【作者】 周笑犁；

【导师】 印遇龙；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2013， 博士

【摘要】 婴幼儿消化生理功能尚未发育成熟,断奶或食物的改变常引起肠道免疫应激和功能紊乱,致使消化吸收不良,导致生长发育迟缓。大豆寡糖来源丰富、无有害残留,且具有明显的益生素作用,但对其调节肠道微生态和免疫功能的作用了解不多。由于仔猪断奶时期的生理特点与婴幼儿非常接近,本研究以断奶环江香猪作为试验动物,通过体内、体外试验分析大豆寡糖对肠道微生态及免疫功能的调节作用,并探讨其机制,为改善婴幼儿的营养供给提供新思路,同时为大豆寡糖的综合开发利用提供理论依据。1.大豆寡糖对小肠形态与营养物质代谢的影响：选用体重为3.19kg左右的21日龄断奶环江香猪,随机分成2组加以不同处理,每个处理6个重复,单栏饲养,分别饲喂基础日粮(对照组)和添加大豆寡糖的基础日粮(大豆寡糖组),自由采食和饮水,试验期为14d。记录每天的采食量,分别于试验开始和结束时称取空腹体重并采集血液,肝素抗凝,离心分离血浆,测定生化参数和游离氨基酸的浓度；采血后,按常规方法进行屠宰,采集小肠组织,测定基因表达、并观察组织形态；再选用10头环江香猪,随机分为2组,处理同前。预试期4d,正试期3d,收集粪样,指示剂法测定粗蛋白和粗脂肪的表观消化率。结果表明,与对照组相比,大豆寡糖组仔猪生长性能无显著变化(P>0.05)；血浆甘油三酯、尿素氮和血氨浓度显著下降(P<0.05),总蛋白浓度显著升高(P<0.05)；血浆甘氨酸、组氨酸、赖氨酸、精氨酸和蛋氨酸的浓度均显著升高(P<0.05)；空肠SGLT1、 GLUT2mRNA的表达量显著高于回肠(P<0.05),并显著高于对照组(P<0.05)；小肠绒毛高度与隐窝深度之比显著升高(P<0.05)；大豆寡糖组仔猪粗蛋白和粗脂肪的表观消化率均有提高趋势(P>0.05)。2.大豆寡糖对肠道微生物区系及其代谢产物的影响：收集空肠、回肠、盲肠和结肠内容物,测定断奶环江香猪肠道微生物区系、肠道内容物中部分细菌数量变化及其代谢产物的含量。结果表明,大豆寡糖组仔猪回肠和结肠内容物中微生物的多样性增加,其中双歧杆菌、短双歧杆菌、罗氏菌属和产丁酸菌(梭杆菌属)等有益菌群的数量及其代谢产物丙酸、丁酸等短链脂肪酸的含量均显著增加(P<0.05),大肠杆菌、梭菌属和链球菌等有害细菌的数量及异丁酸、异戊酸和氨等蛋白质分解代谢产物显著减少(P<0.05)。然后进行体外发酵试验,厌氧收集环江香猪的空肠、回肠、盲肠和结肠内容物作为接种物,分别以0(对照组)、0.5%、1.0%、2.0%、5.0%、8.0%大豆寡糖(大豆寡糖组)、2.0%葡萄糖(葡萄糖组)为底物体外发酵。于接种后0、1、2、4、6、8、10、12、14、16、18、20、22、24、30、36和48h记录产气量,用产气动力学方程分析大豆寡糖的发酵特性,并于发酵48h结束后检测发酵液pH值、NH3-N和短链脂肪酸的浓度以及微生物区系。结果表明：各添加剂量的大豆寡糖组最大理论产气量和产气速率均显著高于对照组、显著低于葡萄糖组(P<0.05),接种回肠内容物的大豆寡糖组产气速率显著低于葡萄糖组,但在结肠内容物发酵液中却显著高于葡萄糖组(P<0.05),接种结肠和盲肠内容物的大豆寡糖组和葡萄糖组的体外发酵延滞产气时间均显著低于对照组(P<0.05)；各肠段内容物的大豆寡糖组发酵液pH值、NH3-N浓度均低于、短链脂肪酸浓度均高于对照组,其中乙酸浓度随大豆寡糖添加剂量的增加呈减少趋势、丙酸和丁酸浓度呈增加趋势,2.0%大豆寡糖组结肠发酵液中丁酸浓度最高；随着大豆寡糖添加量的增加,肠道微生物区系与有益细菌数量先增加再减少,而有害细菌数量呈先减少后增加的趋势。3.大豆寡糖对免疫功能与肠道紧密连接的影响：测定血浆中免疫球蛋白和细胞因子的浓度；采集脾脏、回肠和结肠组织,分析免疫相关基因和肠道紧密连接基因的mRNA表达。结果表明：大豆寡糖增加了血浆免疫球蛋白IgG和IL-10的浓度,减少了血浆中IL-1p的浓度(P<0.05)：脾脏、回肠和结肠TNF-α、IL-1β和IL-8mRNA以及TLR2-NF-KB mRNA的表达降低,结肠和脾脏中IL-10mRNA的表达显著升高(P<0.05)；回肠和结肠紧密连接蛋白ZO-1mRNA的表达水平显著提高(P<0.05)。综上所述,大豆寡糖可显著增加肠道微生态的多样性,提高肠道有益菌的数量、抑制有害菌的增殖,从而减少了蛋白质在大肠中的分解代谢；改善肠道形态结构,调节免疫功能,增强肠黏膜屏障功能,促进了营养物质的代谢。更多还原

【Abstract】 Owing to digestive physiology has not yet mature in infant, after weaning or food changes, the infant often show a series of negative phenomena due to intestinal immune stress and dysfunction, which result in digestive function disorders, growth delay. Soybean oligosaccharide (SBOS) has rich source, no harmful residues and obvious probiotic effect, however, there is much limited information regarding the effects of SBOS on immune function and intestinal microecology. In post-weaning pigs, an experimental model which is considered as more relevant for human nutrition extrapolation than rodents, especially infant. In that overall context, the aim of this study was to investigate the probiotic effect of SBOS as a dietary additive on intestinal microecology and immune function in vivo and in vitro, and its underlying mechanism for weaned piglets were used as animal models. These results provide a new way to improve nutrition supply for the infant and theoretical basis