体外法探究猪空肠和回肠黏膜微生物对低聚半乳糖和低聚甘露糖的发酵特性

【背景】小肠黏膜微生物是肠道菌群的重要组成部分，大量研究表明日粮添加低聚半乳糖（galacto-oligosaccharides，GOS）和低聚甘露糖（manno-oligosaccharides，MOS）能够调控猪的大肠菌群结构，但关于其调控小肠黏膜微生物的研究较少。【目的】通过体外发酵法探究猪空肠黏膜和回肠黏膜微生物发酵GOS和MOS的规律。【方法】以生长猪的空肠黏膜微生物和回肠黏膜微生物作为接种物，以GOS和MOS作为底物进行厌氧发酵，在发酵0、6、12、24 h时采样测定总菌数量、pH、氨态氮（ammonia nitrogen，NH₃-N）、菌体蛋白（microbial crude protein，MCP）和有机酸，在24 h收集微生物提取DNA进行细菌定量分析。【结果】在24 h时，回肠黏膜组的NH₃-N浓度显著低于空肠黏膜组，而MCP浓度显著高于空肠黏膜组（P<0.05）。在发酵的前6 h各组pH无明显变化，有机酸积累较少。在12 h时，MOS组的乳酸、乙酸、丁酸和总短链脂肪酸产量显著高于GOS组（P<0.05），此时只有回肠黏膜组有少量丙酸产生。在24 h时，MOS回肠黏膜组乳酸产量最高而pH值最低（P<0.05）。相较于MOS组，GOS组显著提高了丙酸的产量（P<0.05）。相较于GOS组，MOS组显著提高了乙酸的产量，在空肠黏膜组中显著提高了丁酸和总短链脂肪酸的产量（P<0.05）。定量结果表明，在24 h时，各处理组的厚壁菌门数量都接近总菌数量，属于优势菌门。相较于MOS组，GOS组显著提高了拟杆菌门、链球菌属、韦荣氏球菌属和普拉梭菌细菌的数量，提高了空肠黏膜组中Clostridium cluster IV和回肠黏膜组中Clostridium cluster XIVa的数量（P<0.05）。相较于GOS组，MOS组显著提高了大肠杆菌和乳酸杆菌属的数量，提高了回肠黏膜组中罗氏菌属的数量（P<0.05）。【结论】猪小肠黏膜微生物对GOS和MOS具有不同的发酵模式，主要表现在有机酸的产生和促进细菌的增殖方面。GOS具有产丙酸优势，提高了拟杆菌门和韦荣氏球菌属的数量；MOS促进了乙酸的产生，提高了大肠杆菌和乳酸杆菌的数量。

In vitro fermentation characteristics of galacto-oligosaccharides and manno-oligosaccharides by mucosal microbiota from porcine jejunum and ileum

Background] Mucosal microbiota from small intestine is an important part of gut microbiota. Many studies had shown that galacto-oligosaccharides (GOS) and manno-oligosaccharides (MOS) can regulate the gut microbiota from large intestine of pigs. However, there are few studies on its regulation of mucosal microbiota from small intestine. Objective] This study aimed to explore the in vitro fermentation characteristics of GOS and MOS by mucosal microbiota from porcine jejunum and ileum. Methods] The microbiota samples from the jejunum mucosa and ileum mucosa of growing pigs were used for the anaerobic fermentation of GOS and MOS. Total bacterial number, pH, ammonia nitrogen (NH₃-N), microbial crude protein (MCP), and organic acids were measured at 0, 6, 12, and 24 h during the fermentation. The bacteria were quantified by quantitative PCR after 24 h incubation. Results] After 24 h incubation, the concentration of NH₃-N in the ileum mucosa group was lower than that in the jejunum mucosa group, while the concentration of MCP showed an opposite trend (P<0.05). After 6 h incubation, the pH changed slightly because of the small amount accumulation of organic acids. After 12 h incubation, lactate, acetate, butyrate, and total short-chain fatty acids in the MOS group were significantly higher than those in the GOS group (P<0.05), and only a small amount of propionic acid was produced in the ileum mucosa group. After 24 h incubation, the MOS group with the microbiota from ileum mucosa showed the highest yield of lactate and the lowest pH (P<0.05). The GOS group had higher propionate yield than the MOS group (P<0.05). The MOS group had higher yield of acetate than the GOS group, as well as that of butyrate and total SCFA in the jejunum mucosa group (P<0.05). After 24 h incubation, Firmicutes showed the close number to total bacteria, being the dominant phylum. Compared with MOS, GOS increased the number of Bacteroidetes, Streptococcus,Veillonella, and Faecalibacterium, as well as that of Clostridiumcluster IV in the jejunum mucosa group and Clostridium cluster XIVa in the ileum mucosa group (P<0.05). Compared with GOS, MOS increased the number of Escherichia coli and Lactobacillus, as well as that of Roseburia in the ileum mucosa group (P<0.05). Conclusion] The mucosal microbiota from porcine small intestine had different fermentation patterns for GOS and MOS, as manifested by the varied production of organic acids and bacterial proliferation. GOS had an advantage of propionate production and increased the number of Bacteroidetes and Veillonella. MOS promoted the production of acetate, lactate in the ileum mucosa group and butyrate in the jejunum mucosa group, and increased the number of Escherichia coli and Lactobacillus.