特异性阿拉伯木聚糖降解酶促进肉鸡肠道乳酸菌增殖的机理研究

发布时间：2018-06-18 00:06

本文选题：阿拉伯木聚糖降解酶 + 阿拉伯木聚糖　；参考：《中国农业大学》2017年博士论文

【摘要】：乳酸菌(Lactobacillus)为畜禽肠道重要的共生菌,对于畜禽的肠道健康起着重要作用。小麦是畜禽常用饲料原料之一,但小麦含有大量的阿拉伯木聚糖(arabinoxylan,AX),是小麦中主要的抗营养因子。低聚木糖(xylan oligosaccharides,XoS)是AX的酶解产物,可以有效促进乳酸菌的增殖。研究表明,XOS的益生功能受其结构的影响,但目前关于XOS结构和功能的关系还不清楚,且XOS在乳酸菌内的代谢途径,还缺乏相关报道。因此,为了探明小麦型日粮中是否可以通过添加特异性木聚糖降解酶,降解小麦AX产生XOS,实现促进肉鸡肠道乳酸菌的增殖,从而改善肠道健康的效果,并阐明其机理,本研究设计了以下四个试验:试验一为了研究AX主链降解酶(内切型木聚糖酶))和支链降解酶(阿拉伯呋喃糖苷酶和阿魏酸酯酶)体外酶解木聚糖的协同效应。以可溶性阿拉伯木聚糖(water extractable arabinoxylan,WE-AX)、不可溶性阿拉伯木聚糖(water un-extractable arabinoxylan,WU-AX),榉木木聚糖(beechwoodxylan)为底物时,添加阿拉伯呋喃糖苷酶和阿魏酸酯酶,都可以提高内切型木聚糖酶对底物的酶解作用,提高总还原糖的生成量。与单独添加内切型木聚糖酶相比,阿拉伯呋喃糖苷酶和阿魏酸酯酶分别能与内切型木聚糖在降解WE-AX和WU-AX时发挥协同作用,使还原糖的生成量分别提高24%和17%。以WE-AX为底物时,与对照组相比,同时添加两种支链酶可以使还原糖的生成量提高26%。以WU-AX和榉木木聚糖为底物时,两种支链酶同时添加的连续性协同效应优于同步性协同效应,此时处理后的还原糖产量分别较对照组提高72%和36%。降解WE-AX和WU-AX时,与单独使用内切型木聚糖酶相比,阿拉伯呋喃糖苷酶、阿魏酸酯酶及二者的同时添加,分别可使总XOS的产量提高56.38%、46.06%、137.65%;46.52%、53.03%、94.66%。与葡萄糖和FOS作为碳源时相比,WE-AX和WU-AX来源的XOS均能显著提高稳定期的短乳杆菌的数量(P0.05)。试验二为了探讨AX主链降解酶和支链降解酶在肉鸡体内的协同降解效果,将480只1日龄AA肉公鸡分为5个处理组,饲喂小麦-豆粕配置的日粮,分别为空白对照组、木聚糖酶组、木聚糖酶+阿拉伯呋喃糖苷酶组、木聚糖酶+阿魏酸酯酶组、以及三种酶的同时添加组。研究不同处理对肉鸡生产性能和肠道微生物的影响。试验结果表明,添加木聚糖酶可以增加1-21 d和1-36 d肉鸡的平均日增重,并显著降低肉鸡的料肉比(P0.05);额外添加阿拉伯呋喃糖苷酶和阿魏酸酯酶均可以进一步改善木聚糖酶的效果,提高肉鸡的生长性能。在肉鸡36 d时,与单独添加木聚糖酶组相比,同时添加3种酶制剂可以显著提高肉鸡的平均日增重(P0.05),降低料肉比(P0.05)。此外,在降低36 d肉鸡的空肠食糜粘性和盲肠大肠杆菌数量,提高肉鸡盲肠乳酸菌数量和木聚糖在肉鸡体内的降解率上,2种支链酶均能与木聚糖酶发挥协同增效的作用。试验三为阐明特异性木聚糖酶促进肉鸡肠道乳酸菌增殖的机理。从试验二中所用的肉鸡盲肠中,分离出一株能高效利用XOS的优势乳酸菌,通过16SrDNA技术和生化鉴定方法,鉴定该细菌为乳酸片球菌,命名为BCC-1。de-novo测序技术分析表明,BCC-1包含1条染色体和2个质粒,测序后提交到NCBI数据库,收录号分别为CP018763、CP018764和CP018765。以不添加糖原的培养基为对照组,探讨添加XOS和木糖对细菌基因表达的影响。采用转录组技术分析不同处理的差异基因发现,与空白对照组相比,XOS处理组中有1092个显著差异表达的基因;其中上调表达的基因有559个,下调表达的基因有533个。而XOS与木糖组相比,只有376个差异基因,其中上调的基因有200个,下调的基因有176个。进一步分析差异基因发现,与木糖组相比,XOS处理组中存在5个显著富集的GO term,分别是有机酸的转运、碳水化合物的代谢、磷酸烯醇式丙酮酸依赖的糖磷酸转移酶系统、糖基水解作用以及O-键连接的糖基水解作用。KEGG富集通路分析表明,与木糖组处理组相比,XOS组显著上调了 4个与磷酸化有关的ABC(ATP-binding cassette)转运蛋白基因的表达,同时上调了 8个与糖代谢相关的酶的基因的表达,这些酶主要参与三羧酸循环(tricarboxylic acid cycle,TCA)、丙酮酸代谢以及磷酸戊糖途径。试验四为了验证在饲料中添加Pediococcus acidilactic BCC-1对肉鸡1-21 d肠道健康的改善效果,并阐明其对肉鸡生产性能及肠道微生物的影响。试验采用480只1 d肉鸡,分为4个处理组,分别为对照组,XOS处理组、BCC-1处理组,XOS+BCC-1处理组。21d试验期结束后,测定肉鸡生产性能,肠道形态,盲肠微生物区系以及挥发性脂肪酸。与对照组相比,XOS和BCC-1的添加,均可以提高肉鸡1-21 d的体增重和采食量,显著降低肉鸡的料重比(P0.05)。且XOS及BCC-1的配伍使用可以进一步提高肉鸡的体增重和采食量,并降低料重比。XOS和BCC-1均可以显著降低肉鸡盲肠的pH值(P0.05),而BCC-1的添加,还可以显著降低肉鸡空肠的食糜黏度(P0.05)。与单独添加BCC-1相比,XOS同BCC-1的配伍使用可以进一步降低肉鸡空肠的食糜黏度。与对照组相比,XOS同BCC-1的配伍使用,可以显著的提高肉鸡盲肠食糜中丁酸的含量(P0.05),提高肉鸡回肠绒毛高度(P0.05)和绒毛高度与隐窝深度的比值(P0.01),并降低回肠肌层厚度(P＜0.01)。16SrDNA测序结果表明,XOS和BCC-1都具有提高肉鸡21d盲肠中微生物多样性和厚壁菌门与拟杆菌门比值的作用。盲肠食糜代谢组的结果表明,4个不同处理组的肉鸡盲肠食糜组分差异较大。与对照组相比,添加BCC-1和XOS,分别可以提高肉鸡盲肠中肌醇和4-羟基苯丙酮酸的含量(P0.05)。与单独添加XOS和BCC-1相比,XOS和BCC-1的配伍使用,分别可以提高5-羟基吲哚-3乙酸和葡萄糖酸的含量(P0.05)。以上结果表明,由阿拉伯呋喃糖苷酶、阿魏酸酯酶和内切型木聚糖酶组成的特异性木聚糖降解酶,能有效的降解AX,生成XOC。XOS是通过提高乳酸菌内编码ABC转运蛋白和碳水化合物代谢中的关键酶基因的表达,来促进乳酸菌的增殖,进而改善肉鸡的肠道健康和生产性能。
