【作者】 国春艳；

【导师】 刁其玉；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2010， 博士

【摘要】 本研究从土壤等环境中筛选出产木聚糖酶菌株,分析其中酶活力较高的一株黑曲霉S8-3产酶酶学性质。对该菌株液体发酵后将其与纤维素酶、淀粉酶、蛋白酶、果胶酶等按不同比例复配,在体外对玉米—豆粕—青干草底物进行降解,确定可在反刍动物日粮中添加的复合酶的酶系组成。进一步用体外法研究了外源酶与瘤胃内源酶的协同作用,探讨了外源酶提高饲料利用率的机制。随后研究了复合酶在动物体内的作用效果,以3月龄到7月龄的后备牛为试验动物进行了生长消化代谢试验,分析了添加外源酶对后备牛生长性能、营养消化、血液指标、瘤胃发酵及瘤胃微生物区系变化的影响。本研究系统分析并阐述了外源酶制剂在后备牛日粮中的应用效果及其作用机制。现摘要如下:产木聚糖酶菌株的筛选研究从生长作物、长期堆积草料或腐质物的土壤中筛选产木聚糖酶菌株。通过富集培养定向筛选技术,从土壤中分离获得18株产木聚糖酶菌株,挑选其中5株进行摇瓶培养。对产酶最高的菌株S8-3进行鉴定,同时分析该菌株产生的木聚糖酶的酶学性质。经鉴定该菌株为黑曲霉(Aspergillums Niger);该菌株发酵液的木聚糖酶活性为628.43 U/mL;木聚糖酶的最适作用温度为45聚,最适pH为4.5,在705保温30 min后酶活力仍为60%以上,在pH 3.0-7.0内稳定性较好。预期该菌株产的木聚糖酶具有用于饲料添加剂的潜力。外源酶对玉米-豆粕-青干草型底物人工瘤胃发酵的影响利用体外培养法研究了8种不同外源复合酶组合对玉米-豆粕-青干草型底物瘤胃发酵的影响。外源复合酶由纤维素酶、木聚糖酶、酸性蛋白酶、中性蛋白酶和果胶酶组成。研究表明,各组复合酶处理的产气量、氨态氮浓度、VFA浓度与对照组相比有所增加,均存在显著差异(P<0.05)。研究结果表明,在本试验条件下,添加外源复合酶可以改变瘤胃发酵模式,提高体外发酵产气量、发酵液氨态氮浓度和VFA浓度,提高饲料的消化率。研究结果表明,在本试验条件下,高纤维素酶、低木聚糖酶处理组对玉米-豆粕-青干草类型底物降解效果较好,酸性蛋白酶、中性蛋白酶、果胶酶比例的变化对结果影响较小。外源酶与瘤胃内源酶互作作用的研究在反刍动物日粮中添加外源酶能提高饲料消化率的机制尚不清楚。在瘤胃中直接水解可能是一种方式,但是与瘤胃内庞大的消化酶系相比,外源酶的添加量所占比例较小,因此外源酶在瘤胃内对底物的水解所发挥的作用需要进行量化。本试验采用体外发酵法研究添加外源酶制剂对瘤胃内源酶对羧甲基纤维素、木聚糖及TMR等不同底物降解作用的影响。试验采用7不同完全随机区组设计,研究木聚糖酶、纤维素酶、瘤胃内源酶等3种单酶、复合酶A(木聚糖酶:纤维素酶:瘤胃内源酶=1:1:2)、复合酶B(木聚糖酶:瘤胃内源酶=1:1)、复合酶C(纤维素酶:瘤胃内源酶=1:1)及空白对照组共7个处理组分别在4.5,5.0,5.5,6.0,6.5,7.0等6个pH条件进行体外发酵试验。试验选用的外源纤维素酶和木聚糖酶均为粉状商品酶,使用前进行纯化。瘤胃内源酶通过采集奶牛瘤胃液,经过离心、过硫酸铵沉淀、透析脱盐、琼脂糖凝胶分离纯化等一系列步骤自行制备。在pH4.5-7.0,395条件下测定瘤胃内源酶,外源酶及复合酶对羧甲基纤维素、木聚糖和TMR的降解能力。外源酶制剂与瘤胃内源酶在降解羧甲基纤维素、木聚糖和TMR上具有互作作用。复合酶A降解TMR,葡萄糖和木糖浓度分别较瘤胃内源酶提高了86%和112%;复合酶B降解TMR,木糖浓度较瘤胃内源酶提高了217%,葡萄糖浓度变化无差异。复合酶C降解TMR,葡萄糖浓度和木糖浓度分别较瘤胃内源酶提高了43%和94%。尤其在pH6.0-6.5条件下,复合酶的降解能力更强。研究结果表明,复合酶A中纤维素酶与木聚糖酶的比例更有利于底物的降解。瘤胃内源酶和外源酶之间的互作作用提高了瘤胃内环境中的底物的降解,这可能是添加纤维素酶、木聚糖酶等外源酶促进饲料消化的重要作用机制之一。添加外源酶制剂对后备牛消化代谢及血液生化指标的影响为研究添加外源酶制剂对3-7月龄后备牛生长性能及消化代谢的影响,分别选取16头3月龄、4月龄、5月龄的后备荷斯坦奶牛,共48头。将三月龄牛只随机分为2组,分别标记为CT,ET;将四月龄牛只随机分为2组,分别标记为CA,EA;将五月龄牛只随机分为2组,分别标记为CM,EM,共六个处理组。CT、CA,CM组牛只饲喂对照组TMR,ET、EA,EM组牛只饲喂加酶TMR,剂量为20g/日·头。结果表明:ET、CT组之间,EA、CA组之间,EM、CM组之间,试验牛只体增重和日增重均有显著差异(P<0.05),表明添加复合酶可以增加后备牛的腹围和胸围,促进后备牛的生长。对各处理组牛只瘤胃液中发酵参数测定结果显示,添加酶制剂可以增加瘤胃液中总挥发性脂肪酸和乙酸含量提高乙酸/丙酸比例,改善瘤胃发酵水平。消化试验结果表明,添加酶制剂可以提高后备牛TMR的NDF、ADF和总能表观消化率,但对蛋白质和粗脂肪表观消化率没有显著影响。对各处理组间血清学指标分析结果显示,血浆中甘油三酯、胆固醇、ALP含量差异不显著(P>0.05),白蛋白含量和白球比在个别处理组之间存在显著差异。添加外源酶制剂对后备牛瘤胃发酵参数及瘤胃微生物变化的影响利用PCR-DGGE技术分析三月龄、四月龄、五月龄后备牛在饲喂对照组TMR和加酶处理TMR后瘤胃内细菌和真菌的区系变化的影响。酶制剂处理使3-7月龄后备牛瘤胃微生物DGGE图谱条带数量增加,瘤胃微生物区系发生了变化。序列分析结果表明,酶制剂处理组中有2个克隆分别与普雷沃氏菌和瘤胃黄色球菌相似度高于96%,该处理可以促进瘤胃微生物区系优势菌群的建立。更多还原

【Abstract】 Strains in soil which can produce xylanase were screened from soil. And a strain producing xylanase with highest activity was selected and identified as Aspergillus Niger. This xylanase was studied with its characters such as optimized pH, temperature, and thermal stability. Then it was mixed with cellulose, amylase and protease in different rates to be compound enzymes. Their abilities to degrade corns, bean, and green dry grasses in vitro were detected and the best ratios was determined. Synergetic effect between external enzymes and internal enzymes in rumen was studied in order to understand the mechanism of the higher use of feed when adding external enzymes. More researches were done to know the effects of external compound enzymes influenced in rumor. Experiments of the growth, digestion, and metabolism of replacement cattle aged from three months to seven months were carried out to analyze effects