【作者】 李向阳；

【导师】 董海洲；

【作者基本信息】 山东农业大学 ， 食品科学， 2013， 博士

【摘要】 随着食品工业的飞速发展,需要大量具有卓越功能特性的蛋白质作为食品原料。通过糖基化改性等化学改性方法可以使大豆蛋白原有的许多功能性质,能够更好的满足食品工业的需要。近年来蛋白酶水解大豆蛋白一直是人们研究的热点。传统研究蛋白酶水解大豆蛋白生成大豆肽的方法,是通过三硝基苯磺酸法测定产物的水解度,甲醛滴定法测定氨基氮,三氯乙酸沉淀法测定可溶性氮等的研究,来确定最佳反应条件。试验方法复杂,随意性大,对蛋白酶水解反应的最佳试验条件的确定没有进行定量的研究。本文的研究目的在于通过利用生化反应中普遍存在热效应的微量量热法能克服这些缺点。通过高灵敏度的微量量热仪直接测量酶催化反应过程中微量的热量变化,具有灵敏度高、操作简单等优点。本文针对大豆分离蛋白糖基化改性目前存在的问题,首次采用半干法对其进行改性,明确在适宜的条件下半干法改性能够有效提高大豆分离蛋白的凝胶强度,乳化特性；利用微量量热仪测出蛋白酶对大豆分离蛋白催化反应的热功率——时间曲线,计算出酶催化反应的米凯利斯常数和最大速率,定量得到酶催化反应的最适温度和最适pH。通过金属离子对蛋白酶水解大豆分离蛋白催化反应影响的热功率—时间曲线的分析,率先得出金属离子对酶催化反应的激活或抑制的影响,总结出金属离子在催化反应中对蛋白酶作用的模式；首次测出酶解产物大豆多肽对大肠杆菌、酵母菌、酵母菌代谢作用的热功率—时间曲线,计算出细菌代谢的生长速率常数,得到大豆多肽促菌和抑菌效果。得出如下主要结论。1、通过对添加麦芽糊精进行半干法共价改性处理。最佳改性工艺条件为大豆分离蛋白与麦芽糊精质量比3.7：1,体系含水量为25%,反应温度为71℃,反应时间为30min。在此工艺条件下凝胶强度为1982 g,较未改性大豆分离蛋白提高23.0%。回归方程也能较好地预测大豆分离蛋白凝胶强度随共价改性工艺参数变化的规律。试验证明半干法可有效提高大豆分离蛋白的凝胶强度。2、利用半干法技术制备高乳化活性和乳化稳定性大豆分离蛋白粉。最佳工艺条件为：麦芽糊精添加量39%、体系含水量为18%、反应温度为65℃、反应时间为8.4h条件下,大豆分离蛋白粉的乳化活性71.9ml/g、乳化稳定性91.2%。3、中性蛋白酶对大豆蛋白催化反应的最适温度(319.19K)、最适pH(7.13)。金属离子(Li+, Na+)是属于竞争性抑制类型。其抑制规律是：在相同浓度时Li+的抑制作用>Na+；在不同浓度时,则随着金属离子浓度的增加而抑制作用增加;金属离子(Cd2+,Mn2+)是属于竞争性激活类型,其激活规律是：在相同浓度时Cd2+的激活作用>Mn2+;在不同浓度时,随着金属离子浓度的增加而激活作用增加。4、胰蛋白酶对大豆蛋白催化反应的最适温度(323.27K)、最适pH(7.77)。金属离子(Ca2+,Mg2+)是属于竞争性抑制类型。其抑制规律是：在相同浓度时Ca2+的抑制作用>Mg2+;在不同浓度时,随着金属金属离子浓度的增加而抑制作用增加。金属离子(Na+,K-)是属于竞争性激活类型。激活规律是：在相同浓度时Na+的激活作用>K+；在不同浓度时,随着金属离子浓度的增加而激活作用增加。5、在菠萝蛋白酶最适温度、最适pH条件下,测出了金属离子可逆竞争时菠萝蛋白酶对大豆蛋白催化反应的热功率—时间曲线,得到了金属离子(Li+,Na+)是属于竞争性抑制类型。抑制规律是：在相同浓度时Li+的抑制作用>Na+；在不同浓度时,随着金属金属离子浓度的增加而抑制作用增加；金属离子(Ca2+,Mg2+)是属于竞争性激活类型。激活规律是：在相同浓度时Ca2+的激活作用>Mg2+;在不同浓度时,随着金属离子浓度的增加而激活作用增加。6、碱性蛋白酶对大豆蛋白催化反应的最适温度(329.5K)、最适pH(8.25)。金属离子(Pb2+、Ag+)是属于竞争性抑制类型。抑制规律是：在相同浓度时Pb2+的抑制作用>Ag+；在不同浓度时,随着金属金属离子浓度的增加而抑制作用增加。金属离子(Na+、Mg2+)是属于竞争性激活类型。激活规律是：在相同浓度时Na+的激活作用>Mg2+;在不同浓度时,随着金属离子浓度的增加而激活作用增加。7、大豆多肽对大肠杆菌代谢的作用效果是：当大豆多肽的浓度C<2.06mg/ml时大豆多肽对大肠杆菌的生长代谢的影响为促进作用；在C>2.06mg/ml时,表现出抑菌效果。8、大豆多肽对沙门氏菌代谢的作用效果是：当大豆多肽的浓度C<2.67mg/ml时大豆多肽对沙门氏菌的生长代谢的影响为促进作用；在C>2.67mg/ml时,表现出抑菌效果。9、大豆多肽对酵母菌代谢的作用效果是：在实验浓度范围内,不同浓度的大豆多肽存在时酵母菌的的生长速率常数均大于没有大豆多肽时的酵母菌的的生长速率常数。从作用效果上来看,大豆多肽对酵母菌的生长代谢是起促进作用。本研究为进一步推动蛋白加工行业的发展提供了优化工艺；创新了大豆分离蛋白蛋白酶催化水解热功率类反应的研究模式；明确了蛋白酶对大豆分离蛋白催化反应的最适条件及金属离子的影响效果;补充了大豆分离蛋白蛋白酶水解热动力学反应机理的理论体系；为国内外学者进一步研究和开发大豆多肽生理功能提供了新的理论依据。更多还原

【Abstract】 Soybean is one of the main economic crops in China, which has been extensively cultivated in north China. Concerning its characteristics of the edible oils and food protein resources, it is generally acknowledged that soybean becomes an indispensable source of food for human life. High protein, containing all essential amino acids, are part of advantages of soybean and it contains eight kinds of essential amino acids, the content of which is close to or higher than that of the United Nations food and agriculture organization (FAO)’s and the world health organization (WHO)’s ideal recommendations. Compared with animal protein, the nutritional value of soybean is able to meet the nutritional needs of human beings. And with the improvement of people’s living standard, modern diseases such as obesity, high blood pressure, the animal protein intake gradually reduce, plant protein intake increases gradually.With the rapid development of food industry, a lot of excellent functional properties of protein as raw materials for the food are needed. High protein, but complex structure, which leads to the limit of using scope and usage. Through chemical methods, many functions of the original nature of soy protein has been modified, such as solubility, water and emulsification, foaming, oily, have significant degree of improvement. The functional properties of soy protein has important application value in the food industry, meanwhile, it better meets the needs of the food industry. Soybean protein isolate is an important protein product, hydrolyzed by protease producing soybean peptides, peptides are much more absorbable than the protein and amino acid, at the same time, Soybean protein isolate have functions of reducing blood press, reducing blood fat and cholesterol, anti thrombosis, promoting the absorption of calcium, pHospHorus and trace elements, regulating