猪模拟小肠液的制备及仿生消化法测定饲料可消化养分含量的研究

发布时间：2018-07-05 17:14

本文选题：生长猪 + 模拟小肠液　；参考：《中国农业科学院》2015年硕士论文

【摘要】：本试验首先研究生长猪空肠液中消化酶的特性、空肠液消化酶的纯化与模拟肠液的制备方法,在此基础上探讨了基于单胃动物仿生消化系统(SDS-Ⅱ)和模拟小肠液的仿生消化法测定日粮消化能和可消化氨基酸含量的可行性。从5个方面开展工作:试验一旨在研究不同生长阶段及日粮条件下猪空肠液消化酶的活性,为模拟小肠液的制备参数提供参考。试验采用2×2两因素完全随机设计,其中日粮设两个水平,即玉米-豆粕型日粮和玉米-玉米干酒糟(DDGS)-米糠粕型日粮,猪生长阶段设两个水平,即生长期(60-100天)和育肥期(100-140天),测定空肠液中淀粉酶、胰蛋白酶、糜蛋白酶、脂肪酶的活性、总蛋白含量和p H。结果表明:日粮类型与生长阶段对生长猪空肠液中各消化酶体积比活性及p H均无显著影响(P0.05),猪空肠液单位体积主要消化酶平均活性和p H分别为:淀粉酶189.79 U/ml、胰蛋白酶62.10U/ml、糜蛋白酶7.36 U/ml、脂肪酶4.91U/ml和p H 6.44。试验二旨在探讨空肠消化液中主要消化酶的活性与日粮养分消化率间的相关性,为模拟肠液制备中消化酶活性的误差控制标准提供参考。玉米-豆粕型日粮和玉米-DDGS-米糠粕型日粮分别设16个重复,每个重复1头猪,测定日粮及粪样的干物质、盐酸不溶灰分(AIA)、粗蛋白、总能及肠液中主要消化酶的活性,然后对每种日粮下的养分消化率与主要消化酶的活性进行简单相关分析和典型相关分析。结果表明:1)两种日粮下,生长猪个体间4种主要消化酶活性的变异系数均在20%以上,而日粮干物质、粗蛋白、能量消化率的变异系数均在1.5%以内;2)猪空肠液中淀粉酶、胰蛋白酶和糜蛋白酶活性与日粮养分消化率无显著相关性(P0.05),而脂肪酶的活性与日粮养分消化率有一定的相关性,但对养分消化率变异的贡献是有限的。试验三分为两部分进行:首先探讨猪空肠液中消化酶的纯化方法,然后比较以空肠液纯化粉剂制备的模拟小肠液与以试剂酶制备的模拟小肠液在消化能力上的差异。采用2×2两因素完全随机设计,即日粮和猪生长阶段各设两个水平。采用冰乙醇沉淀-冻干纯化空肠液中的消化酶,测定纯化过程中消化酶的活性损失和肠液纯化粉剂的比活性。然后,根据四个处理下的空肠液中消化酶的活性,以试剂酶、处理组内肠液纯化粉剂+试剂酶、所有处理肠液混合液纯化粉剂+试剂酶制备模拟小肠液分别测定日粮的水解物能值(EHGE),并与生物学法测值进行相关性分析。结果表明:1)乙醇沉淀-冻干获得的肠液消化酶活性的回收率均在60%以上,日粮类型和生长阶段对纯化粉剂的消化酶活性无显著影响;2)乙醇沉淀过程中消化酶的活性损失显著的高于冻干过程(P0.01);3)以肠液纯化粉剂制备的模拟小肠液测定日粮的EHGE均显著高于以试剂酶制备模拟肠液的相应测值(P0.05),且与生物学法测值的相关性更高。以上结果表明,乙醇-沉淀冻干法提纯的肠液消化酶粉剂可以更好地模拟小肠液。试验四旨在研究基于胃蛋白酶-模拟猪肠液-Viscozyme复合酶的仿生消化法估测猪棉籽粕日粮的消化能值。分别采用肠液纯化粉剂和试剂酶模拟猪小肠液测定11种脱酚棉籽蛋白和19种棉籽粕日粮的酶水解物能值,并与生物学法测值进行简单相关分析。结果表明:1)采用肠液纯化粉剂模拟小肠液测定脱酚棉籽蛋白的表观酶水解物能值(AEHGE)和真酶水解物能值(TEHGE)与试剂酶测值间均具有很高的相关性(P0.05,r=0.96);2)采用肠液纯化粉剂比试剂酶制备的模拟小肠液测定的棉籽粕日粮的酶水解物能值与生物学法测值的相关性更高(r=0.94 VS 0.87)。这表明基于猪源性消化酶粉剂制备的模拟小肠液在棉籽粕酶水解物能值的测定上优于试剂酶。试验五研究基于胃蛋白酶-肠液纯化粉剂两阶段仿生消化法测定棉籽粕可消化氨基酸的含量。以仿生消化法分别测定10种不同来源棉籽粕的可消化氨基酸含量,并与生物学法测值进行相关性分析。结果表明:仿生消化法测定的棉籽粕除蛋氨酸外的其他氨基酸的可消化含量均与体内法测值显著相关(P0.05),这表明猪源性消化酶粉剂制备的模拟小肠液适用于棉籽粕可消化氨基酸含量的测定。
[Abstract]:In this experiment, we first studied the characteristics of digestive enzymes in the empty intestines of growing pigs, the purification of digestive enzymes in the jejunum and the preparation of simulated intestinal liquid. On the basis of this, the feasibility of the bionic digestion method based on the bionic digestive system (SDS- II) and the simulated intestinal fluid for the determination of the digestible energy and the content of digestible amino acids in the diet were discussed in 5 aspects. The experiment is to study the activity of digestive enzyme of pig jejunum in different growth stages and diets and provide reference for simulating the preparation parameters of small intestinal fluid. The experiment adopts 2 x 2 two factors completely random design, of which there are two levels of diet, namely corn soybean meal type diet and corn corn dry distiller's grains (DDGS) rice bran meal type diet. At the growth stage, there were two levels, namely, the growth period (60-100 days) and the fattening period (100-140 days). The activity of amylase, trypsin, chymotrypsin and lipase in the jejunum fluid, total protein content and P H. results showed that there was no significant effect on the volume ratio activity of digestive enzymes and P H in the growth stage and the growth stage (P0.05). The average activity of the main digestive enzyme and P H in the unit volume of the jejunum were: amylase 189.79 U/ml, trypsin 62.10U/ml, chymotrypsin 7.36 U/ml, lipase 4.91U/ml and P H 6.44. test two to explore the correlation between the activity of the main digestive enzymes in the jejunum digestive juice and the nutrient digestibility in the diet, to simulate digestive enzyme activity in the preparation of intestinal liquid The diet of corn soybean meal and corn -DDGS- rice bran meal had 16 repetitions, each repeated 1 pigs, measured the dry matter of the diet and feces, the insoluble ash (AIA), the crude protein, the total energy and the activity of the main digestive enzymes in the intestinal juice, and then the nutrient digestibility and the main elimination of each diet. The activity of the enzyme was analyzed by simple correlation analysis and canonical correlation analysis. The results showed that: 1) under two diets, the coefficient of variation of 4 main digestive enzyme activities among 4 growing pigs was more than 20%, while the coefficient of variation of dry matter, crude protein and energy digestibility were