【作者】 金龙；

【导师】 张永根；

【作者基本信息】 东北农业大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 紫色达利菊（Petalostemun purpureum）是一种适应性极强,适口性较好,并广泛分布于北美草原上的一种豆科植物。紫色达利菊含有较高的缩合单宁并具有极强的抗致病性大肠杆菌的能力。但关于其缩合单宁对大肠杆菌的抑菌活性及可能机制,是否具有保护植物蛋白过瘤胃的作用以及对瘤胃微生物的影响并不清楚。因此,设计三个实验来研究紫色达利菊缩合单宁对反刍动物的影响:实验一、紫色达利菊所含缩合单宁对肉牛后肠大肠杆菌抑制能力及其作用机理。在致病性大肠杆菌O157:H7的体外培养基中加入十一种不同植物缩合单宁（添加量: 400μg/ml）,结果发现仅紫色达利菊所含缩合单宁表现出较强的抗致病性大肠杆菌O157:H7生物活性。同时,在厌氧条件下添加200μg/ml该缩合单宁表现出对大肠杆菌（ATCC 25922）的抑制作用。穿透电子显微镜观察发现大肠杆菌细胞膜在缩合单宁的作用下增厚,扫描电子显微镜观察发现当添加较高浓度缩合单宁时,大肠杆菌菌体表面被一层膜外物质包裹。大肠杆菌细胞膜的通透性在200μg/ml缩合单宁添加量下显著降低。为进一步确定紫色达利菊所含缩合单宁对反刍动物后肠消化及后肠大肠杆菌的影响,分别在2009和2010年进行放牧试验,试验采用随机区组设计,处理为对照组（不含紫色达利菊以雀麦草为主的草地）,试验组Ⅰ（以紫色达利菊与雀麦草为主的草地）和试验组Ⅱ（以紫色达利菊与针茅草和雀麦草混合的草地）。于2009和2010年夏季和秋季在试验地采集植物样品和粪样,用于分析植物和粪样的化学成分及粪样中的大肠杆菌数量。缩合单宁含量在试验组Ⅰ和Ⅱ显著高于对照组（P<0.01）。试验组Ⅰ和Ⅱ动物粪样中大肠杆菌的数量在2009年秋季和2010年夏秋季放牧中显著低于对照组（P<0.05）。秋季放牧中,对照组粪样pH、总氮、有机物消化率、氨态氮和挥发性脂肪酸显著高于试验组Ⅰ和Ⅱ而乙酸/丙酸低于对照组（P<0.05）。夏季放牧动物粪样各指标差异不显著。因此,紫色达利菊所含缩合单宁可能通过改变大肠杆菌细胞膜或与细胞膜相互作用来抑制大肠杆菌的生长,从而减少肉牛粪便中的大肠杆菌数量。实验二、紫色达利菊及其与禾本科和豆科牧草混合氮代谢规律的研究。为确定紫色达利菊中缩合单宁的分布和体外瘤胃降解率,分别在营养生长期和盛花期采集整株植物,测定叶、茎和花所占的比例以及总酚,可提取单宁、蛋白结合单宁和纤维结合单宁的含量。花中总缩合单宁的含量显著高于茎和叶的含量（P<0.05）。营养生长期叶中的总酚和总缩合单宁都高于盛花期。紫色达利菊不同部位可提取单宁的量远高于蛋白结合单宁和纤维结合单宁。盛花期整株植物缩合单宁含量高于营养生长期（P<0.001）。体外试验采用2 x 2析因试验设计,将不同草地采集的两期植物放入三套DAISYII发酵器中,同时每个发酵器中的两个发酵罐添加聚乙二醇（单宁活性抑制剂）,分别在0, 1, 2, 4, 8, 12, 24, 48和72 h取样。营养生长期具有较高的真干物质和真氮消化率,同时干物质和氮素中潜在降解部分（b）均高于盛花期。营养生长期干物质中快速降解部分（a）要高于盛花期（P<0.001）,而氮素中的快速降解部分却低于盛花期（P<0.01）。添加聚乙二醇后,营养生长期干物质的慢速降解部分速率常数（C）增加（P<0.01）但对盛花期无影响。添加聚乙二醇增加盛花期真氮素潜在降解部分,对营养生长期无影响。两期紫色达利菊体外12h残留饲料δ¹⁵N的含量在添加聚乙二醇后显著增加（P<0.01）。这表明紫色达利菊在营养生长期拥有较高的瘤胃消化率而在盛花期其缩合单宁对氮素的消化率有一定影响,同时两期植物所含缩合单宁均在消化过程中对微生物的附着有一定影响。但总的来看,高缩合单宁对紫色达利菊真干物质和真氮的消化率影响不大。为确认紫色达利菊与冷季禾本科植物混合对瘤胃发酵和菌体蛋白合成的影响。测定了紫色达利菊与混合冷季植物样品（冷季植物以雀麦草为主）,并用缩合单宁含量在7, 14, 29和42 g /kg DM的样品进行体外发酵试验。试验采用4×2析因试验设计,重复两次。δ¹⁵N硫酸氨用来测定菌体蛋白合成效率。添加聚乙二醇并未对最大产气量以及延迟期产生影响,但能够增加产气率（P<0.01）。随着紫色达利菊含量的增加,12和48h体外真干物质消化率均有所提高（P<0.01）但菌体蛋白产量在12h有所下降（P<0.01）。添加聚乙二醇增加12h真干物质消化率（P<0.01）。随着缩合单宁含量的增加,添加聚乙二醇增加氨态氮和支链挥发性脂肪酸的含量并降低菌体蛋白的合成效率（P<0.01）。这表明冷季植物中混合紫色达利菊能够增加瘤胃降解率。含量为4.2%的缩合单宁对干物质降解率无影响但能增加菌体蛋白合成效率。为了确定紫色达利菊所含缩合单宁对保护豆科植物蛋白不被微生物降解成氨态氮的能力,同时确定对甲烷气体产量以及瘤胃微生物的影响。试验采用5×2析因试验设计,用含δ¹⁵N标记的苜蓿与紫色达利菊组成五个比例:100:0、75: 0、50:50、25: 75和0:100,同时添加或不添加聚乙二醇,重复三次。利用Ammoniaδ¹⁵N diffusion技术和Real-time PCR测定苜蓿蛋白向氨态氮中的转化效率和微生物的菌体拷贝数。随着紫色达利菊比例的增加,添加聚乙二醇能够显著提高氨态氮和氨态氮中δ¹⁵N含量以及苜蓿δ¹⁵N向氨态氮的转化效率（P<0.01）。添加聚乙二醇对48h内消耗单位干物质的甲烷气体产量无影响。12h时,三种纤维降解菌,产琥珀酸丝状杆菌,瘤胃白球菌和瘤胃黄球菌随着紫色达利菊的比例增加而降低但差异不显著。非纤维降解菌中甲烷菌,瘤胃链球菌和嗜淀粉瘤胃杆菌的拷贝数同样随着紫色达利菊的比例增加而降低但差异不显著。这表明,缩合单宁能够减少瘤胃发酵氨态氮的浓度,同时减少混合苜蓿氮向氨态氮的转化,从而保护苜蓿中的氮不以氨态氮的形式损失。实验三、缩合单宁对主要瘤胃微生物的影响以及微生物对缩合单宁的适应性微生物纯培养试验选用3种纤维降解菌并在含缩合单宁0和25μg/ml培养液中以纤维素为底物,在试验开始前适应21天,然后在含缩合单宁0、75、150、300和450μg/ml中进行滤纸消化试验。同时选用四种非纤维降解菌在含缩合单宁0和50μg/ml中培养并适应15天,然后添加0、100、200、400和600μg/ml缩合单宁并测定生长曲线。当缩合单宁含量为75μg/ml时,降低产琥珀酸丝状杆菌,瘤胃白球菌和瘤胃黄球菌的滤纸消化率（P <0.01）。扫描电镜观察结果发现当缩合单宁含量高于150μg/ml时,纤维降解菌停止生长,纤维表面出现大量絮状物质。而非纤维降解菌中,瘤胃普雷沃氏菌,嗜淀粉瘤胃杆菌以及瘤胃链球菌对缩合单宁比较敏感,在含量为200μg/ml即显著改变其生长曲线,而反刍动物半月形单胞菌在缩合单宁含量为600μg/ml仍能较好的生长。试验选用三种纤维降解菌和四种非纤维降解菌均未表现出对缩合单宁的适应性。因此,高剂量缩合单宁对瘤胃微生物有抑制作用并影响纤维降解菌的附着。综上所述,紫色达利菊是一种高缩合单宁、高蛋白的豆科植物,其缩合单宁能够通过与细胞膜相互作用抑制大肠杆菌,并能够降低反刍动物后肠大肠杆菌的数量。同时,紫色达利菊与其它冷季草地植物混合后能够提高真干物质消化率。紫色达利菊与苜蓿混合时,其所含缩合单宁具有保护苜蓿蛋白不被瘤胃微生物降解成氨态氮的能力,但低浓度的缩合单宁就可能造成纤维降解菌的纤维附着能力受抑制。更多还原

