固定化蛋白酶的制备及松仁多肽加工工艺研究
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摘要
红松(Pinus koraiensis Sieb.et Zucc),是主产于中国小兴安岭的珍贵树种。红松仁是一种营养价值很高的果品,富含蛋白质、氨基酸、脂肪酸、维生素、矿物质及其它生理活性物质,具有广阔的应用开发潜力。
本文以红松松仁为原料,利用现代分离分析手段,对松仁蛋白的提取工艺、酶解条件进行优化,又对最佳蛋白酶进行固定化研究再用其制备松仁多肽,主要结果如下:
1、采用超声波辅助碱提酸沉法提取松仁蛋白,最佳工艺条件依次为:料液比1:24.61、pH值10、提取温度48.3℃、超声时间100min。松仁蛋白提取率为233.23 mg/g。超声波辅助提取法可以有效地提高松仁蛋白质的提取率,缩短反应周期。
2、选择国产碱性、中性蛋白酶、Alcalase2.4L碱性蛋白酶和风味蛋白酶作为松仁蛋白酶解备用酶,对松仁蛋白进行酶解效果研究,根据其水解程度,确定Alcalase蛋白酶为松仁蛋白酶解的最佳用酶。通过响应面法优化得到此酶作用下的最佳酶解条件为:pH值8.0、水解温度53℃、酶与底物比8800 U/g蛋白、底物浓度1.2%、酶解的时间50min。在最佳酶解的条件下水解度(DH)为43.15%。酶与底物比对其酶解过程具有极显著性影响,其次是pH。
3、使用交联-吸附法对Alcalase2.4L蛋白酶进行固定化,研究Alcalase蛋白酶固定化工艺,得到了载体与交联剂浓度、载体交联时间、给酶量、pH、固定化时间的最佳固定化条件:壳聚糖浓度2.5%、戊二醛浓度0.4%、载体交联时间4h、给酶量915U/g载体、pH9.4、固定化时间8h,固定化酶活力回收率为54.35%。同时对固定化酶及溶液酶的米氏常数、最适温度、pH、热稳定性等性质进行了研究和比较。固定化Alcalase蛋白酶重复使用6次,相对活力仍在60%以上。说明固定化Alcalase蛋白酶具使用特性,可用作工业化生产。
4、研究了固定化Alcalase2.4L蛋白酶酶解松仁蛋白的工艺。对酶解条件进行了优化,得到固定化Alcalase2.4L蛋白酶最佳酶解条件:底物浓度1.2%、pH值9.0、温度50℃、给酶量0.2g/g底物、反应时间2h,水解度达到25%。说明用固定化Alcalase蛋白酶酶解松仁蛋白制备多肽的方法可行。
Pinus koraiensis Sieb. et Zuee is a rare tree species of Xiao xing an ling in China. The pine nut is a high nutritional value of fruits, rich in protein, amino acids, fatty acids, vitamins, minerals and other substances physical activity has a wide application development potential.
In this research, pine nuts were used as material, the use of modern analytical tools from the pine nut protein extraction technology, hydrolysis condition optimization; also research the best immobilized protease. The main results are as follows:
1. Use ultrasound-assisted alkali extraction method from pine nut protein, optimum conditions were: Solid to liquid ratio (W/V) 1:24.61、pH10.0、extraction temperature48.3℃、ultrasonic time 100min. Pine nut protein extraction rate was 233.23 mg/g. Ultrasound-assisted extraction method can be effective in raising the pine nut protein extraction rate, shorten response cycle.
2. Select Domestic the alkaline protease、neutral proteinase、the Alcalase2.4L alkalin proteinase and the Flavourzyme proteinase as a pine nut protein enzymolysis preparation enzyme, the enzyme effect pine nut protein research, in accordance with its hydrolysis of Alcalase2.4L alkalin proteinase identified as the best use of protease enzyme. Through the Response Surface Method to optimize the best hydrolysis conditions were: hydrolysis temperature 53℃, pH value 8.0, enzyme concentration 8800U/g pro, substrate concentration of 1.2%, hydrolysis time 50min. Under this best condition, the pine nut protein hydrolysis degree reached 43.15%. Its ratio of protease to protein has significant impact, followed by the pH value.
3. To immobilize Alcalase2.4L alkalin proteinase with the cross-linking method, studied the process of immobilized Alcalase and the best conditions of immobilization, such as chitosan concentration 2.5%, glutaraldehyde concentration0.4% carrier cross-linking time4h, the weight of enzyme915U/g carrier, pH value 9.4, immobilization time8h, immobilization enzyme activity recovery rate was54.35%. Some characters of immobilized and free Alcalase2.4L, such as Michaelis constant, optimum temperature, pH, and thermal stability such as the nature were studied and compared. Repeated 6 times hydrolysis processes by immobilized Alcalase2.4L protease, the relative activity was still more than 60%. Illuminate immobilized Alcalase protease with the use of properties, can be used as industrial production.
4. Studied immobilized Alcalase2.4L protease hydrolysis pine nut protein process. The optimal hydrolysis conditions were pH value9.0, temperature50℃, reaction time2h enzyme dosage0.2g/gsubstracte, substrate concentration 1.2%, hydrolysis degree is 25%. The use of immobilized Alcalase2.4L protease hydrolysis pine nut protein peptide preparation method is feasible.
