【作者】 吴非；

【导师】 霍贵成；

【作者基本信息】 东北农业大学 ， 食品科学， 2003， 博士

【摘要】 大豆富含蛋白质，是重要的蛋白资源。其中的皂甙、异黄酮、磷脂等成分对人体还有医疗保健作用。但大豆含有多种抗营养因子，如胰蛋白酶抑制剂、血球凝集素、致敏因子等，有碍营养素的吸收，造成豆制品感官上的缺陷，危害人体健康。目前，失活抗营养因子主要采用热处理，但长时间高热会降低大豆蛋白质的营养价值，而且某些抗营养因子的耐热性很强，不足以使其完全失活。基于多数抗营养因子为蛋白质类物质，采用蛋白酶失活是很有前途的方法。 本课题对外源蛋白酶失活胰蛋白酶抑制剂进行了系统研究，采用体外及体内试验方法，共计八个实验，包括豆制品中胰蛋白酶抑制剂活性的调查、外源蛋白酶的筛选、碱性蛋白酶失活胰蛋白酶抑制剂的研究、低胰蛋白酶抑制剂活性大豆分离蛋白的研制与生产、低胰蛋白酶抑制剂活性大豆分离蛋白的加工特性研究、低胰蛋白酶抑制剂活性大豆分离蛋白对大鼠生长性能影响的研究、低胰蛋白酶抑制剂活性保健豆乳的研制以及低胰蛋白酶抑制剂活性发酵豆乳的研制。 实验结果表明，水解蛋白、乳用蛋白、组织蛋白、水豆腐、干豆腐中胰蛋白酶抑制剂活性均被钝化了90％以上；腐乳、豆酱中胰蛋白酶抑制剂活性为未检出水平。无糖豆粉、×××豆粉、豆浆以及内酯豆腐中胰蛋白酶抑制剂钝化率低于90％。巴氏杀菌可钝化胰蛋白酶抑制剂14.07％；高压杀菌可钝化91.1％，超高温灭菌可钝化65.9％。 碱性蛋白酶失活胰蛋白酶抑制剂能力最强，其次为酸性蛋白酶，中性蛋白酶和木瓜蛋白酶失活能力较弱。水解度在最初的5～10min内迅速提高，以后提高速度减慢，30min时，碱性蛋白酶达到的水解度最高，为16.34％。 碱性蛋白酶失活胰蛋白酶抑制剂的最优参数为pH值8.88～9.05、温度43.40～44.70℃、酶用量10.44～11.29μL/g、底物浓度为6.51～7.18％。酶处理10min以内时，失活率提高迅速；30min时，达到95.01％；30min后，失活率趋于平稳。水解度在10min以内迅速提高，达到10.87％；10min～50min期间，水解度变化缓和，50min以后，水解度变化趋于稳定，在20％左右。 碱性蛋白酶对胰蛋白酶抑制剂的失活率随着底物浓度的上升而加大，当底物浓度为7％时，失活率最高，为76.24％。底物浓度高于7％，随着浓度增加，失活率变化平稳，当底物浓度为10％时，失活率仍接近最高值，为75.40％。采用10％的工业化底物浓度博士学位论文 外源蛋白酶失活大豆胰蛋白酶抑制剂的研究 东北农业大学一生产出的低胰蛋白酶抑制剂活性大豆分离蛋白，检测结果符合国家一级品的标准。酶处理时间长的产品，大豆蛋白分子的裂解程度加大，短肽的平均分子量趋于变小，酶处理50min时，肋平均分子量为2135D。。产物中存在着各种大小不等的肽分子，中低肤分子占相当大的比例，未水解的大豆蛋白或大肽分子及氨基酸只占少数。 低胰蛋白酶抑制剂活性大豆分离蛋白的氮溶指数明显改善，酶处理smin内，氮溶指数增加显著。而且，PH4．5时氮溶指数的改善要比pH7．0时明显。酶处理30min内水解度与氮溶指数呈线性关系，PH4．5时这种相关性比较严格。氮溶指数随PH值的变化呈“铃形”曲线形式，产品在碱性范围内比在酸性范围内有较高的氮溶指数。产品的起泡性优于大豆分离蛋白，酶处理 50min时起泡性是大豆分离蛋白的 171倍。是酶处理 30min时的1．07倍，但产品的泡沫稳定性低于大豆蛋白。酶处理10min时产品的热凝集性可提高4倍以上，10min后，热凝集性变化趋于平稳。大豆分离蛋白的体外消化指数为 30％，酶处理 30min时为 43．26％，处理 50mln为 45．72％。 用胰蛋白酶抑制剂活性不同的饲料饲喂大鼠35天，发现随着饲喂时间的延长，各组别的大鼠日增重都有所下降，生豆粉组下降幅度最大，为47．6％，酶处理50min组日增重下降幅度最小，为 45 6％。在不同饲喂时期，酶处理组日增重都高于其它组，饲喂 35天时，酶处理组日增重为 3．16克；’只·日，显著高于生豆粉组门．旧克／只·日）、豆粕组和无糖豆粉组（分别为 2．4g克；’只·日和 2 64克；’只·日）。在饲喂期间，酶处理50mn组采食量几乎没有变化，其它组大鼠采食量都不同程度降低，其中生豆粉组采食量下降最明显，下降幅度为5．5％。饲喂％天时，酶处理组大鼠采食量最大，为18．4克／只·日，而生豆粉组采食量仅为m．8克．’只·日。在饲喂期间，各组大鼠的饲料转化率均呈上升趋势，上升幅度差异不明显，在饲喂H大时，各组间饲料转化率差异显著，酶处理孤m山组为 5．85，而生豆粉组为 7 73。生豆粉组大鼠胰腺相对重和脾相对重显著高于其它组，分别为0．348和0．375。而且，胰腺上皮组织缺乏完整性，具有明显的增生性变性，而酶处理组未见增生。生豆粉组和豆粕组大鼠小肠上皮组织细胞崩解，肠绒毛完整性受到破坏，而酶处理组小肠绒毛完好，结构完整，绒毛长度极显著高于其它组。各组别大鼠的胸腺相对重差别不显著。酶处理50min组大鼠小肠中的乳杆菌数量极显著高于其它组，为 1．7 X 10’个；。’g。各组别大鼠血清中肿瘤坏死因子，白细胞介素一 2和白细胞更多还原

