【作者】 李勇；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本试验的目的是通过研究玉米淀粉结构(直链淀粉与支链淀粉比例,下称“直/支比”)、玉米膨化和酶制剂添加对仔猪玉米—豆粕日粮体外和体内消化率的影响,以及玉米膨化和酶制剂添加对仔猪肠道菌群多样性的作用,为建立更加准确有效的玉米品质控制方案,减小由玉米品质差异引起的动物生产性能的变异,改善玉米的饲用价值提供理论依据。本研究开展了三部分试验,首先探讨了玉米淀粉结构和膨化玉米储藏时间对体外抗性淀粉的形成以及对淀粉消化率的影响；其次通过胃蛋白酶—胰蛋白酶两步消化-透析法(下称“体外法”),以干物质、粗蛋白、总能、淀粉和NDF的消化率为指标,筛选出仔猪玉米—豆粕日粮的淀粉酶、蛋白酶和木聚糖酶的适宜配比；并研究了玉米膨化、添加植酸酶或植酸酶与淀粉酶、蛋白酶和木聚糖酶组合对玉米—豆粕日粮体外消化率的影响；在此基础上采取体外法和简单T-型瘘管法(下称“体内法”)相结合的方法,进一步研究膨化和酶制剂添加对提高仔猪玉米—豆粕日粮体内消化率及对肠道菌群多样性的影响,并建立了体内消化率和体外消化率之间的回归关系,以评价体外法筛选酶制剂的可靠性；第三,研究不同淀粉结构的玉米膨化或在玉米—豆粕日粮中添加酶制剂对仔猪日粮体内消化率的影响。主要研究结果如下：第一部分试验：采用3×5因子设计,研究玉米淀粉结构和储藏时间对膨化玉米中抗性淀粉的形成以及对淀粉体外消化率的影响,为寻求适合于膨化的饲用玉米提供参考。从78个玉米样品中筛选出3种玉米,LA(低直/支链比)、MA(中直/支链比)和HA(高直/支链比),然后进行膨化并在温度15℃和湿度80%RH条件下储藏不同时间(0w、1w、2w、3w和4w)。用体外测定的方法,以抗性淀粉的形成和淀粉消化率为指标,评估淀粉的直/支比对膨化玉米质量的影响。结果表明：直/支比的提高极显著地提高了抗性淀粉的含量,降低了淀粉体外消化率(P<0.01)。随着储藏时间的增加抗性淀粉的含量极显著地提高(P<0.01),同时淀粉消化率显著降低(P<0.05)。本试验结论：高直/支比显著地提高膨化玉米储藏后抗性淀粉的含量以及降低了淀粉的体外消化率。随着储藏时间延长,膨化玉米抗性淀粉含量极显著增加,淀粉消化率显著降低(P<0.05)。,第二部分试验：试验一：采用完全随机性设计,应用体外法探讨淀粉酶、蛋白酶和木聚糖酶的高(2500U/kg、1250U/kg、2200U/kg)、中(1500U/kg、750U/kg、2200U/kg)、低(1000U/kg、500U/kg、260U/kg)三个不同剂量组合的联合添加对玉米—豆粕日粮干物质、粗蛋白、总能、淀粉和NDF体外消化率的影响,筛选并确定各种成员酶的适宜配比。试验结果表明：高酶水平组和中酶水平组的淀粉和NDF的消化率均显著高于低酶水平组和对照组(P<0.05)。虽然高酶水平组和中酶水平组的干物质、总能、淀粉和NDF的消化率差异不显著,但粗蛋白消化率高酶水平组显著高于中酶水平组(P<0.05)。。本试验结论：中等剂量的淀粉酶(750U/kg)和木聚糖酶(2200U/kg)是该玉米-豆粕日粮的适宜添加水平。试验二采用2×6因子设计,应用体外法进一步考察玉米膨化、添加植酸酶(500U/kg,下同)或植酸酶与中和高酶水平组合联合对玉米-豆粕日粮体外消化率的影响,为提高玉米的营养价值进一步提供理论依据。本试验高、中酶水平组含试验一(第三章)的中等水平的木聚糖酶和淀粉酶,蛋白酶水平在两个组保持不变。试验结果表明：膨化玉米组日粮干物质、蛋白、总能、淀粉和NDF的消化率都显著高于未膨化玉米组(P<0.05)。在未膨化玉米组,植酸酶和高、中酶水平联合使日粮总能,蛋白和NDF和淀粉消化率显著高于单独添加植酸酶组(P<0.05)。本试验结论：玉米膨化可以提高所有考察的营养物质的消化率；植酸酶和淀粉酶、蛋白酶、木聚糖酶组合之间存在协同效应。试验三采用体外和体内两种方法结合考察玉米膨化和添加植酸酶、中、高酶水平组合对玉米—豆粕日粮消化率的影响,并用DGGE技术考察膨化和酶制剂添加对肠道菌群多样性的影响,从而评估体外法对筛选酶制剂的有效性。体外试验采用单因子设计,以膨化玉米组为正对照,以未膨化组为负对照,在未膨化组添加植酸酶或中、高水平酶组合(酶水平同试验二)共五个试验组。体内试验选择5头安装简单T-型瘘管的断奶仔猪进行5×5拉丁方试验,日粮同体外试验。评估膨化和添加酶制剂对日粮体外、回肠末端及粪干物质、总能、淀粉和NDF消化率的影响,并应用PCR/DGGE技术评估膨化和酶制剂添加对回肠末端菌群多样性的影响。结果表明：和试验二结果相似,膨化玉米组较未膨化玉米组有较高的干物质、蛋白、淀粉、总能、淀粉和NDF的消化率(P<0.05)。中、高酶水平的添加提高了未膨化玉米组体内干物质和蛋白的消化率(P<0.05)。除蛋氨酸、苏氨酸、色氨酸和胱氨酸外,高酶水平组体内氨基酸消化率达到膨化玉米组的水平(P<0.05),而且显著高于负对照组。体外营养消化率、能量消化率和体内的营养消化率、能量消化率之间存在较强的线性相关(P<0.05)。回肠食糜中细菌PCR/DGGE电泳图谱结果相似性聚类分析表明,处理组间相似性很差,说明膨化和酶制剂的添加影响了肠道菌群的建立。与对照组相比,高水平酶组降低了回肠末端食糜细菌DGGE图谱条带数和多样性指数(P<0.05)。本试验结论：玉米膨化或酶制剂添加是提高仔猪日粮营养价值的有效方法。第三部分试验：应用体内法研究了玉米淀粉结构、玉米膨化和添加酶制剂对玉米—豆粕日粮的仔猪体内消化率的影响,为选择适用于仔猪的玉米提供依据。选择6头安装简单T-型瘘管的断奶仔猪进行6×6拉丁方试验。以第一部分试验选出的低直伎比玉米(LA)组为正对照,高直／支比玉米(HA)组为负对照,在两个对照组中分别添加高水平酶(同试验三)或将两种玉米膨化,共六个试验组,以淀粉和能量的消化率为指标,评估玉米淀粉结构、膨化和添加酶制剂对仔猪日粮消化率的影响。结果表明：玉米膨化极显著提高了两种不同直／支链比玉米—豆粕日粮的回肠末端淀粉和能量表观消化率(P<0.01)；低直／支比玉米—豆粕日粮仔猪回肠末端淀粉和能量表观消化率极显著高于高直／支比玉米—豆粕日粮(P<0.01)；加酶并没有显著提高两种玉米的仔猪回肠末端淀粉和能量表观消化率(P>0.05)。本试验结论：低直／支比玉米的淀粉和能量消化率显著高于高直／支比玉米；膨化能够极显著提高玉米回肠末端淀粉和能量消化率。本研究通过以上试验得到如下结论：①玉米中淀粉的结构(玉米淀粉直／支比)对仔猪回肠淀粉消化率和消化能具有显著影响,因此,玉米淀粉直／支比是影响玉米品质的重要因素。②玉米膨化加工可以显著提高玉米—豆粕日粮营养物质的消化率；适宜的酶制剂种类和配比有利于提高仔猪玉米—豆粕日粮回肠末端干物质、蛋白和大部分氨基酸的消化率,并且降低回肠末端菌群的多样性。③体外两步酶解法可以用于预测仔猪对营养物质的消化率,从而提高对猪日粮中酶制剂进行筛选的速度和效率。更多还原