immune function, antioxidant effect, low sensitivity to tranquilize mind, etc. Therefore the protease hydrolysis of soybean protein has been the hot topics in the study of people in recent years.Traditional research about the method of producing soybean peptides generated by protease hydrolysis of soybean protein is to determine the degree of hydrolysis product by trinitrobenzene sulfonic acid, formaldehyde titration determination of amino nitrogen, soluble nitrogen determination of trichloroacetic acid precipitation, to determine the optimal reaction conditions, such as temperature, acidity. Test method is complex and randomness. There is no precise quantitative research made to test the optimum experiment conditions for protease hydrolysis reaction. The purpose of this study is to overcome the shortcomings through microcalorimetry produced by the use of biochemical reactions. Directly measured by high sensitivity micro calorimeter trace amounts of heat in the process of enzyme catalytic reaction changes, which has the advantages of high sensitivity, simple operation.To increase the gel strength of soybean protein isolate, the semi-dry method was used to modify it by covalent modification with malt dextrin. Based on the results of single factor experiment, the Box-Behnken mode was applied to obtain the optimum conditions of modification. The Optimum reaction conditions were that the mass ratio of soy protein isolate and maltodextrin 4:1、the system water content 25%、reaction temperature 71℃, and the reaction time 30 min. Under the conditions, the gel strength of modified soybean protein isolate was 1982 g, which increased by 23.0% compared with that of unmodified one. The regression equation could also well predict the changes in the gel strength of the soy protein isolate with the changes in technological parameters of covalent modification. The results proved that semi-dry method can effectively increase the gel strength of soybean protein isolate.To increase the emulsifying properties of soybean protein isolate (SPI), the semi-dry method was used to prepare high emulsifying activity and emulsion stable soybean protein by cross-linking malt dextrin (MD). The central composite design based on the response surface methodology was applied to study the effects of MD content added, water content added, reaction temperature and time on the emulsifying properties of SPI, and the regression models were established between the emulsifying properties and different factors. The results showed that the emulsifying activity of SPI was 71.9 mL/g, and its emulsion stability was 91.2% under the conditions of 39% MD added,18% water content at 65℃ for 8.4 h.Based on soybean protein isolate as the research object, using the trace calorimetry as the research method, measured by micro calorimeter protease to soy protein catalytic reaction of thermal power-time curve, according to the thermal dynamic theory and comparative schedule method to resolve the different temperature, acidity and metal ions exist michalis faloutsos of enzyme catalytic reaction constant (Km, K’) and the maximum rate (Vmax), establish m kelly’s constant with temperature, pH, and metal ions, the relation between enzymatic reaction to obtain the optimum temperature, pH and metal ions on the influence of enzyme catalytic reaction activation or inhibit effect.Studies have shown that the optimum temperature(319.19K) for neutral protease to soy protein catalytic reaction, the optimum pH (7.13). Metal ions (Li+, Na+) belongs to the type of competitive inhibition.The rule of Inhibition is:in the same concentration of Li+> Na+; In different concentrations, with the increase of metal ion concentration,the inhibition increased. Metal ions (Cd2+, Mn2+) is belong to the type of competitive activation. The rule of activate is:in the same concentration of Cd2+>Mn2+. In different concentrations, with metal ion concentration increasing, the activation increased.Studies have suggested that optimum temperature (323.27K)for soy protein with trypsin catalytic reaction, the optimum pH (7.77). Metal