【作者】 亓宏伟；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 动物肠道内的微生物与动物生长及其健康存在密切的关系,动物所进食的日粮不仅是影响其生产性能的主要因素,也是影响动物肠道内微生物区系的主要因素。动物采食的日粮不但为动物本身提供所需的各种营养物质,同样也是动物肠道内的微生物的发酵底物。蛋白质作为影响动物生产性能和健康状况的最重要的营养物质之一,同样会对动物肠道微生物的组成产生影响。本研究旨在探明不同来源蛋白对动物肠道微生态环境以及肠道结构功能的影响,揭示不同来源蛋白存在差异可能的原因。首先以SD大鼠为动物模型,初步考察不同来源蛋白对大鼠盲肠微生物及代谢产物的影响；然后以21日龄断奶仔猪为模型系统考察不同来源蛋白对生产性能、肠道健康、氨基酸的吸收转运以及肠道微生物的影响；最后将所选大豆分离蛋白与玉米醇溶蛋白分别添加合成氨基酸,考察氨基酸比例以及来源蛋白对断奶仔猪肠道微生物的影响。具体包括以下三个试验：试验一：不同来源蛋白对SD大鼠盲肠微生物及代谢产物的影响为了考察不同来源蛋白对单胃动物肠道微生物及其代谢产物的影响,本试验选择100只SD清洁级雄性断奶大鼠,随机分为5组,每组20只,分别以酪蛋白、大豆分离蛋白、玉米醇溶蛋白为唯一蛋白源配制日粮以及无氮日粮组和三种蛋白按照一定比例(三种蛋白比例为SPI:ZEIN:CAS=65:25:10)进行配比的复合蛋白日粮,进行为期14天的饲养试验,比较不同来源蛋白对SD大鼠盲肠内容物中总细菌、乳酸杆菌、大肠杆菌、双歧杆菌、芽孢杆菌的数量及比例以及微生物代谢产物—挥发性脂肪酸含量的影响。结果表明,酪蛋白显著提高大鼠盲肠中乳酸杆菌、双歧杆菌及芽孢杆菌等有益菌的数量,同时也提高了微生物代谢产物—乙酸、丙酸、丁酸的含量；复合蛋白日粮组和大豆分离蛋白组对益生菌的促进作用仅次于酪蛋白处理组；玉米醇溶蛋白组和无氮日粮组对肠道总细菌以及各种益生菌的促进作用最差,大豆分离蛋白组显著提高了大鼠盲肠大肠杆菌的数量。以上结果表明,不同来源蛋白对SD大鼠盲肠内容物中的微生物数量及比例均产生不同程度的影响,酪蛋白组以及复合蛋白组能够提高大鼠盲肠内容物中总细菌以及乳酸杆菌、双歧杆菌、芽孢杆菌等有益微生物的数量和比例,玉米醇溶蛋白组对有益菌的促进作用最差。试验二：不同来源蛋白对断奶仔猪肠道微生物及肠道健康的影响本试验旨在考察不同来源蛋白对断奶仔猪生产性能、肠道健康、肠道氨基酸转运载体mRNA表达量以及整个肠道(十二指肠、空肠、回肠、盲肠、结肠、直肠)内容物中微生物数量及组成的影响。试验选用21日龄断奶仔猪36头,采用单因子试验设计,随机分为6个处理,每个处理6个重复。六个处理分别饲喂用大豆分离蛋白、玉米醇溶蛋白、酪蛋白以及复合蛋白(三种蛋白比例为SPI:ZEIN:CAS =65:25:10)作为蛋白源配制的半纯合日粮以及玉米豆粕型实用日粮和无氮日粮。试验期14天。试验结果表明：1.日粮配方中不同来源蛋白对断奶仔猪生产性能产生不同的影响,其中,酪蛋白组在日增重方面显著高于大豆分离蛋白组和玉米醇溶蛋白组,其料肉比显著低于大豆分离蛋白组和玉米醇溶蛋白组。玉米醇溶蛋白组生产性能最低。以玉米、豆粕和鱼粉为主要成分的实用日粮组的采食量显著高于其余各半纯合日粮组。2.酪蛋白处理组与实用日粮组显著增加了盲肠、结肠和直肠中短链脂肪酸(乙酸、丙酸、丁酸)的含量,玉米醇溶蛋白组显著增加了后肠挥发性盐基氮的含量。大豆分离蛋白组短链脂肪酸含量介于酪蛋白组和玉米醇溶蛋白组之间。3.酪蛋白组、实用日粮组以及复合蛋白组胃蛋白酶和胰蛋白酶活性显著高于大豆分离蛋白组和玉米醇溶蛋白组,趋势与各处理生产性能一致。4.各处理组血液中白蛋白、球蛋白和总蛋白含量以实用日粮组、酪蛋白组以及复合蛋白组最高,大豆分离蛋白组和玉米醇溶蛋白组次之,无氮日粮组最低。对于血清尿素氮含量则以玉米醇溶蛋白组和大豆分离蛋白组最高,实用日粮组、酪蛋白组以及复合蛋白组次之,无氮日粮组最低。各处理组对血清中的细胞因子IL-2, IL-6无显著影响,大豆分离蛋白组提高了血清中TNF-a的量。5.仔猪各肠段(胃、十二指肠、空肠、回肠、盲肠、结肠)内容物pH值以玉米醇溶蛋白组和无氮日粮组最高,其次是大豆分离蛋白组,实用日粮组、酪蛋白组以及复合蛋白组最低。6.各处理组粗蛋白及氨基酸消化率以酪蛋白组和实用日粮组最高,其次是复合蛋白组和大豆分离蛋白组,玉米醇溶蛋白组最低,趋势与各处理生产性能一致。玉米醇溶蛋白组粗蛋白及氨基酸消化率最低,可能与其不平衡的氨基酸组成相关。7.酪蛋白组、实用日粮组以及复合蛋白组显著提高了小肠(十二指肠、空肠、回肠)的绒毛长度,同时增加了绒毛长度与隐窝深度的比值,各处理组对小肠隐窝深度的影响表现不一,无显著规律性。8.各处理组氨基酸及肽转运载体在小肠(十二指肠、空肠、回肠)粘膜中的表达量以酪蛋白组和实用日粮组最高,各处理对十二指肠CAT-1的表达量无显著影响,氨基酸转运载体与二肽转运载体mRNA表达量以无氮日粮组和玉米醇溶蛋白组最低,各处理对IGF-1 mRNA表达量酪蛋白组和实用日粮组显著高于其余各处理组。9.实用日粮组与酪蛋白组显著提高了各个肠段总细菌以及乳酸杆菌、双歧杆菌、芽孢杆菌等有益菌的数量和比例,显著降低了各个肠段内容物中大肠杆菌的数量和比例。大豆分离蛋白组显著提高了后肠(盲肠、结肠、直肠)大肠杆菌的数量和比例。以上结果表明,优质的的蛋白源—酪蛋白可以提高断奶仔猪的生产性能,降低肠道内的pH值,维持肠道组织形态的健康,增加肠道内有益菌的数量和比例。而植物蛋白,尤其是氨基酸组成不平衡的玉米蛋白则降低断奶仔猪的生产性能,对肠道健康具有一定的破坏作用,不利于动物以及其肠道内有益菌的生长繁殖。试验三：不同来源蛋白平衡氨基酸后对断奶仔猪肠道微生物及肠道健康的影响为进一步探讨不同来源蛋白质对仔猪肠道微生态环境和健康的影响差异是否与其氨基酸组成有关,本试验考察了不同来源蛋白添加合成氨基酸使其氨基酸模式与酪蛋白一致后对肠道微生物、肠道组织形态学以及与肠道健康和氨基酸转运密切相关的基因表达的影响。选用21日龄断奶仔猪36头,随机分为6个处理,分别饲喂以酪蛋白、大豆分离蛋白、玉米醇溶蛋白、大豆分离蛋白添加合成氨基酸、玉米醇溶蛋白添加合成氨基酸为唯一蛋白来源的半纯合日粮以及玉米豆粕型实用日粮。