摘要
氨基酸及其代谢产物对人和动物的健康有重要意义。由于小肠在解剖结构上处于优先利用摄入氨基酸的地位,小肠日粮氨基酸的代谢可直接影响氨基酸进入门静脉循环的模式,从而影响整个机体对氨基酸的利用。目前,关于小肠内氨基酸代谢情况存在许多争议,关于小肠内氨基酸的微生物代谢和转化情况鲜有报道。因此,本文旨在探究猪小肠微生物体外代谢氨基酸情况,分离鉴定猪小肠氨基酸代谢菌,为研究小肠内氨基酸的微生物代谢机理、转化途径和调控机制提供理论基础。
第一部分：体外法研究猪小肠微生物对氨基酸的代谢。通过体外培养研究猪小肠各肠段微生物对精氨酸、谷氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、蛋氨酸、苯丙氨酸、脯氨酸、苏氨酸、色氨酸和缬氨酸的代谢情况。分别接种十二指肠、空肠、回肠微生物处理液至含有10 mmol／L上述不同氨基酸的培养基中,体外发酵24h。0h、6h、12h、24h取样,用HPLC测定各组发酵液中游离氨基酸浓度,计算氨基酸消失率,分析各肠段微生物对氨基酸的代谢规律。试验结果表明,体外发酵24h,十二指肠组、空肠组和回肠组中赖氨酸、苏氨酸、组氨酸、精氨酸、谷氨酸和支链氨基酸的消失率显著高于蛋氨酸、苯丙氨酸、色氨酸和脯氨酸(P<0.01);十二指肠、空肠和回肠微生物对同种氨基酸的代谢情况存在差异。提示,猪小肠微生物可能对小肠内赖氨酸、苏氨酸、精氨酸、谷氨酸、组氨酸和支链氨基酸的代谢有重要影响。
第二部分：猪小肠氨基酸代谢菌的分离和筛选。为了研究猪小肠氨基酸代谢菌的物种资源,该试验对赖氨酸、苏氨酸、组氨酸、谷氨酸和精氨酸代谢菌进行分离和筛选。利用改良型的Hungate滚管技术,根据菌落形态差异挑选出氨基酸代谢菌疑似菌株30株。将疑似菌株菌液接种到含有其对应氨基酸的培养基中,体外发酵24h。通过HPLC测定发酵液中游离氨基酸浓度,计算氨基酸消失率,筛选可代谢氨基酸的菌株。将筛选出的氨基酸代谢菌菌液接种到含有其对应氨基酸的培养基中,进行复筛。本试验共分离氨基酸代谢菌13株。其中,赖氨酸代谢菌3株,命名为L1、L2和L3,单菌发酵液中赖氨酸消失率分别为83.17%、47.17%和85.70%。苏氨酸代谢菌2株,命名为T1和T2,单菌发酵液中苏氨酸消失率分别为14.34%和2.09%。组氨酸代谢菌2株,命名为H1和H8,单菌发酵液中组氨酸消失率分别为8.72%和4.60%。精氨酸代谢菌3株,命名为A1、A3和A4,单菌发酵液中精氨酸消失率分别为5.81%、21.41%和15.23%。谷氨酸代谢菌3株,命名为G4、G9和G10,单菌发酵液中谷氨酸消失率分别为15.44%、33.26%和5.75%。试验结果表明,除赖氨酸代谢菌外,其余单菌的氨基酸代谢率显著低于小肠各肠段混合菌的代谢率。
第三部分：猪小肠氨基酸代谢菌的鉴定与分类。为了进一步探究猪小肠氨基酸代谢菌的种类和特征,本试验对菌株L1、L2、L3、T1、H1、H8、A3、A4、G4和G9进行了形态、生化和16SrRNA基因序列鉴定。形态和生化鉴定结果表明,菌株L1、L2、L3和G9为克雷伯属(Klebsiella)细菌；T1、G4和H1为大肠杆菌(Escherichiacoli)；H8、A3和A4为链球菌属(Streptococcus)细菌。系统进化树分析结果表明,L2、L3与Klebsiella pneumoniae strain K42和Klebsiella pneumonia ATCC13883T位于同一个分枝内,相似性达99%。G9和Klebsiella pneumoniae strain ECU-15位于同一个分枝内,相似性达99%。A3与Streptococcus bovis位于同一个分枝内,相似性达99%。
Amino acids and their metabolites are important to both animals and humans. Considering the anatomic priority of small intestine in amino acids utilization, intestinal amino acid metabolism plays a significant role in modulating amino acid systemic availability and profile for the utilization of extraintestinal tissues. Up to now, the intestinal metabolism of amino acids is still controversial. Knowledge about the microbial metabolism of dietary amino acids in the foregut is still unknown. Thus, this thesis aimed to investigate the in vitro microbial metabolism of amino acids, isolate amino acids utilizing bacteria and provide theoretical basis for further research on microbial metabolism of intestinal amino acids. It was described in the following three sections.
