本文選題：阿拉伯木聚糖降解酶 + 阿拉伯木聚糖��； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：乳酸菌(Lactobacillus)為畜禽腸道重要的共生菌,對(duì)于畜禽的腸道健康起著重要作用。小麥?zhǔn)切笄莩Ｓ蔑暳显现?但小麥含有大量的阿拉伯木聚糖(arabinoxylan,AX),是小麥中主要的抗?fàn)I養(yǎng)因子。低聚木糖(xylan oligosaccharides,XoS)是AX的酶解產(chǎn)物,可以有效促進(jìn)乳酸菌的增殖。研究表明,XOS的益生功能受其結(jié)構(gòu)的影響,但目前關(guān)于XOS結(jié)構(gòu)和功能的關(guān)系還不清楚,且XOS在乳酸菌內(nèi)的代謝途徑,還缺乏相關(guān)報(bào)道。因此,為了探明小麥型日糧中是否可以通過(guò)添加特異性木聚糖降解酶,降解小麥AX產(chǎn)生XOS,實(shí)現(xiàn)促進(jìn)肉雞腸道乳酸菌的增殖,從而改善腸道健康的效果,并闡明其機(jī)理,本研究設(shè)計(jì)了以下四個(gè)試驗(yàn):試驗(yàn)一為了研究AX主鏈降解酶(內(nèi)切型木聚糖酶))和支鏈降解酶(阿拉伯呋喃糖苷酶和阿魏酸酯酶)體外酶解木聚糖的協(xié)同效應(yīng)。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),櫸木木聚糖(beechwoodxylan)為底物時(shí),添加阿拉伯呋喃糖苷酶和阿魏酸酯酶,都可以提高內(nèi)切型木聚糖酶對(duì)底物的酶解作用,提高總還原糖的生成量。與單獨(dú)添加內(nèi)切型木聚糖酶相比,阿拉伯呋喃糖苷酶和阿魏酸酯酶分別能與內(nèi)切型木聚糖在降解WE-AX和WU-AX時(shí)發(fā)揮協(xié)同作用,使還原糖的生成量分別提高24%和17%。以WE-AX為底物時(shí),與對(duì)照組相比,同時(shí)添加兩種支鏈酶可以使還原糖的生成量提高26%。以WU-AX和櫸木木聚糖為底物時(shí),兩種支鏈酶同時(shí)添加的連續(xù)性協(xié)同效應(yīng)優(yōu)于同步性協(xié)同效應(yīng),此時(shí)處理后的還原糖產(chǎn)量分別較對(duì)照組提高72%和36%。降解WE-AX和WU-AX時(shí),與單獨(dú)使用內(nèi)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時(shí)添加,分別可使總XOS的產(chǎn)量提高56.38%、46.06%、137.65%;46.52%、53.03%、94.66%。與葡萄糖和FOS作為碳源時(shí)相比,WE-AX和WU-AX來(lái)源的XOS均能顯著提高穩(wěn)定期的短乳桿菌的數(shù)量(P0.05)。試驗(yàn)二為了探討AX主鏈降解酶和支鏈降解酶在肉雞體內(nèi)的協(xié)同降解效果,將480只1日齡AA肉公雞分為5個(gè)處理組,飼喂小麥-豆粕配置的日糧,分別為空白對(duì)照組、木聚糖酶組、木聚糖酶+阿拉伯呋喃糖苷酶組、木聚糖酶+阿魏酸酯酶組、以及三種酶的同時(shí)添加組。研究不同處理對(duì)肉雞生產(chǎn)性能和腸道微生物的影響。試驗(yàn)結(jié)果表明,添加木聚糖酶可以增加1-21 d和1-36 d肉雞的平均日增重,并顯著降低肉雞的料肉比(P0.05);額外添加阿拉伯呋喃糖苷酶和阿魏酸酯酶均可以進(jìn)一步改善木聚糖酶的效果,提高肉雞的生長(zhǎng)性能。在肉雞36 d時(shí),與單獨(dú)添加木聚糖酶組相比,同時(shí)添加3種酶制劑可以顯著提高肉雞的平均日增重(P0.05),降低料肉比(P0.05)。此外,在降低36 d肉雞的空腸食糜粘性和盲腸大腸桿菌數(shù)量,提高肉雞盲腸乳酸菌數(shù)量和木聚糖在肉雞體內(nèi)的降解率上,2種支鏈酶均能與木聚糖酶發(fā)揮協(xié)同增效的作用。試驗(yàn)三為闡明特異性木聚糖酶促進(jìn)肉雞腸道乳酸菌增殖的機(jī)理。從試驗(yàn)二中所用的肉雞盲腸中,分離出一株能高效利用XOS的優(yōu)勢(shì)乳酸菌,通過(guò)16SrDNA技術(shù)和生化鑒定方法,鑒定該細(xì)菌為乳酸片球菌,命名為BCC-1。