基于多级逆流固液萃取的大豆分离蛋白提取工艺研究
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摘要
大豆分离蛋白(SPI)是以脱脂豆粕为原料经过碱溶酸沉等方式制备而成的蛋白质产品,传统的提取工艺存在用水量高、提取率低、乳清废水排放量大等问题。本文以豆粕中蛋白质为研究对象,在基于质谱的蛋白质种类识别基础上,考察了提取条件对豆粕中不同种类蛋白质的释放行为的影响,建立基于多级逆流固液提取技术的SPI提取新工艺,在此基础上研究并建立大豆深加工行业乳清废水中高附加值蛋白的提取方法与综合利用,旨在降低提取过程用水量并提高蛋白质收率。主要结论如下:
1.利用HPLC对蛋白提取物及乳清废水进行了分离,收集了分离出的主要蛋白质组分;采用酶解技术结合液质联用技术对分离出的蛋白质进行了种类识别。结果表明不同种类蛋白质的保留时间分布范围较宽,提取物中7S和11S组分中的蛋白质在色谱图中的保留时间主要集中在24 min以后,含有少量2S组分,乳清废水中主要是2S组分,其保留时间集中在24 min以前。采用飞行时间质谱和凝胶过滤色谱分析了蛋白提取物和乳清废水;结果表明,SPI的分子量范围为10 kDa-320 kDa,乳清废水的分子量范围为10 kDa-30 kDa。
2.在蛋白质组分识别的基础上,对豆粕中的蛋白质溶出过程进行了监测,并对SPI提取过程中豆粕内各蛋白质组分的释放行为进行了研究。结果表明,2S组分释放速率较快,经过三次提取的相对释放量可达到90%以上;7S和11S组分释放速率较2S组分的释放速率低,四次提取的相对释放量达84%,提高提取过程中固相和液相之间的浓度梯度和延长7S和11S组分相对提取时间有利于提高7S和11S组分的释放速率。
3.考察了多级逆流固液提取过程中提取条件对蛋白质总提取率、高分子量组分的酸沉率的影响。结果表明,采用多级逆流固液提取技术可在相同的蛋白质收率条件下用水量节省16%,同时由于提取液中较高的7S和11S浓度,在酸沉阶段7S和11S的收率较传统工艺中的酸沉阶段的收率提高3%。实现多级逆流固液提取工艺中试放大,获得的SPI中粗蛋白含量和氮溶解指数为分别为94.9%、99.4%,均高于传统提取工艺获得的SPI。
4.利用疏水层析法对乳清废水中的胰蛋白酶抑制剂进行纯化,并利用HPLC和MALDI-TOF对纯化结果进行表征。结果表明,疏水层析法可对胰蛋白酶抑制剂进行有效的分离纯化,并以纯化的胰蛋白酶抑制剂为配基,环氧活化的琼脂糖凝胶为载体制取亲和介质,以标准的胰蛋白酶溶液为样品对亲和层析介质进行表征。结果表明,制取的两种亲和介质均能实现对胰蛋白酶的纯化。
本研究将多级逆流固液萃取技术应用于SPI提取,并考察了不同提取条件对蛋白质收率及释放速率的影响,同时对乳清废水中的胰蛋白酶抑制剂进行了分离纯化,对建立节水和提高蛋白收率的SPI提取工艺具有重要的参考价值。
Soybean protein isolate (SPI) is a mixture of different types of protein obtained from soybean by method of basic extraction and isoelectric precipitation. For traditional extraction process, there are several disadvantages such as high water consumption, low protein recovery, protein degradation. In this study, a new process based on multi-stage countercurrent solid-liquid extraction (MSCSLE) was investigated to decrease water consumption and optimize the process of protein release. The main proteins were characterized and the release behavior and dynamic change of these proteins were investigated. Then, the effect of operational parameters on MSCSLE process was investigated. The possibility for separation of trypsin inhibitor in whey waste water was also investigated. The main results are summarized as follows:
1. The proteins in the SPI and whey waste water were separated using HPLC and chromatographic peaks were collected. Proteins were identified by mass spectrometric analysis coupled with tryptic digestion. The retention times of proteins in 7S and 11S components were after 24 min. Most of the proteins in whey waste water were 2S protein, which had a retention time below 24 min. The range of molecular weight was obtained by high performance gel filtration chromatography. The molecular weight ranges in SPI and whey waste water were from 10 kDa to320 kDa and from 10 kDa to30 kDa respectively.
