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30170687 30270969 30771524

负责人顾宪红项目批准号 30170687 学科代码 C120104
项目名称用分子生物学方法研究菊粉调节仔猪大肠发酵的机理
申请书摘要    菊粉作为天然植物提取物，在动物大肠中能选择性地促进双歧杆菌增殖，优化其微生态环境，提高营养素的利用；菊粉能值很低，可作为食糖和脂肪的替代品。且富含菊粉的菊芋在夜种埠芄恪１鞠钅恳宰兄碜魑锬Ｐ停捎梅肿由镅Х椒ǎ教志辗鄣鹘谧兄泶蟪Ψ⒔偷幕恚⑽夜笄萋躺繁Ｐ吞砑蛹烈约叭死嗍秤孟宋＝∑诽峁├砺垡谰荨
申请书主题词
结题中文摘要    本项目建立了定量测量细菌的Q-PCR方法，并用该法分3个实验研究了菊粉对培养液和仔猪、肉仔鸡肠道微生物的影响规律。离体实验设0、1、2、4、6、8g/L六个菊粉水平，厌氧培养猪鲜粪24h。结果表明：随着菊粉水平的增加，培养液中双歧杆菌、乳酸杆菌、总细菌以及丙酸升高(P<0.05)，大肠杆菌、氨及pH降低(P<0.05)。仔猪实验选用90头28d断奶仔猪，分成五组，分别饲喂0、0.5、1.0、1.5%菊粉±抗生素五种日粮28天。结果表明：1%菊粉组猪盲肠乳酸杆菌和回肠、盲肠和直肠双歧杆菌以及盲肠内容物中丙酸增加 (P<0.05)；盲肠、结肠、直肠内容物中pH和血糖下降(P<0.05)。超额完成的肉仔鸡实验选用3w肉仔鸡120只，分成四组，分别饲喂含0、0.2、0.4 和0.8%菊粉的日粮28天。结果表明：随着菊粉水平的增加，盲肠双岐杆菌数增加(P<0.05)；血浆、盲肠、结肠和粪便pH以及血糖下降（P<0.05）。三个实验一致证实了菊粉促进有益菌增殖、抑制有害菌生长的益生作用，首次发现菊粉对动物胰腺和小肠中各主要消化酶活以及盲肠菊粉酶、纤维素酶活影响显著，对开发菊粉添加剂提供了理论基础。
结题英文摘要    The present project was conducted to establish a molecular biological method for detecting and quantifying bacteria, and to investigate the effects of inulin on bacterial communities in culture medium, the digestive tracts of weaned piglets and broilers with three experiments (experiments in vitro, in weaned piglets, in broilers). First, PCR amplification for 16S rDNA of standard bacterial strains (Escherichia coli, Lactobacillus, Bifidobacterium, Salmonell), and 1.2% Agarose gel electrophoresis of 16S rDNA amplified productions were conducted to establish a biological method for detecting and quantifying Escherichia coli, Lactobacillus, Bifidobacterium, Salmonella and bacteria in pure cultures, in vitro and in vivo samples by Alphalmager TM2200 Electrophoresis Gel Figure Image Quantity Analyse System. The Q-PCR method developed had a detection limit per reaction: 102-103 cells for Escherichia coli, 103-104 cells for Lactobacillus, 104-105 cells for Bifidobacterium, 103-104 cells for Salmonella and 102-103 cells for bacteria, which accuracy of the Q-PCR method was acceptable, but need improving. In vitro experiment was conducted to allot 6 treatments of 0, 1, 2, 4, 6 and 8g/L inulin each with 6 replicates for anaerobe incubation 24h at 37℃ and measure pH, NH3, volatile fatty acids and the populations of Escherichia coli, Lactobacillus, Bifidobacterium, Salmonella and bacteria in the culture medium. The results showed that, as inulin level rose from 4g/L to 8g/L in the culture medium, the populations of Bifidobacterium, Lactobacillus and bacteria, and propionic acid content increased (P<0.05). However, the populations of Escherichia coli and pH, NH3 decreased (P<0.05). In piglet experiment, 90 28-day-old weaned piglets were conducted to 5 diets of 0, 0.5, 1.0, 1.5% inulin ± antibiotics for 28 days and blood, intestinal fluid and feces samples were collected to measure pH, bacterial communities, volatile fatty acids and digestive enzyme activities. The results showed that the supplement of 1.0 % inulin could increase the populations of Lactobacillus in cecum and of Bifidobacterium in ileum, cecum and rectum, and propionic acid content in cecum (P<0.05), and decrease pH in cecum, colon and rectum and plasma glucose content (P<0.05). In broiler experiment overfulfilled, 120 21-day-old Arbor Acre broilers were randomly allocated into 4 treatments with 6 replicates / treatment and fed diets each supplemented with 0, 0.2, 0.4 and 0.8% inulin for 28 days. The performance, bacterial communities, digestive enzymes and blood biochemical indexes were measured. The results showed that the populations of Bifidobacterium in ileum rose and pH in plasma, cecum, colon and feces and plasma glucose fell as inulin supplemented (P<0.05). Three experiments identically approved prebiotic action that was to promote the multiplication of Bifidobacterium and restrain the proliferation of Escherichia coli, which changed microbial metabolites compounds and specially increased propionic acid content (P<0.05), dropped pH in plasma and large intestine and blood glucose (P<0.05). It was concluded that inulin could have significant effects on the activities of digestive enzymes such as amylase, trypsin, lipase in pancreas and small intestine and inulinase and cellulase in cecum for the first time.These results can provide good basis for further research of inulin as feed addtive.
结题中文主题词    16S rDNA扩增法；肠道微生物区系；菊粉；断奶仔猪；肉仔鸡
结题英文主题词    PCR method for 16S rDNA; Intestinal bacterial communities; Inulin; Weaned piglet; Broiler