that external enzymes influenced the growing performance, digestion, indexes in blood, fermentation in rumor and changes of micro-organisms in rumor. The article explained the use of external enzymes and their mechanism of action in ruminants.1. Screen acidic xylanase-producing microbes from natural environment for potential use in feed industry. Twenty xylanase-producing strains were isolated from soil by enrichment culture techniques. Five strains were chosen to ferment in shaking flasks. The strain S8-3, which had the highest production of xylanase, was preliminarily identified by standard methods, and the property of the xylanase produced by the strain was studied. Strain S8-3 was identified as Aspergillus Niger. The maximal enzymatic activity of xylanase in culture liquid was 628.43 U/mL. The xylanase activity was optimal at 45 degrees C and pH 4.5. Xylanase was stable at pH ranging from 3.0 to 7.0. Xylanase retained more than 60 % of its original activity after incubation for 30 min at 70 degrees C. The xylanase produced by strain S8-3 in this study may have the potential in feed industry.2. An in vitro system was conducted to investigate the effects of eight different levels of exogenous complex enzymes in a corn-soybean meal-hay diet on rumen fermentation. Exogenous complex enzyme consisted of cellulose, xylanase, acid protease, neutral protease and pectinase. The results showed gas production, ammonia-N (NH3-N), volatile fatty acids (VFA) concentration in each treatment were significantly higher than those in Control treatment (P<0.05). It also showed that the rumen fermentation pattern was changed with the addition of exogenous complex enzyme, and gas production in vitro, NH3-N and VFA concentration and the degradability were improved. Under the conditions of this experiment, high cellulose, low xylanase pattern group had better degradation on the corn - soybean meal - hay substrate; acid protease, neutral protease, pectinase had no significant effects on the degradation.3. The mechanism by which enzyme additives improve feed digestion in ruminants is not fully understood. Direct hydrolysis of feed in the rumen is a potential mode of action, but the importance of this mode needs to be quantified because of the relatively low exogenous hydrolyses activity added compared with the total activity added compared with the total activity present in the rumen. We examined the interactions between ruminal and exogenous enzymes on substrate (CMC, xylan and TMR) degradation using a completely randomized experimental design, with a 6(enzyme preparations and their combinations)×5(assay pH) arrangement of treatments. Ruminal enzymes were extracted from cattle fitted with rumen cannulae. Exogenous enzymes were commercial enzyme include cellulose and xylanase. The complex enzymes A, which was composed with the xylanase, cellulose and ruminal enzymes at the ratio of one third each. The complex enzymes B, which was composed with the xylanase and ruminal enzymes at the ratio of 0.5 to 0.5. The complex enzymes C, which was composed with the cellulose and ruminal enzymes at the ratio of 0.5 to 0.5. Ruminal and exogenous enzymes preparations and their combinations were tested for the ability to degrade soluble cellulose, xylan, and TMR over a range of pH form 4.5 to 7.0 at 39℃. Exogenous enzymes acted synergistically