Enzymatic Modification of Whey Protein Gels at low pH

Acidic;Transglutaminase;Gel;Whey Proteins

Eissa, Ahmed Sherif ; Christopher Daubert, Committee Member,Saad Khan, Committee Chair,Allen Foegeding, Committee Member,Jason Haugh, Committee Member,Eissa, Ahmed Sherif ; Christopher Daubert ; Committee Member ; Saad Khan ; Committee Chair ; Allen Foegeding ; Committee Member ; Jason Haugh ; Committee Member

University:North Carolina State University

关键词: Acidic;    Transglutaminase;    Gel;    Whey Proteins;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/3195/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Whey proteins are widely used in a variety of food products due to their functional and nutritional properties. This study focuses on modifying whey protein gel properties at acidic conditions by enzymatic treatment. Enzymes offer a powerful tool to modify biopolymers in general, and proteins in specific. Transglutaminase enzyme is used in this study to induce ε-(γ-glutamyl)lysine bonds between whey protein molecules at alkaline or neutral conditions, followed by subsequent acidification using glucono-δ-lactone (GDL) to form gels at the desired acidic pH. We examine the viability of β-lactoglobulin and α-lactalbumin for crosslinking and conclude that β-lactalbumin is easily crosslinked in its native state, while β-lactoglobulin needs partial or complete denaturation to undergo the enzymatic catalysis. Denaturation of β-lactoglobulin is done either by raising the pH to 8, or by thermal treatment (80 oC for 1 hr) or by chemical denaturation using dithiothreitol (DTT). Crosslinks induced by transglutaminase increase the molecular weight of the whey proteins considerably to exceed 107 Da. In the first part of this study, we investigate the cold-set gel formation by initially conducting the enzymatic reaction at pH 8 and 50 oC then following up with acidulation by GDL to pH 4. The resulting gel exhibits superior rheological properties with higher elastic modulus and substantially higher fracture/yield stress and strain compared to cold-set gels with no enzyme. In the second part of this study, we examine an alternative route for crosslinking, in which we preheat the whey protein first at 80 oC for 1 hr at pH 7, and then conduct the crosslinking at 50 oC. This procedure induces both disulfide and ε-(γ-glutamyl)lysine bonds. The elastic modulus of the final gels shows a modest increase for samples treated with enzyme while the fracture strain and stress reveal significant increase with enzyme treatment. Interestingly, the microstructures of both gels, with and without enzyme treatment, are found to be similar with a fractal dimension of ~2 obtained independently using rheology and confocal microscopy. This suggests that the nonlinear fracture/yield properties are not reflected in the microstructure of the gels in the length scales probed using confocal microscopy.In the third part of this study, we investigate the relative roles of physical and chemical interactions that affect the properties of protein polymers and cold-set gels at pH 4, prepared using the same protocol as in the second part of the thesis. We examine the role of hydrogen bonding, hydrophobic interactions, disulfide bonds and ε-(γ-glutamyl)lysine bonds. Physical interactions do not play a major role in the molecular weight or the size of the polymer, prior to acidification. However, they affect the gel rheological properties profoundly. The disruption of the hydrogen bonds inhibits gelation, while the disruption of the hydrophobic interactions causes extensive syneresis and result in weak and fragile gels. Chemical bonding affects the gel elastic modulus mildly and plays a detrimental role in the fracture/yield properties. The introduction of ε-(γ-glutamyl)lysine bonds increases the fracture strain and enhances the rheological properties of the gels that lack the disulfide bonds.In the fourth part of the thesis, we investigate the conformational characteristics of β-lactoglobulin — the main constituent of whey proteins - subject to enzymatic crosslinking, using Fourier Transform Infrared (FTIR) spectroscopy. We find major differences between thermal denaturation and chemical denaturation (using DTT or β-mercaptoethanol). Crosslinking by transglutaminase of the thermally denatured β-lactoglobulin does not change the spectra in the amide I region but alters the C-H stretching mode, suggesting modification of the hydrophobic interactions. On the other hand, crosslinking by transglutaminase of the chemically denatured β-lactoglobulin changed the structure of the α-helix and induced intermolecular β-sheets In the fifth part of the study, we derive a transglutaminase-catalyzed polymerization model of β-lactoglobulin based on probabilistic approach of non-linear polymers. Derived equations show critical gelation conversion of 5.8%. Although the model is based on several assumptions, we believe it represents the starting point towards a more realistic model taking into account the intramolecular crosslinks and the unequal functionalities of the lysine and glutamine residues along the β-lactoglobulin backbone.In the last part of the study, we present a brief discussion of the effect of transglutaminase on hydrophobic associations in chemically (using DTT) denatured whey proteins. Interestingly, we find that crosslinking of whey proteins by transglutaminase can modulate and screen the hydrophobic associations. This modulating effect is attributed to the compactness of the crosslinked protein chains that limit exposure of hydrophobic moieties to form hydrophobic associations.