[Abstract]:Lactic acid bacteria (Lactobacillus), an important symbiotic bacteria in the intestines of livestock and poultry, plays an important role in the intestinal health of livestock and poultry. Wheat is one of the common feed raw materials for livestock and poultry. But wheat contains a large number of Arabia xylan (arabinoxylan, AX). It is the main anti nutritional factor in wheat. Xylan oligosaccharides (XoS) is the enzymatic hydrolysis of AX. The study shows that the probiotic function of XOS is affected by its structure, but the relationship between the structure and function of XOS is not clear, and the metabolic pathways of XOS in lactic acid bacteria are not reported. Degradation of wheat AX to produce XOS to promote the proliferation of lactic acid bacteria in broiler intestinal tract and improve the effect of intestinal health and clarify its mechanism. The following four experiments were designed in this study: one was to study the enzymatic hydrolysis of AX main chain degrading enzyme (endonuclease xylanase) and branched chain degrading enzyme (Arabia furanosidase and ferulic acid ester enzyme) in vitro With soluble Arabia xylan (water extractable arabinoxylan, WE-AX), insoluble Arabia xylan (water un-extractable arabinoxylan, WU-AX), and beech xylan (beechwoodxylan) as the substrate, the addition of Arabia furanosidase and ferulic acid ester enzyme can increase the substrate of the xylanase to the substrate. Compared with the single added xylanase, Arabia furanosidase and ferulic acid ester enzyme can play a synergistic effect on the degradation of WE-AX and WU-AX, respectively, and the production of reducing sugar is increased by 24% and 17%. to WE-AX, respectively, compared with the control group. Adding two kinds of branched chain enzymes can increase the yield of reducing sugar by increasing the amount of 26%. with WU-AX and beech xylan as substrate. The synergistic effect of the two branched chain enzymes at the same time is better than the synergetic synergism. At this time, the yield of reduced sugar after treatment is increased by 72% and 36%. to WE-AX and WU-AX respectively compared with the control group. Compared with glucosidase, the simultaneous addition of furanosidase, ferulosidase and two in Arabia can increase the yield of total XOS by 56.38%, 46.06%, 137.65%, 46.52%, 53.03%, compared with glucose and FOS as carbon sources, XOS of WE-AX and WU-AX sources can significantly increase the number of Lactobacillus brevis (P0.05) in the stable period (P0.05). Test two for the purpose of discussion. The synergistic degradation effect of AX main chain degrading enzyme and branched chain degrading enzyme in broilers, 480 1 day old AA meat roosters were divided into 5 treatment groups, and the diet of wheat soybean meal was fed to