【Abstract】 The native legume Purple prairie clover （PPC, Petalostemon purpureum） is well adapted to the prairie region and is considered an important palatable component of prairie hay. PPC contained high concentration of condensed tannin （CT） that possesses strong antimicrobial activity against Escherichia coli O157:H7. Therefore, PPC could be a valuable diet component for sustainable beef production and food safety. However, there is no information available on the nutritive value of PPC and the effects of PPC tannin on nutrient metabolism of ruminant. The overall objectives of this research included （1） evaluating the in vivo effect of incorporating PPC into mixed forages on the reduction of fecal shedding of E. coli and possible antibiotic mechanism. （2）Determine the effect of condensed tannin on PPC two growth stages rumen nitrogen digestibility and on rumen microbial protein synthetic efficiency by mixed cool season grass. Meanwhile,δ¹⁵N alfalfa mixed PPC were used for assess of condensed tannin capacity in reducing nitrogen from alfalfa to ammonia, and （3） rumen bacteria reaction by condensed tannin and adaptation were evaluated by pure culture technique.For further understanding the effect of CT on E. coli, In vitro study was conducted to assess the inhibition of Condensed tannins isolated from PPC on E. coli or E. coli O157:H7 and possible mechanism. Tannin-mediated alterations in E. coli cell walls were detected by transmission electron microscopy （TEM）, and scanning electron microscopy （SEM） revealed large amounts of extra cell material present on E. coli. The permeability of bacteria membrane was reduced when CT were added at levels of 200μg/ml. Grazing studies were conducted at Swift Current, Saskatchewan during two seasons （summer and fall） in two consecutive years （2009 and 2010） to assess the effect of including PPC in cool season pastures on fecal shedding of E.coli in beef cattle. Twenty five steers were allocated into five paddocks distributed in three treatments. One paddock containing pure brome grass （Check; C）, two paddocks （Simple） and two paddocks （Complex）. Purple prairie clover was mainly in vegetative/early flowering stage during summer and in later flowering/early seeding stage during fall. Fecal samples were collected from rectum of the cattle by hand grabbing. Fecal samples collected in 2010 were mixed for each animal into a single sample for summer and fall grazing periods and analyzed for organic matter （OM）, total N, ammonia-N （NH3-N） and volatile fatty acids （VFA）. Check and PPC mixed plants collected from all paddocks were harvested during summer and fall periods for CT analysis. Concentration of CT in PPC mixed pasture