【作者】 张富新；

【导师】 陈锦屏；

【作者基本信息】 陕西师范大学 ， 动物学， 2003， 博士

【摘要】 本实验以关中奶山羊皱胃为原料，对羔羊哺乳状况、屠宰年龄、皱胃处理方式和不同提取方法对凝乳酶活性的影响，凝乳酶原的激活，凝乳酶纯化与分离以及凝乳酶和胃蛋白酶特性进行了系统的研究，为羔羊凝乳酶开发以及在干酪生产中的应用提供理论基础。 羔羊哺乳状况研究表明，全哺乳组凝乳活性最高(26108SU／g)，蛋白水解活性与随机哺乳组无明显差别(P＞0.05)，而全喂草料组蛋白水解活性最高(1925U／g)，这表明哺乳能够刺激皱胃中凝乳酶的持续分泌。屠宰年龄实验表明，羔羊出生5天时，凝乳活性与蛋白水解活性比率(C／P)最大，是提取羔羊凝乳酶的最佳年龄。 皱胃处理方式研究结果表明，冷冻处理对酶活性影响最小，盐渍风干处理影响较大，自然风干处理酶活性损失最多。同时还发现盐渍风干处理和自然风干处理可使皱胃中一部分酶原自动激活。 用传统方法和不同pH缓冲液方法提取羔羊凝乳酶时，食盐浓度、提取时间、提取温度、提取液与皱胃比例、提取次数对凝乳活性有重要的影响。随着食盐浓度增大，凝乳活性逐渐提高，当达到一定浓度后，凝乳活性又逐渐降低；在提取初期，提取速度较快，凝乳活性明显提高，当提取达到最大值后，凝乳活性又逐渐下降；随着提取温度的升高，凝乳活性逐渐增大，但温度过高时，会导致酶变性失活；随着提取液与皱胃比例的增大，酶溶出速度加快，提取次数越多，皱胃中酶提取越充分，提取2次后，皱胃中绝大部分酶被提出。正交试验表明，用传统方法提取时，当食盐浓度8％，皱胃与提取液比例1：20，提取温度30℃，提取时间48h，提取最大凝乳活性为65700SU／g；用pH4.5缓冲液提取时，当食盐浓度6％，皱胃与提取液比例1：10，提取温度30℃，提取时间60h，提取最大凝乳活性为58200SU／g；用pH5.4缓冲液提取时，当食盐浓度8％，皱胃与提取液比例为1：20，提取温度30℃，提取时间48h，提取最大凝乳活性为72300SU／g；用pH6.0缓冲提取时，当食盐浓度8％，皱胃与提取比例1：10，提取温度25℃，提取时间48h，提取最大凝乳活性为63850SU／g。由此可见，用pH5.4缓冲液提取，可获得最大的酶活性产量。 本实验还对超声波在羔羊凝乳酶提取中的应用进行探讨，结果表明，超声提取可大大缩短提取时间，超声提取40min可达到传统方法48h的提取效果。在食盐浓度8％，皱胃与提取液比例1：15时，用30W／cm2超声强度提取40min，可获得最大的凝乳活性。羔羊凝乳酶以无活性的酶原形式分泌，pH，食盐和温度对其激活有重要的影响小H越低，激活速度越快，在 pHZ．0激活时仅需 5—10ruin，pH3．0时需要30min，在较低pH下激活，己激活酶不稳定，易失活；随着缓冲液中NaCI浓度的增加，激活速度随之加快；在5℃下激活速度非常缓慢，25℃以上激活速度明显加快。 干酪生产中应用的酶是凝乳酶和胃蛋白酶的粗提物，应用凝胶过滤层析和离子交换层析对羔羊凝乳酶粗提物纯化分离结果表明，凝胶过滤层析不能使两种酶分离，仅除去一部分杂蛋白；利用DEAE纤维素（DE32）离子交换层析，可除去大部分杂蛋白，使凝乳酶和胃蛋白酶完全分离。此外，在对样品浓缩时发现，羔羊凝乳酶粗提物中含有分子量小于 10000的活性小肽物质，这在凝胶过滤层析时出现峰＊和峰I［l亦得到证实。应用SDS——PAGE凝胶电泳和PAGE凝胶等电聚焦电泳对纯化羔羊凝乳酶和胃蛋白酶测定结果表明，羔羊凝乳酶分子量为30700，等电点为pH4．5；羔羊胃蛋白酶分子量为29900，等电点为pH4．4。 羔羊凝乳酶特性研究表明，最适凝乳温度为45 ’C；45 ’C处理60Inin，酶活性开始降低，60’C处理60min，酶活性完全丧失；酶最适pH为2．（kr刀，在pHZ．（h刀范围内保持24h，具有较好的稳定性；在凝乳过程中，随着原料乳pH下降，凝乳活性逐渐增大；随着酶溶液中离于强度的增加，凝乳活性逐渐降低；乳中＊”具有明显的促凝作用，当CaC12浓度大于0刀3％时，凝乳活性趋势于稳定；AI叶可明显提高酶的凝乳活性，Cu2＋和Zn”对酶活性有抑制作用；随着乳液浓度增大，凝乳活性逐渐增加，当浓度大于 15％时，凝乳活性趋于稳定；以酪蛋白为底物，随着时间延长，蛋白水解活性逐渐增大，动力学特性研究表明，米氏常数llilll为6刁 g／L。 羔羊胃蛋白酶最适凝乳温度为45 ’C；在45℃处理30Inin，酶活性开始下降，60C处理30lliln，酶活性完全丧失；酶的最适pH为1．G－3刀，在此pH范围内具有很好的稳定性；原料乳pH、CaZ”浓度、乳液浓度和离子强度对凝乳活性的影响与凝乳酶规律一致：A13＋明显提高其凝乳活性，FC卜具有一定的促凝作用；以酪蛋白为底物，蛋白水解活性明显高于凝乳酶，米氏常数Km为234．sg／L。更多还原

