Deamidation And Enzymatic Glycosylation And Crosslinking Of Caseinate And Soybean Protein As Well As Functional Properties Of The Products

Glycosylation is an important method of protein modification, which can effectively improve functional properties of the treated proteins via conjugating saccharides into the molecules of the protein substrates. The glycosylated protein can be widely used in food processing due to their improved functional properties. Caseinate and soybean protein are two important ingredients in food industry. Glycosylation of caseinate and soybean protein also can improve their fuctional properties and thereof provide better application in food processing.In the recent work, a new application of transglutaminase (TGase, EC 2.3.2.13) catalyzed glycosylation and crosslinking between protein and glucosamine were proposed. The evaluation results showed that the obtained product contained glucosamine and exhibited better properties. In the present research, the expansion studies were carried out. Deamidation and glycosylation were combined, and the functional properties of parent protein and the modified proteins were evalued.Two deamidated caseinates and soybean proteins with different deamidation degree were generated and modified by TGase in the presence of glucosamine under a fixed molar ratio of acyl acceptor to donor to carry out glycosylation and crosslinking. SDS-PAGE electrophoresis was used to demonstrate the glycosylation and crosslinking occured, and the HPLC analyse was used to measure the glucosamine amount. The free amino content was evaluated to reflect the crosslinking extent. Circular dichroism (CD) and FT-IR spectroscopy were used to show the strucure changes of the proteins and further demonstrate the glycosylation and crosslinking. Some properties including hydrated properties, reheological properties, and in vitro digestibility were measured, and show the impacts of protein deamidation on the glycosylation and crosslinking of two proteins.1. Deamidation as well as glucosamine conjugation and crosslinking of two proteinsTwo deamidated caseinates with degrees of deamidation of 16.6% and 28.1% were prepared, and then were used to generate the glycosylated and crosslinked products. The reaction was carried out at fixed molar ratio of acyl acceptor to donor of 3:1 (based on acyl of the protein substrates) and with the optional caseinate concentration elevated from 30 to 50 g L-1. The glucosamine amount of the glycosylated and crosslinked caseinate detected by HPLC analysis was 12.6 g kg protein, while that of two glycosylated and crosslinked deamidated caseinated were 9.8 and 6.9 g kg-1 protein. The reactable amino groups of caseinate were 0.61 mol kg-1 proteins, and those of the glycosylated and crosslinked caseinate were 0.51 mol kg-1 protein. Two glycosylated and crosslinked deamidated caseinates contained slight higher reactable amino groups (0.54-0.56 mol kg-1 protein). The results declared that the protein crosslinking occurred and the glycosylated and crosslinked deamidated caseinates were less crosslinked than the glycosylated and crosslinked caseinate.Two deamidated soybean proteins with degrees of deamidation of 12.2% and 27.4% were prepared, and then were used to generate the glycosylated and crosslinked products. The reaction was also carried out at fixed molar of acyl accepteor to donor of 3:1 (based on acyl of the protein substrates). The glucosamine amount of the glycosylated and crosslinked soybean protein detected by HPLC was 19.7 g kg-1 protein, while that of two glycosylated and crosslinked deamidated soybean proteins were 12.0 and 4.4 g kg-1 protein. The reactable amino groups of soybean protein were 0.49 mol kg-1 proteins, and those of the glycosylated and crosslinking soybean protein were 0.36 mol kg-1 protein. Two glycosylated and crosslinked deamidated soybean proteins contained reactable amino groups of 0.38 mol kg-1 protein. The results declared that the protein crosslinking occurred and the deamidated soybean proteins were less crosslinked than soybean protein.2. Modification in side chain and secondary structure of the proteinsSDS-PAGE analysis revealed that glycosylation and crosslinking occurred simultaneously during the reaction. FT-IR analysis indicated that glucosamine was covalently conjugated into