was higher （P < 0.01） in fall than in summer. Compared to the Check, counts of E. coli in feces of cattle grazing pasture containing PPC was lower （P < 0.05） in fall for both years, and in summer during 2010. There was no difference in fecal E. coli counts between cattle grazing the simple and complex PPC-containing pastures in 2009 and 2010. Fecal pH, total N, NH3-N, VFA and acetate: propionate ratio （A: P） did not change during summer grazing in 2010. . However, cattle grazing C in fall had higher （P < 0.05） pH, N, NH3-N, VFA, and lower （P < 0.05） A:P than cattle grazing simple and complex pastures containing PPC. Determination of microbial population revealed that total 16S rDNA gene copies were higher in C, but not significant. Amount of 16S rDNA gene copies of Fibrobacter succinogenes, Ruminococcus albus, and Ruminobacter amylophilus in fecal samples from animals grazing C or PPC were low but not different. Other main rumen bacteria species （Ruminococcus flavefaciens, Prevotella bryantii, Streptococcus bovis and Selenomonas ruminantium） were not detectable. These results suggest that inhibitory effects of CT on E. coli are related to shifts in the cell membrane. Incorporation of PPC into forage has potential to reduce the prevalence of fecal E. coli.For understanding the chemical changes in PPC at different growth stage. The chemical composition of the whole plant and condensed tannin content from leaf, stem and flower were determined in two stages. Whole plants of PPC were harvested from artificial pastures at Swift Current, SK at vegetative （VEG） and full-flowering/early seeding （FL） stages. Proportions of leaf, stem and flower were determined after freeze drying. Whole plants were analyzed for OM, total N, neutral detergent fibre （NDF） and acid detergent fibre （ADF）. Leaf, stem, flower and whole plant were also analyzed for total phenolics, total tannins and condensed tannin （CT）. Condensed tannins were detected in all tissues （i.e. leaf, stem and flower） of the plant, with flower containing the highest （198-213 g/kg DM） and stem the lowest （17-18 g/kg DM） contents. Concentrations of total phenolics, total extractable tannins and total CT （extractable and protein- and fibre- bound） in leaf were higher （P<0.01） in VEG than FL stage, but similar in stem and flowers at both growth stages.The following three in vitro were conducted to evaluate the effect of condensed tannin on rumen fermentation and nitrogen digestion by mixed cool season grass andδ¹⁵N lablled alfalfa. The first In vitro experiment was conducted to assess the effects of condensed tannins （CT） on the ruminal degradability of PPC by using three DAISYII fermentor units. Whole PPC plants were harvested at vegetative （VEG） and full-flowering/early seeding （FL） stages from pastures located at three different sites.