玉米黄粉酶法制备高F值低聚肽的研究
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摘要
高F值低聚肽具有高支低芳的特殊氨基酸构成，能够作为肝病患者、高付出病人和运动员的膳食补充发挥重要的生理功能。玉米湿法淀粉厂的副产物玉米黄粉支链氨基酸含量高，非常适宜于高F值低聚肽的制备。因此，本文以玉米黄粉为原料，研究了高F值低聚肽的酶法制备过程和技术路线。主要研究内容包括玉米黄粉蛋白的分离和增溶、蛋白酶的选择、酶解条件优化及过程控制、超滤及吸附条件的确立，并且对酶解产物进行相对分子质量分布范围和F值的分析鉴定，拟定了质量标准。主要研究结果如下：
1、确定了以玉米黄粉为原料酶法制备高F值低聚肽的基本工艺路线，其关键在于玉米黄粉蛋白的预处理、玉米黄粉蛋白的酶解和高F值低聚肽的分离。
2、玉米黄粉的预处理对酶法制备高F值低聚肽是必需的，工艺涉及三个方面：第一，黄粉的脱色。选择丙酮作溶剂，以浸提条件料液比1：3、30℃浸提4h，重复3次操作可基本除去玉米黄色素。第二，脱除淀粉。脱色黄粉与水1：1混合90℃糊化1h后以1.0％的α—淀粉酶在pH5.5、60℃反应至碘试验不变蓝，再加入2.0％糖化酶在pH4.8、45℃酶解8h后得到蛋白质含量为83.5％的玉米黄粉蛋白。第三，玉米黄粉蛋白的增溶。添加还原剂Na_2SO_3、碱性条件和升高温度可在不同程度上提高玉米黄粉蛋白的溶解度。本试验选择加入0.10％Na_2SO_3作还原剂，pH10.0，90℃处理15min的方法进行增溶可使玉米黄粉蛋白溶解度达到14.24％，可溶性氮指数NSI达到12.70％。
3、单酶水解在酶解产物组分相对分子质量分布范围上无法达到预定要求，采用双酶水解法。2709碱性蛋白酶对玉米黄粉蛋白酶解作用最强，最佳工艺参数是：底物浓度5.0％，10.0％的酶用量，pH10.0，45℃，水解时间8h，其结果为DH达到了33.5％，产物中肽的含量为2.86g/100mL，氨基态氮含量为102.94mg/100mL，产物组分中相对分子质量分布范围为：5,000以上占21.5％，2,000～5,000占24.4％，200～2,000占51.9％，200以下仅占2.2％。利用木瓜蛋白酶进行二次酶解，最佳工艺参数是：2.0％的木瓜蛋白酶用量，pH7.0，60℃，水解时间为4h，产物中肽的含量为2.10g/100mL，氨基态氮含量为230.05mg/mL，产物组分相对分子质量分布范围为：5,000以上占10.6％，2,000～5,000占16.6％，200～2,000占67.1％，200以下占3.7％。
4、高F值低聚肽的分离和脱苦研究表明：分离小分子肽方法选择截留相对分子质量为5,000的膜超滤，多肽和蛋白质被除去，流出液组分中相对分子质量分布范围为5,000以上占6.4％，2,000～5,000占12.2％，200～2,000占76.9％，200以下占4.5％，达到低聚肽含量要求。为提高F值，研究了以吸附剂脱除AAA的方法，选择粉末状活性
碳作吸附剂，最佳工艺参数是：料液比13，pH3刀，20℃，吸附时间为3h，产物含氮量
为 2．7g／L，F值为 22．59，产物组分中相对分子质量分布范围为：5，000以下的肽占94石％，
2，000以下的低聚肽占70．5％，符合高F值低聚肽的质量要求。由本研究确立的酶法制
备高F值低聚肽混合物工艺所获得的高F值低聚肽Mw为1厂90，Mn为920，得率以玉
米黄粉蛋白计为 34．l％。
5、根据初步研究，制定了以玉米黄粉为原料双酶法制备高F值低聚肽混合物的质
量标准，分别从感官指标、理化指标和微生物指标三个方面规范产品质量，可作为生产
中的质量控制参考。
活性肽的酶法制备是生物技术蛋白质工程和酶工程的重要内容。本研究所制得的高
F值低聚肽混合物，低聚肽含量高，相对分子质量小，能够作为特殊生理、病理及运动
状态下人群的膳食营养供给。制备工艺各步骤的工艺参数和质量管理指标都已经基本确
定，有必要进一步中试、生产和推广应用。
（本论文共计图ZI个，表15个＊
High Fischer ratio oligopeptides with a special amino acids composition of high branched chain amino acids (BCAA) and low aromatic amino acids (AAA) could play important physiologic functions on liver disease patients, high expend patients and athletes. CGM was a by-product in a wet corn starch plant, which was suitable for preparation of high Fischer ratio oligopeptides for its high branched chain amino acids content. It was utilized as raw material to prepare for high Fishcher ratio oligopeptides by enzyme methods. Protein of CGM isolating, protease and hydrolysis condition selecting, hydrolysis process controlling, ultrafiltration and adsorption condition selecting were studied in this dissertation. The relative molecular mass (Mr) range and Fischer ratio were studied, too. The main results indicated:
1. The basic processing was affirmed. Key facets concerned pre-treat of CGM, hydrolysis of protein of CGM, isolating of high Fischer ratio oligopeptides.
