Effects Of TGase-induced Oligochitosan Glycation And Cross-linking On The Properties Of Two Proteins

Caseinate and soy protein(SPI) are two important food proteins, as they have some advantages such as essential amino acids composition. However, all food protein don’t have all the required properties in food processing. Protein modification thus is necessary to food industry. The Maillard reaction(i.e. protein glycation) is an effective and powerful way to covalent attachment of saccharides into protein molecules, which can improve protein properties. However, there are some disadvantages for this type of protein glycation, such as diffcult in reaction control, the extended reaction time, and potential formation of some undesired(e.g. color or mutagenic) products. In recent, transglutaminase(TGase, E.C. 2.3.2.13) is proved able to conjugate the amino-containing saccharides into proteins, resulting in the products as glycated and cross-linked proteins. This enzymatic approach is an interesting alternative besides the Maillard-type glycation, as it can modify the properties of food proteins safely and effectively.In this study, TGase was applied to cross-link caseinate or SPI in the presence of oligochitosan of 5 k Da to prepare glycated and cross-linked proteins. Polyacrylamide gel electrophoresis(SDS-PAGE) analysis, high performance liquid chromatograph(HPLC), and assay of free amino groups content were used to confirm the glycation and cross-linking occurred in the prearation. Fourier transform infrared spectroscopy(FT-IR) and circular dichroism(CD) were used to reveal the side chain and secondary structural changes of the modified products, respectively. After that, the impacts of glycation and cross-linking on properties of the two proteins were evaluated. The main results of this study are listed as following.(1) Glycation and cross-linking of caseinate and soy proteinWith the optimal preparation conditions from the previous studies, caseinate or SPI was modified to obtain three modified products(GC-caseinate I, GC-caseinate II and GC-SPI). SDS-PAGE analysis results showed that the modified products contained protein polymers and were glycoproteins. HPLC analysis results further indicated that GC-caseinate I, GC-caseinate II and GC-SPI were glycated, as they were detected to have respective glucosamine amount of 12.8, 30.8 and 13.6 g/kg protein. In comparison with caseinate, GC-caseinate I and GC-caseinate II contained less reactable –NH2(0.50 and 0.52 versus 0.62 mol/kg protein), indicating caseinate cross-linking. Cross-linked SPI(CL-SPI) and GC-SPI had reactable –NH2 of 0.43 and 0.42 mol/kg protein, respectively, which were clearly lower than that of SPI(0.50 mol/kg protein), which indicated soy protein cross-linking.(2) Side chain and secondary structural characteristics of the modified productsThe results from FT-IR spectra indicated that the oligochitosan was covalently conjugated into caseinate or SPI. CD analysis results also showed that GC-caseinates had less random coil but more α-helix and β-sheet structure, which enabled them an order secondary structure. However, GC-SPI exhibited a more open secondary structure, as it had less α-helix and β-sheet but more random coil structure.(3) Properties of the modified products1) Glycation and cross-linking of proteins caused the modified products with changed properties in terms of water-binding capacity, oil-binding capacity and in vitro digestibility. In comparison with caseinate, GC-caseinate I and GC-caseinate II showed higher water but lower oil binding capacity. In comparison with SPI and CL-SPI, GC-SPI showed enhanced water and oil binding capacity. Glycation and cross-linking of caseinate resulted in higher in vitro digestibility for GC-caseinate II but lower one for GC-caseinate I as they had higher and lower glaction extents. GC-SPI also had lower in vitro digestibility than SPI due to the protein cross-linking.2) The carried modification decreased emulsifying activity index of the modified products, but they showed higher emulsifying stability index. GC-caseinate I and GC-caseinate II showed higher surface hydrophobicity than caseinate, while CL-SPI and GC-SPI showed higher surface hydrophobicity than SPI. In comparison with caseinate, GC-caseinate I and GC-caseinate II in dispersions showed improved apparent viscosity, viscoelastic modulus. GC-caseinate I and GC-caseinate II could form acidified gels with stronger strength, enhanced water-holding capacity and bulk density. GC-caseinate I and GC-caseinate II had shorter gelation time and lower gelation temperature, at the same time, the structures of GC-caseinate gels were significantly modified.3) In comparison with caseinate, GC-caseinate I and GC-caseinate II showed larger hydrodynamic radius(173.9 and 168.2 versus 145.3 nm), and larger negative zeta-potential(-32.9 and-30.9 versus-27.6 m V) in dispersions. In comparison with SPI and CL-SPI, GC-SPI could form aggregates with enlarged hydrodynamic radius(180.2 versus 82.9 and 101.0 nm) and negative zeta-potential(-31.2 versus-27.7 and-30.7 m V) in dispersion. Thermogravimetric analysis demonstrated the modified products exhibited lower decomposition temperature and greater mass loss, indicating the modified products with lower thermal stability.