【作者】 刘晶；

【导师】 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 果胶为一类天然高分子化合物,属结构性碳水化合物。苜蓿、羊草、玉米秸以及稻秸等粗饲料中均含有果胶,但以苗蓿中最为丰富。果胶与淀粉虽然同属非纤维性碳水化合物,但发酵特性差异较大。迄今为止有关果胶影响瘤胃发酵、微生物蛋白合成的报道不多,而对果胶影响瘤胃微生物群落结构以及果胶降解菌这类功能微生物的研究与认知更加有限。本研究首先探索的苜蓿日粮与玉米秸日粮下奶牛瘤胃中的差异微生物菌种或菌群,并建立果胶分解菌检测的分子技术,从而获取更多的果胶降解菌信息；然后以湖羊为模式动物,系统研究了以玉米秸为粗饲料的日粮中补充添加果胶对瘤胃发酵、微生物蛋白合成以及微生物菌群结构的影响。一、果胶对瘤胃微生物菌群的影响及果胶菌检测1.饲喂不同果胶含量日粮对奶牛徽生物菌群的影响。本试验检测了苜蓿、玉米秸以及羊草三种粗饲料中的果胶含量,依次为9.3%,2.8%和3.7%(干物质基础)。通过454焦磷酸测序技术分析苜蓿日粮与玉米秸日粮下奶牛瘤胃液中的微生物16S rRNA基因,发现Selenomonas和Treponema菌属包括菌株T.saccharophilum是苜蓿日粮下的特有菌群,Selenomonas ruminantium的比例在苜蓿日粮中也有高于玉米秸日粮的趋势,分析发现T. saccharophilum与S.ruminantium都是降解果胶、淀粉的微生物。2.不同果胶含量粗饲料组合对瘤胃微生物分布的影响。为了验证测序发现的微生物差异是由日粮中果胶或/和淀粉含量差异引起的,本试验分别对苜蓿、玉米秸、果胶、淀粉以及秸秆与果胶、淀粉的组合为底物的体外发酵液中微生物菌群进行PCR-DGGE分析,发现玉米秸与果胶组合的DGGE条带分布与苜蓿组相似度由67.5%增加至72%,而玉米秸与淀粉组合后与苜蓿条带相似度从67.5%降至65%,从微生物学角度证实了果胶是秸秆与苜蓿的重要差异养分之一。对差异条带克隆测序发现了与T. saccharophilum相似度为99%的克隆,进一步证实该菌对果胶降解的特异性。Real-time分析还发现整个Treponema菌属(密螺旋菌菌属)在以淀粉为底物的条件下数量大大降低,而果胶的添加显著促进该菌属的生长(P<0.05),提示了以T. saccharophilum为代表的Treponema菌属都与果胶降解密切相关。而S. ruminantium在果胶或淀粉为底物的发酵液中丰度差异不显著。3.果胶分解菌T. saccharophilum实时定量方法构建与体内外丰度检测。由于迄今缺乏准确、可靠的检测方法,T. saccharophilum在瘤胃中的数量未知。因此,本研究设计了能够特异性扩增T. saccharophilum16S rRNA基因的引物,验证结果表明引物灵敏、可靠,可以应用于未来瘤胃生态研究。利用设计的引物进一步从体外、体内角度检测了T. saccharophilum与不同果胶含量粗饲料的关系。体外结果表明T. saccharophilum在苜蓿以及添加果胶的底物中丰度显著高于玉米秸、羊草以及淀粉为底物(P<0.05)时,而淀粉不支持该菌的生长；体内结果表明T. saccharophilum在苜蓿日粮饲喂的奶牛瘤胃中显著高于羊草、玉米秸以及稻草秸日粮(P<0.05)。通过对比瘤胃其他细菌的丰度发现,T. saccharophilum在瘤胃中的相对丰度较高,提示了这个菌在瘤胃中重要性,并可能扩展今后与瘤胃中的果胶代谢相关的研究。二、果胶对湖羊生产性能、瘤胃发酵和微生物菌群结构的影响1.果胶对体外瘤胃发酵参数与微生物蛋白合成的影响。为了评估果胶与淀粉对瘤胃发酵的影响,首先进行了体外发酵,以700mg的玉米秸与150mg豆粕为底物的基础上设置4个处理：(1)淀粉150mg+果胶Omg(Pe0),(2)淀粉100mg+果胶50mg (Pe50),(3)淀粉50mg+果胶100mg (Pe100),(4)淀粉Omg+果胶150mg (Pe150)。结果表明,果胶体外发酵速率显著高于淀粉(P<0.05),尤其是6h前的早期发酵,此外24h总产气量果胶也显著高于淀粉(P<0.05)。果胶为底物的发酵属于乙酸型,而淀粉发酵属于丙酸型,本试验中乙丙比随着果胶的增加而线性增加。Pe0与Pe50的pH值低于Pe100和Pe150(P<0.05),P150的MCP产量显著高于Pe0(P<0.05),提示果胶可优化发酵速率、pH等瘤胃发酵参数,并促进微生物蛋白合成。2.果胶对湖羊生长性能和瘤胃发酵的影响。为了深入研究果胶对反刍动物生产性能的影响,本试验选取45只健康无病的7月龄公湖羊,按照随机区组设计和同栏大小相近原则,根据体重分为5个处理：基础日粮(精粗比50：50的全混合日粮)为对照组(Ctrl),在基础日粮上分别添加2.5%和5%果胶的低果胶日粮(PeL)和高果胶日粮(PeH),分别添加2.5%和5%淀粉的低淀粉日粮(StL)和高淀粉日粮(StH)。结果发现,添加果胶可以增加采食量,而添加淀粉则线性降低采食量(P<0.05)。日粮PeH的MCP产量在各组中最高(P<0.05),且果胶的添加对MCP的生成有线性增加效应(P<0.01),而淀粉的添加则对MCP产量具有曲线降低效应,即高淀粉日粮反而抑制了MCP的合成。果胶添加线性增加日增重(P<0.05), PeH在各组中日增重最高(P<0.05),与其MCP合成量一致。此外,饲喂Pe日粮可显著降低血清中甘油三脂(TG)水平(P<0.05)。这些结果表明,与淀粉相比,添加果胶有助于瘤胃微生物蛋白合成,改善湖羊的生产性能。3.果胶对湖羊瘤胃微生物菌群结构的影响。为了深入研究与果胶降解相关的瘤胃细菌,采用Real-time以及Miseq高通量测序,深度分析上述试验中各组日粮下湖羊瘤胃微生物菌群分布情况。经过菌门、菌科、菌属以及OTU水平的多重比较分析,证实日粮中添加果胶形成的微生物分布与对照或淀粉处理组有显著差异。实时定量结果表明,瘤胃已知的典型微生物中除了Treponema以及Tsaccharophilum被果胶富集,多数菌偏好淀粉利用。然而,通过高通量测序发现,果胶的添加主要富集了厚壁菌门的微生物成员,这些相关微生物多数为未培养或者尚未有特异引物能够准确定量的微生物,包括该门类下梭菌纲下的Mogibacteriaceae科和Lachnospiraceae (毛螺菌科)以及Erysipelotrichi(产芽胞菌纲)下产芽胞菌科的成员。其中毛螺菌科下Butyrivibrio(丁酸弧菌属)、Coprococcus(粪球菌属)、Moryella以及一未分类属,产芽胞菌科下Holdemania(霍尔德曼氏菌属)、L7A_E11以及Bulleidia属都表现出显著的果胶特异性。综上所述,作为结构性碳水化合物的日粮果胶,其营养效果优于淀粉,能促进瘤胃微生物蛋白合成,进而提高动物生产性能。日粮果胶还可促进瘤胃中Treponema菌属、厚壁菌门微生物的生长,提示这些菌群可能是高果胶含量日粮下参与微生物蛋白生成的关键菌群。更多还原

【Abstract】 Pectin is a natural complex polysaccharides and one of the major components of the primary cell wall of plants, thus it is a structural carbohydrate present in plant feedstuffs of ruminants. Pectins commonly exist in fiber feeds such as alfalfa hays, Chinese wild rye, corn stover and rice straw, with alfalfa especially rich in pectin content. Pectin and starch belong to non-fibrous carbohydrate but with remarkable different fermentation characteristics. Up to date, there seems to be very few studies focused on the effect of pectin on ruminal microbial structure and the knowledge of pectinolytic bacteria in the rumen is very limited. Therefore, the objectives of this study were to evaluate effects of pectin on ruminal fermentation and microbial protein pply and especially, to explore the microbial species and group associated with pectin-rich diets. This study