【作者】 庄苏；

【导师】 王恬；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2011， 博士

【摘要】 外源酶制剂用于反刍动物的研究始于20世纪60年代,但由于作用不稳定,直到90年代人们才开始对外源酶制剂对反刍动物作用进行深入研究,取得一些成效,但与单胃动物方面研究相比,外源酶制剂应用与研究还存在一定差距。本研究以国产4种纤维素酶与4种木聚糖酶为研究对象,从不同pH与不同酶剂量研究入手,筛选最适酶活力的pH反应条件以及最高酶活力外源酶制剂产品,在此基础上进一步研究纤维水解酶以及纤维水解酶与甲酸在牧草青贮过程中对青贮品质的影响,以及用体外方法研究纤维水解酶与瘤胃液共培养后对相关酶活力和瘤胃发酵特性的影响,探索纤维水解酶与瘤胃液共培养下对瘤胃微生物菌群的影响,为纤维水解酶在反刍动物生产中应用提供依据。具体研究内容与结果如下：1不同pH值与不同酶添加水平对酶活力及还原糖生成量影响在pH5.0、5.5、6.0、6.5与7.0条件下,4种木聚糖酶中,A酶活力最高。在pH5.0、5.5、6.0下，A酶活力稳定,在pH7.0时,保持65%以上酶活；B酶活力最适pH范围为5.0与5.5,当pH上升到6.0以上时,酶活力迅速下降。C酶活力最适pH为6.0,单位酶活力最低；D酶活力在所有pH条件下,酶活力变化最小,稳定性强。4种纤维素酶,在pH5.5与5.5时,羧甲基纤维素酶酶活高,随pH上升,其酶活力下降。b酶与d酶的羧甲基纤维素酶活力显著高于a酶与c酶；a、b、c与d酶中结晶纤维素酶活力最适pH分别为6.0、5.0、6.0与5.5；b与d酶中的结晶纤维素酶活力高于a酶与c酶。在pH6.0与等量纯底物条件下,B木聚糖酶与a、b纤维素酶均随着酶添加水平增加而增加反应体系中还原糖生成量。仅A酶在使用水平为0.008 mg时木糖生成量最高。木聚糖酶与纤维素酶混合后并不能进一步提高还原糖生成量。2纤维水解酶对象草青贮发酵品质的影响分别以250 mg·kg-1、500 mg·kg-1纤维素酶、木聚糖酶以及250 mg·kg-1木聚糖酶+250 mg·kg-1纤维素酶处理象草在实验室小型发酵瓶中研究外源酶制剂对象草青贮过程中发酵品质的影响。结果显示,与对照组相比,酶处理显著降低青贮pH值(P<0.01),增加青贮料WSC含量(P<0.01)与乳酸含量(P<0.01)。青贮30d结束时,除了复合酶处理组外,其他酶处理组NH3-N含量显著低于对照组(P<0.05)；乙酸含量除高剂量纤维素酶组外,其他各组差异不显著(P>0.05)。酶处理改善了象草青贮品质。3甲酸与纤维水解酶对黑麦草与三叶草混合青贮发酵品质的影响本试验研究甲酸与纤维水解酶对黑麦草与三叶草混合青贮品质的影响。试验共设8组：对照组、0.3%甲酸、1.0 g·kg-’纤维素酶、0.3%甲酸+1.0 g·kg-1纤维素酶、1.0 g·kg-1木聚糖酶、0.3%甲酸+1.0 g.kg-1木聚糖酶、1.0 g·kg-1纤维素酶+1.0 g·kg-1木聚糖酶与0.3%甲酸+1.0 g·kg-1纤维素酶+1.0 g·kg-1木聚糖酶。试验结果显示：在青贮过程中,与对照组相比,甲酸或甲酸+酶复合处理牧草后显著降低青贮的pH值以及LA含量与氨氮含量(P<0.05),增加WSC含量(P<0.05)；酶处理组显著降低青贮pH值,增加青贮中WSC含量、乳酸含量、乙酸含量(P<0.05)以及降低了氨氮含量、NDF与ADF含量(P<0.05)。综合分析得出,纤维水解酶处理混合牧草后改善青贮品质,其效果优于甲酸或甲酸+酶处理组。4纤维水解酶对羊草或玉米青贮体外瘤胃发酵酶活力及发酵特性的影响试验评价了纤维素酶与木聚糖酶复合处理羊草或玉米青贮后与瘤胃液体外共培养下有关酶活力及瘤胃发酵特性的影响。试验设计为三个各含纤维素酶与木聚糖酶0,10.0与50 mg酶水平,与0与24h两个预处理牧草时间。结果显示,纤维水解酶处理羊草后与瘤胃液共培养能提高0与8h培养液中木聚糖酶活性、内切葡聚糖酶活性以及开始时培养液中结晶纤维素酶活性；同时提高以玉米青贮为底物时培养液中木聚糖酶、内切葡聚糖酶及结晶纤维素酶活性。酶活水平与酶添加剂量呈相关关系。当培养时间延长至24 h及48 h,外源酶并不能提高培养液中相关酶酶活。外源酶增加24 h与48 h时发酵液中乙酸产量(P<0.05)以及8h、24h与48 h时TVFA产量(P<0.05),具有增加乙酸与丙酸比的趋势(P＝0.138)以及增加48 h体外发酵累积产气量(P<0.05),但对戊酸与异戊酸含量无影响。结果指出添加纤维水解酶能提高体外培养下前8h时发酵液中木聚糖酶、纤维素酶酶活,并能改善体外瘤胃发酵的特性,增加TVFA产量及产气量,但对后期发酵液中相关酶活力没有影响。5纤维水解酶对体外发酵中瘤胃微生物菌群多样性影响应用PCR-DGGE技术分析纤维水解酶处理羊草或玉米青贮后对体外培养下瘤胃微生物菌群的影响。结果显示,纤维水解酶处理并不影响细菌菌群数量,培养液微生物菌群呈现多样性,所测得的细菌与基因库中的细菌相似性达89%-99%。其中被鉴定的菌群有反刍兽新月形单胞菌(Selenomonas ruminantium)、丁酸弧菌(Butyrivibrio fibrisolvens, Butyrivibrio hungatei)、真细菌(Eubacterium uniforme)、琥珀酸弧菌(Succinivibrio dextrinosolvens)、罗斯伯里氏菌(Roseburia faecis)、瘤胃杆菌(Bovine ruminisbacterium)。其余均为未培养瘤胃细菌。所测菌群均进行Genebank number的申请,取得相应的编号(HQ645848-HQ645865)。更多还原

【Abstract】 The exogenous enzymes in ruminants were used in the 1960s, but the responses were variable. Until to 1990s, researchers reexamined in detail the role of exogenous enzymes in ruminant production and get good results, but compared with norumiants, the quantity of exogenous enzymes products used in ruminants were smaller.4 cellulases and 4 xylanases made in china were selected to determine the effects of pH and dosage on enzyme activities in order to get optimum pH condition of enzymes action and the best enzymes products, then a series studies were conducted to determine the effects of exogenous fibrolytic enzymes, enzymes+formic acid on the fermentation quality of forage silages and to investigate the effects of exogenous fibrolytic enzymes treated forage on rumen fermentation and ruminal bacterial community in continuous culture