【Abstract】 This experiment is to probe the factors, such as the effect of the kid’s suckling, the butchering age and the ay of the abomasums treatment on the chymosin activity;the activation of the prochymosin; the purification and separation of chymosin and the characterization of chymosin and pepsin, using Cuan Zhong dairy kid’s abomasums as material, which would provide the theoretic base for the exploitation of the kid chymosin and its application to the cheese production.The research on the condition of kid’s suckling indicates that the highest milk-dotting activity(26108su/g)was obtained for the whole suckling section, whereas proteolytic activity were no significant different between the whole suckling and random section(p>0.05),but proteolytic activity(1925u/g) was highest for the complete grass-feeding, which demonstrated suckling could stimulate the continuous secretion of chymosin in the abomasum. The experiment on butchering age showed that it was 5d after the kid birth that the ratio of clotting activity and proteolytic activity(C/P)was the highest, which was a best age for extracting kid chymosin.The methods of the abomasums treatment suggested that the freezing method had least influence on milk-clotting activity, while the salting air-drying and natural air-drying went to another extreme. Meanwhile, the partial proenzyme from the abomasums would be activated automatically by the latter two methods.As the kid chymosin was extracted by the traditional way and the buffering way at different pH values, its activity mainly depended on the salt concentration, extraction time and temperature, the ratio of buffer and abomasums and extraction times. With the increasing salt concentration, milk-clotting activity became higher and higher, then reached its peak, and then decreased gradually; In the early extraction, the speed was quick, the milk-clotting activity was increased obviously. After it amounted to the maximum, the activity became steadily lower steadily; the increasing temperature in extracting could improve the extraction activity, but too high temperature could result in the denaturation and inactivation; The greater the ratio of abomasums and butter and was, the quicker speed was when the enzymes was drawn out, and after extracting for twice, most of the enzymes in the abomasums could be drawn out. The result oforthogonal experiment were as follows:1.by the traditional method, using 8% salt concentration, 1:20 ratio of abomasums and butter, 30℃extraction temperature and 48h extraction time, the highest activity could be obtained; 2.In buttering way at pH4.5, pH5.4and pH6.0 respectively, using 6%,8%and8% salt concentration,1: 15,1:20and 1:15 ratio of abomasums and butter,30℃ ,3 0℃ and25℃ extracting temperature,60h,60h and 48h extraction time, the highest activity could be obtained. From above it could be see that it was the buffering way at pH5.4 that the largest enzyme yield could be obtained.In addition, the application of the supersonic wave to the extraction of kid’s chymosin was studied in the experiment. And the results showed that the supersonic extraction could shorten the extraction time greatly, which could have the same effect for 40min as the traditional way of 48h. The greatest activity could be obtained on the condition of 8% salt concentration, 1:20 ratio of abomasums and butter, 30W/cm2 supersonic strength and 40min extraction time.The kid chymosin was secreted as being the form of non-activated prochymosin, and its activation depended on pH value, salt concentration and temperature. The lower the pH value was, the higher activated speed become. It needed only 5-10min to activate at pH2.0, 30min at pH3.0. But the activated chymosin wasn’t somewhat stable enough at the lower pH value. The activated speed was improved by the increasing salt concentration. It was very slow at 5℃ but become higher above 25℃.The enzyme from abomasums used in cheese production was rough extracts containing chymosin and pepsin. Through the gel filtration chromatography and ion exchange c更多还原