International Journal of Biological Macromolecules
Rheology of protein gels synthesized through a combined enzymatic and heat treatment method
Introduction
The gelation of polymers into a network structure with specific macroscopic properties has long been an area of research in polymer science and engineering. Of particular interest is the area where specific, biological based modifications can be made, such as through the use of enzymes. Enzymes can provide highly specific modifications in the microstructure of a biopolymer, which can result in significant changes in the macroscopic properties [1].
Enzymes, such as transglutaminase (EC 2.3.2.13), are used to cross-link proteins to form a biopolymer gel. An example of this use is the cross-linking of whey proteins that are used in food products. Transglutaminase catalyzes the formation of cross-links between two proteins by creating an ε-(γ-glutamyl) lysine bond between the γ-carboxyamide group and the ε-amino group of a peptide bound lysyl residue [2]. This allows the formation of new chemical cross-links that would be insensitive to pH. Increases in the number of chemical cross-links would allow control (enhancement) of the fracture stress/strain, vis-à-vis, texture diversity and shelf life, in proteins. Unfortunately, a limitation of this technique is the requirement of prior denaturation of the whey proteins, usually by the addition of chaotropes or denaturants. Therefore, whey protein gels are typically synthesized via heat treatment, which denatures the proteins and promotes thermal reconstruction of the proteins to develop physical and chemical (disulfide) intermolecular cross-links [3], [4]. Proteases are another class of enzymes, which can modify the characteristics of whey protein gels [5]. Pre-treatment of the whey protein with proteases before heat treatment shows an increase in the cohesiveness and hardness of the gels. One advantage of proteases is the reduced cost and availability, in-contrast to transglutaminase.
Four types of intermolecular interactions control whey protein gel formation: hydrophobic interactions (5–10 kJ/mol), hydrogen bonds (10–40 kJ/mol), electrostatic interactions (25–80 kJ/mol) and covalent bonds (200–400 kJ/mol) [2]. Intermolecular hydrophobic interactions arise when previously hidden non-polar amino acid side-chains are exposed as the macromolecular structure uncoils during thermal denaturation. Hydrogen bonding results from the interaction of polar amino acid side-chains. Repulsive electrostatic interactions aid in the production of fine-stranded network structures with good water-holding characteristics. Intermolecular covalent bonding results in permanent cross-links and is most commonly formed by sulfydryl–disulfide interchange [3]. The texture of whey protein gels is controlled by the balance between the weak, reversible ‘physical’ cross-links (hydrophobic, hydrogen bonding, electrostatic) and the strong, covalent ‘chemical’ cross-links [3]. Unfortunately, there is no way to tailor the extent of chemical cross-linking during heat treatment [3].
Many fundamental issues related to heat treatment, gelation, and disulfide linkages are known as well as how to develop gels with controlled and enhanced properties [6], [7], [8]. However, an overlooked area is the development and utilization of whey protein gels at acidic or low pH (∼4) because of their weak and brittle nature compared with elastic gels produced at neutral or basic conditions. Presumably, low pH prevents the formation of intermolecular disulfide bonds, which is the only form of chemical cross-links produced during heat treatment. However, another hypothesis is that the electrostatic interactions in whey proteins affect gelation at this pH. Whey proteins have an approximate pI of 4–5 and gels produced by heat treatment at this pH are weaker than those produced at lower pH such as a pH of 1–2 [6]. Although a fundamental understanding of this problem is not complete, the ability to produce a whey protein gel at low pH with desirable texture and rheology would enhance its utility, particularly due to the fact that low pH food products are desirable due to their shelf stability [6] and less stringent sterilization processes [9].
A combination of enzymatic and heat treatments may provide a solution to creating low pH whey protein gels, because it eliminates the weakness found in both methods. A weakness of the enzymatic treatment method is that whey proteins must be denatured to some extent to allow the enzyme access to key amino acids. Acidic pH may partially, if not completely, denature the whey proteins. A weakness of the heat treatment method at low pH is the lack of chemical cross-linking. This may be overcome by enzyme modification.
The aim of this study was to gain physical insight to whey protein gels formed at low pH (∼4) by using enzyme modification followed by heat treatment. The rationale is as follows. By working at low pH, whey protein should denature thereby facilitating enzyme-catalyzed bond formation. Based on the enzyme selected, modification could be independent of pH. The heat treatment method might serve to deactivate the enzyme as well as allow formation of additional physical cross-links. By understanding the factors affecting enzyme modification and controlling the balance of enzyme and heat treatment, we envision development of low pH gels with tailored textures that mimic neutral and basic pH gels.
Section snippets
Materials
Samples of whey protein isolate (BiPRO Lot JE 017-8-420) were provided by Davisco Foods International, Inc. (Le Sueur, MN). Microbial ‘broad-range’ transaminase (T-7684), SealPlate film (Z36,965-9) and pyridoxal 5-phosphate (P-9255) were purchased from Sigma (St. Louis, MO). Costar 12 well Cell Culture plates were used as molds for the gels. Degassed 20% (w/w) whey protein solutions were prepared in water, pH was adjusted with NaOH or HCl (0.1 or 1 M) and NaN3 added (0.02%) to prevent bacterial
Enzyme selection and analysis
Enzyme selection is a key factor in designing a process that can produce a protein gel which has industrial relevance. For an enzyme to be successful in the manufacturing of whey protein gels, it must be produced from a microbial source. The enzyme must also either not require a cofactor or the cofactor must be benign to the gelation process. Thus, a microbial ‘broad-range’ transaminase was selected that could meet these two conditions and affect the rheology of whey protein gels at pH 4. A
Conclusions
Enzyme modification of whey proteins is an effective tool to tailor the final rheological properties of a whey protein gel at low pH. A methodology was developed to combine enzyme and heat treatment to produce whey protein gels at a low pH. Several process parameters including enzyme concentration, enzyme treatment time and waiting time used in this methodology were modulated to determine their effect on the final gel properties. After whey protein dissolution at pH 4, the elastic modulus
Acknowledgements
The authors gratefully acknowledge the Southeast Dairy Food Research Center for partial support of this work. We also thank Professor E.A. Foegeding for discussions and comments during the preparation of this manuscript.
References (37)
Trends Food Sci. Technol.
(1997)J. Biol. Chem.
(1975)- et al.
Int. J. Biol. Macromol.
(1992) - et al.
Food Chem.
(1991) - et al.
Carbohydr. Polym.
(2002) - et al.
Colloids Surfaces
(1990) - et al.
Adv. Colloid Interf. Sci.
(1993) - et al.
Macromolecules
(2000) Food Biotech.
(1995)- Gezimati J, Singh H, Creamer LK. In Parris N, Kato A, Creamer LK, editors. Macromolecular interactions food technol,...
Nahrung
J. Agric. Food Chem.
J. Food Sci.
J. Agric. Food Chem.
Food science
Appl. Environ. Microbiol.
Protein Eng.
Eur. J. Biochem.
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