2. The release rates of different types of protein in soybean meal were determined according to the peak areas in chromatogram. The results indicated that proteins in 2S had a higher release rate than that in 7S and 11S. The almost 90% of total 2S protein could be released when the soybean meal was washed three times. The total released proteins in 7S and11S was 84% after four-time extraction. The release rate of 7S and 11S proteins increased with the extraction time and ratio of water to solid increasing during extraction process.
3. MSCSLE was carried out and effect of water/solid ration and extraction time on the release rate, total released proteins, and precipitation ratio of high MW proteins were investigated. Compared to the traditional extraction method, 16% of water consumption could be saved by method of MSCSLE. At the same time, MSCSLE could decrease the degradation velocity of 7S and 11S, resulting in higher 7S and 11S content in SPI. Further analysis indicated that the crude protein content and nitrogen solution index of the product were 94.9% and 99.4% respectively.
4 The trypsin inhibitor in whey waste water was purified using hydrophobic chromatography. The obtained trypsin inhibitor was verified based on HPLC-MS and MALDI-TOF MS analysis. The result indicated that hydrophobic chromatography was an effective method to separate trypsin inhibitor from whey waste water. Then, trypsin inhibitor was coupled on agarose gel, which was activated by 1, 4-butanediol diglycidyl ether, and affinity chromatographic media was prepared. The efficacy of affinity media was test by trypsin solution. The result showed the trypsin could be purified by the affinity medium, indicating that the whey waste water can be used a resource to separate high value proteins.
MSCSLE was used in the extraction of SPI, and the impact of extraction conditions to the recovery ratio and release action was studied. The extraction conditions were optimized. Besides, the TI in whey waste water was purified by hydrophobic chromatography. This was benefit to construct a new SPI extraction method with low water consumption and high protein recovery.
1.利用HPLC对蛋白提取物及乳清废水进行了分离,收集了分离出的主要蛋白质组分;采用酶解技术结合液质联用技术对分离出的蛋白质进行了种类识别。结果表明不同种类蛋白质的保留时间分布范围较宽,提取物中7S和11S组分中的蛋白质在色谱图中的保留时间主要集中在24 min以后,含有少量2S组分,乳清废水中主要是2S组分,其保留时间集中在24 min以前。采用飞行时间质谱和凝胶过滤色谱分析了蛋白提取物和乳清废水;结果表明,SPI的分子量范围为10 kDa-320 kDa,乳清废水的分子量范围为10 kDa-30 kDa。
2.在蛋白质组分识别的基础上,对豆粕中的蛋白质溶出过程进行了监测,并对SPI提取过程中豆粕内各蛋白质组分的释放行为进行了研究。结果表明,2S组分释放速率较快,经过三次提取的相对释放量可达到90%以上;7S和11S组分释放速率较2S组分的释放速率低,四次提取的相对释放量达84%,提高提取过程中固相和液相之间的浓度梯度和延长7S和11S组分相对提取时间有利于提高7S和11S组分的释放速率。
3.考察了多级逆流固液提取过程中提取条件对蛋白质总提取率、高分子量组分的酸沉率的影响。结果表明,采用多级逆流固液提取技术可在相同的蛋白质收率条件下用水量节省16%,同时由于提取液中较高的7S和11S浓度,在酸沉阶段7S和11S的收率较传统工艺中的酸沉阶段的收率提高3%。实现多级逆流固液提取工艺中试放大,获得的SPI中粗蛋白含量和氮溶解指数为分别为94.9%、99.4%,均高于传统提取工艺获得的SPI。
4.利用疏水层析法对乳清废水中的胰蛋白酶抑制剂进行纯化,并利用HPLC和MALDI-TOF对纯化结果进行表征。结果表明,疏水层析法可对胰蛋白酶抑制剂进行有效的分离纯化,并以纯化的胰蛋白酶抑制剂为配基,环氧活化的琼脂糖凝胶为载体制取亲和介质,以标准的胰蛋白酶溶液为样品对亲和层析介质进行表征。结果表明,制取的两种亲和介质均能实现对胰蛋白酶的纯化。
本研究将多级逆流固液萃取技术应用于SPI提取,并考察了不同提取条件对蛋白质收率及释放速率的影响,同时对乳清废水中的胰蛋白酶抑制剂进行了分离纯化,对建立节水和提高蛋白收率的SPI提取工艺具有重要的参考价值。
