NSP复合酶优化及在肉鸡日粮中高剂量添加效果研究
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摘要
本论文主要研究了在选取的最佳饲用NSP单酶制剂基础上，通过体外模拟消化的方式来确定不同NSP单酶制剂与不同饲料原料之间的最佳酶解量关系，以建立起一套能针对不同饲料配方而得到相应变化的复合酶配方预测模型，并应用肉鸡养殖试验研究了高剂量添加复合酶对肉鸡生产性能、养分利用率、器官指数、血液指标、肠道黏膜形态、食糜黏度和微生物菌群等重要指标的影响。本论文试验共分三部分。
试验一：选取了不同菌株来源的木聚糖酶、纤维素酶、β-甘露聚糖酶、-半乳糖苷酶、果胶酶样品各3种，通过研究不同单酶制剂在不同温度（30、37、40、45、50、55、60、65℃）和pH值（3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0）条件下酶活变化情况，以及各单酶制剂对酸碱性和胃蛋白酶、胰蛋白酶的耐受性，以期获得本研究所需的最佳饲用单酶制剂。试验结果表明：选用各单酶制剂的3种样品中，木聚糖酶-Ⅰ、纤维素酶-Ⅱ、β-甘露聚糖酶-Ⅰ、-半乳糖苷酶-Ⅰ和果胶酶-Ⅲ最适合在动物生产上应用，所选得的各单酶最适温度和pH分别为45、50、55、40、45℃和6.0、4.5、4.5、4.5、6.0，且在不同酸碱缓冲液和胃蛋白酶、胰蛋白酶处理后仍能保存很高的相对酶活。
试验二：根据不同饲料原料中非营养物质或抗营养物质的含量，选取相应的NSP酶制剂，采用肉鸡体外消化模型，进行梯度酶解试验，以酶解产物为指标，确定不同饲料原料与不同酶制剂添加关系曲线，并推导其最佳添加量；同时，确定不同饲料配方中NSP复合酶制剂的添加关系。试验结果得到了木聚糖酶、纤维素酶、β-甘露聚糖酶、-半乳糖苷酶、果胶酶分别酶解玉米、豆粕、小麦、麸皮、棉粕、菜粕、米糠粕、DDGS的关系曲线，并得到了各酶解关系的产物生成量及其占底物比例，确定了各单酶酶解各饲料原料所需最佳酶活量，其中木聚糖酶为0.336、0、6.0、1.22(3.66)、2.24、0、0.44、5.0(20.0) U/g，β-甘露聚糖酶为1.68、3.90、0.276、6.28、4.08、1.72、7.04、0.84U/g，纤维素酶为4.98、28.62、8.88、19.44、44.48、27.78、22.8、9.72U/g，-半乳糖苷酶为1.11、27.78、1.94、1.48、13.34、5.37、4.45U/g，果胶酶为0.234、37.3、0.474、4.26、7.56、10.68、0.64U/g）。通过肉鸡养殖试验表明，体外模拟消化试验得到的不同单酶酶解不同饲料原料添加量结果与肉鸡养殖试验存在正比关系，在酶制剂添加量达到一定量之后，酶解效果不再呈显著性增加，这为饲料配方中添加复合酶配方的制作提供了理论数据。
试验三：研究了在降低饲粮能量时，添加常规和高剂量复合酶对肉鸡生产性能、养分利用率、器官指数、血液指标、肠道黏膜形态、食糜黏度和微生物菌群的影响。试验将320只1日龄爱拔益加肉鸡分为4组，每组8个重复，每个重复10只。第Ⅰ组为正对照组，饲喂常规饲粮；第Ⅱ组为负对照组，配方中增加了杂粕和麦类原料的用量，同时降低210KJ ME/kg；第Ⅲ组和第Ⅳ组为常规剂量加酶组和高剂量加酶组，分别在负对照组饲粮的基础上添加0.02%和0.2%复合酶，试验期为42d。结果表明，肉鸡全期负对照组料重比显著高于高剂量加酶组(P<0.01)，常规剂量加酶组和正对照组料重比显著低于负对照组(P<0.05)；高剂量加酶组的蛋白质、脂肪和能量表观代谢率极显著高于负对照组(P<0.01)，高剂量加酶组的干物质表观代谢率和表观代谢能显著高于负对照组(P<0.05)；各试验组之间小肠和脾脏器官指数无显著性差异(P>0.05)，高剂量加酶组的胃和法氏囊器官指数和负对照组有极显著性差异(P<0.01)；负对照组甲状腺素显著低于其它三组(P<0.05)；高剂量加酶组胰岛素含量显著高于其它三组(P<0.05)；正对照组和高剂量加酶组绒毛高度极显著高于其它两组(P<0.01)，负对照组绒毛高度显著短于常规剂量加酶组(P<0.05)；负对照组隐窝深度极显著高于其它三组(P<0.01)，高剂量加酶组隐窝深度显著低于正对照组和常规剂量加酶组(P<0.05)；高剂量加酶组V/C值极显著高于其它三组(P<0.01)，负对照组V/C值显著低于正对照组和常规剂量加酶组(P<0.05)；各组之间黏膜厚度无显著差异(P>0.05)；高剂量加酶组相对黏度显著低于负对照组(P<0.05)；两个加酶组乳酸杆菌数显著高于两个对照组(P<0.05)。试验说明在降低210KJ/kg代谢能的饲粮中添加0.02%和0.2%复合酶对肉鸡生产性能、养分利用率、器官指数、血液指标、肠道黏膜形态、食糜黏度和微生物菌群，均具有很好的改善作用；相对于0.02%的添加量，0.20%复合酶添加量可进一步提高酶的应用效果。