δ¹⁵N labelled ammonium sulfate was included in the inoculum to assess microbial protein synthesis and feed colonization. Half of the jars in each unit were supplemented with polyethylene glycol （PEG）, yielding a 2 x 2 factorial arrangement of treatments in each unit. Plants harvested at VEG stage had higher （P<0.001） TDMD, total nitrogen degradability （TND） and potential degradable fraction （b） of DM and N than those harvested at FL stage. Inclusion of PEG increased （P<0.01） TND and the potentially degradable N fraction of PPC harvested at FL, but not at VEG stage.Effect of PPC tannins on ruminal fermentation and ture dry matter disappearance of mixed forages from grazing trial were determined in vitro （48-h batch culture） by incubating mixtures of PPC and cool season grasses containing 7, 14, 29 and 42 g CT/kg DM with mixed rumen microbes.δ¹⁵N labelled ammonium sulfate was added to quantify microbial protein （MP） synthesis. Polyethylene glycol （PEG） was included in half of the vials for each mixture yielding a 4 x 2 factorial arrangement of treatments. Substrate, PEG and substrate x PEG interaction had no effect on potential gas production （A） or Lag time. However, rate of gas production was increased （P<0.05） by the inclusion of PEG. As the proportion of PPC increased in the forage mixtures, true dry matter disappearance （TDMD） increased （P < 0.01） at both 12 and 48-h incubation, whereas efficiency of MP synthesis （mg/g truly digested DM） decreased （P<0.01） at 12 but not at 48 of the incubation. Inclusion of PEG increased （P<0.01） TDMD at 12-h but reduced （P<0.01） the efficiency of MP synthesis at 12 and 48h incubation. As the concentration of CT increased, efficacy of PEG treatment in increasing ammonia accumulation and decreasing MP synthesis was increased. The results indicated that incorporation of PPC into cool season grasses improved ruminal digestibility. Condensed tannins in PPC/grasses mixture at concentrations up to 42 g/kg DM had no negative effects on the extent of DM digestibility, but increased MP synthesis.Effect of PPC on retard of alfalfa protein transferred into ammonia, methane production and rumen bacteria were determined in vitro （48-h batch culture） by incubating mixtures of PPC andδ¹⁵N labeled alfalfa at different ratios （100:0, 75: 0, 50:50, 25: 75, and 0:100）. Half of the vials were supplemented with polyethylene glycol （PEG）, yielding a 5 x 2 factorial arrangement of treatments in each batch culture. The experiment was conducted at three time and incubations were carried out at 6, 12, 24 and 48h. Determination of alfalfa protein conversion to ammonia and changes in main ruminal bacteria were detected usingδ¹⁵N diffusion and real-time polymerase chain reaction techniques, respectively. Inclusion of PEG did not affect methane produced per unit of digested DM （DDM）. As the tannin concentration increased, addition of PEG increased （P<0.01） ammonia and NH3-15N accumulation, and the amount of 15N from alfalfa converted into ammonia. Bacterial gene copies of cellulolytic （Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus）, non-cellulolytic （Streptococcus bovis and Ruminobacter amylophilus） bacteria and Archae were reduced as PPC ratio increased, but these changes were statistically similar. Overall, the alfalfa protein can be protected by CT from PPC to convert into ammonia, but the CT also has a negative effect on ruminal bacteria.Our previous study showed PPC contained high concentration of tannins. However, the ecological role of PPC in the mixed forage pasture and the effects of PPC tannins on the nutritive value of mixed forages have not been assessed. Thus, two of in vitro experiments were conducted to assess the effect of CT from PPC on ruminal fermentation, degradability and bacteria population.A study using pure cultures of main ruminal bacteria was conducted to assess if ruminal bacteria were able to overcome the negative effects of isolated CT from PPC. Three cellulolytic bacteria （Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus） and four non-cellulolytic bacteria （Prevotella bryantii, Ruminobacter amylophilus, Streptococcus bovis, and Selenomonas ruminantium） were used. Ruminal bacteria was initially cultured in a media containing 0 or 25μg CT/ml （cellulolytic） during 21-d and 0 or 50μg CT/ml （non-cellulolytic） during 15-d doing transferences every three days. At these levels of CT, cellulolytic and non-cellulolytic bacteria were able to overcome the potential negative effects of tannins. To observe the persistence of this effect, cellulolytic bacteria were then cultured for 4-d on Whatman N°1 filter paper in medium containing 0, 75, 150, 300 or 450μg CT/ml, whereas non-cellulolutic bacteria were cultured for 24-h in ruminal fluid medium containing 0, 100, 200, 400 or 600μg CT/ml. The growth curve showed that among non-cellulolytic bacteria, P. bryantii, R. amylophilus, and S. bovis were more sensitive to CT than S. ruminantium, which grew well up to 600μg CT/ml. The fiber digestion of F. succinogenes, R. flavefaciens, and R. albus was markedly reduced by CT at 75μg CT/ml and little bacteria grow was observed at CT concentrations up to 150μg/ml （P <0.01）. Scanning electron micrographs indicated that the attachment of cellulolytic bacteria was depressed by CT and no adaptation was found in this group.Overall, Purple prairie clover could be characterized as good quality forage with higher N content and uique bioactive compounds and also has the potential to alter membrane of E coli and reduce fecal shedding of E. coli in cattle. Condensed tannins in PPC/grasses mixture at concentrations up to 42 g/kg DM had no negative effects on the extent of DM digestibility, but increased MP synthesis. The CT can protect alfalfa nitrogen from ruminal degradation into ammonia but, the attachment of cellulolytic bacteria can be affected by CT even at very low concentration. The cellulotic bacteria were very sensitive to CT from PPC and their attachment can be affected.更多还原