the protein substrates, reflected by the enhanced absorption of some typical peaks. CD analysis results showed that after reactions the secondary structure of the glycosylated and crosslinked proteins was modified. Glycosylation and crosslinking led to the the glycosylated and crosslinked deamidated caseinates more order structure (i.e. less random-coil but more a-helix, p-sheet and β-turn); at the same time, the two reactions also resulted in the glycosylated and crosslinked deamidated soybean proteins more open structure (i.e. less a-helix and P-sheet but more random-coil).3. Fuctional properties of the proteinsThe solubility of deamidated proteins was higher than that of parent protein. Increasing the degree of deamidation, improved the solubility of the proteins.The glycosylated and crosslinked deamidated caseiante showed a better solubility than caseinate. Water-binding capability of two glycosylated and crosslinked deamidated caseinates, GC-DCN1 and GC-DCN2 were 4.4 and 3.0 fold of caseinate, and oil-binding capability 1.5 and 1.2 fold higher than those of casinate. EAI of GC-DCN1 and GC-DCN2 were 7.3% and 1.6% lower than caseinate, while ESI were 10.8% and 4.6% higher. The in vitro digestibility of glycosylated and crosslined deamidated caseinate was decreased than caseinate. The glycosylated and crosslinked deamidated caseinate showed a weaker solubility than glycosylated and crosslinked caseiate. Water-binding capability of GC-DCN1 and GC-DCN2 were 8.6% and 37.0% lower than glycosylated and crosslinked caeinate, while oil-binding capability were 25.0% and 38.6% lower. EAI of GC-DCN1 and GC-DCN2 were 7.8% and 14.4% higher, while ESI were 4.4% and 9.7% higher than that of glycosylated and crosslinked caseinate. The in vitro digestibility of glycosylated and crosslinked caseinate was higher than caseinate. The surface hydrophobic of deamidated caseinate was lower than that of caseinate. The surface hydrophobic of the glycosylated and crosslinked caseinate and deamidated caseaintes was lower than that of caseinate and deamidated caseinate. The glycosylated and crosslinked caseinate showed the lowest surface hydrophobic.The solubility of glycosylated and crosslinked soybean protein was lower than that of soybean protein. Water-binding capability of two glycosylated and crosslinked deamidated soybean proteins, GC-DSP1 and GC-DSP2 were 41.5% and 32.3% higher than soybean protein, and oil-binding capability were 1.4 and 1.0 fold of soybean protein. EAI of GC-DSP1 was 7.2% lower than soybean protein, and GC-DSP2 was not insignificant. ESI of GC-DSP1 and GC-DSP2 were 16.1% and 2.1% higher than that of soybean protein. The surface hydrophobic of GC-DSP1 and GC-DSP213.8% and 30.1% lower than soybean protein. The in vitro digestibility of glycosylated and crosslinked deamidated soybean protein was lower than that of soybean protein. The glycosylated and crosslinked deamidated soybean protein showed a better solubility than glycosylated and crosslinked soybean protein. Water-binding capability of GC-DSP1 and GC-DSP2 were 15.9% and 37.5% lower, while oil-binding capability were 23.3% and 38.4% lower than that of glycosylated and crosslinked soybean protein. EAI of GC-DSP1 and GC-DSP2 were 5.7% and 14.1% higher than that of glycosylated and crosslinked soybean protein, while ESI were 17.2% and 6.2% lower. The surface hydrophobic of GC-DSP1 and GC-DSP2 were 41.5% and 74.6% higher than that of glycosylated crosslinked soybean protein. The in vitro digestibility was higher than that of glycosylated crosslinked soybean protein.TPA textural analysis of the acid-induced gels showed that hardness, adhesion, and gumminess of the glycosylated and crosslinked caseinate, soybean protein and their deamidated products have been significantly improved compared to parent proteins. Based on the mechanical spectra of the acid-induced gels, the glycosylated and crosslinked caseinate showed shorter gelation time than caseinate.Rheological properties have also been significantly improved. Apparent viscosity, the viscous modulus and the elastic modulus of the glycosylated and crosslinked caseinate dispersion was significantly higher than that of caseinate. Meanwhile, the properties of glycosylated and crosslinked deamidated caseinate were lower than that of glycosylated and crosslinked caseinate.