负责人霍贵成项目批准号 30270969 学科代码 C120104
项目名称益生菌定植及其增强免疫机理的研究
申请书摘要
申请书主题词    益生菌定植非特异性免疫
结题中文摘要    项目以益生菌中最为重要的青春双歧杆菌、婴儿双歧杆菌、长双歧杆菌、短双歧杆菌和嗜酸乳杆菌为研究对象，通过益生菌对病原菌的防御功能、对抗消化道逆环境特性、仔猪小肠粘液糖蛋白和模拟人肠道粘膜上皮细胞的粘附定植机理及其免疫功能的研究表明，婴儿双歧杆菌和长双歧杆菌具有良好的抗逆性条件性状；所筛选益生菌对病原菌有一定的拮抗作用；同时粘附试验证实不同菌株双歧杆菌所分泌的粘附素具有菌株差异性，介导双歧杆菌粘附过程可能是不只一种机制在起作用，青春双歧杆菌的粘附素为菌体分泌到培养基中的一种蛋白质，受体为细胞表面的一种糖蛋白；嗜酸乳杆菌的粘附素是菌体细胞壁上的一种蛋白质，其在肠道上皮细胞上的粘附受体为蛋白；经热处理失活后的青春双歧杆菌仍具有粘附能力，而嗜酸乳杆菌基本丧失粘附能力；婴儿双歧杆菌和长双歧杆菌增强正常BALB/C小白鼠的非特异性免疫功能，并具有良好调节肠道菌群能力，婴儿双歧杆菌效果优于长双歧杆菌；青春双歧杆菌与嗜酸乳杆菌菌细胞壁肽聚糖对昆明小鼠免疫功能具有促进作用。
结题英文摘要    The Bifidobacteria adolescent , Bifidobacteria baby, Bifidobacteria tall, Bifidobacteria short and Lactobacillus acidophilus were tested to study defensible function of pathogen, intestine tolerance,the mechanism of adhesion and immunologic enhancement. The results were showed that adhered substances which different Bifidobacteria bacteria excreted had bacterial significance. And there was not only a mechanism. The adhesin of Bifidobacteria adolescent was confirmed to be a certain kind of protein produced and excreted to the media by bacteria, and the receptor on intestinal epithelia should be some kind of glycoprotein. While the adhesin of Lactobacillus. acidopholus is protein on the surface of bacteria cell wall and the receptor is protein. The inactived Bifidobacteria. adolescent still can adhere to the intestinal epithelia while Lactobacillus. acidophilus cann't. The result approved that the adhesin of them were different from each other.In animal experiment , Bifidobacteria baby and Bifidobacteria tall had specific modulation on immune function of normal BALB/C rats.And there was good modulation of intestinal microflora.In comparison of Bifidobacteria tall , Bifidobacteria baby had the advantage .Mice were injected with bacteria suspension or Peptidoglycan and examed the influence on immunity. The result showed that Peptidoglycan could increase the immunity of animals tested.
结题中文主题词    益生菌；定植；非特异性；粘附
结题英文主题词    probiotics, colonization, natural immunity,?adhension

负责人陈庆森项目批准号 30771524 学科代码 C120204
项目名称酪蛋白糖巨肽(CGMP)对小鼠肠粘膜免疫调节作用的研究
申请书摘要    根据我们在酪蛋白糖巨肽(CGMP)的分离纯化技术及对该物质生物活性等方面的前期研究，本研究利用荧光原位杂交技术、免疫组化技术、放射免疫分析、显微观察以及图像分析等方法，对饲喂不同剂量的BALB/c小鼠肠道内各类微生物区系(microflora)分布及消长规律、腹膜巨噬细胞(peritoneal macrophages)的吞噬活性、肠粘膜中S-IgA含量的变化、CD4+/CD8+T细胞比率以及相关细胞因子的变化进行系统的研究，确定和证实CGMP对BALB/c小鼠肠粘膜内各类微生物区系的变化和肠粘膜免疫系统的调控作用；揭示乳源CGMP如何调控肠粘膜的免疫应答过程及其机制。对评价乳源生物活性肽的功能以及人与动物的营养健康具有重要指导意义，也将为用乳源生物活性肽调理、预防和治疗肠道疾病以及对乳品资源的利用与深加工奠定理论基础。
申请书主题词    CGMP，肠粘膜免疫，S-IgA，细胞因子，微生物区系