每个处理6头猪,进行为期14天的饲养试验。结果表明,不同来源蛋白添加合成氨基酸后,增加了断奶仔猪的生产性能,降低了空肠和盲肠的pH值,其对肠道环境的改善作用和对有益微生物的促进作用要优于单纯使用大豆分离蛋白和玉米醇溶蛋白作为唯一蛋白源的应用效果,但其改善程度不及动物性蛋白—酪蛋白。CHOP是反映氨基酸平衡状况的重要指标,玉米醇溶蛋白在添加合成氨基酸后,CHOPmRNA的表达量显著下降,表明添加合成氨基酸改善了玉米醇溶蛋白的氨基酸平衡情况。本试验结果表明,不同来源蛋白质对仔猪肠道微生态环境和健康的影响差异与其氨基酸组成有关,平衡氨基酸后可以缩小但不能完全消除蛋白质来源的差异。总之,不同来源蛋白对断奶仔猪肠道微生态环境和健康具有不同的影响,饲喂优质蛋白质有利于改善肠道微生态环境,增加有益菌的数量,提高仔猪生产性能。蛋白质来源差异的部分原因是氨基酸组成和模式不同,平衡氨基酸可以缩小不同来源蛋白质的微生态调解效应的差异。更多还原

【Abstract】 Intestinal microflora plays an important role in animal growth and health. The diet is the main factor for both growth performance and intestinal flora. The protein is one of important nutrients that are essential for growth and intestinal flora. However, what are the effects of different dietary protein sources such as soybean, zein and animal protein on intestinal microflora? What are the effects of different dietary protein sources on microbial quantity and ratio of primary bacteria (eg: Lactobacillus) and what are the effects of different dietary protein sources on weaning piglets growth performance, intestinal flora and amino acid transport? All these questions remain to be studied. Therefore, this study was conducted to answer above questions. Firstly, the effects of different dietary protein sources on cecal microflora in SD rats were investigated; then, the effects of different dietary protein sources on growth performance, gut health and amino acid transport in weaned piglets were researched; thirdly, the effects of selected soya isolate protein and zein diet supplemented with amino acids on intestinal microflora and amino acid transport in weaned piglets were evaluated.Experiment 1:Effects of different protein sources on cecal microflora and metabolites of SD ratsTo determine the effects of different dietary protein sources on cecal microflora and metabolites in monogastric animals, one hundred SD rats were randomly assigned into 5 diet treatments. Diets were respectively formulated with casein, soya protein isolate (SPI), and zein as the only protein source, and Nitrogen free diet (NFD) as well as combined protein (COM) (SPI:casein: zein=65:25:10) diet were applied as controls. All rats were fed for 14 days. The results showed that casein significantly increased the number of total bacteria, Lactobacillus, Bifidobacterium and Bacillus in cecum contents of rats, and significantly lowered the number and ratio of E. coli, and increased the concentrations of microorganism metabolic products:acetic acid, propionic acid and butyric acid. The COM and SPI treatments were lower than casein treatment in the effect of promoting probiotics. The