The study was conducted to determine the enzymolysis relationship between different NSPenzymes and different feedstuffs by in vitro digestion method after the optimum NSP enzymes wereselected, and establish a prediction model of complex enzyme formula correspond to different feedformulations. An experiment was also conducted to study the effects of super dose complex enzyme onthe growth performance, nutrient availability, organ indexes, serum hormones concentration, smallintestinal mucosa, digesta viscosity and microbial community of broilers. The study had threeexperiments.
The experiment1was to determine optimum enzymes among three different sources of xylanase,cellulose, β-mannanase,-galactosidase and pectase by studying the properties of temperature (30,37,40,45,50,55,60,65℃), pH (3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0) and tolerance toacid-base, pepsin and trypsin. The results showed that the optimum enzymes are xylanase-I, cellulose-Ⅱ, β-mannanase-I,-galactosidase-I and pectase-Ⅲ which optimum temperature and pH are45,50,55,40,45℃and6.0,4.5,4.5,4.5,6.0, and have high relative activity treated by acid-base, pepsin andtrypsin.
The experiment2was to determine the optimal enzymatic hydrolysis dose of different NSPenzymes to different feedstuffs by gradient in vitro digestion method simulating broiler, and therelationship between added complex enzyme and different feed formulation. The enzymatic hydrolysiscurves of different NSP enzymes to different feedstuffs were also deduced by testing the enzymatichydrolysis products based on the content of non-starch polysaccharides in different feedstuffs. Theresults showed that the enzymatic hydrolysis curves of different enzymes to different feedstuffs havenormal linear relationship. The amount of product, which degraded from different feedstuffs by differentenzymes, and its percentage of substrate are calculated. The optimal enzymatic hydrolysis dose ofxylanase, β-mannanase, cellulose,-galactosidase and pectase to maize, soybean meal, wheat, wheatbran, cottonseed meal, rapeseed meal, rice bran meal and DDGS are0.336,0,6.0,1.22(3.66),2.24,0,0.44and5.0(20.0) U/g,1.68,3.90,0.276,6.28,4.08,1.72,7.04and0.84U/g,4.98,28.62,8.88,19.44,44.48,27.78,22.8and9.72U/g,1.11,27.78,1.94,1.48,13.34,5.37and4.45U/g,0.234,37.3,0.474,4.26,7.56,10.68and0.64U/g. The broiler trials showed that enzymolysis effect was no longersignificant increase after added enough enzyme and there is proportional relationship between in vitroand breeding broiler test. The results provided theoretical data for complex enzyme added to differentfeed formulae.