the blank control group, xylanase group, xylanase + Arabia furanosidase group, xylanase + feruloesterase group and three enzymes simultaneously. The experimental results showed that adding xylanase could increase the average daily weight gain of 1-21 D and 1-36 D broilers and significantly lower the ratio of meat and meat (P0.05) to broilers, and the addition of Arabia furanosidase and ferulic acid esterase could further improve the efficiency of xylanase. The growth performance of broilers was improved. Compared with the individual adding xylanase group at 36 D, the average daily weight gain (P0.05) and the ratio of meat and meat (P0.05) were significantly increased by the addition of 3 enzymes, and the number of lactobacilli and the number of lactobacilli in the cecum of broilers were increased and the number of Lactobacillus in the broilers and the number of Lactobacillus in the broilers were increased. On the degradation rate of chitosan in broilers, 2 kinds of branched chain enzymes can all play synergistic effects with xylanase. Experiment three is to elucidate the mechanism of specific xylanase to promote the proliferation of lactic acid bacteria in broiler intestinal tract. From the broiler cecum used in the second experiment, a dominant lactic acid bacteria which can efficiently use XOS is isolated by 16SrDNA technology and birth. The method of identification was identified as Staphylococcus Lactococcus, which was named as BCC-1.de-novo sequencing technology. The analysis showed that BCC-1 contained 1 chromosomes and 2 plasmids. After sequencing, the bacteria were submitted to the NCBI database, and the numbers were CP018763, CP018764 and CP018765. were used as the control group without glycogen adding, and the gene table of XOS and xylose was added to the bacterial gene table. There were 1092 significant differentially expressed genes in the XOS treatment group compared with the blank control group. There were 559 genes up to up and 533 genes down regulated, while XOS and xylose group had only 376 differentially different genes, of which there were 200 genes up to be up to 200. There are 176 down-regulated genes. Further analysis of differential gene discovery, compared with the xylose group, there are 5 significant enriched GO term in the XOS treatment group, which are the transport of organic acids, carbohydrate metabolism, the phosphoenolpyruvate dependent glycosyltransferase system, the glycosyl hydrolysis and the glycosyl hydrolysis of the O- bond,.K EGG enrichment pathway analysis showed that, compared with the xylose group, the XOS group significantly increased the expression of 4 ABC (ATP-binding cassette) transporter genes related to phosphorylation, and increased the expression of 8 genes related to glucose metabolism. These enzymes were mainly involved