【作者】 欧宇；

【导师】 李胜利；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2003， 硕士

【摘要】 本论文通过三部分试验就四种处理组日粮在绵羊瘤胃的肽释放规律，及五种抗瘤胃降解小肽进行了研究。 第一章 不同预处理条件对瘤胃液含氮物质分离结果的影响 通过两个实验研究了不同预处理条件对瘤胃液含氮物质分离结果的影响，并筛选出最佳的离心分离条件。实验一以三只成年羯羊为实验动物，于清晨空腹时采集瘤胃液，设100g、150g、200g、250g、300g五个离心力处理组，以原虫数为评定指标，初选出200g，10min的分离条件，再以200g为离心力，设5min、10min、15min、20min四个离心时间处理，以离心沉淀中瘤胃液氮含量(N％)、N％与原虫数的比值为评定指标，分析认为，200g，10min是较为适宜的低速离心条件。实验二在试验一的基础上，设10,000g、15,000g、20,000g、25,000g四个高速离心力处理组，以瘤胃液有机物含量、瘤胃液氮含量和瘤胃液有机物中氮的百分含量为评定指标，分析认为，20,000g，15min是较为适宜的高速离心条件。 第二章 不同蛋白质饲料在瘤胃内的肽释放规律的研究及对瘤胃pH值、氨氮、菌体蛋白氮、游离氨基酸氮的影响 本试验目的是评价不同蛋白源对瘤胃液肽浓度的变化规律的影响。采取4×4拉丁方设计，以豆粕、酪蛋白、鱼粉、玉米蛋白粉四种蛋白质原料设计四个处理组日粮。在晨饲后0、1、2、4、6小时采集瘤胃液，对瘤胃的pH值、肽、氨基酸及其它蛋白质成分进行分析。各处理组的瘤胃pH值在采食后均呈下降趋势。豆粕、鱼粉、玉米蛋白粉三组的氨氮浓度在采食后1h达到峰值，酪蛋白组在采食后4h达到峰值，各处理组的氨氮浓度在采食后6h均回复至接近初始值。各处理组的菌体蛋白氮均在采食后1h达到峰值，然后在2h或4h恢复到初始水平。除酪蛋白组外，各处理组的瘤胃液游离氨基酸浓度随时间的变化差异不大。各处理组的瘤胃液肽浓度均在采食后1h达到峰值，但释放程度及6h内在瘤胃的平均释放量不同；在采食后1小时和6小时瘤胃所释放的肽中，氨基酸残基相对比例较高的有天冬氨酸、谷氨酸、酪氨酸、组氨酸。瘤胃内肽的释放与氨氮值和菌体蛋白氮有一定的正相关关系。其中豆粕处理组肽的释放与氨氮值、菌体蛋白氮的变化呈显著正相关。 第三章 抗瘤胃降解小肽的研究 通过两个试验对具有特别氨基酸结构的小肽在瘤胃内的累积情况作了研究。试验一用高效液相色谱法测定了Gly-Gly、Gly-Gly-Gly、Pro-Ala、Val-Val、Pro-Leu、Met-Met六种小肽的标准品。经单标肽的测定得到六种小肽的HPLC出峰保留时间，依次分别为：2.71、2.74、3.62、16.38、33.74、35.65min。Pro-Ala、Val-Val、Pro-Leu、Met-Met四种小肽在6～60ug／ml的浓度中国农业大学硕士学位论文：中文摘要一之间，其峰面积＊）对质量浓度（y3 fig／thl）回归得到方程分别为 y－（X＋85733）／17568，时一09998；y一（X－20138）／26865，／一0．9992；y一（ ＋363．56）／16979，才一0．9899；y一（X一16887）／55499，RZ—0．994。分析认为，HPLC测定小肽的含量快速、简便和准确，结果稳定，可以用来测定己知的小肽在瘤胃中的释放情况。试验二以鱼粉、豆粕、酪蛋白、玉米蛋白粉四种原料为主要蛋白源设计四种日粮处理组，采集不同时间点的绵羊瘤胃液，并进行超滤截留分子量小于3000Da 以下的瘤胃降解物，利用高效液相色谱技术（HPLC）测定这四种处理组瘤胃中小肽 （Pro－Ala、ValVal、Pro－Leu、MetMet）在不同时间点的瘤胃含量。玉米蛋白组、鱼粉组、豆粕组在瘤胃内6h的小肽累积量顺序为：Pro．Leu＞ValVa＞Pro．la＞MetMet，酪蛋白组瘤胃内6h的小肽累积量顺序为：Pro－Leu＞Pro－la＞ValValDeMet．Met。本试验研究证实，从氮端起第一位氨基酸残基是Pro，又有疏水性氨基酸结构的小肽，或者两种结构特点居其一的小肽，有抗瘤胃降解的特性。更多还原