The experiment3was conducted to investigate the effects of low energy diets supplemented withdifferent dosage of compound enzyme on the growth performance, nutrients utilization, organ index,serum hormones concentration, intestinal mucosa morphology, digesta viscosity and microbialcommunity of broilers. Three hundred and twenty broilers (1day-old) were randomly assigned to fourtreatments with eight replicates with ten broilers each. The positive control (PC) treatment was fed a corn-soybean meal diet. The diet of negative control (NC) treatment was reduced by210KJ ME/kg, andthe content of miscellaneous meal and wheat was increased in this treatment. The conventional dose ofenzyme (CDE) and super dose of enzyme (SDE) treatment was respectively fed the NC diet withdifferent dosages of compound enzyme0.02%and0.2%. The trial lasted for42d. The results showedthat the FCR of broilers fed the SDE treatment was significantly lower than those fed the NC (P<0.01);the FCR of broilers fed the CDE and PC treatment was significantly lower than the NC (P<0.05). TheCP, EE and energy apparent metabolizability of the SDE treatment diet were higher than the NC diet(P<0.01); the DM apparent metabolizability and AME of the SDE treatment diet were higher than theNC diet (P<0.05). There were no significant differences in the organ index of small intestine and spleenof broilers fed different diets (P>0.05); the organ index of stomach and bursa of fabricius of broilers fedthe SDE diet had difference significantly with those fed the NC diet (P<0.01). The thyroxine of broilersfed the NC treatment was significantly lower than other three treatments (P<0.05); the insulin ofbroilers fed the SDE diet was significantly higher than other three treatments (P<0.05). As for villiheight, the PC and SDE treatment were significantly higher than other two treatments (P<0.01); the NCtreatment was significantly lower than the CDE treatment (P<0.05). As for crypt depth, the NCtreatment were significantly lower than other three treatments (P<0.01); the SDE treatment wassignificantly lower than the PC and CDE treatment (P<0.05). As for V/C ratio, the SDE treatment weresignificantly higher than other three treatments (P<0.05); the NC treatment was significantly lower thanthe PC and CDE treatment (P<0.05). As for mucosa thickness, there was no significantly difference(P>0.05). As for digesta viscosity, the SDE treatment was significantly lower than the NC treatment(P<0.05). As for Lactobacillus of microbial community, two added enzyme treatments weresignificantly higher than the NC and PC treatment (P<0.05). The experiment prove that the growthperformance, nutrients utilization, organ index, serum hormones concentration, intestinal mucosamorphology, digesta viscosity and microbial community of broilers were improved significantly as thebroilers fed the diet (reduce210KJ ME/kg) containing complex enzyme at the level of0.02%and0.2%.The effect of complex enzyme was increased significantly while the dosage of enzyme was enhancedfrom0.02%to0.2%.