effect of promoting probiotics in zein treatments and NFD were lower than other treatments. The treatment of SPI significantly increased the number arid ratio of E.coli. The treatment of zein was worst in the effect of promoting the role of probiotics, the possible reason might be the unbalance amino acids of zein protein.Experiment 2:Effects of different protein sources on intestinal microflora and intestinal health of weaned pigletsThe aim of this experiment was to approach the effect of different proteins on intestinal microflora, intestinal health and intestinal environment in weaned piglets. Thirty-six piglets were randomly allotted into 6 treatment groups with 6 replicates in each. The SPI, casein, zein and COM were applied as protein source. Regular corn-soybean diet and N-free diet were used as control groups. The trial was conducted for 14 days. The results showed that:1. The treatment of casein was higher than SPI and zein treatments in ADG, and was lower than SPI and zein treatments in F/G. The treatment of zein was lowest in 6 treatments. The ADFI of normal treatment was higher than other treatments.2. The treatments of casein and normal increased the VFA in the cecum, colon and rectum contents of weaned pigs. Zein treatment increased the VBN of cecum, colon and rectum. The SPI treatment was between zein and casein treatment.3. The effects of casein, normal and COM treatments on enzyme activities (units/mg prot) were higher than other treatments in the weaned pigs. The trend was consistent with the performance.4. The serum albumin, globulin and total protein content in the normal, casein and COM treatments were the highest among all groups, and these in the soy protein isolate and zein group were lower. The lowest was NFD treatment. The serum urea nitrogen was higher in zein and SPI treatments. There is no significant different in the serum cytokines IL-2, IL-6 among groups. SPI increased serum TNF-αconcentration.5. The pH-value of zein and NFD treatments was higher than other treatments in all parts of gastrointestine (stomach, duodenum, jejunum, ileum, cesium and colon). The pH-value of casein, COM and normal treatments were lower than other treatments.6. Protein and amino acid digestibilities of casein and normal treatments were higher than other treatments. Zein treatment was lower than other treatments. The trend was consistent with the performance. The possible reason might be the imbalance amino acids of zein.7. Casein, normal and COM treatments significant increased the height of villus and increased the ratio of villus to crypt depth. The crypt depths were