in vitro. The results were as follows.1 Effects of the different pH value and adding dosages on enzyme activities and reducing sugar releaseUnder the condition of pH5.0,5.5,6.0,6.5 and 7.0, A xylanase activity was the higher compared with other enzymes. A enzyme activity was very stable at pH ranging from 5.0, to 6.0 and retained more 65% at pH 7.0. The optimum range of pH were 5.0 to 5.5 for B xylanase, with pH up to 6.0, the B xylanase activity was decreased rapidly. The optimum pH for C xylanase activity was 6.0, but the enzymes activity was lowest among enzymes. D xylanase activity was very stable at testing pH condition. CMCase activities were very higher at pH 5.0 and 5.5, but with pH increasing, the activity was decreased for a, b, c and d enzyme. CMCase activities for b and d enzymes were higher than a and c enzymes. The optimum pH of Avicelase activities for a, b, c and d enzymes was 6.0,5.0,6.0 and 5.5 respectively. Avicelase activities of b and d enzymes were higher than a and c enzymes activities.With increasing enzymes level, the reducing sugar release was high significant for A, B xylanase and a, b cellulase under pH6.0 and the same substrate level, but the release quantity of reducing xylose was greatest at the 0.008 mg level of A xylanase. The release quantity of reducing sugars was not enhanced further in addition of mixture of cellulase +xylanase in the substrate of xylan or CMC-Na.2 Effects of fibrolytic enzymes on the fermentation quality of elephant grass (Pennisetum purpureum Schumach.) silagesThis study was conducted to research the effects of the fibroltyic enzymes on the fermentation quality of elephant grass silages. Chopped elephant grass were ensiled in laboratory mini-silos either untreated (control) or treated with cellulase(250 or 500 mg-kg-1), cellulase(250 mg-kg-1) plus xylanase(250 mg-kg’), xylanase(250 or 500 mg-kg-1), respectively. The results showed that the enzymes treatments reduced rapidly the pH value(P<0.01), and enhanced significantly content of lactic acid(P<0.01) and water soluble carbohydrate(P<0.01) of silages compare with control. The content of ammonia nitrogen in the enzymes treated silages, expect for cellulase plus xylanase treated, was lower than the untreated silage (P<0.05) and the content of acetic acid, except high level of cellulase treatment, were no difference (P>0.05) in the silages at the end of ensiling. The exogenous fibrolytic enzymes improved the fermentation quality of elephant grass silage during ensiling.3 Effects of formic acid, fibrolytic enzymes on the fermentation quality of Italian ryegrass (Lolium multiflorum Lam.) and white clover(Trifolium repens L.) mixture silageThis experiment was conducted to examine the effects of formic acid, fibrolytic enzymes on the fermentation quality of Italian ryegrass and white clover mixture silages. The mixture material of 80% Italian ryegrass+20% white clover was ensiling in the plastic bags either untreated (control) or treated with formic acid (0.3%), cellulase(1.0 mg-kg-1), formic acid+cellulase(0.3%+1.0 mg·kg-1), xylanase(1.0 mg·kg-1), formic acid+xylanase (0.3%+1.0 mg·kg-1), cellulase+xylanase(1.0 mg-kg-1, respectively) and formic acid+ cellulase+xylanase (0.3%+1.0 mg·kg-1+1.0 mg·kg-1). The results showed that formic acid