【Abstract】 Soybean is the most important sources of protein for animals and humans. It contains healthful such substances as soyasaponin, isoflavone, fibrin, unsaturated fatty acid, etc. are medicinally healthful. However, raw beans contain a number of antinutritional factors (ANFs), such as trypsin inhibitors, lectin and allergic proteins, producing adverse effects on the animal growth and health. It is usual to subject the raw soybean to some types of heat treatments. But the prolonged high temperature treatment required to destroy all ANFs is likely to damage the availability of certain essential amino acids. Since some ANFs are chemically proteins, it seems that proteolysis is a promising approach to inactivate them.The effective activation techniques of TIA and its usage in food processing were systematically investigated in this trial. Seven in vitro trials and one in vivo trials were conducted, including: Investigation of the TIA in soybean products; activation of TIA by various protease; activation of TIA by Alcalase; The exploitation of SPI with low TIA; The processing properties of SPI with low TIA; The effect of SPI with low TIA on the growth in rats; The exploitation of nutritional soybean milk with low TIA and The exploitation of fermented soybean milk with low TIA.TIA in Hydrolysed Soybean Protein, Soybean Protein for Milk Use, Soybean Protein Textures and Tofu was activated more than 90%. There was nearly no TIA in Fermented Tofu and Soybean Sauce. TIA in Soybean Meal without Sugar, X X X Soybean powder, Soya milk and lactone bean curd were activated less than 90%. Pasteurization could activate 14. 07% of the TIA. High pressure sterilization can activate 91.1% while UHT 65.9%.The relative activation activity to TIA of Alcalase was the highest. The acidic protease was secondary. The neutral protease and papain had the weakest relative activation activity. DH of soybean protein rose rapidly within 5-10 minutes, and then the proteolysis rate slowed down. DH by the Alcalase was the highest 30 minutes later, reaching 16.34%.The optimum activation parameters of Alcalase was pH8. 88-9. 05, temperature 43. 40-44. 70 , the amount of Alcalase 10. 44~11. 29 L/g, substrate concentration 6. 5l-7. 18%, activation time 30min. The activation rate of TIA was high within the premier ten minutes, reaching 95. 01% and being unchangable 30 minutes later. The DH of soybean protein rose rapidly within the premier 10 minutes, reaching 10. 87%, and changed slowly from 10 to 50 minutes. It was nearly stable 50 minutes later, reaching about 20%.The activation rate to TIA of Alcalase rose with the increase of substrate concentration. It was the highest (76. 24%) when the substrate concentration was 7% and became stable when the substrate concentration was more than 7%. It remainedthe highest (75.40%) when the substrate concentration was 10%. The test results of SPI with low TIA. accorded to those of the best soybean products in our country. With the proteolysis time extended, the degree of the soybean protein cleavage became great, and the average molecular weight of the peptides decreased. The average molecular weight was 2135Da. There were different sizes of soybean peptides in the product, and the majority of peptides was in small and middle size. There were less intact soybean protein, big size of peptides and amino acid in the product.The NSI of the SPI with low TIA rose with the proteolysis time extended. It rose rapidly within the premier 5 minutes. The NSI at pH4. 5 improved remarkably than that at pH7. 0. The NSI of enzyme-modified SPIs was strongly correlated with their DH. The pH. -solubility curves were bell-shaped for all samples. The NSI was higher at alkaline pH than that at acidic pH. The foam property of SPI with low TIA (50mins) reached about 1. 71 times improvement than that of SPI. And it was 1. 07 times than that of the product (30 minutes). But the foam stability was lower than that of the SPI. Enzyme treatment was effective in increasing thermal aggregation of SPI, amounting to more th更多还原