not significantly different among groups.8. The expression of amino acid and peptide transportation related genes in casein and normal treatments were higher than other treatments. There was no significant difference in CAT-1 mRNA expression among all treatments. The expression of amino acid and peptide transportation related genes in zein and NFD treatments were lower than other treatments. The expressions of IGF-1 mRNA in casein and normal treatments were higher than other treatments.9. The treatments of normal and Casein significantly increased the number and ratio of Lactobacillus, Bifidobacterium and Bacillus, and significantly reduced the number and ratio of E.coli. The treatment of SPI significantly increased the number and ratio of E.coli. The possible reason was that SPI contained antigen and increased diarrhea of weaned piglets.Casein could enhance the performance of weaned piglets, reduce intestinal pH value, maintain intestinal morphology of health, and increase the number as well as the scale of beneficial bacteria in the intestines. However, the plant protein especially zein impaired the intestinal health and blunted the growth of beneficial bacteria in the gut.Experiment 3:Effects of different protein sources on intestinal microflora and intestinal health of weaned piglets after amino acids supplementation.The aim of this experiment was to approach the effect of different proteins supplemented with amino acid on intestinal microflora, intestinal health and intestinal environment of weaned piglets. Thirty-six piglets were randomly allotted into 6 treatment groups with 6 replicates each. The trial was conducted for 14 days. The results showed that supplementation of amino acids promoted intestinal environment, intestinal health and intestinal microflora, but the improvement was lower than the treatment of casein. CHOP was an important marker indicating the balance of amino acids. The expression of CHOP mRNA was lower after supplementing amino acid to zein diet, which suggested the balance of amino acids was improved after amino acids supplementation in zein diet.In summary, effects of different proteins on intestinal microflora in weaned piglets were distinct. Casein could effectively increase intestinal Lactobacilli, Bifidobacteria and Bacillus, and reduced the number of E. coli. The effect of SPI was between casein and zein treatments. Zein treatment was the worst one. Effect of plant protein was improved after amino acid supplementation on intestinal microflora in weaned piglets. The supplementation of amino acids could partially improve plant protein quality.更多还原