| Recent researches found out that gut microbiota participated in the nutrient metabolism, especially nitrogen containing compounds such as amino acids. Nearly 30-60% dietary essential amino acids were disappeared in the first pass intestinal metabolism by gut epithelial cells. However, researchers reported that gut epithelial cell could only metabolize branch chain amino acids, rather than other essential amino acids.So the substantial utilization of essential amino acids in the first pass intestinal metabolism might mainly due to the gut bacteria. Recent in vitro research showed luminal bacteria from pig small intestine had the ability to metabolize essential amino acids. However, the small intestinal bacteria contain luminal bacteria and gut wall associate bacteria. It is still not clear whether their metabolism on amino acids are similar or not. Large intestinal bacteria were different to small intestinal bacteria on not only population but also composition, thus its metabolism on amino acids might be also not the same. The role of large intestinal bacteria in protein fermentation in the hindgut was still not clear. This study used piglet and rat as subjects, aimed to investigate the role of small intestinal bacteria in protein metabolism and large intestinal bacteria in protein fermentation.1 Variations in the Metabolism of Amino Acids by Bacteria Derived from Different Compartment of the Pig Small IntestineSamples from the digesta, gut wall washes and gut wall of the jejunum and ileum were used as inocula, which represent luminal, loosly attached and tightly attached bacteria, respectively. Anaerobic media containing single AA were used and cultured for 24 h. The 24 h culture was served as inocula for the subsequent 30 times of subcultures. Results showed that for the luminal bacteria, all AA concentrations except phenylalanine in ileum significantly decreased during the 24 h in vitro incubation, while 4 AA (glutamate, glutamine, arginine and lysine) in jejunum decreased, with the disappearance rate at 60-95%. For tightly attached bacteria, all AA concentrations were generally increased during the first 12 h and then decreased, suggesting a synthesis first and then catabolism pattern. Among them, glutamate in both segments, histidine in jejunum and lysine in ileum increased significantly during the first 12 h and then decreased significantly at 24 h. Concentrations of glutamine and arginine did not change during the first 12 h but went through a significantly decrease at 24 h. Jejunal lysine and ileal threonine were significantly increased for the first 6 or 12 h. For the loosely attached bacteria, there was no clear pattern for all the AA metabolism.However, glutamate, methionine and lysine in jejunum significantly decreased after 24 h cultivation, while glutamine and threonine in jejunum, and glutamine and lysine in ileum significantly increased for the first 12 h. During subculture, AA metabolism,either utilization or synthesis, was generally decreased as the time of transfer increased.However, disappearance rate of lysine in each group was around 90% throughout the subculture, suggesting highly utilization of lysine by bacteria from all three compartments. Analysis of the microbial community during the 24 h in vitro cultivation revealed that bacteria composition in most AA cultures varied between different niches(lumen and wall adherent fractions) in jejunum, while relatively similar in ileum.However, for isoleucine and leucine cultures, bacteria composition diversity was similar between luminal fraction and tightly attached fraction, but significantly higher than in loosely attached fraction. For glutamine and valine cultures, bacteria diversity was similar between luminal and loosely attached fraction, but significantly lower than that of tightly attached bacteria. After 30 subcultures, bacteria diversity in arginine,valine, glutamine, and leucine cultures varied between niches in jejunum while relatively stable in ileum, in consistent with those in 24 h in vitro cultures. The above findings provide basic knowledge of the compartmental AA metabolism in small-intestinal bacteria, which will help to improve the utilization and function of dietary AA in the intestine of both human and animal.2 Antibiotic Influences Amino Acids Utilization by Pig Small Intestinal BacteriaSixteen litters of weaned piglets were randomly allocated into two groups feeding control diet and antibiotic containing diet (oxytetracycline, olaquindox, kitasamycin,50 mg/kg each). The piglets were slaughtered after 18 days. Blood biochemical parameters,organ weight,amino acids concentration in jejunum,ileum and blood were measured. The population of total bacteria, Firmicutes and Bacteroidetes in jejunum and ileum were quantified. The results showed that antibiotic significantly increase feed conversion ratio, but had no effect on feed intake and weight gain. Antibiotic administration significantly decreased jejunal and ileal amino acids concentration,while significantly increased amino acids and ammonia concentration in the blood. Ileal total bacteria and Firmicutes population were significantly lower in antibiotic group compared to control group, while jejunal population of Escherichia coli were significantly increased after antibiotic treatment. Antibiotic has no effect on organ index.These results suggested that antibiotic treatment significantly altered bacteria composition in the small intestine and also amino acids metabolism, which further indicated that small intestinal bacteria can complete amino acids with the host.3 Antibiotic Influence Bacteria Composition in Pig Stomach and Small IntestineBased on the previous chapter, digesta samples from stomach, duodenum, jejunum,and ileum and mucosal samples from duodenum, jejunum and ileum were used for high throughput deep sequencing. The results showed that antibiotic significantly altered the bacterial composition in the duodenum and jejunum, but not in the ileum. Antibiotic treatment significantly decreased the population of beneficial bacteria in the lumen and digesta such as Clostridium and Lactobacillus, while significantly increased the abundance of conditional pathogen such as Escherichia coli and Streptococcus. Besides,antibiotic could inhibit the interaction between luminal and mucosal bacteria. The abundance of Eubacteria contortum, Gemmiger formicilis, E. biforme and E.coprostanoligene were significantly correlated with the ileal amino acids concentration,suggesting they might be the dominant amino acids metabolizing bacteria. These results improve our understanding on the role of antibiotic in the small intestine. Antibiotic could not only alter the abundance and composition of the luminal and mucosal bacteria in the small intestine, but also interfered with their interaction, which might increase the risk of pathogen colonization4 Antibiotic Influence Amino Acids Fermentation by Colonic Microbiota in PigletsBased on the animal trial in chapter 2, colonic samples were used for high-throughput sequencing and fermentation products analysis. The results showed that antibiotic had no effect on the colonic microbial composition. Antibiotic treatment significantly increased colonic valerate and isovalerate level, together with the relative level of propionate, butyrate, valerate, isobutyrate and isovalerate. Beside, antibiotic significantly increased the concentration of biogenic amine (putrescine, cadaverine and spermin) and phenolic and indolic compound in the colon. Meanwhile, colonic ammonia level was significantly increased in antibiotic group. These results showed that antibiotic increased the level of nitrogenous compound entered into the hindgut and their metabolism by colonic microbiota, which will produce large amount and different kinds of toxic compounds.5 Colonic Bacterial Inhibition Influence Amino Acids Metabolism in Rat Large IntestineA total of 20 healthy SPF wistar rat were randomly assigned into 2 groups. One feeding basal diet, the other supplementing BCM in drinking water. After 42 d, the rats were killed. Blood and digesta samples from cecum and colon were collected for further analysis. The results showed that BCM supplementation has no influence on rat growth performance, but significantly decreased blood glucose and uracil. Cecal methylamine,putrescine,phenylalanine,tryptamine and skatole in BCM treated rat was significantly lower than control group, which indicated enhancement in protein metabolism in the large intestine. BCM administration significantly decreased the level of fatty acids and carbohydrate in the colon. BCM significantly decreased the population of methanogen while increased the level of sulfur reducing bacteria. Beside, BCM altered the bacterial composition in the colon through reducing the Actinobacteria,Acidobacteria and Proteobacteria. These results indicated that BCM administration could alter the bacterial structure in colon, inhibit hindgut bacteria activity, enhance bacterial protein fermentation and decrease the metabolism of carbohydrate and fatty acids. |
