Effects Of Two Different Casein Hydrolysates On The Fermentation Characteristics And Microbial Community In The Small Intestine Of Pigs

There are large number of microbes living in gastrointestinal tract of monogastric animals including pigs.The gut microbes formed a complex microecological system that regulates the metabolism of protein in the entire intestine of pigs,which not only affect animal health and production performance,but also affect the environment.It was once believed that the small intestine was only an organ for nutrients digesting and absorbing,but in recent years with the well-understood of the gut microbiota,researchers now believe that small intestinal bacteria play important roles in protein and amino acids metabolism.Therefore,understanding the metabolism mechanism of protein in the small intestine and the function of gut microbes is meaningful for improving nutrient utilization and regulating nitrogen excretion of animal.In recent years,studies have shown that some anaerobic bacteria in the rumen preferentially take up small peptides instead of free amino acids or even unable to utilize free amino acids.At present,information on the different utilization of free amino acids and small peptide by small intestine bacteria of the pig is limited.Therefore,this study was to investigate the effects of different nitrogen forms of casein hydrolysates on the fermentation characteristcs and microbial community in the small intestine of pigs,amining to explore the role of microbiota in protein metabolism,in order to provide a theoretical basis for making nutrition policy to improved protein utilization and regulated nitrogen excretion in swine industry.This paper is divided into four parts:1.Effect of pig small intestinal bacteria on utilization of two casein hydrolysate by in vitro methodIn this part we investigated the effect of pig small intestinal bacteria on utilization of casein hydrolysate with different nitrogen structure.Samples from the digesta,and gut wall of the duodenum,jejunum and ileum from Duroc,Landrace and Yorkshire goods hybridization pigs were used as inocula.Casein acid hydrolysates and casein enzymatic hydrolysates used as substrates,respectively,for 12 hours fermentation at 37 degree Celsius Samples of fermentation were collected at 0,3,6 and 12 hours for free amino acid,peptide-amino acid and microbial amino acid analysis.Compare the disappearance of total free amino acid and total peptide-amino acid of the two groups,we found that the utilization of total peptide-amino acid was significantly higher than that of total free amino acid in the casein enzymatic hydrolysate group(P<0.05).In the case of casein acid hydrolysate,the utilization of total peptide-amino acid was significantly higher than that of total free amino acid after 3 hours incubation by duodenum lumial bacteria,tightly-attached bacteria and ileum luminal bacteria(P<0.05).With the time goes by,this difference became slightly and at the end of the fennentation,the utilization of total free amino acids was significantly higher than that of total peptide-amino acids.Compared with the different casein hydrolysates on the synthesis of microbial amino acids,we found that after 6 hours incubation the amount of microbial amino acids was synthesized by duodenum lumial bacteria,ileum luminal bacteria and the tightly-attached bacteria from casein enzymatic hydrolysate was significantly higher than that in the casein acid hydrolysate.We concluded that some of the bacteria in duodenum and ileum are able to use small peptides,and may be use the small peptides at the first priority if they exposure to the amino acid at the same time.2.Effect of two different casein hydrolysates on pig small intestinal bacteria fermentation characteristics by in vitro incubationIn this study,the in vitro method was used to investigate the fermentation characteristics of luminal bacteria and tightly-attached bacteria of pig small intestine on two different casein hydrolysates.Materials and methods in this experiment are based on Experiment 1.Samples of fermentation were collected at 0,3,6 and 12 hours,which were used to measure ammonia nitrogen,microbial protein and short chain fatty acids.Real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid.The results showed that,for the luminal bacteria,the concentration of MCP and SCFAs in casein enzymatic hydrolysate group were higher than the concentration of casein acid hydrolysate group in duodenum and ileum after 12 hours of anaerobic incubation(P<0.05).The quantity of total bacteria and Firmicutes in casein enzymatic hydrolysate group was higher than that in casein acid hydrolysate in duodenum.In addition,the quantity of E.coli and Firmicutes in the casein enzymatic hydrolysate group was higher than that in the casein acid hydrolysate group after 6 hours of incubation in the ileum(P<0.05).Compare with the casein enzymatic hydrolysate group,the quantity of total bacteria,Lactobacillus and E.coli were higher than those of casein acid hydrolysate group after 12 hours incubation.