ions (Ca2+, Mg2+) belongs to the type of competitive inhibition. The rule of Inhibition is:in the same concentration of Ca2+>Mg2+ In different concentrations, with the increase of metal ion concentration,the inhibition increased. Metal ion (Na+, K+) is belong to the type of competitive activation. The rule of activate is:in the same concentration Na+>K-; In different concentrations, with the metal ion concentration increasing, activation increased.Under the condition of optimum temperature, the optimum pH, to measure pineapple protease catalytic soy protein reaction of thermal power-time curve when the metal ions reversible competitive, by comparing the quantitative,metal ions (Li+, Na+) belongs to the type of competitive inhibition. The rule of Inhibition is:in the same concentration of Li+> Na+; In different concentrations, with the increase of metal ion concentration,the inhibition increased; Metal ion (Ca2+, Mg2+) belong to the type of competitive activation. The rule of activate is:in the same concentration Ca2+>Mg2+; In different concentrations, with the increase of metal ion concentration,the activation increased.Studies have shown that the optimum temperature (329.5 K)for alkaline protease to soy protein catalytic reaction, the optimum pH (8.25). Metal ions (Pb2+, Ag+) belongs to the type of competitive inhibition. The rule of inhibition is:in the same concentration of Pb2+> Ag+ In different concentrations, with the increase of metal ion concentration,the inhibition increased. Metal ions (Na+, Mg2+) is belong to the type of competitive activation. The rule of activate is:in the same concentration of Na+>Mg2+; In different concentrations, with the increase of metal ion concentration,the activation increased.Measuring thermal power-time curve of the soybean peptides to the metabolism of e. coli, we get the effects of soybean peptides on metabolism of e. coli. Research shows that when the concentration of soybean peptides C< 2.06 mg/ml, soybean peptides can promote the growth metabolism of e. coli in order to role; When C> 2.06 mg/ml, bacteriostatic effect.Measuring thermal power-time curve of the soybean peptides to salmonella metabolism, we get the effects of soybean peptides on salmonella metabolism. Research shows that when the concentration of the soybean polypeptide C< 2.67 mg/ml, soybean peptide’s influence on the growth of salmonella metabolism is in promote role; In C> 2.67 mg/ml, bacteriostatic effect.Measuring thermal power-time curve of soybean peptides to yeast metabolism, we get the effect on soy peptide to yeast metabolism. In the experimental concentration range. In different concentrations of soybean peptides,the growth rate of yeast is greater than that of yeast without soybean peptides. On the effect of it, soybean peptide is a promoting role to the growth of yeast metabolism.The innovation of this article is for the first time using the method of trace calorimeter to measure protease to soy protein catalytic reaction of thermal power-time curve, calculating michalis faloutsos constant and the maximum rate in enzyme catalytic reaction, to get optimum temperature and the optimum pH of enzyme catalytic reaction by quantitative. First measure of metal ions to the influence of protease hydrolysis of soybean separation protein catalytic reaction heat power-time curve, obtained the effect of enzymatic reaction of metal ions to activate or inhibit. First measure of thermal power-time curve of soybean peptides on escherichia coli, yeast, the yeast metabolism, calculated the growth of bacteria metabolic rate constant, got effect of the soy peptide on promoting bacteria and bacteriostatic. In this paper, the study of protease catalytic hydrolysis reaction to provide quantitative research methods can be found. For the domestic and foreign scholars study,the reaction conditions of protease hydrolysis of soybean protein can be used and the paper provides theoretical basis. Besides, the reaction mechanism for the development of the pHysiological functions of soybean peptides can be provided deep theoretical support.更多还原