for comprehensive development and utilization of SBOS.1. Effects of SBOS on small intestinal morphology and nutrient metabolism: Twelve Huanjiang mini-piglets weaned at21d of age with an average body weight of3.19kg were randomly allocated into two groups with six replicates in the feeding trial. The piglets were fed with a0.5%of corn starch (control group), or0.5%of SBOS in a corn-and soybean meal-based diet for14days. Feed intake per piglet was recorded daily, and the body weight was got at the beginning or end of this trail, as well as collecting the blood. The plasma were separated by centrifugation and used for measuring biochemical parameters and concentrtions of free amino acids. And then, the jejunum and ileum were collected for histological analysis, the mRNA expression abundances of sodiuml glucose cotransporter (SGLT1) and glucose transporfer (GLUT2) in jejunum and ileum were determined by relative quantitative RT-PCR respectively. And then Ten Huanjiang mini-piglets were randomly assigned to two groups receiving either dietary supplementation with0.5%of corn starch (control group), or0.5%of SBOS in a corn-and soybean meal-based diet as same as the feeding trial in the digestion trial. After4d pretrial and3d official test, the feces were collected to determine the digestibility of dry matter, crude protein and crude fat by indicator method. The results showed:compared with the control group, dietary supplementing SBOS decreased (P<0.05) the plasma concentrtions of triglyceride, urea nitrogen and ammonia, while increased (P<0.05) the plasma content of total protein, and the plasma contents of glycine, histidine, lysine, arginine and metione; the mRNA expression of SGLT1and GLUT2in jejunum of SBOS group were higher (P<0.05) than ileum, as well as than that of the control group (P<0.05); SBOS increased the ratio of villus height and fossae depth in small intestinal (P<0.05); SBOS increased (P>0.05) the apparent digestibility of crude protein and crude fat in comparison with the control group (P>0.05).2. Effects of SBOS on intestinal microflora and its metabolite:the jejunum, ileum, cecum and colon and their contents were collected to determine the concentrations in the luminal contents of ammonia and short-chain fatty acid (SCFA), as well as gut microflora composition. The results showed that dietary supplementation with SBOS increased the diversity of ileum and colon microflora, and promoted (P<0.05) the amounts of some presumably beneficial intestinal bacteria (Bifidobacterium, Faecalibacterium prausnitzii, Fusobacterium prausnitzii and Roseburia) as well as the amounts of SCFAs in the intestinal luminal content, while diminishing (P<0.05) bacteria with pathogenic potential(Escherichia Coli, Clostridium and Streptococcus) and the amount of several protein-derived catabolites (isobutyrate, isovalerate and NH3-N).And then in vitro, sterile jejunum, ileum, cecum or colon content as