within 1.5%; 2) amylase, trypsin and chymotrypsin in pig's empty intestines. There was no significant correlation between sex and dietary nutrient digestibility (P0.05), but the activity of lipase had a certain correlation with the nutrient digestibility of the diet, but the contribution to the variation of nutrient digestibility was limited. The experiment was divided into two parts. First, the purification method of digestive enzymes in the pig jejunum solution was discussed, and then the preparation of the purified powder was prepared by the jejunum solution. The difference between the simulated small intestinal fluid and the simulated intestinal fluid prepared by the reagent enzyme was different. The 2 x 2 two factors were designed completely, and there were two levels in the diet and the growth stage of the pig. The digestive enzymes in the jejunum solution were purified by the ice ethanol precipitation and freeze drying. The loss of the activity of the elimination enzyme in the purification process and the ratio of the purified powder in the intestinal liquid were measured. Then, according to the activity of digestive enzymes in the four treated jejunum fluid, the reagent enzyme, the purified powder + reagent enzyme in the intestinal liquid of the treatment group, the hydrolysate energy value (EHGE) of the simulated small intestine liquid were prepared by the purified powder and reagent of the intestinal liquid mixture, and the correlation analysis was carried out with the biological method. The results showed that: 1) The recovery of the activity of digestive enzyme obtained by ethanol precipitation and freeze drying was above 60%, and the diet type and growth stage had no significant effect on the digestive enzyme activity of the purified powder; 2) the activity loss of digestive enzymes in the process of ethanol precipitation was significantly higher than that of the freeze drying process (P0.01); 3) the EH of the simulated intestinal fluid for the determination of the diet prepared by the purified powder of intestinal fluid GE was significantly higher than the corresponding measured value of the simulated intestinal liquid (P0.05) with the reagent enzyme, and the correlation was higher with the biological method. The above results showed that the enteric digestive enzyme powder purified by the ethanol precipitation freeze dry method could better simulate the small intestinal fluid. Four the experiment was designed to study the bionics based on the gastric protease mimic pig intestinal fluid -Viscozyme complex enzyme. Digestion method was used to estimate the digestibility of pig cottonseed meal diet. The enzyme hydrolysate values of 11 dephenolates and 19 kinds of cottonseed meal were measured by intestinal liquid purification powder and reagent enzyme simulated pig small intestine solution. The results showed that 1) used intestinal liquid to simulate small intestine liquid to determine dephenolol. The apparent enzyme hydrolysate value (AEHGE) and the value of the true enzyme hydrolysate (TEHGE) and the measured value of the reagents had a high correlation (P0.05, r=0.96); 2) the correlation between the hydrolysate of the hydrolysate of the cottonseed meal measured by the intestinal solution and the measured values of the cottonseed meal was higher than that of the reagents (r=0.94 VS 0.). 87). This shows that the simulated intestinal fluid based on porcine digestive enzyme powder is superior to the reagent enzyme in the determination of the enzyme hydrolysate of cottonseed meal. In test five, the content of digestible amino acids in cottonseed meal was determined by the two stage bionic digestion method based on pepsin - intestinal liquid purification powder. 10 different sources of cottonseed were determined by bionic digestion method. The results showed that the digestible content of other amino acids in the cottonseed meal, except methionine, was significantly correlated with the body method (P0.05), which showed that the simulated intestinal fluid prepared by the porcine digestible powder was suitable for the digestibility of cottonseed meal. Determination of the content of amino acids.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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