristics,which might further act as a key factor on the effect of body health.It has been reported that long-term intervention of different dietary proteins could differentially shape the microbial composition.However,the changes of microbial composition during a short-term intervention and its impact on intestinal health are less investigated and remain unclear.Therefore,it is necessary to explore the short-term effect of dietary protein on gut microbiota and the corresponding effect on gut and body health.The content of this study includes four parts in order to answer the above questions:1.Short-term effects of different dietary proteins on the development of gut microbiota and metabolites in ratsPCR-DGGE analysis was performed to compare changes in gut microbial composition in rats cecum after they were fed with diets composed of proteins from different sources including soy,pork,beef,chicken,fish and casein(control)for 2,7 and 14 days.Dietary proteins rapidly altered the microbial composition in rat cecum.On day 7,the new microbial composition was developed,and tended to become stable during the rest time of intervention.Clear differences were observed between soy and animal-derived protein groups.Blautia wexlerae appeared more frequently in soy protein group,while A.muciniphila was much less frequently observed when compared with other groups.In addition,the concentration of butyric acid,isovaleric acid and NH3-N in the cecum of rats fed soy protein were significantly higher than those animal derived protein groups.2.Short-term effects of different dietary protein sources on functions of gut microbial in ratsBased on the result of first chapter,samples from the rat fed different dietary proteins for 7 days were selected for non-amplified metagenomics sequencing analysis to get a deeper understanding of microbial composition and functions.The results indicated that dietary proteins differentially affected the composition of both bacteria and fungi,however,the response of bacteria to dietary proteins was more sensitive than fungi.Casein induced a significant higher abundance of Lactococcus,and enhanced galactose metabolism and branched chain amino acid metabolism than other groups.This could be due to the galactose glycosylation in kappa casein and the higher concentration of branched amino acids.Compared with animal derived proteins,soy protein induced a significant higher abundance of Fusobacterium,this was then found to be positive correlated with the concentration of butyric acid,indicating that the higher amount of butyric acid in soy group could be produced by metabolism of these Fusobacterium species.In addition,soy protein group showed significant lower abundance of A.muciniphila than all the other groups,which is consistant with the DGGE results from chapter one.A.nmciniphila was most abundant in the chicken protein group.Meanwhile the abundance of genes involved in citrate cycle and oxidative phosphorylation metabolism of microorganisms in the chicken protein group was significantly higher than other groups,suggesting a relatively enhanced oxygen-resistant metabolism.3.Regulation of the metabolism of A.muciniphila by dietary chicken and soy proteinBased on the previous results,dietary chicken protein resulted in the highest abundance of A.muciniphila,while soy protein resulted in an extremely low abundance.Since A.muciniphila has been reported to be a potential probiotics that could well utilize gut mucus to grow in gut and is closely related to the host metabolism and immune response,therefore it is of great importance to clarify the effects of dietary proteins on the regulation of growth and metabolism of this bacterium.Studies have shown that the metabolic characteristics of A.muciniphila can vary due to different nutritional environment.Therefore,germ-free mice fed with either chicken or soy protein were colonized by A.muciniphila.With a combination of metatranscriptome anaylsis,the metabolism profile of A.muciniphila was explored.The results showed that dietary soy protein significantly reduced the relative abundance of A.muciniphila in the intestinal tract,accompanied by a significantly enhanced two-component system metabolic process on mRNA level.Dietary chicken protein kept the abundance at a relatively high and stable level,accompanied by significantly enhanced oxidative phosphorylation,represented by the highly expressed NADH dehydrogenase genes such as Amic₁614/K00333(nadh-quinone oxidoreductase subunit D)and Amuc₁612/K00335(nadh-quinone oxidoreductase subunit F),which require the participation of oxygen.This is consistent with the fact that A.muciniphila may benefit from the presence of nanomolar concentrations of oxygen.4.The effect of dietary chicken or soy protein together with A.muciniphila on gut barrier and immune cells in spleenThe profile of gut mucus barrier and immune cells were analyzed to explore the influence of dietary proteins together with the regulated A.muciniphila on host health.The results showed that the number of colon goblet cells was significantly regulated.Compared with soy protein,chicken protein induced a significant higher number of colonic goblet cells both in SPF and germ-free mice,indicating that this regulation is independent on the presence of gut microbes.The Muc2 gene expression level and thickness of mucus layer were higher in the colon of SPF mice fed chicken protein diet than that fed soy protein diet,while no difference was observed in germ free mice.When colonizing with A.muciniphila,the expression of Muc2 gene in colon of germ free mice fed chicken protein significantly increased than that was not colonized,but no difference in the thickness of mucus layer was observed,which may be due to the relatively short feeding period.None of the two parameters changed in the germ free mice fed soy protein before and after colonization.Taken together,chicken protein combined with A.muciniphila could promote the secretion of mucus in colon and help maintain homeostasis of gut barrier.Measurement of immue cells showed that the main difference was found between SPF and germ free mice.This was expected since long-term lack of antigen stimulation could lead to an almost dormant immune system in germ free mice.After colonization of A.muciniphila,the proportion of immune cells in germ free mice fed both dietary proteins had a trend to reach the levels of SPF mice.However,when compared to the soy protein diet,a chicken protein diet together with the colonization of A.muciniphila was more beneficial to restore the proportion of immune cells to the same level as SPF mice.Taken together,our results indicate that dietary chicken and soy proteins differentially regulate the growth and metabolism of A.muciniphila,which would futher potentially affect the gut mucus barrier and body health.Chicken protein performs better than soy protein on these effects.In conculsion,this study elucidated that the gut microbiota and their metabolites could respond rapidly to the regulation of different dietary proteins,among which,soy protein varied the most from animal derived proteins.The abundance of A.muciniphila was significantly lower than all the other protein groups,while it was the highest in chicken protein group.With the combination of colonizing A.muciniphila in germ free mice fed either chicken or soy protein diet,chicken protein increased the number of goblet cells,and could promote mucus secretion together with the colonized A.muciniphila.This regulation could further play a critical role to enhance the growth and metabolism of A.muciniphila in mucus layer.In addition,a chicken protein diet together with the colonization of A.muciniphila tended to restore the proportion of immune cells more close to the same level as SPF mice than the soy protein diet.Therefore,this study indicated that a chicken protein diet is more beneficial for colonization and metabolism of A.muciniphila,which could further play a positive role in the regulation of host immunity when compared with soy protein diet.