【Abstract】 In vitro and in vivo studies were conducted to investigate the effect of corn starch structure (amylase amylopectin ratio, AM/AP), corn extrusion and different enzyme blends on digestibilities of pig diets and ileal digesta bacterial community, which may provide reference to reduce corn nutritive variation and improve nutritive value of corn. This study has three parts. In first part, the effects of corn starch structure and the strorage duration of extruded corn on resistant starch formation and starch digestibility were investigated initially. In second part, the diets were incubated in sextuple with the in vitro two-stage enzyme incubation and dialysis procedures (in vitro method), and digestibilities of proximal nutrients of corn/soya based diets in terms of of dry matter, crude protein, starch, NDF and energy were determined to decide the optimal dosage of three enzymes. Further investigation was done on the effects of corn extrusion, phytase and its combination with amylase, protease and xylanase blend on the digestibilities of proximal nutrients of corn/soya based diets. Following experiment by in vitro and T-canula (in vivo) were conducted to find the effects of enzyme addition and corn extrusion on ileal digestibilities of proximal nutrients of corn/soya based diets and ileal digesta bacteria comunity, and also established regression relationships between digestibilities in vitro and in vivo to validate the effect of enzyme screened out by in vitro. In third part, based on the experiments above, the effects of corn extrusion and enzyme inclusion on the digestibilities of different statch structure (AM/AP) corn/soya based diets were compared. The main results are as followings:In First part, the effects of AM/AP and storage duration on formation of resistant starch and starch digestibility of extruded corn were investigated. Three corn samples with high, medium and low AM/AP (0.39,0.44, and 0.6) selected from 76 corn samples are extruded and stored in Ow, 1w,2w,3w and 4w at temperature 15℃and humidity 80%RH. In this experiment, extruded corn with higher AM/AP has the higher resistant starch formation and lower starch digestibility (P<0.01). And with the extended storage period from Ow to 4w, the resistant starch content of extruded corn was increased significantly and starch digestibility was decreased (P<0.05). Conclusion:corn AM/AP and extrude corn storage condition increased the resistant starch content and decreased the corn starch digestibility in vitro. In second part, Exp.1 the effects of enzyme blends (amylase, protease and xylanase) at different dosage rates [(high (H) vs medium (M) vs low (L)] on the digestibility were investigated in vitro using a two-stage enzyme incubation method to decide the optimal dosage of enzymes. The digestibilities of starch and NDF were both higher in H-and M-enzyme diets than in L-enzyme and control diets (P<0.05). Though the CAD of DM, GE, starch and NDF did not differ between H-and M-enzyme diets, the CP digestibilities was higher in H-enzyme than in M-enzyme diets(P<0.05). Conclusion:Medium level of amylase and xylanase is optimal to the corn/soya based diets.Experiment 2 was designed to examine the effects of corn extrusion, the addition of phytase and its combination with H-and M-enzyme on the digestibilities of dietary components based on the results of exreriment 1 to provide theory basis for improving corn nutritive value. In this experiment M-levels of xylanase and amylase were included in both the H-and M-enzyme blends, with protease dosage being held the same in each blend as in Exp.1. The digestibilities of all dietary components evaluated was higher (P<0.05) in Extruded than in nEXd corn