| 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. |
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