in the three carboxylic acid cycle (tricarboxylic acid cycle, TCA), pyruvate generation. In order to verify the improvement effect of Pediococcus acidilactic BCC-1 on the intestinal health of broiler 1-21 D, and to clarify its effect on the production performance of Broilers and the intestinal microorganism, 480 1 D broilers were divided into 4 treatment groups, the control group, the BCC-1 treatment group, the BCC-1 treatment group, XOS+, XOS+, XOS+, XOS+, XOS+, XOS+, XOS+, and XOS+. After the end of the.21d test period of BCC-1 treatment group, the production performance, intestinal morphology, cecum microbial flora and volatile fatty acids were measured. Compared with the control group, the addition of XOS and BCC-1 could increase the weight gain and feed intake of 1-21 D in broilers, and significantly reduce the ratio of feed to weight (P0.05). And the compatibility of XOS and BCC-1 can be further proposed. The weight gain and feed intake of the high broiler, and the lower feed weight than.XOS and BCC-1, can significantly reduce the pH value of the cecum of broilers (P0.05), while BCC-1 addition can significantly reduce the chying viscosity (P0.05) of the broiler jejunum. Compared with the single addition of BCC-1, the compatibility of XOS with BCC-1 can further reduce the viscosity of chic in broiler jejunum. The combination of XOS and BCC-1 can significantly increase the content of butyric acid (P0.05) in broiler checum, increase the ratio of the height of the chorionic villus (P0.05) and the height of the chorionic villus to the depth of the recess (P0.01), and reduce the thickness of the ileum (P < 0.01).16SrDNA sequencing. The results show that XOS and BCC-1 all have the enhancement in the middle intestine of broiler 21d. The results of the cecum metabolism group showed that the chylum chylic components of broilers with 4 different treatments were different. Compared with the control group, adding BCC-1 and XOS could increase the content of inositol and 4- hydroxypyruvic acid (P0.05) in the cecum of broiler, respectively, and the addition of XOS and BCC-1 alone. Compared with the compatibility of XOS and BCC-1, the content of 5- hydroxyl indole -3 acetic acid and gluconic acid (P0.05) can be increased respectively. The above results show that the specific xylan degrading enzyme composed of furanosidase, feruloesterase and endogenous xylanase in Arabia can effectively degrade AX, and the formation of XOC.XOS is by improving the encoded ABC in lactic acid bacteria. The expression of key enzymes genes in transporters and carbohydrate metabolism promotes the proliferation of lactic acid bacteria and improves the intestinal health and performance of broilers.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831.5

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