Soybean Whey Proteins-polysaccharide Complexation And The Selective Extraction Of Proteins

The ability to control or manipulate the macromolecular interactions between protein and polysaccahrides would allow selective purification of target proteins without affecting their natural biological activity. Soybean whey are a by-product of manufacturing of soy protein isolates(SPI) from soybean seeds(Glycine max), and the reocovery of protein of which can improve the utilization of soybean as well as reduce the the environment pollution. In this thesis, Bsed on the standpoint of thermodynamics and kinetics, the selective complexation behavior between soybean whey proteins and natural charged polysaccharides is extensively investigated with regard to the formation mechanism and control factors affecting the formation of complexes. The major results are as follows:In order to purify KTI, the complex behavior of soybean whey proteins(SWP) with chitosan(Ch) was firstly studied by turbidimetric titration and Tricine-SDS-PAGE. Four critical pH values can be observed, which indicated the phase transitions upon changing pH of the system. An initial slight increase in turbidity occurred at pHc, which corresponded to the soluble complex formation. A sharp elevation in turbidity was found at pH?1, signifying the onset of phase separation. At pHmax, maximum turbidity was obtained, whereas at pH?2 the coacervate or precipitate started to redissolove. By controlling pH to pHmax, the biggest amounts of complexes can be obtained with the highest protein content(32%) at SWP/Ch=4:1, pH 6.3. In the protein-chitosan complex, KTI and the BBI were higher in contents, than those of SBA and ?-amylase. At the condition of SWP/Ch=100:1, pH 4.8 and low ionic strength, KTI was found to be selectively complexed. After selective complexation with chitosan, a high purity KTI(>90% by SEC-HPLC) could be obtained, TIA was 5901.3 U/mg.The selective complex behavior of soybean whey proteins(SWP) with carrageenan(CG) was studied to purify the KTI, BBI, and SBA. Obviously, the strength of complexation for SWP-CG system was stronger than SWP-Ch system. At mixing weight ratio ?5:1, a competitive binding of KTI versus BBI, as well as SBA versus ?-amylase was observed, KTI and SBA complexed firstly with CG, then BBI and ?-amylase. ITC results showed that, at the same pH, the CG-affinity(Kb) of SBA was 43.1 ± 13.1×106 M-1, higher than that of ?-amylase(8.39 ± 1.55×106 M-1); the CG-affinity of KTI was 14.5 ± 4.03 × 106 M-1, than that of BBI(4.05 ± 1.13 ×106 M-1). Therefore, a purified KTI and SBA can be obtained by controlling to pH 3.57, 4.48, mixing weight ratio of 15:1, respectively. After KTI completely transferred into coacervates at 10:1, pH 3.69, purified BBI(92.5%) was enriched in the supernatant, and CIA was 690 U/mg.The effect of polymer charge density on the selective purification of protein was studied when KTI-BBI mixture(KBM) complexing with carboxylated polysaccharides(CPS) and sulphated polysaccharides(SPS). It was found that, the complexes formed between CPS and KBM was more like “coacervates”, whereas SPS–KBM system formed “precipitates” complexes, might related to the higher polymer charge density of SPS as compared with CPS. Dextran sulfate has the highest charge density, the dosage required to purify BBI was the lowest(protein/ polysaccharide of 10:1); ?-carrageenan, chondroitin sulfate, and sodium alginate have medium charge density, the required dosage became 5:1(protein/polysaccharide ratio); xanthan gum has the lower charge density, the required dosage was the most(protein/ polysaccharide of 2:1). Binding of KBM to the lowest charge density polysaccharide, arabic gum expectedly cannot realize the purification of BBI under conditions where binding to the more highly charged polysaccharides do.From the view of thermodynamics, the complex behavior of BBI binding to ?-carrageenan(CG) was fully investigated. ITC results showed that BBI-CG binding contained two different stages: heat enthalpy(-?H) growing process and heat enthalpy(-?H) attenuation process. It was found that, the critical transition concentration(rcritical) was independent of protein concentration, ionic strength and temperature. Electrostatic interaction and non-electrostatic interaction, such as hydrophobic contribution were involved in this binding process. When BBI binding to carboxylated polysaccharides, such as sodium alginate, xanthan gum and gum arabic, ITC titration generated sigmoidal saturation curves, whereas binding to sulfated polysaccharides, such as ?- and ?-carrageenan, dextran sulfate, chondroitin sulfate, the similar two-step binding behavior was observed. After mild acid hydrolysis at 80 °C for 3 h, fully disordered ?-carrageenan fragments was obtained, and BBI-LC fragments binding process showed a typical sigmoidal saturation curves. This results indicated that the sulfate groups of carrageenan plays a key role in the formation of two-stage binding.In this part, we turn our attention to actual soybean whey, which is a multicomponent system consisting of carbohydrates, nonprotein nitrogen, proteins not precipitated, and mineral salts, etc. To purify these soybean whey protein, two successive and selective coacervations induced by chitosan(Ch) and carrageenan(CG) were investigated as a function of pH, mixing weight ratios, polysaccharides types. In the presence of high concentrations of carbohydrates mineral salts, and non-protein nitrogen, the required Ch for removal of protease inhibitors was half of protein content(50%), much higher than single soybean whey proteins(5%, in the first part). The binding constants for the interaction increased on the order Ch-66.7 < Ch-200 < Ch-510. At the second selective complexation, we observed a competitive binding behavior between KTI/BBI and CG. At a mixing weight ratio of 3:1(pH 3.0 for ?-CG, and pH 3.11 for ?-CG), the preferential binding of KTI to CG led to the single enrichment of BBI in the supernatant. After two successive coacervations, the obtained BBI was purified to ~92.7% as analyzed by SEC-HPLC and showed a good bioactivity(CIA: 669.5 CIU/mg).Finally, we assemble a quick shearing experimental reactor to recover soybean whey proteins by adding high concentration of polysaccharides(4%). Among six kinds of polysaccharides, ?-carrageenan(LC) and dextran sulfate(DS) are the most suitable polysaccharide for protein recovery. At SWP/LC 2:1 or SWP/DS 3:1, pH 3.5, the highest protein recovery were 91% and 89.2%, respectively. LC can be added in powder form, whereas DS can be prepared by higher concentration up to 10%(w/v). CLSM results showed that, before shearing, the microstructure of protein-polysaccharides complexes exhibited inhomogenous due to the high concentration of polysaccharide. After shearing, LC-SWP and DS-SWP complexes formed a large amounts of aggregate particles; whereas microstructure of Na A-SWP and XG-SWP complexes is more likely largely flocculent structure, with no obvious aggregate particles formed. For GA-SWP and CMC-SWP complexes, there were almost no aggregate particles or largely flocculent structure can be observed, thus no phase separation was formed. The separation of protein and polysaccharide can be achieved by ultrafiltration using membrane with MW cutoff of 100 k Da, and more than 75%–80% proteins and 90% of polysaccharide were existed in the filtrates and retentates respectively.