微波辅助菜籽饼粕蛋白水解产物的高效制备及其功能特性研究
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摘要
菜籽饼粕蛋白是重要的植物蛋白源之一,但由于菜籽饼粕中的一些有毒和抗营养成分的存在,限制了菜籽饼粕蛋白的应用。研究表明通过水解蛋白质获得多肽是一条有效的改善蛋白质功能特性的途径。本课题的研究目的是利用微波辅助酶解技术,在前人研究的基础上建立一种高效的制备菜籽多肽的工艺,并对获得的菜籽多肽的功能特性和抗氧化活性进行研究。研究的方法是借助微波辅助技术,利用碱性蛋白酶,风味蛋白酶以及碱性蛋白酶-风味蛋白酶双酶酶解脱脂菜籽饼粕蛋白,通过对酶解工艺重要参数的优化,建立最佳工艺条件,并对酶解后获得的菜籽多肽的部分功能特性和抗氧化活性进行了考察。实验结果表明:
1通过响应面分析法优化出最佳碱性蛋白酶酶解脱脂菜籽饼粕蛋白的工艺条件为:微波温度46℃,微波功率500 W,微波辅助酶解时间7 min(水解度为12.57%)可以达到普通水浴酶解4 h(水解度为14.14%)的效果。由于适当的加热处理可以使得蛋白质结构打开,酶解过程中更多的蛋白质分子酶切位点与蛋白酶结合,酶解反应易于进行。所以,在酶解反应进行前对菜籽饼粕进行预处理是非常有必要的。因此,本实验研究了用微波辅助加热代替普通水浴加热对菜籽饼粕蛋白进行预处理,短时间内达到普通水浴预处理菜籽饼粕蛋白的效果。通过优化得出:微波预处理功率600 W,温度75℃,微波预处理菜籽饼粕3 min,可以达到普通水浴加热预处理30 min的效果。
2优化出微波辅助风味蛋白酶酶解脱脂菜籽饼粕的最佳工艺条件为:微波温度50℃,微波功率500 W,水解时间13 min,水解度可达25.64%。酶解后获得的菜籽多肽具有良好的功能特性:当水解度达到25%时,溶解度可以达到55%;水解度为15%时持油性达到最大,而持水性在水解度达到5%时即可达到最大;在水解度为10%时乳化能力指数和乳化稳定性指数均达到最大;菜籽饼粕蛋白酶解液的起泡性及起泡稳定性要好于菜籽饼粕蛋白,且随着水解度的不断增加,起泡性不断增大。通过对菜籽多肽抗氧化活性的研究表明菜籽饼粕蛋白酶解液中的不同分子量的多肽片段都具有一定程度的抗氧化活性,同时也说明不同强度的抗氧化活性由不同水解度的酶解产物中存在得不同分子量的多肽片段产生的。
3利用微波辅助技术,采用正交试验用碱性蛋白酶和风味蛋白酶对脱脂菜籽粕进行分步酶解。优化出酶与蛋白的质量比为0.225 g/g,两种酶的质量比为2/1,即:碱性蛋白酶加量为E:S为9 000 U/g;风味蛋白酶加量为E:S为37.5 LAPU/g,碱性蛋白酶酶解3 min,风味蛋白酶酶解13 min,此时的水解度为53.67%。同时在最优化条件下进行验证试验,此时得到的水解度和氮收率分别为50.94%和96%,多肽为分子量在1 000 Da左右的短肽。
Rapeseed protein is one of the plant protein resources. But because of the existence of toxic substances and anti-nutritional factors in rapeseed meal, the application of rapeseed meal was limited. Some researches show that it is an effective method to modify the functional properties through hydrolyzing protein to get peptide. So the purpose of this subject is establishing a high efficiency peptide preparation technology using microwave technology on the base of previous study and studying the functional properties and antioxidative characteristic of the rapeseed peptide. The method is microwave assisted hydrolyzing the rapeseed protein by Alcalase, Flavourzyme and the two enzymes respectively. Through optimizing the important process parameters, the optimal microwave assisted enzyme hydrolysis condition is established. And then in the optimal condition the rapeseed peptide is prepared and study their functional properties and antioxidative characteristic. The research result is showing as following:
1 By the analysis of response surface methodology (RSM), the optimal microwave assisted Alcalase hydrolysis condition is: microwave temperature 46℃, microwave power 500 W and microwave time 7 min. And in this condition the degree of hydrolysis (the DH) is 12.57% which is equal to the hydrolysis degree of hydrolysis in common water bath for about 4 hours (the DH is 14.14%). And because of the suitable heating pretreatment can make unfold of protein structure and make more hydrolysis sites contact with the enzyme, the reaction can process easily. Hence, it is very necessary to pretreat the rapeseed meal before the hydrolysis reaction. In this experiment, the water bath pretreatment of rapeseed meal is substituted by microwave heating and the result show that several minutes pretreatment using this method can achieve to the effect of 30 minutes water bath pretreatment. And the optimal pretreatment condition is microwave power 600 W, microwave temperature 75℃and microwave pretreatment time 3 min.