(2)For the tightly-attached bacteria:After 12 hours incubation,the concentration of MCP and total SCFAs in casein enzymatic hydrolysate group were higher than those of casein acid hydrolysate group in duodenum and ileum(P<0.05).The number of Lactobacillus and Firmicutes in the casein enzymatic hydrolysate group was higher than that in the casein acid hydrolysate group in duodenum(P<0.05),and the number of Firmicutes was higher in the casein enzymatic hydrolysate group than in the casein acid hydrolysate group(P<0.05).We concluded that some of the bacteria in duodenum and ileum are able to use small peptides,and may be use the small peptides at the first priority if they exposure to the amino acid at the same time.3.Effects of two casein hydrolysates on the microbial composition in the ileum of the pigsThe aim of this part was to investigate the effects of casein hydrolysates with different nitrogen structures on the microbial composition in the ileum of the pig.Materials and methods in this experiment are based on Experiment 1.Samples of fermentation were collected at 0,3,6 and 12 hours for microbial analysis.Pyrosequencing of the V1-V3 region of the 16S rRNA genes showed that at the end of fermentation the diversity of ileum microbiota in the two groups were significantly decreased(P<0.05).At the phylum level,casein enzymatic hydrolysates significantly increased the abundance of Firmicutes in the ileum digesta and tightly-attached bacteria at the end of fermentation(P<0.05),however,the abundance of proteobacteria was significantly decreased in the casein enzymatic hydrolystate group(P<0.05).At the genus level,as for the ileum digesta,the abundance of Escherichia-Shigella in the casein acid hydrolysate was higher than that in casein enzymatic hydrolysates(P<0.05).However,the abundance of the Lactobacillus,Terrisporobacter and Turicibacter was lower than that in casein enymaztic hydrolysates(P<0.05).As for the ileum tightly-attached bacteria,the abundance of Escherichia-Shigella in casein acid hydrolysate was higher than that in casein enzymatic hydrolysates(P<0.05).However,the abundance of the Lactobacillus and Fusobacterium lower than that in casein acid hydrolysate(P<0.05).In conclusion,different casein hydrolysates may modulate the microbial composition of the ileum.The bacteria in the ileum can degradation both small peptide and amino acid which were distribute in different phylum and genus.4.Effects of different nitrogen form diets on the ileal bacteria fermentation characteristics and composition in pigDietary protein is the main source of animal amino acids,however,in current animal husbandry industry,high crude protein(CP)level and imbalance of amino acids in diets result in enormous waste of protein source and severe nitrogen pollution.Based on the previous study.Twenty-seven Duroc x Landrace x Large White growing barrows(45 days of age)were randomly allocated to 16%protein diet(C),13%protein + casein enzymatic hydrolysate(P)and 13%protein + free amino acids groups.At the age of 85 days,digesta was collected from ileal T-cannulas at 0,3,6,12 hours after feeding for fermentation pattern and microbial analysis.The result showed that the concentration of NH3-N in group P was significantly higher than that in group A(P<0.05).The concentration of MCP,total SCFAs and acetate in group P was significantly higher than that in group A and group C(P<0.05).Pyrosequencing of the V1-V3 region of the 16S rRNA genes showed that the dietary treatment in the condition of this study only modulated several genera without changing the diversity of gut microbiota.At the phylum level,Firmicutes,Proteobacteria were the dominant bacteria.At the genus level,Lactobacillus,Escherichia-Shigella,Enterococcus were the dominat bacteria.Dietary nitrogen forms significantly affected the abundance of Lactobacillus,Enterococcus,Escherichia-Shigella,Holdemanella,Erysipelatoclostridium,Lachnospiraceae_Unclassified and Collinsella(P<0.05).These results suggest that the addition of small peptides into low protein diet can promote the production of NH3-N,MCP and SCFAs.The addition of different nitrogen forms at low protein diet can affect the fermentation characteriastics and reshape the composition of microbiota in ileum of pig.These results indicate that there is a difference in the use of two different nitrogen forms of casein hydrolyzate by the small intestine bacteria.The bacteria tend to use small peptide synthesis of bacterial protein,to promote the fermentation and promote the growth of Firmicutes.In vivo experiments showed that the addition of small peptides to a low protein diets can promote ileal bacteria fermentation,promote the bacterial protein synthesis.