The aim of the first experiment was to investigate the in vitro metabolism of amino acids, including arginine, glutamate, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine, by microorganisms from different segment of pig small intestine. Fresh samples from duodenum, jejunum and ileum were used as inocula for cultivation of individual amino acid. Samples were takend after incubation of 0 h,6 h,12 h and 24 h. Concentrations of free amino acid in the culture media were detected by HPLC, which calculated and expressed as disappearance rates. Results showed that the disappearance rate of lysine, threonine, histidine, arginine, glutamate and BCAA in all treatments is higher than that of methionine, phenylalanine, proline and tryptophan (P<0.01) after incubation of 24 h. For each amino acid, the metabolic rates by microorganisms from duodenum, jejunum and ileum are different. The above results revealed the importance of microbial metabolism of lysine, threonine, arginine, glutamate, histidine, valine, leucine and isoleucine in small intestine.
In order to research and analyze species resource of amino acid utilizing bacteria, lysine, threonine, histidine, arginine and glutamate utilizing strains were isolated by modified Hungate technique from pig small intestine.30 suspected strains were isolated by discriminating colonial morphology. Each suspected isolate was used to inoculate culture bottles with corresponding amino acid. After 24 h incubation, the cultures were taken to detect the concentration of free amino acid by HPLC and analyze amino acid disappearance rate to select amino acids utilizing strains. Each selected strains was used to inoculate culture bottles with corresponding amino acid. After 24 h incubation, the cultures were taken to detect the concentration of free amino acid by automatic amino acid analyzer and analyze amino acid disappearance rate. Thirteen strains were isolated, of which three lysine utilizing strains were named after L1, L2 and L3, with lysine disappearance rate of 83.17%, 47.17% and 85.70% respectively. Two threonine utilizing strains were named after T1 and T2, with threonine disappearance rate of 14.34% and 2.09% respectively. Two histidine utilizing strains were named after H1 and H8, with histidine disappearance rate of 8.72% and 4.60% respectively. Three arginine utilizing strains were named after A1, A3 and A4, with arginine disappearance rate of 5.81%,21.41% and 15.23% respectively. Three glutamate utilizing strains were named after G4, G9 and G10, with glutamate disappearance rate of 15.44%,33.26% and 5.75% respectively. Results showed that except lysine utilizing strains, the amino acid metabolic rate of the other stains are lower than that of mixing microorganisms from duodenum, jejunum and ileum.
The study was designed to identify the morphology, biochemical and molecular traits of L1, L2, L3, T1, H1, H8, A3, A4, G4 and G9. Based on morphological and physiological characteristics, L1, L2, L3 and G9 were identified as Klebsiella, T1 and G4 were identified as Escherichia coli, and H8, A3 and A4 were identified as Streptococcus. The 16S rRNA gene sequence analysis showed that L2 and L3 had a closest relative with Klebsiella pneumoniae strain K42 and Klebsiella pneumonia ATCC13883T, G9 had a closest relative with Klebsiella pneumoniae strain ECU-15, and A3 had a closest relative with Streptococcus bovis.
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