本文以红松松仁为原料,利用现代分离分析手段,对松仁蛋白的提取工艺、酶解条件进行优化,又对最佳蛋白酶进行固定化研究再用其制备松仁多肽,主要结果如下:
1、采用超声波辅助碱提酸沉法提取松仁蛋白,最佳工艺条件依次为:料液比1:24.61、pH值10、提取温度48.3℃、超声时间100min。松仁蛋白提取率为233.23 mg/g。超声波辅助提取法可以有效地提高松仁蛋白质的提取率,缩短反应周期。
2、选择国产碱性、中性蛋白酶、Alcalase2.4L碱性蛋白酶和风味蛋白酶作为松仁蛋白酶解备用酶,对松仁蛋白进行酶解效果研究,根据其水解程度,确定Alcalase蛋白酶为松仁蛋白酶解的最佳用酶。通过响应面法优化得到此酶作用下的最佳酶解条件为:pH值8.0、水解温度53℃、酶与底物比8800 U/g蛋白、底物浓度1.2%、酶解的时间50min。在最佳酶解的条件下水解度(DH)为43.15%。酶与底物比对其酶解过程具有极显著性影响,其次是pH。
3、使用交联-吸附法对Alcalase2.4L蛋白酶进行固定化,研究Alcalase蛋白酶固定化工艺,得到了载体与交联剂浓度、载体交联时间、给酶量、pH、固定化时间的最佳固定化条件:壳聚糖浓度2.5%、戊二醛浓度0.4%、载体交联时间4h、给酶量915U/g载体、pH9.4、固定化时间8h,固定化酶活力回收率为54.35%。同时对固定化酶及溶液酶的米氏常数、最适温度、pH、热稳定性等性质进行了研究和比较。固定化Alcalase蛋白酶重复使用6次,相对活力仍在60%以上。说明固定化Alcalase蛋白酶具使用特性,可用作工业化生产。
4、研究了固定化Alcalase2.4L蛋白酶酶解松仁蛋白的工艺。对酶解条件进行了优化,得到固定化Alcalase2.4L蛋白酶最佳酶解条件:底物浓度1.2%、pH值9.0、温度50℃、给酶量0.2g/g底物、反应时间2h,水解度达到25%。说明用固定化Alcalase蛋白酶酶解松仁蛋白制备多肽的方法可行。
Pinus koraiensis Sieb. et Zuee is a rare tree species of Xiao xing an ling in China. The pine nut is a high nutritional value of fruits, rich in protein, amino acids, fatty acids, vitamins, minerals and other substances physical activity has a wide application development potential.
In this research, pine nuts were used as material, the use of modern analytical tools from the pine nut protein extraction technology, hydrolysis condition optimization; also research the best immobilized protease. The main results are as follows:
1. Use ultrasound-assisted alkali extraction method from pine nut protein, optimum conditions were: Solid to liquid ratio (W/V) 1:24.61、pH10.0、extraction temperature48.3℃、ultrasonic time 100min. Pine nut protein extraction rate was 233.23 mg/g. Ultrasound-assisted extraction method can be effective in raising the pine nut protein extraction rate, shorten response cycle.
2. Select Domestic the alkaline protease、neutral proteinase、the Alcalase2.4L alkalin proteinase and the Flavourzyme proteinase as a pine nut protein enzymolysis preparation enzyme, the enzyme effect pine nut protein research, in accordance with its hydrolysis of Alcalase2.4L alkalin proteinase identified as the best use of protease enzyme. Through the Response Surface Method to optimize the best hydrolysis conditions were: hydrolysis temperature 53℃, pH value 8.0, enzyme concentration 8800U/g pro, substrate concentration of 1.2%, hydrolysis time 50min. Under this best condition, the pine nut protein hydrolysis degree reached 43.15%. Its ratio of protease to protein has significant impact, followed by the pH value.
3. To immobilize Alcalase2.4L alkalin proteinase with the cross-linking method, studied the process of immobilized Alcalase and the best conditions of immobilization, such as chitosan concentration 2.5%, glutaraldehyde concentration0.4% carrier cross-linking time4h, the weight of enzyme915U/g carrier, pH value 9.4, immobilization time8h, immobilization enzyme activity recovery rate was54.35%. Some characters of immobilized and free Alcalase2.4L, such as Michaelis constant, optimum temperature, pH, and thermal stability such as the nature were studied and compared. Repeated 6 times hydrolysis processes by immobilized Alcalase2.4L protease, the relative activity was still more than 60%. Illuminate immobilized Alcalase protease with the use of properties, can be used as industrial production.
4. Studied immobilized Alcalase2.4L protease hydrolysis pine nut protein process. The optimal hydrolysis conditions were pH value9.0, temperature50℃, reaction time2h enzyme dosage0.2g/gsubstracte, substrate concentration 1.2%, hydrolysis degree is 25%. The use of immobilized Alcalase2.4L protease hydrolysis pine nut protein peptide preparation method is feasible.
引文
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