体外法研究猪后肠道对芳香族氨基酸的发酵特性

Metabolic pattern of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method

【目的】采用体外发酵技术探究芳香族氨基酸在猪后肠的发酵特性。【方法】以杜×长×大育肥猪回肠、盲肠和结肠食糜为接种物,接种于10 mmol/L单一氨基酸的培养基中,37 o C培养24 h,测定4、8、12、16和24 h的产气量(GP),采集0 h和24 h样品,测定样品中的氨氮(NH3-N)和微生物蛋白(MCP)浓度,利用变性梯度凝胶电泳技术(DGGE)和Real-time PCR定量技术分析体外培养中参与代谢特定氨基酸的细菌组成及数量。【结果】不同芳香族氨基酸的发酵液中NH3-N和MCP浓度存在极显著差异(P<0.01),肠段对GP、NH3-N和MCP影响极显著(P<0.01),且芳香族氨基酸与肠段对GP、NH3-N和MCP浓度均存在交互作用(P<0.01)。定量PCR表明,芳香族氨基酸和肠段均显著影响发酵液中总菌数量(P<0.05)。DGGE分析显示,同一肠段不同芳香族氨基酸组的细菌群落结构具有高度的相似性,其中回肠Phe组和Tyr组、结肠Tyr组和Trp组的相似性分别高达87.9%和80.5%,盲肠和结肠微生物香农指数变化显著(P<0.05)。【结论】不同芳香族氨基酸的肠道代谢菌具有差异性,与Trp和Phe相比,Tyr的盲肠和结肠代谢菌多样性较低,与Trp和Tyr相比,Phe更多地合成菌体;特定芳香族氨基酸的不同肠道发酵去向不同,与回肠和盲肠比,结肠中芳香族氨基酸更多地合成菌体。

[Objective] This experiment was conducted to study different metabolic patterns of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method. [Methods] Ileum, cecum and colon chyme in Duroc,Landrace and Yorkshire goods hybridization pigs were taken as inoculum. The single aromatic amino acid concentration was kept 10 mmol/L in fermentation flask. Then the fermentation flask was incubated at 37 ℃for 24 h.Gas production was measured at 4, 8, 12, 16 and 24 h, and samples of fermentation collected at 0 h and 24 h were used to measure ammonia nitrogen NH3-N and microbial crude protein（MCP）. Denaturing gradient gel electrophoresis（DGGE） and real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid. [Results] The concentrations of NH3-N and MCP were significantly affected by aromatic amino acids and intestinal segments（P〈0.01）. Intestinal segments also affected gas production（GP） significantly（P〈0.01）. NH3-N, MCP and GP were affected by interaction of aromatic amino acids and intestinal segments. DGGE analysis showed bacteria of aromatic amino acids shared amount of bands together, especially similarity analysis of DGGE profile of Phe and Tyr in ileum, Tyr and Trp in colon were 87.9% and 80.5% separately. Shannon diversity indices analysis revealed that aromatic amino acids in cecum and colon varied significantly（P〈0.05）. Real-time PCR results showed that the quantity of total bacteria were affected by aromatic amino acids and intestinal segments significantly（P〈0.05）. [Conclusion] The potential as proportion of different aromatic amino acids are different.Compared with Trp and Phe, the diversity of bacteria utilizing Tyr in cecum or colon is low; compared with Tyr and Trp, a large number of Phe participated in synthesizing bacteria.The fermentation pattern of specific aromatic amino acids in different intestinal segment was unique. Compared with ileum and cecum, much more aromatic amino acids participated in the synthesis of bacteria in colon.