Enzymatic Glycosylation And Crosslinking Of Two Proteins With A Degraded Chitosan And Some Properties Of The Products

Caseinate and soybean protein are important food proteins, which contain a variety of essential amino acids. Glycosylation of proteins is an approach in which saccharide is covalently conjugated into proteins. Glycosylation of proteins be Maillard reaction could improve functional properties of proteins, but there exists safety issues and undesired browning in the modified products. Preparation of glycosylated proteins by transglutaminase is different from that by Maillard reaction, and could improve the functional properties of proteins safely and effectively.In the present work, transglutaminase was applied to crosslink caseinate or soybean protein in the presence of degradaed chitosan to prepare glycosylated and crosslinked proteins. Amount of the conjugated degradaed chitosan (expressed in glucosamine) was analysed and used as an indicator to select optimal preparation conditions of modified proteins including protein concentration, TGase addition and reaction time via single factor trials. SDS-PAGE analysis was used to confirm glycosylation and crosslinking occurred in the modification, and free amino groups content was used to evaluate the extent of glycosylation. Structural changes of proteins were analyzed by the Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). Finally, original proteins and crosslinked proteins were used as controls, to evaluate the impact of glycosylation and crosslinking on some functional properties of the two proteins. The main results are listed in following.1. Preparing conditions andd glycosylation extend of the modified proteinsThe glycosylated and crosslinked caseinate was prepared at molar ratio of acyl donor/ acceptor of 1:3, the temperature of 37℃ and pH of 7.5. The selected caseinate concentration, TGase addition and reaction time from single-factor trials were 50 g/L,15 U/g protein and 4 h, respectively. The glycosylated and crosslinked caseinate had glucosamine amount of 14.99 g/kg protein, and less reactable amino groups than caseinate (0.58-0.58 versus 0.64 mol/kg protein), but statistically insignificant content of reactable amino groups than the crosslinked caseinate.The glycosylated and crosslinked soybean protein was prepared at molar ratio of acyl donor/ acceptor of 1:3, temperature of 37℃ and pH of 7.5. The selected protein concentration, TGase addition and reaction time were 30 g/L,10 U/g and 4 h, respectively. The glycosylated and crosslinked soybean protein had glucosamine amount of 19.40 g/kg protein, lower reactable amino group (0.38 mol/kg protein) than soybean protein (0.49 g/kg protein), but statistically insignificant content of reacrable amino groups than the crosslinked soybean protein.SDS-PAGE revealed that glycosylated and crosslinked caseinate and soybean protein consisted of many protein polymers, because some new peptide bands were observed on the top of stacking gel and separating gel; at the same time, glycoprotein-specific staining results revealed that glycosylated and crosslinked caseinate and soybean protein contained saccharides, as these protein polymers appeared as the positive staining bands.2. Modified structure of the glycosylated and crosslinked proteinsFT-IR spectra indicated that the degraded chitosan were covalently conjugated into caseinate and soybean protein. CD analysis result showed that the modified caseinate had less random-coil but an order structure, but the modified soybean protein had less a-helix and (3-sheet but an open random-coil structure.3. Functional properties of the glycosylated and crosslinked proteinsCaseinate and its modified product had the lower solubility around pH 4.0-4.5. Totally, glycosylated and crosslinked caseinate exhibited slightly higher solubility than cross-linked caseinate but lower solubility than caseinate. Glycosylated and crosslinked caseinate or crosslinked caseinate showed insignificant difference in their surface hydrophbicity, which were much higher than that of caseinate (11.0 and 11.6 vs.8.8). Glycosylated and crosslinked soybean protein and crosslinked soybean protein were also higher in surface hydrophobicity than soybean protein (18.4 and 18.3 vs.14.9).The modification impaired the emulsifying properties of caseinate. The glycosylated and crosslinked caseinate showed lower emulsifying activity index (EAI) and emulsifying stability index (ESI) about 35.3% and 25.6% than caseinate, but the higher EAI and ESI about 13.8% and 30.0% than that of crosslinked caseinate, respectively. The glycosylated and crosslinked soybean protein showed lower EAI but the higher ESI than soybean protein. The digestibility of caseiante and soybean protein was improved and impaired by modification, respectively.Rheological properties and water holding capacity of caseinate were improved significantly. Apparent viscosity, storage modulus and viscous modulus of the glycosylated and crosslinked caseinate were obviously improved. Based on the mechanical spectra of the acid-induced gels, the glycosylated and crosslinked caseinate showed shorter gelation time. Water holding capacity of the glycosylated and crosslinked caseinate was nearly 6 times higher than caseinate. The glycosylated and crosslinked caseinate exhibited higher oil binding capacity than the crosslinked caseinate but similar oil binding capacity than caseinate. The glycosylated and crosslinked soybean protein showed higher water (54.7%) and oli (85%) binding capacity than soybean protein.