2. Pre-treating was necessary for high Fischer ratio oligopeptides preparation. It concerned with 3 facets. First, decolor of material. The parameters of the pigment extractin: adding acton into CGM in proper ratio of substance to solvent (1:3), extracting 4h at 30℃, repeating this operation 3 times were the economical, high qualitative way. Second, remove starch. Decolor CGM mixed with water at 1:1, cultured Ih at 90℃, then added 1.0% a-amylase at pH 5.5, 60℃, after it didn't change to blue in the iodine experiment, given 2.0% p-amylase to culture 8h at pH 4.8, 45℃, protein of CGM with 83.5% protein content was obtained. Third, increase the solubility of protein of CGM. Dealing with alkaline, rising temperature and adding reductant Na2SO3 were good way. Adding 0.10% Na2SO3, heating 15 minutes at pH10.0, 90℃ was applied to increasing solubility to 14.24%, the index of nitrogen solubility (NSI) to 12.70%.
3. The Mr range of hydrolysates was far away from predetermined only treated by one protease. Two proteases were used in hydrolysis. 2709 alcalase had powerful enzymatic hydrolysis. Results indicated that the optimum parameters for 2709 alcalase were 5.0% substance, 10.0% enzyme to substance, pH10.0, 45℃ and 8h. In such condition, the degree of hydrolysis was 33.5%, peptides content was 2:86g/100mL, ammoniac nitrogen content was
102.94mg/100mL, the Mr ranges of hydrolysates above 5,000, from 2000 to 5,000, from 200 to 2000 and below 200 were 21.5%, 24.4%, 51.9% and 2.2% respectively. The optimum parameters for the second enzymatic hydrolysis by papain indicated 2.0% papain to substance, pH 7.0, 60℃ and 4h. Peptides and ammoniac nitrogen were 2.1g/100mL, 230mg/100mL respectively, the Mr ranges above 5,000, from 2,000 to 5,000, from 200 to 2,000 and below 200 were 10.6%, 16.6%, 67.1%, 3.7% respectively.
4. The research of high Fischer ratio oligopeptides isolating and debittering indicated: The Mr 5,000 withholded ultrafiltration was used to remove peptides and proteins. Mr ranges of hydrolysats passed through the membrane above 5,000, from 2,000 to 5,000, from 200 to 2,000 and below 200 were 6.4%, 12.2%, 76.9%, 4.5% respectively. It attained the demand of oligopeptides. In order to promote Fischer ratio, powdered activated carbon was studied as AAA adsorbent. The optimum parameters were adsorbent to the volume of hydrolysates 1:3, pH3.0, 20C and 3h. The product had 2.7g/L nitrogen content, Fischer ratio was 22.59, Mr ranges below 5,000 and below 2,000 were 94.6% and 70.5% respectively. The yield was 34.1% on protein of CGM by weight. The bitter weakened by the means of hiding bitter peptides in this research.
