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30170687

负责人顾宪红项目批准号  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