试验一：选取了不同菌株来源的木聚糖酶、纤维素酶、β-甘露聚糖酶、-半乳糖苷酶、果胶酶样品各3种，通过研究不同单酶制剂在不同温度（30、37、40、45、50、55、60、65℃）和pH值（3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0）条件下酶活变化情况，以及各单酶制剂对酸碱性和胃蛋白酶、胰蛋白酶的耐受性，以期获得本研究所需的最佳饲用单酶制剂。试验结果表明：选用各单酶制剂的3种样品中，木聚糖酶-Ⅰ、纤维素酶-Ⅱ、β-甘露聚糖酶-Ⅰ、-半乳糖苷酶-Ⅰ和果胶酶-Ⅲ最适合在动物生产上应用，所选得的各单酶最适温度和pH分别为45、50、55、40、45℃和6.0、4.5、4.5、4.5、6.0，且在不同酸碱缓冲液和胃蛋白酶、胰蛋白酶处理后仍能保存很高的相对酶活。
试验二：根据不同饲料原料中非营养物质或抗营养物质的含量，选取相应的NSP酶制剂，采用肉鸡体外消化模型，进行梯度酶解试验，以酶解产物为指标，确定不同饲料原料与不同酶制剂添加关系曲线，并推导其最佳添加量；同时，确定不同饲料配方中NSP复合酶制剂的添加关系。试验结果得到了木聚糖酶、纤维素酶、β-甘露聚糖酶、-半乳糖苷酶、果胶酶分别酶解玉米、豆粕、小麦、麸皮、棉粕、菜粕、米糠粕、DDGS的关系曲线，并得到了各酶解关系的产物生成量及其占底物比例，确定了各单酶酶解各饲料原料所需最佳酶活量，其中木聚糖酶为0.336、0、6.0、1.22(3.66)、2.24、0、0.44、5.0(20.0) U/g，β-甘露聚糖酶为1.68、3.90、0.276、6.28、4.08、1.72、7.04、0.84U/g，纤维素酶为4.98、28.62、8.88、19.44、44.48、27.78、22.8、9.72U/g，-半乳糖苷酶为1.11、27.78、1.94、1.48、13.34、5.37、4.45U/g，果胶酶为0.234、37.3、0.474、4.26、7.56、10.68、0.64U/g）。通过肉鸡养殖试验表明，体外模拟消化试验得到的不同单酶酶解不同饲料原料添加量结果与肉鸡养殖试验存在正比关系，在酶制剂添加量达到一定量之后，酶解效果不再呈显著性增加，这为饲料配方中添加复合酶配方的制作提供了理论数据。
试验三：研究了在降低饲粮能量时，添加常规和高剂量复合酶对肉鸡生产性能、养分利用率、器官指数、血液指标、肠道黏膜形态、食糜黏度和微生物菌群的影响。试验将320只1日龄爱拔益加肉鸡分为4组，每组8个重复，每个重复10只。第Ⅰ组为正对照组，饲喂常规饲粮；第Ⅱ组为负对照组，配方中增加了杂粕和麦类原料的用量，同时降低210KJ ME/kg；第Ⅲ组和第Ⅳ组为常规剂量加酶组和高剂量加酶组，分别在负对照组饲粮的基础上添加0.02%和0.2%复合酶，试验期为42d。结果表明，肉鸡全期负对照组料重比显著高于高剂量加酶组(P<0.01)，常规剂量加酶组和正对照组料重比显著低于负对照组(P<0.05)；高剂量加酶组的蛋白质、脂肪和能量表观代谢率极显著高于负对照组(P<0.01)，高剂量加酶组的干物质表观代谢率和表观代谢能显著高于负对照组(P<0.05)；各试验组之间小肠和脾脏器官指数无显著性差异(P>0.05)，高剂量加酶组的胃和法氏囊器官指数和负对照组有极显著性差异(P<0.01)；负对照组甲状腺素显著低于其它三组(P<0.05)；高剂量加酶组胰岛素含量显著高于其它三组(P<0.05)；正对照组和高剂量加酶组绒毛高度极显著高于其它两组(P<0.01)，负对照组绒毛高度显著短于常规剂量加酶组(P<0.05)；负对照组隐窝深度极显著高于其它三组(P<0.01)，高剂量加酶组隐窝深度显著低于正对照组和常规剂量加酶组(P<0.05)；高剂量加酶组V/C值极显著高于其它三组(P<0.01)，负对照组V/C值显著低于正对照组和常规剂量加酶组(P<0.05)；各组之间黏膜厚度无显著差异(P>0.05)；高剂量加酶组相对黏度显著低于负对照组(P<0.05)；两个加酶组乳酸杆菌数显著高于两个对照组(P<0.05)。试验说明在降低210KJ/kg代谢能的饲粮中添加0.02%和0.2%复合酶对肉鸡生产性能、养分利用率、器官指数、血液指标、肠道黏膜形态、食糜黏度和微生物菌群，均具有很好的改善作用；相对于0.02%的添加量，0.20%复合酶添加量可进一步提高酶的应用效果。
The study was conducted to determine the enzymolysis relationship between different NSPenzymes and different feedstuffs by in vitro digestion method after the optimum NSP enzymes wereselected, and establish a prediction model of complex enzyme formula correspond to different feedformulations. An experiment was also conducted to study the effects of super dose complex enzyme onthe growth performance, nutrient availability, organ indexes, serum hormones concentration, smallintestinal mucosa, digesta viscosity and microbial community of broilers. The study had threeexperiments.
The experiment1was to determine optimum enzymes among three different sources of xylanase,cellulose, β-mannanase,-galactosidase and pectase by studying the properties of temperature (30,37,40,45,50,55,60,65℃), pH (3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0) and tolerance toacid-base, pepsin and trypsin. The results showed that the optimum enzymes are xylanase-I, cellulose-Ⅱ, β-mannanase-I,-galactosidase-I and pectase-Ⅲ which optimum temperature and pH are45,50,55,40,45℃and6.0,4.5,4.5,4.5,6.0, and have high relative activity treated by acid-base, pepsin andtrypsin.