【Abstract】 A serial of experiments in this article were conducted to investigate the release rule of peptides in sheep rumen, which were given four diets, and small peptides (SP) resistant to rumen degradation.Chapter 1 Effects of different pre-processing on separation result of rumen nitrogen contentsIn this study, different separation pre-condition of nitrogen contents in rumen were investigated, and the prime condition of centrifugation was determined. Experiment 1 was conducted on three sheep. Rumen contents was sampled in the morning, which was centrifuged by five levels (100g, 150g, 200g, 250g , 300g) .Evaluating by protozoa number, it was roughly determined that 200g is better. Then, rumen contents was centrifuged at 200g for 5 min> 10min> 15min> 20min respectively. Evaluating by percent of nitrogen in deposition (N%) , ratio between N% and protozoa number, 200g,10min was considered the best low speed of centrifuge. Experiment 2 is based on the preceding one, designing four groups of high speed centrifugationC 10,000g, 15,000g>20,000g,25,000g). Evaluating by organic matter of rumen contents, percent of nitrogen, percent of nitrogen in organic matter, it was determined that 20,000g, 15min was the best condition of high speed centrifugation.Chapter 2 Research of peptides release in sheep rumen giving different protein ration and elfect on pH^ NH3-N> MCP and FAA-N in rumen contentsThe objective of this experiment is to evaluate the effect of different protein resource on peptide concentration in rumen. This experiment is used in a 4x4 Latin square design, and four diets, of which main protein resources are soybean, casein, fish meal and corn gluten meal respectively. At 0, 1, 2, 4 and 6 h after morning meal, rumen contents were collected, and pH, peptides, ammo acid and other nitrogen-containing compounds in the rumen were analyzed. PH value of all treatments tended to go down. NH3-N concentration of SBM, FM, CGM attained peak value at 1h after meal, and of casein did at 4h after feeding. The NH3-N concentration of all treatments came back to initial value at 6h. MCP of all treatments attained peak value at Ih after meal, then came back to initial level at 2h or 4h. Except casein group, the FAA-N value of rest ones changed not much.Peptide concentration in rumen of all treatments attained peak value at 1h after meal. However, it is various that the release extent of rumen peptide and average peptide concentration within 6h. In the peptides from rumen at Ih and 6h after meal, contain predominantly asparticx glutamic, tyrosine and histidine residues. There is correlation between peptide release and NH3-N, MCP. The rumen peptide release of SBM group is significantly correlated with change of NH3-N, MCP.Chapter 3 Research of small peptides that resistant to rumen degradationWith two experiments, it was studied that accumulation of rumen SP with special amino acidconstruction. Experiment 1 was conducted to analyze the standard product of Gly-Gly, Gly-Gly-Gly, Pro-Ala, Val-Val, Pro-Leu, Met-Met, with high-performance liquid chromatography (HPLC). The retention time (RT) of these six standard peptides is: 2.71, 2.74, 3.62, 16.38, 33.74, 35.65min respectively. Due to the close molecular structure, it is hard to separate the chromatogram wave between Gly-Gly and Gly-Gly-Gly, so this two peptides were not analyzed in following research. The results indicated that between 6~60ug/ml, the regression equation of peak area (x) against concentration (y, ug/ml) of the rest four dipeptides was y = (x +857.33)7 17568, R2=0.9998; y = (x -2013.8)/ 26865, R2=0.9992; y = (x +363.56)7 16979, R2=0.9899; y = (x - 16887)7 55499, R2=0.994 respectively. HPLC method is rapid, simple, accurate and stable, and which is proposed to be used for measuring known small peptides in rumen contents. Experiment 2 designed four diets as those in chapter 2. Rumen contents was sampled from sheep at different time, and after pre-processing, was ultra filtered and intercepted mixture of SP, MW<3000Da. With HPLC, SP (Pro-Ala, Val-Val更多还原