treatment or formic acid plus enzyme treatments decreased the pH value (P<0.05), LA content and NH3-N content (P<0.05), increased WSC contents (P<0.05) of silages compared with untreated silage; the enzyme treatments enhanced significantly WSC contents, LA contents and Ac contents (P<0.05) of silages and decreased the pH value, NH3-N contents, NDF and ADF content (P<0.05) of silages, compared with control during ensiling. The formic acid and exogenous fibrolytic enzymes can improve mixture silages quality of ryegrass and white clover and the results treated with enzymes were better than formic acid or formic acid plus enzymes treatment.4 Effects of fibrolytic enzymes on enzymes activities and fermentation of Chinese wildrye or maize silages by mixed ruminal microorganisms in vtiroTwo of in vitro studies were conducted to determine the effects of mixture treatment of cellulase and xylanase on enzymes activities and fermentation of Chinese wildrye or maize silages by mixed ruminal microorganisms. Three levels of fibrolytic enzyme supplements (0,10.0 and 50.0 mg, respectively) and two treatment times (0 and 24 h before incubation) were tested. The enzyme treatment increased xylanase and CMCase activities 0 and 8 h postincubation and Avicelase activity 0 postincubation for Chinese wildrye. The level of enzyme affected enzyme activities. Then, the exogenous enzyme addition did not affect enzymes activities in incubation fluid at 24 and 48 h of incubation. The AA content at 24 and 48 h of incubation, the total VFA contents at 8,24 and 48 h of the incubation were increased (P<0.05) by the enzyme treatment. Enzyme treatment increased (P<0.05) the cumulative gas production and tended to increase the ratio of acetate to propionate (P=0.138) at the end of incubation. Enzyme treatment had no effects valerate and isovalerate concentrate (P>0.05). In the experiment of enzymes treated maize silages, the enzymes treatment increased xlyanase activity at 0 h, CMCase activity at 0 and 8 h and Avicelase activity at 0,8 and 24 h of the incubation. Date from this study suggest that addition of fibrolytic enzymes to Chinese wildrye or maize silage by mixed ruminal microorganisms can enhance xylanase and cellulase activities in the initial 8 h of the incubation and had no effect the enzyme activity at 24 and 48 h of the incubation and improved rumainal fermentation characteristic in vitro. 5 Effects of the fibrolytic enzymes on ruminal bacterial community in vitro incubationThe effect of the fibrolytic enzymes on ruminal bacterial community in vitro incubation was analyzed by PCR-DGGE technology. The analysis of DGGE profiles revealed that the fibrolytic enzyme treatment did not affect the composition of the rumen bacterial community. The sequence similarity analyses of the all clone sequences in 16S rDNA library resealed that 6 clones showed similarities with database sequences over 97%, the rest similarities in a range of 84%-96%.7 clone sequences were similar to Selenomonas ruminantium, Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Eubacterium uniforme, Succinivibrio dextrinosolvens, Roseburia faecis, Bovine ruminisbacterium. The rests were uncultured rumen bacterium. All of clone sequences get a Genebank number (accession No: HQ645848-HQ645865).更多还原