The experiment2was to determine the optimal enzymatic hydrolysis dose of different NSPenzymes to different feedstuffs by gradient in vitro digestion method simulating broiler, and therelationship between added complex enzyme and different feed formulation. The enzymatic hydrolysiscurves of different NSP enzymes to different feedstuffs were also deduced by testing the enzymatichydrolysis products based on the content of non-starch polysaccharides in different feedstuffs. Theresults showed that the enzymatic hydrolysis curves of different enzymes to different feedstuffs havenormal linear relationship. The amount of product, which degraded from different feedstuffs by differentenzymes, and its percentage of substrate are calculated. The optimal enzymatic hydrolysis dose ofxylanase, β-mannanase, cellulose,-galactosidase and pectase to maize, soybean meal, wheat, wheatbran, cottonseed meal, rapeseed meal, rice bran meal and DDGS are0.336,0,6.0,1.22(3.66),2.24,0,0.44and5.0(20.0) U/g,1.68,3.90,0.276,6.28,4.08,1.72,7.04and0.84U/g,4.98,28.62,8.88,19.44,44.48,27.78,22.8and9.72U/g,1.11,27.78,1.94,1.48,13.34,5.37and4.45U/g,0.234,37.3,0.474,4.26,7.56,10.68and0.64U/g. The broiler trials showed that enzymolysis effect was no longersignificant increase after added enough enzyme and there is proportional relationship between in vitroand breeding broiler test. The results provided theoretical data for complex enzyme added to differentfeed formulae.
The experiment3was conducted to investigate the effects of low energy diets supplemented withdifferent dosage of compound enzyme on the growth performance, nutrients utilization, organ index,serum hormones concentration, intestinal mucosa morphology, digesta viscosity and microbialcommunity of broilers. Three hundred and twenty broilers (1day-old) were randomly assigned to fourtreatments with eight replicates with ten broilers each. The positive control (PC) treatment was fed a corn-soybean meal diet. The diet of negative control (NC) treatment was reduced by210KJ ME/kg, andthe content of miscellaneous meal and wheat was increased in this treatment. The conventional dose ofenzyme (CDE) and super dose of enzyme (SDE) treatment was respectively fed the NC diet withdifferent dosages of compound enzyme0.02%and0.2%. The trial lasted for42d. The results showedthat the FCR of broilers fed the SDE treatment was significantly lower than those fed the NC (P<0.01);the FCR of broilers fed the CDE and PC treatment was significantly lower than the NC (P<0.05). TheCP, EE and energy apparent metabolizability of the SDE treatment diet were higher than the NC diet(P<0.01); the DM apparent metabolizability and AME of the SDE treatment diet were higher than theNC diet (P<0.05). There were no significant differences in the organ index of small intestine and spleenof broilers fed different diets (P>0.05); the organ index of stomach and bursa of fabricius of broilers fedthe SDE diet had difference significantly with those fed the NC diet (P<0.01). The thyroxine of broilersfed the NC treatment was significantly lower than other three treatments (P<0.05); the insulin ofbroilers fed the SDE diet was significantly higher than other three treatments (P<0.05). As for villiheight, the PC and SDE treatment were significantly higher than other two treatments (P<0.01); the NCtreatment was significantly lower than the CDE treatment (P<0.05). As for crypt depth, the NCtreatment were significantly lower than other three treatments (P<0.01); the SDE treatment wassignificantly lower than the PC and CDE treatment (P<0.05). As for V/C ratio, the SDE treatment weresignificantly higher than other three treatments (P<0.05); the NC treatment was significantly lower thanthe PC and CDE treatment (P<0.05). As for mucosa thickness, there was no significantly difference(P>0.05). As for digesta viscosity, the SDE treatment was significantly lower than the NC treatment(P<0.05). As for Lactobacillus of microbial community, two added enzyme treatments weresignificantly higher than the NC and PC treatment (P<0.05). The experiment prove that the growthperformance, nutrients utilization, organ index, serum hormones concentration, intestinal mucosamorphology, digesta viscosity and microbial community of broilers were improved significantly as thebroilers fed the diet (reduce210KJ ME/kg) containing complex enzyme at the level of0.02%and0.2%.The effect of complex enzyme was increased significantly while the dosage of enzyme was enhancedfrom0.02%to0.2%.
引文
[1]鲍恩东，等.肉鸡热应激病理损伤与应激蛋白相关性研究[J].中国农业科学，2004，37（2）：301-305.
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