5. The quantity standard was formulated according to the preliminary study on preparation of high Fischer ratio oligopeptides by two enzymes method from CGM. It could keep the quantity on sensory organ level, physical and chemical level and microorganism level in production controlling.
Preparation high Fischer ratio oligopeptides by enzymatic hydrolysis belonged to protein engineering and enzyme engineering of biology techn
1、确定了以玉米黄粉为原料酶法制备高F值低聚肽的基本工艺路线，其关键在于玉米黄粉蛋白的预处理、玉米黄粉蛋白的酶解和高F值低聚肽的分离。
2、玉米黄粉的预处理对酶法制备高F值低聚肽是必需的，工艺涉及三个方面：第一，黄粉的脱色。选择丙酮作溶剂，以浸提条件料液比1：3、30℃浸提4h，重复3次操作可基本除去玉米黄色素。第二，脱除淀粉。脱色黄粉与水1：1混合90℃糊化1h后以1.0％的α—淀粉酶在pH5.5、60℃反应至碘试验不变蓝，再加入2.0％糖化酶在pH4.8、45℃酶解8h后得到蛋白质含量为83.5％的玉米黄粉蛋白。第三，玉米黄粉蛋白的增溶。添加还原剂Na_2SO_3、碱性条件和升高温度可在不同程度上提高玉米黄粉蛋白的溶解度。本试验选择加入0.10％Na_2SO_3作还原剂，pH10.0，90℃处理15min的方法进行增溶可使玉米黄粉蛋白溶解度达到14.24％，可溶性氮指数NSI达到12.70％。
3、单酶水解在酶解产物组分相对分子质量分布范围上无法达到预定要求，采用双酶水解法。2709碱性蛋白酶对玉米黄粉蛋白酶解作用最强，最佳工艺参数是：底物浓度5.0％，10.0％的酶用量，pH10.0，45℃，水解时间8h，其结果为DH达到了33.5％，产物中肽的含量为2.86g/100mL，氨基态氮含量为102.94mg/100mL，产物组分中相对分子质量分布范围为：5,000以上占21.5％，2,000～5,000占24.4％，200～2,000占51.9％，200以下仅占2.2％。利用木瓜蛋白酶进行二次酶解，最佳工艺参数是：2.0％的木瓜蛋白酶用量，pH7.0，60℃，水解时间为4h，产物中肽的含量为2.10g/100mL，氨基态氮含量为230.05mg/mL，产物组分相对分子质量分布范围为：5,000以上占10.6％，2,000～5,000占16.6％，200～2,000占67.1％，200以下占3.7％。
4、高F值低聚肽的分离和脱苦研究表明：分离小分子肽方法选择截留相对分子质量为5,000的膜超滤，多肽和蛋白质被除去，流出液组分中相对分子质量分布范围为5,000以上占6.4％，2,000～5,000占12.2％，200～2,000占76.9％，200以下占4.5％，达到低聚肽含量要求。为提高F值，研究了以吸附剂脱除AAA的方法，选择粉末状活性
碳作吸附剂，最佳工艺参数是：料液比13，pH3刀，20℃，吸附时间为3h，产物含氮量
为 2．7g／L，F值为 22．59，产物组分中相对分子质量分布范围为：5，000以下的肽占94石％，
2，000以下的低聚肽占70．5％，符合高F值低聚肽的质量要求。由本研究确立的酶法制
备高F值低聚肽混合物工艺所获得的高F值低聚肽Mw为1厂90，Mn为920，得率以玉
米黄粉蛋白计为 34．l％。
5、根据初步研究，制定了以玉米黄粉为原料双酶法制备高F值低聚肽混合物的质
量标准，分别从感官指标、理化指标和微生物指标三个方面规范产品质量，可作为生产
中的质量控制参考。
活性肽的酶法制备是生物技术蛋白质工程和酶工程的重要内容。本研究所制得的高
F值低聚肽混合物，低聚肽含量高，相对分子质量小，能够作为特殊生理、病理及运动
状态下人群的膳食营养供给。制备工艺各步骤的工艺参数和质量管理指标都已经基本确
定，有必要进一步中试、生产和推广应用。
（本论文共计图ZI个，表15个＊
High Fischer ratio oligopeptides with a special amino acids composition of high branched chain amino acids (BCAA) and low aromatic amino acids (AAA) could play important physiologic functions on liver disease patients, high expend patients and athletes. CGM was a by-product in a wet corn starch plant, which was suitable for preparation of high Fischer ratio oligopeptides for its high branched chain amino acids content. It was utilized as raw material to prepare for high Fishcher ratio oligopeptides by enzyme methods. Protein of CGM isolating, protease and hydrolysis condition selecting, hydrolysis process controlling, ultrafiltration and adsorption condition selecting were studied in this dissertation. The relative molecular mass (Mr) range and Fischer ratio were studied, too. The main results indicated:
1. The basic processing was affirmed. Key facets concerned pre-treat of CGM, hydrolysis of protein of CGM, isolating of high Fischer ratio oligopeptides.
