Aggregation Mechanisms of a Whey Protein Model System at Low PH

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Title: Aggregation Mechanisms of a Whey Protein Model System at Low PH
Author: Mudgal, Prashant
Advisors: Dr. Orlin D. Velev, Committee Member
Dr. Brian E. Farkas, Committee Member
Dr. Allen E. Foegeding, Committee Co-Chair
Dr. Christopher R. Daubert, Committee Chair
Abstract: MUDGAL, PRASHANT. Aggregation Mechanisms of a Whey Protein Model System at Low pH. (Under the direction of Dr. Christopher R. Daubert and Dr. E. Allen Foegeding) Recently, there has developed increasing interest in cold-thickening whey protein ingredients for food applications where heat may not be desirable and because of their nutritional benefits over conventionally-used starches and hydrocolloids. In previous studies, a procedure allowing for the production of a cold-thickening whey protein ingredient, without any addition of salt or heat, was developed. This procedure involved pH adjustment to 3.35, thermal gelation, followed by drying and milling to a powder. Originally, this procedure was applied to whey protein isolates, but also worked with concentrates. Although, these modified powders provide certain benefits, such as instant thickening without addition of heat or salts, these ingredients were prepared at low pH and yield astringent flavors that limit use in practical applications. To effectively tailor the original modification process to expand the functionality and utility of cold-thickening modified whey protein ingredients, the basic mechanism behind the cold-thickening must be explained. β-Lactoglobulin (β-lg) is the major component of whey protein products and was selected as a model system to investigate the cold-thickening mechanisms. Concentration effects on β-lg modification were studied at low pH using capillary and rotational viscometry, transmission electron microscopy (TEM), and high performance liquid chromatography coupled with multi-angle laser light scattering (HPLC-MALS). From the results of the capillary viscometry, a critical concentration (C<sub>c</sub> ~ 6.4 % w/w protein) was identified below which no significant thickening functionality could be achieved. Microscopy revealed formation of flexible fibrillar network at pH 3.35 during heating at all concentrations. These flexible fibrils had a diameter of approximately 5 nm and persistence length of about 35 nm as compared to more linear and stiff fibrils formed at pH 2 and low ionic strength (<i>I</i>) conditions. Under similar heating conditions at concentrations above C<sub>c</sub>, larger aggregates similar to microgels were observed. However, at concentrations below C<sub>c</sub>, isolated fibrils were observed with an average contour length of about 130 nm. In further investigations, concentration effects were studied together with ionic strength (CaCl<sub>2</sub>) effects on the β-lg cold-thickening mechanism using light scattering, microscopy, and rotational viscometry. A slight increase in <i>I</i> (up to 20 mM) with β-lg concentrations above the critical concentration resulted in an increased thickening function from modified powders. The network characteristics remained fine stranded with small addition of CaCl<sub>2</sub>, and increased aggregation was observed at all concentrations. The aggregates formed during heating persisted in dried powders; however, freeze concentration effects during freezing further enhanced thickening function as obtained from the reconstituted modified powders. The size of the aggregates formed during the modification process was found to be dependent on the initial protein concentration from kinetic studies using light scattering and rheology. A nucleation and growth mechanism best fit the aggregation at pH 3.35, and nucleation was found to be a rate limiting step below the critical concentration. Above the critical concentration, nucleation occurred rapidly leading to a higher degree of aggregation and formation of large aggregates. By manipulating concentration and heating times, it seemed feasible to control the degree of aggregation and the size of aggregates between 10<sup>6</sup> to 10<sup>8</sup> Da. According to electrophoretic studies, disulfide linked aggregates were formed upon heating during the modification procedure. However, disulfide interactions alone could not explain the observed concentration dependent thickening differences. Some acid hydrolysis occurred at pH 3.35 and with additional hydrolysis of β-lg with the enzyme pepsin, solution viscosities increased by about two logs. Likely, formation of pre-aggregates during hydrolysis may explain increased aggregation and higher viscosities.
Date: 2009-12-14
Degree: PhD
Discipline: Food Science
URI: http://www.lib.ncsu.edu/resolver/1840.16/3871


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