2 The optimal microwave assisted Flavourzyme hydrolysis condition for rapeseed protein is microwave temperature 50℃, microwave power 500 W, microwave hydrolysis time 13 min, and the DH is 25.64%. The rapeseed peptide obtained from the rapeseed meal protein has good functional properties: the solubility can reach 55% when the DH is 25%; fat absorption and water absorption of the hydrolysate reach peak when the DH is 15% and 5%, respectively; the emulsifying activity and emulsifying stability could reach maximum when the DH was 10%; the foaming capacity of the hydrolysate is better than the rapeseed protein and the foaming capacity increase with the accelerate of the DH. And the study of antioxidative characteristic displayed that hydrolysate of different DH all had a certain extent antioxidative characteristic, which was determined by the molecular weight and the species of peptide in the hydrolysate.
3 In this part experiment, the microwave assisted technology is used and the step enzymatic hydrolysis process of defatted rapeseed meal protein is studied. After the Alcalase hydrolysis, the Flavourzyme is used to hydrolyze the rapeseed meal protein continuously. The optimal dosage ratio of enzyme and rapeseed meal is 0.225 g/g, the ratio of the two enzyme is 2/1 (Alcalase 9 000 U/g, Flavourzyme 37.5 LAPU/g), the hydrolysis time is just 3 min for Alcalase and 13 min for Flavourzyme, and in this condition the DH is 53.67%. The verification test for the optimal condition show the DH and the nitrogen recovery ratio is 50.94% and 96%. And the molecular weight of mainly hydrolysate was 1000 Da.
1通过响应面分析法优化出最佳碱性蛋白酶酶解脱脂菜籽饼粕蛋白的工艺条件为:微波温度46℃,微波功率500 W,微波辅助酶解时间7 min(水解度为12.57%)可以达到普通水浴酶解4 h(水解度为14.14%)的效果。由于适当的加热处理可以使得蛋白质结构打开,酶解过程中更多的蛋白质分子酶切位点与蛋白酶结合,酶解反应易于进行。所以,在酶解反应进行前对菜籽饼粕进行预处理是非常有必要的。因此,本实验研究了用微波辅助加热代替普通水浴加热对菜籽饼粕蛋白进行预处理,短时间内达到普通水浴预处理菜籽饼粕蛋白的效果。通过优化得出:微波预处理功率600 W,温度75℃,微波预处理菜籽饼粕3 min,可以达到普通水浴加热预处理30 min的效果。
2优化出微波辅助风味蛋白酶酶解脱脂菜籽饼粕的最佳工艺条件为:微波温度50℃,微波功率500 W,水解时间13 min,水解度可达25.64%。酶解后获得的菜籽多肽具有良好的功能特性:当水解度达到25%时,溶解度可以达到55%;水解度为15%时持油性达到最大,而持水性在水解度达到5%时即可达到最大;在水解度为10%时乳化能力指数和乳化稳定性指数均达到最大;菜籽饼粕蛋白酶解液的起泡性及起泡稳定性要好于菜籽饼粕蛋白,且随着水解度的不断增加,起泡性不断增大。通过对菜籽多肽抗氧化活性的研究表明菜籽饼粕蛋白酶解液中的不同分子量的多肽片段都具有一定程度的抗氧化活性,同时也说明不同强度的抗氧化活性由不同水解度的酶解产物中存在得不同分子量的多肽片段产生的。
3利用微波辅助技术,采用正交试验用碱性蛋白酶和风味蛋白酶对脱脂菜籽粕进行分步酶解。优化出酶与蛋白的质量比为0.225 g/g,两种酶的质量比为2/1,即:碱性蛋白酶加量为E:S为9 000 U/g;风味蛋白酶加量为E:S为37.5 LAPU/g,碱性蛋白酶酶解3 min,风味蛋白酶酶解13 min,此时的水解度为53.67%。同时在最优化条件下进行验证试验,此时得到的水解度和氮收率分别为50.94%和96%,多肽为分子量在1 000 Da左右的短肽。
Rapeseed protein is one of the plant protein resources. But because of the existence of toxic substances and anti-nutritional factors in rapeseed meal, the application of rapeseed meal was limited. Some researches show that it is an effective method to modify the functional properties through hydrolyzing protein to get peptide. So the purpose of this subject is establishing a high efficiency peptide preparation technology using microwave technology on the base of previous study and studying the functional properties and antioxidative characteristic of the rapeseed peptide. The method is microwave assisted hydrolyzing the rapeseed protein by Alcalase, Flavourzyme and the two enzymes respectively. Through optimizing the important process parameters, the optimal microwave assisted enzyme hydrolysis condition is established. And then in the optimal condition the rapeseed peptide is prepared and study their functional properties and antioxidative characteristic. The research result is showing as following:
1 By the analysis of response surface methodology (RSM), the optimal microwave assisted Alcalase hydrolysis condition is: microwave temperature 46℃, microwave power 500 W and microwave time 7 min. And in this condition the degree of hydrolysis (the DH) is 12.57% which is equal to the hydrolysis degree of hydrolysis in common water bath for about 4 hours (the DH is 14.14%). And because of the suitable heating pretreatment can make unfold of protein structure and make more hydrolysis sites contact with the enzyme, the reaction can process easily. Hence, it is very necessary to pretreat the rapeseed meal before the hydrolysis reaction. In this experiment, the water bath pretreatment of rapeseed meal is substituted by microwave heating and the result show that several minutes pretreatment using this method can achieve to the effect of 30 minutes water bath pretreatment. And the optimal pretreatment condition is microwave power 600 W, microwave temperature 75℃and microwave pretreatment time 3 min.