Soybean protein isolate (SPI) is a mixture of different types of protein obtained from soybean by method of basic extraction and isoelectric precipitation. For traditional extraction process, there are several disadvantages such as high water consumption, low protein recovery, protein degradation. In this study, a new process based on multi-stage countercurrent solid-liquid extraction (MSCSLE) was investigated to decrease water consumption and optimize the process of protein release. The main proteins were characterized and the release behavior and dynamic change of these proteins were investigated. Then, the effect of operational parameters on MSCSLE process was investigated. The possibility for separation of trypsin inhibitor in whey waste water was also investigated. The main results are summarized as follows:
1. The proteins in the SPI and whey waste water were separated using HPLC and chromatographic peaks were collected. Proteins were identified by mass spectrometric analysis coupled with tryptic digestion. The retention times of proteins in 7S and 11S components were after 24 min. Most of the proteins in whey waste water were 2S protein, which had a retention time below 24 min. The range of molecular weight was obtained by high performance gel filtration chromatography. The molecular weight ranges in SPI and whey waste water were from 10 kDa to320 kDa and from 10 kDa to30 kDa respectively.
2. The release rates of different types of protein in soybean meal were determined according to the peak areas in chromatogram. The results indicated that proteins in 2S had a higher release rate than that in 7S and 11S. The almost 90% of total 2S protein could be released when the soybean meal was washed three times. The total released proteins in 7S and11S was 84% after four-time extraction. The release rate of 7S and 11S proteins increased with the extraction time and ratio of water to solid increasing during extraction process.
3. MSCSLE was carried out and effect of water/solid ration and extraction time on the release rate, total released proteins, and precipitation ratio of high MW proteins were investigated. Compared to the traditional extraction method, 16% of water consumption could be saved by method of MSCSLE. At the same time, MSCSLE could decrease the degradation velocity of 7S and 11S, resulting in higher 7S and 11S content in SPI. Further analysis indicated that the crude protein content and nitrogen solution index of the product were 94.9% and 99.4% respectively.
4 The trypsin inhibitor in whey waste water was purified using hydrophobic chromatography. The obtained trypsin inhibitor was verified based on HPLC-MS and MALDI-TOF MS analysis. The result indicated that hydrophobic chromatography was an effective method to separate trypsin inhibitor from whey waste water. Then, trypsin inhibitor was coupled on agarose gel, which was activated by 1, 4-butanediol diglycidyl ether, and affinity chromatographic media was prepared. The efficacy of affinity media was test by trypsin solution. The result showed the trypsin could be purified by the affinity medium, indicating that the whey waste water can be used a resource to separate high value proteins.
MSCSLE was used in the extraction of SPI, and the impact of extraction conditions to the recovery ratio and release action was studied. The extraction conditions were optimized. Besides, the TI in whey waste water was purified by hydrophobic chromatography. This was benefit to construct a new SPI extraction method with low water consumption and high protein recovery.
引文
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