the inoculum were collected from Huanjiang pigs to conduct in vitro fermentation using SBOS at the concentrations of0,0.5,1.0,2.0,5.0and8.0%, and2.0%glucose as the substrates. The gas production at0,1,2,4,6,8,10,12,14,16,18,20,22,24,30,36and48h, pH, and the contents of NH3-N, acetic acid, propanoic acid, butyric acid and microflora in the fermentation broth were determined after48h fermentation in an anaerobic system. Results indicated that the maximum amount and rate of gas production in the fermentation broth supplemented with SBOS were higher than those of the control group, lower than glucose group (P<0.05), while an obvious lag in gas-producing time was observed in fermentation broth of colon or cecal group supplemented with SBOS and glucose were lower than the control group (P<0.05); The pH and NH3-N concentration in fermentation broth supplemented with2.0%SBOS were lower (P<0.05) than the control group (P<0.05); The concentration of short chain fatty acids (SCFA) in the groups supplemented with SBOS and glucose was higher than that of the control group (P<0.05), the concentration of acetic acid was decreased due to the addition of SBOS, while the concentration of propionate acid and butyrate acid exhibited a gradual increase. The maximum content of butyrate acid was observed in the fermentation broth of colon group supplemented with2.0%SBOS; With the additive amount of SBOS increased, the microflora diversity and beneficial bacteria have increased and then decreased, while bacterial pathogens has suppressed as the concentration of SBOS increased and then enriched. Therefore, SBOS is beneficial to gut health as functional food or feed additives to promote the intestinal microbial fermentation, increase the proportion of butyric acid in the fermented broth, reduce the pH and concentration of NH3-N.3. Effects of SBOS on the immune function and intestinal barrier:the spleen, ileum and colon were collected to determine the expression levels in mRNA of cytokines and tight junctions-associated proteins, as well as the concentrations of immune globulin and cytokines in the plasma. The results indicated that dietary supplementation with SBOS increased (P<0.05) concentrations of immunoglobulins and IL-10in plasma, expression levels of IL-10mRNA in colon and spleen, and ZO-1mRNA in ileum and colon; while decreased (P<0.05) concentrations of IL-1β in plasma, expression levels of TNF-a, IL-1β and IL-8mRNA in spleen, ileum and colon, as well as TLR2-NF-κB mRNA levels.These findings suggested that dietary supplementation with SBOS could effectively promote the diversity of intestinal microflora, enrich beneficial intestinal microflora and suppress bacterial pathogens in intestine, and resulte to reduce the protein catabolism in the large intestine; improve the intestinal morphology, immune function, enhance the barrier function of the intestinal mucosa, promote metabolism of intestinal nutrients.更多还原