diets. For nEXd corn diets, the combination of phytase with M-and H-enzymes resulted in a higher (P<0.05) digestibilities of GE, CP, NDF and starch than did phytase addition alone. In Extruded or nEXd the digestibilities of starch and NDF was higher (P<0.05) in phytase, H-and M-and phytase combined with H-and M-enzyme compared to the control diet. Conclusion:extrusion improved digestibility of the diets. The combination phytase and xylanase, amylase, protease was demonstrated to be more effective in improving the nutrient digestibility than did any of the single enzyme addition.In Exp.3, both in vitro and in vivo trials were conducted to evaluate responses of corn/soya based diets to the corn extrusion and the addition of phytase, H-and M-enzyme blends. Five cannulated pigs were fed five diets according to a 5×5 Latin square design. Similar to the results observed in vitro and in vivo trial. The Extruded corn diet had higher (P<0.05) digestibilities of DM, CP, starch, NDF and GE than nEXd corn diets. M-and H-enzyme addition both resulted in increased DM and CP digestibilities of nEXd corn diets. The digestibilities of amino acids of nEXd corn diets with H-enzyme addition was comparable with that of the Extruded corn diet (P<0.05), and was higher (P<0.05) than that of the nEXd corn diet except for Met, Thr, Trp and Cys. Compared to the nEXd, extrusion of corn improved the digestibilities of CP, starch and GE in feces. These results suggest that extrusion and multiple enzyme addition are-effective ways to improve the nutritional value of corn-based diets for pigs. There was a close linear relationship between the digestibilites in vitro and in vivo.Similarity analysis of bacterial community DGGE profiles from ileal digesta indicated low similarity of among the treatments. The diversity index of bacterial community in H-enzyme was significantly lower than the control (P<0.05), suggesting the enzyme inclusion had a noticeable effect on bacterial community of pig ileum.In third part, an in vivo trial was conducted to evaluate responses of corn/soya based diets to the starch structure, corn extrusion and the addition of H-enzyme blend in Exp.3 of the second part. Six cannulated pigs were fed-six diets according to a 6×6 Latin square design. Regardless of enzyme inclusion or AM/AP, corn extrusion increased the ileal starch and GE digestibilities corn/soya based diets of pigs. The lower AM/AP corn/soya based diets has higher ileal starch and GE digestibilities. The results showed that corn extrusion and lower AM/AP ratio increased the ileal starch and GE digestibilities of cron/soya based diets of pigs, but Enzyme addition has no effects on the ileal starch and GE digestibility.Conclusions can be drawn from results above:Corn starch amylose/amylopectin ratio has significant effects on the pig ileal starch and energy digestibility. Corn starch amylose/amylopectin ratio is an important factor that affects corn quality. Corn extrusion improved the digestibilities of the weaned pig corn/soya based diets. Optimal Enzyme type and inclusion rate increased the ileal DM, CP and most AA digestibilities and decreased ileal bacterial community of weaned pigs. The in vitro method can be used to predict the nutrient digestibilities by pigs to increase the speed and efficiency of enzyme selection更多还原