de-novo測(cè)序技術(shù)分析表明,BCC-1包含1條染色體和2個(gè)質(zhì)粒,測(cè)序后提交到NCBI數(shù)據(jù)庫(kù),收錄號(hào)分別為CP018763、CP018764和CP018765。以不添加糖原的培養(yǎng)基為對(duì)照組,探討添加X(jué)OS和木糖對(duì)細(xì)菌基因表達(dá)的影響。采用轉(zhuǎn)錄組技術(shù)分析不同處理的差異基因發(fā)現(xiàn),與空白對(duì)照組相比,XOS處理組中有1092個(gè)顯著差異表達(dá)的基因;其中上調(diào)表達(dá)的基因有559個(gè),下調(diào)表達(dá)的基因有533個(gè)。而XOS與木糖組相比,只有376個(gè)差異基因,其中上調(diào)的基因有200個(gè),下調(diào)的基因有176個(gè)。進(jìn)一步分析差異基因發(fā)現(xiàn),與木糖組相比,XOS處理組中存在5個(gè)顯著富集的GO term,分別是有機(jī)酸的轉(zhuǎn)運(yùn)、碳水化合物的代謝、磷酸烯醇式丙酮酸依賴(lài)的糖磷酸轉(zhuǎn)移酶系統(tǒng)、糖基水解作用以及O-鍵連接的糖基水解作用。KEGG富集通路分析表明,與木糖組處理組相比,XOS組顯著上調(diào)了 4個(gè)與磷酸化有關(guān)的ABC(ATP-binding cassette)轉(zhuǎn)運(yùn)蛋白基因的表達(dá),同時(shí)上調(diào)了 8個(gè)與糖代謝相關(guān)的酶的基因的表達(dá),這些酶主要參與三羧酸循環(huán)(tricarboxylic acid cycle,TCA)、丙酮酸代謝以及磷酸戊糖途徑。試驗(yàn)四為了驗(yàn)證在飼料中添加Pediococcus acidilactic BCC-1對(duì)肉雞1-21 d腸道健康的改善效果,并闡明其對(duì)肉雞生產(chǎn)性能及腸道微生物的影響。試驗(yàn)采用480只1 d肉雞,分為4個(gè)處理組,分別為對(duì)照組,XOS處理組、BCC-1處理組,XOS+BCC-1處理組。21d試驗(yàn)期結(jié)束后,測(cè)定肉雞生產(chǎn)性能,腸道形態(tài),盲腸微生物區(qū)系以及揮發(fā)性脂肪酸。與對(duì)照組相比,XOS和BCC-1的添加,均可以提高肉雞1-21 d的體增重和采食量,顯著降低肉雞的料重比(P0.05)。且XOS及BCC-1的配伍使用可以進(jìn)一步提高肉雞的體增重和采食量,并降低料重比。XOS和BCC-1均可以顯著降低肉雞盲腸的pH值(P0.05),而B(niǎo)CC-1的添加,還可以顯著降低肉雞空腸的食糜黏度(P0.05)。與單獨(dú)添加BCC-1相比,XOS同BCC-1的配伍使用可以進(jìn)一步降低肉雞空腸的食糜黏度。與對(duì)照組相比,XOS同BCC-1的配伍使用,可以顯著的提高肉雞盲腸食糜中丁酸的含量(P0.05),提高肉雞回腸絨毛高度(P0.05)和絨毛高度與隱窩深度的比值(P0.01),并降低回腸肌層厚度(P＜0.01)。16SrDNA測(cè)序結(jié)果表明,XOS和BCC-1都具有提高肉雞21d盲腸中微生物多樣性和厚壁菌門(mén)與擬桿菌門(mén)比值的作用。盲腸食糜代謝組的結(jié)果表明,4個(gè)不同處理組的肉雞盲腸食糜組分差異較大。與對(duì)照組相比,添加BCC-1和XOS,分別可以提高肉雞盲腸中肌醇和4-羥基苯丙酮酸的含量(P0.05)。與單獨(dú)添加X(jué)OS和BCC-1相比,XOS和BCC-1的配伍使用,分別可以提高5-羥基吲哚-3乙酸和葡萄糖酸的含量(P0.05)。以上結(jié)果表明,由阿拉伯呋喃糖苷酶、阿魏酸酯酶和內(nèi)切型木聚糖酶組成的特異性木聚糖降解酶,能有效的降解AX,生成XOC。XOS是通過(guò)提高乳酸菌內(nèi)編碼ABC轉(zhuǎn)運(yùn)蛋白和碳水化合物代謝中的關(guān)鍵酶基因的表達(dá),來(lái)促進(jìn)乳酸菌的增殖,進(jìn)而改善肉雞的腸道健康和生產(chǎn)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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S831.5

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