2. Pre-treating was necessary for high Fischer ratio oligopeptides preparation. It concerned with 3 facets. First, decolor of material. The parameters of the pigment extractin: adding acton into CGM in proper ratio of substance to solvent (1:3), extracting 4h at 30℃, repeating this operation 3 times were the economical, high qualitative way. Second, remove starch. Decolor CGM mixed with water at 1:1, cultured Ih at 90℃, then added 1.0% a-amylase at pH 5.5, 60℃, after it didn't change to blue in the iodine experiment, given 2.0% p-amylase to culture 8h at pH 4.8, 45℃, protein of CGM with 83.5% protein content was obtained. Third, increase the solubility of protein of CGM. Dealing with alkaline, rising temperature and adding reductant Na2SO3 were good way. Adding 0.10% Na2SO3, heating 15 minutes at pH10.0, 90℃ was applied to increasing solubility to 14.24%, the index of nitrogen solubility (NSI) to 12.70%.
3. The Mr range of hydrolysates was far away from predetermined only treated by one protease. Two proteases were used in hydrolysis. 2709 alcalase had powerful enzymatic hydrolysis. Results indicated that the optimum parameters for 2709 alcalase were 5.0% substance, 10.0% enzyme to substance, pH10.0, 45℃ and 8h. In such condition, the degree of hydrolysis was 33.5%, peptides content was 2:86g/100mL, ammoniac nitrogen content was
102.94mg/100mL, the Mr ranges of hydrolysates above 5,000, from 2000 to 5,000, from 200 to 2000 and below 200 were 21.5%, 24.4%, 51.9% and 2.2% respectively. The optimum parameters for the second enzymatic hydrolysis by papain indicated 2.0% papain to substance, pH 7.0, 60℃ and 4h. Peptides and ammoniac nitrogen were 2.1g/100mL, 230mg/100mL respectively, the Mr ranges above 5,000, from 2,000 to 5,000, from 200 to 2,000 and below 200 were 10.6%, 16.6%, 67.1%, 3.7% respectively.
4. The research of high Fischer ratio oligopeptides isolating and debittering indicated: The Mr 5,000 withholded ultrafiltration was used to remove peptides and proteins. Mr ranges of hydrolysats passed through the membrane above 5,000, from 2,000 to 5,000, from 200 to 2,000 and below 200 were 6.4%, 12.2%, 76.9%, 4.5% respectively. It attained the demand of oligopeptides. In order to promote Fischer ratio, powdered activated carbon was studied as AAA adsorbent. The optimum parameters were adsorbent to the volume of hydrolysates 1:3, pH3.0, 20C and 3h. The product had 2.7g/L nitrogen content, Fischer ratio was 22.59, Mr ranges below 5,000 and below 2,000 were 94.6% and 70.5% respectively. The yield was 34.1% on protein of CGM by weight. The bitter weakened by the means of hiding bitter peptides in this research.
5. The quantity standard was formulated according to the preliminary study on preparation of high Fischer ratio oligopeptides by two enzymes method from CGM. It could keep the quantity on sensory organ level, physical and chemical level and microorganism level in production controlling.
Preparation high Fischer ratio oligopeptides by enzymatic hydrolysis belonged to protein engineering and enzyme engineering of biology techn
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