contained two parts. In the first part, specific bacterial species or group associated with pectin-rich alfalfa diet compared in the rumen of (?)y cows was explored and quantitative assay method of specific pectinolytic species was developed. In the second part, the effects of pectin supplementation on rumen fermentation, microbial protein supply and microbial structure in the rumen of Hu sheep was systematically investigated.1. Effects of dietary pectin on ruminal bacteria and detection of pectinolytic bacteria1.1Effects of dietary pectin content on bacterial group in the rumen of dairy cows. The yields of chemical-extracted pectin from alfalfa hay, corn stover and (?)se wild rye were determined to be9.3,2.8and3.7%, respectively. With454pyrosequencing method, the abundance of Selenomonas, Treponema and T. saccharophilum were found to be significant higher in the rumen of dairy cows fed on alfalfa hay as main forage source compared to those fed on corn stover. Pure culture based studies demonstrated that these species are soluble carbohydrates fermenters.1.2Effects of combination of pectin with different feeds on the distribution of ruminal bacteria. To evaluate whether Selenomonas and Treponema are pectinolytic bacteria, in vitro rumen fermentation was conducted using alfalfa, corn stover, pectin, corn starch or their combination as the substrates. The cluster analysis of DGGE banding patterns showed that combination of corn stover with pectin as the substrate resulted in the band patterns being closely grouped with those of alfalfa. On the contrary, combinations of corn stover with starch diverted the pattern away from alfalfa. It is indicated from a microbial ecological aspect that pectin is one of the important nutritional differences that exist between corn stover and alfalfa. The specific bands associated with pectin-rich treatments were identified to be dominated by the members of Treponema genus, with one clone showing99%similarity with T. saccharophilum. Real-time PCR analysis demonstrated that the growth of Treponema genus was remarkably supported by inclusion of pectin but not starch, highlighting their specific ability to degrade pectin. However, the relative abundances of S. ruminantium were not significantly different between the fermentation fluids with pectin and starch as substrate.1.3Development a real-time PCR assay targeting at the ruminal pectinolytic T. saccharophilum to access its abundance in both in vitro and in vivo. Due to lack of a reliable and accurate detection method, the abundance of T. saccharophilum in the rumen has not been well determined. Thus, the specific primers targeting at the16S rRNA gene of T. saccharophilum was developed in the present study and the specificity and accuracy of the primers was confirmed, which might also be used in further ruminal ecological studies. The results obtained in the present study clearly demonstrate that quantification of T. saccharophilum in vivo and in vitro is possible by real-time PCR using our designed primers. In vitro results showed that the population of T. saccharophilum was significantly higher with alfalfa or pectin as substrates than with corn stover or Chinese wild rye (P<0.05). However, addition of starch did not support the growth of T. saccharophilum. Similarly, the in vivo results also showed that the relative abundance of T. saccharophilum in the rumen of cows fed alfalfa hay diet was significantly higher (P<0.05) than those in the rumen of cows fed the diet containing Chinese wild rye, corn stover or rice straw. The population of T. saccharophilum species, particularly with alfalfa diet, is comparable or even higher than those of other common rumen bacterial species. The relatively high proportion of T. saccharophilum in the rumen might expand our knowledge of the importance of this bacterium in the rumen, especially for