2 The optimal microwave assisted Flavourzyme hydrolysis condition for rapeseed protein is microwave temperature 50℃, microwave power 500 W, microwave hydrolysis time 13 min, and the DH is 25.64%. The rapeseed peptide obtained from the rapeseed meal protein has good functional properties: the solubility can reach 55% when the DH is 25%; fat absorption and water absorption of the hydrolysate reach peak when the DH is 15% and 5%, respectively; the emulsifying activity and emulsifying stability could reach maximum when the DH was 10%; the foaming capacity of the hydrolysate is better than the rapeseed protein and the foaming capacity increase with the accelerate of the DH. And the study of antioxidative characteristic displayed that hydrolysate of different DH all had a certain extent antioxidative characteristic, which was determined by the molecular weight and the species of peptide in the hydrolysate.
3 In this part experiment, the microwave assisted technology is used and the step enzymatic hydrolysis process of defatted rapeseed meal protein is studied. After the Alcalase hydrolysis, the Flavourzyme is used to hydrolyze the rapeseed meal protein continuously. The optimal dosage ratio of enzyme and rapeseed meal is 0.225 g/g, the ratio of the two enzyme is 2/1 (Alcalase 9 000 U/g, Flavourzyme 37.5 LAPU/g), the hydrolysis time is just 3 min for Alcalase and 13 min for Flavourzyme, and in this condition the DH is 53.67%. The verification test for the optimal condition show the DH and the nitrogen recovery ratio is 50.94% and 96%. And the molecular weight of mainly hydrolysate was 1000 Da.
引文
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74. Shuang Lin, Dong Yun, Dawei Qi, Chunhui Deng, Yan Li, Xiangmin Zhang. (2008). Novel microwave-assisted digestion by trypsin-immobilized magnetic nanoparticles for proteomic analysis. Proteome, 7(3),1297-1307
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80. Vioque J, Sánchez-Vioque R, Clemente A, Pedroche J, Millán F. (2000). Partially Hydrolyzed rapeseed protein isolates with improved functional properties. J Am Oil Chem Soc, 77,447-450
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66. Naveeda Khatoon,Jamuna Prakash. (2006). Nutrient retention in microwave cooked germinated legumes. Food chemistry, 97(1),115-121
67. Nichole Cumbuy, Ying Zhong, Marian Naczk, Fereidoon Shahidi. (2008). Antioxidant activity and water-holding capacity of canola protein hydrolysates. Food chemistry, 109(1),144-148
68. Niranjan Rajapakse, Eresha Mendis, Won-Kyo Jung, Jae-Young Je, Se-Kwon Kim. (2005). Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Res Int, 38(2),175-182.
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72. Sadeghi AA, Shawrang P. (2007). Effects of microwave irradiation on ruminal protein degradation and intestinal digestibility of cottonseed meal. Livest Sci, 106,176-181
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74. Shuang Lin, Dong Yun, Dawei Qi, Chunhui Deng, Yan Li, Xiangmin Zhang. (2008). Novel microwave-assisted digestion by trypsin-immobilized magnetic nanoparticles for proteomic analysis. Proteome, 7(3),1297-1307
75. Shuang Lin, Guoping Yao, Dawei Qi, Yan Li, Chunhui Deng, Pengyuan Yang, et al. Fast and efficient proteolysis by microwave-assisted protein digestion using trypsin-immobilized magnetic silica microspheres. Analytical chemistry, 80(10),3655-3665
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79. Vesper H W. (2005). Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin Al by mass spectrometry. Mass sepctrom, 19,2865-2870
80. Vioque J, Sánchez-Vioque R, Clemente A, Pedroche J, Millán F. (2000). Partially Hydrolyzed rapeseed protein isolates with improved functional properties. J Am Oil Chem Soc, 77,447-450
81. Wanasundara PKJ, Ross ARS, Amarowicz R. (2002). Preparation and characterization of hydrolyzed proteins from defibrinated Bovine Plasma. J. Agric. Food Chem, 67,623-630
82. Xi-qun Zheng, Li-te Li, Xiao-lan Liu, Xiao-jie Wang, Jie Lin, Di Li. (2006). Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten. Applied Microbiology and Biotechnology, 73(4), 763-770.
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