with enzymes from mixed rumen microorganisms in degrading soluble cellulose, xylan and TMR. The exogenous enzymes can interact with the ruminal enzymes when depredating CMC, Xyaln and TMR under the conditions of pH4.5-7.0, 39℃. Then the hydrolysis products of TMR were detected. The concentrate of glucose and xylan of the complex A group were 86 percent and 112 percent higher than ruminal enzymes group; the concentrate of xylan of the complex B group were 217 percent higher than ruminal enzymes group, but of the glucose was no different; The concentrate of glucose and xylan of the complex C group were 43 percent and 94 percent higher than ruminal enzymes group. And hydrolysis ability was much higher under pH 6.0-6.5, 39℃conditions. The synergistic effect between ruminal and exogenous enzymes increased the hydrolytic potential within the rumen environment and is likely a significant mechanism by which enzyme additives improve feed digestion.4. The effect of supplementation of exogenous enzymes on growth performance, nutrition digestibility and metabolism, rumen fermentation and microbiota diversity of 3 to 7 month heifers was studied. 16 herds heifer were selected on 3 month old, 4month old, 5 month old, respectively, add up to 48 herbs. The age of heifers in 3 month were randomly divided into two groups, labeled CT, ET; aged at 4 month heifers were randomly divided into two age groups, labeled CA, EA; aged at 5 month heifers were randomly divided into 2 groups were labeled as CM, EM. CT, CA, CM group of heifers fed control group TMR, ET, EA, EM group cows fed TMR supplement enzyme in a dose of 20g / day. head. The results showed that between ET and CT groups, between EA and CA groups, between EM and CM groups, body weight gain and average daily gain were significantly different. Adding enzyme can promote the growth of bovine abdomen and chest. Rumen fermentation parameters of each group heifers were measured. The results show that compound enzyme can increase the total rumen volatile fatty acids and acetic acid content, as well as acetic acid / propionic acid ratio. Digestion experiment results showed that compound enzyme preparations can increase the NDF, ADF apparent digestibility, total energy apparent digestibility, but crude fatty and crude protein digestibility did not significantly affected by compound enzyme treatment. The analysis results showed that plasma triglycerides, cholesterol, ALP levels were not significantly different. It could conclusion that TMR of exogenous enzymes can improve the feed digestion and promote the development of the rumen, improve ruminal fermentation, have no effects on lipid metabolism and protein metabolism, and thus promote their growth on 3 to 7 months heifers.5. Using sequence analysis of DGGE combined PCR to evaluate the microbiota diversity of heifers feed TMR treated by compound enzyme. The results showed that all groups of bacteria DGGE profiles reserve rumen similarity is not high, but enzyme preparation treatments increased numbers of DGGE bands, the results show that the rumen microbiota diversity changed. Sequence analysis showed that there were 2 clones, respectively Prevotella sp. and Ruminococcus flavaciens similarity higher than 96% in enzyme treatment group. Enzyme treatment can promote the establishing of advantages of ruminal microflora.更多还原