pectin digestion.2. Effects of pectin on production performance, rumen fermentation and microbial structure in the rumen of Hu sheep2.1Effects of pectin on rumen fermentation parameters and microbial protein synthesis in vitro. An in vitro fermentation was conducted to investigate the effects of pectin and starch on ruminal fermentation. With700mg corn stover and150mg soybean meal as basic substrates, the following treatments were used:(1)150mg corn starch+0mg pectin (PeO),(2)100mg corn starch+50mg pectin (Pe50),(3)50mg corn starch+100mg pectin (Pe100), and (4)0mg corn starch+150mg pectin (Pel50). The rate of gas production increased with the increasing ratio of pectin, with highest value in Pe100and Pe150and lowest in Pe0(P<0.05), especially during the early fermentation before6h. Total gas production in24h was significantly higher with Pe100and Pe150compared to PeO and Pe50. Substituting pectin for starch linearly increased the ratios of acetate to propionate. The pH of fermentation fluid increased with the increasing pectin ratio, significantly lower in PeO and Pe50than in e100or Pe150(P<0.05). The MCP production was highest in Pe150and lowest in Pe0(P<0.05). The results obtained here indicated that inclusion of pectin optimized the fermentation rate, environmental pH and further promoted the MCP production.2.2Effects of pectin on production performance and rumen fermentation of Hu sheep. To evaluate the effects of pectin on animal growth,45male sheep were divided into five groups of nine animals each, and randomly assigned to five dietary treatments with supplementation of0,2.5and5%pectin or corn starch, respectively, to a basal diet consisted of corn stover and concentrate mixture (50:50, DM basis). Dry matter intake was increased quadratically or decreased linearly with the increasing level of pectin or starch, respectively. Increasing level of pectin led to a linear increase (P<0.05) in microbial protein synthesis and daily weight gain, with highest values at5%pectin among all treatments (P<0.05). The TG levels in the serum of sheep fed with pectin diets were significant lower compared to control diet and starch diets (P<0.05).2.3Effects of pectin on the bacterial structure in the rumen of Hu sheep. To obtain a deep understanding of ruminal bacteria involved in pectin digestion, Illumina MiSeq sequencing platform were used to generate large-scale16S rRNA gene sequencing data from genomic DNA of ruminal bacteria associated with the above experimental diets in section2.2. Multi-comparison on phylum, family, genus and OTU level demonstrated that the distribution patterns of bacteria among diets Ctrl, Pe and St were significantly different. Real-time PCR quantification showed that, besides Treponema genus and T. saccharophilum, most of typical rumen bacteria were higher when starch was supplemented. However, with high-throughput sequencing technology, it is found that the majority of bacteria groups that enriched by pectin were remain uncultured or not well studied, most of them belonging to Firmicutes, including Mogibacteriaceae, Lachnospiraceae and Erysipelotrichi. Genus such as Butyrivibrio, Copromccus, Moryella, Holdemania, L7A_E11and Bulleidia were all associated with pectin digestion to a different extent.In summary, compared to starch, pectin is a more desirable carbohydrate, which could promote the rumen MCP synthesis and thus improve the growth performance of ruminants. The growth of ruminal bacteria Treponema and Firmicutes were enhanced by pectin, indicating that they may play an important role in microbial protein synthesis under a pectin-rich diet. A better understanding of microbial populations that accompany dietary differences will expand our knowledge of ecological importance of bacteria in the rumen and may further lead to beneficial strategies to improve ruminant production performance.更多还原