Aggregation of alpha-Lactalbumin at PH 3.5-6.0

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Title: Aggregation of alpha-Lactalbumin at PH 3.5-6.0
Author: Schneider, Paula Anne
Advisors: Dr. John Cavanagh, Committee Member
Dr. Brian Farkas, Committee Member
Dr. E. Allen Foegeding, Committee Chair
Abstract: Heat-induced protein aggregation is an important reaction in food processing because it causes undesirable appearance changes, which may lead the beverage to phase separate. Recently, there has been an increased demand for and growth in the area of sports beverages. Sports beverages can be categorized several different ways; although the category of current interest is that of beverages having a low pH (from 2.8 to 3.5) and representing a good source of protein. Generally these beverages derive a high proportion of their protein from bovine whey, since it is a source for branched chain amino acids which can benefit muscle building and maintenance. These low pH beverages can be astringent. Astringency is positively related to low pH where the degree⁄intensity of perceived astringency increases as the pH of the beverage decreases. One way to minimize astringency is to increase the pH; although aggregation readily increases the nearer the pH is to the protein's isoelectric point. The selection of a less heat sensitive protein source (ex. α-lactalbumin) and/or the use of molecular aggregation blockers can provide ways to control aggregation. The first objective of this research was to study the aggregation of α-lactalbumin over a pH range 3.5 to 6.0. Protein solubility and turbidity development were used to monitor aggregation. Turbidity was evaluated using a spectrophotometer set to monitor at 400 nanometers, and a turbiditimeter that measures scattering at a 90° angle from the incident light. Change in protein solubility in response to pH adjustment with 1M phosphoric acid and thermal (120 seconds in a 85°C water bath) treatment was determined by measuring dispersed protein after centrifugation at 11950 x g (18-24°C) for 60 to 90 minutes. As expected, the lowest protein solubility and highest degree of turbidity resulted near the isoelectric point of α-lactalbumin. The affect of calcium content was also evaluated. In the holo-form, α-lactalbumin was more stable to native aggregation; although no difference was seen in stability after heat treatment. Indirect determination of the mechanism for α-lactalbumin aggregation was facilitated through the use of various compounds that had previously demonstrated aggregation-blocking or reduction abilities in different systems (ex. protein, pH, heating conditions, etc.). Blocking agents included amyloid/β-sheet blockers (ex. thioflavin-T and quercetin), hydrophobic amino acid-interacting molecules (ex. Hydroxyl-propyl-β-cyclodextrin), and the use of proteins as blockers (ex. αs-casein) were evaluated. The amyloid/β-sheet blockers were not effective in suppressing turbidity development or maintaining/increasing protein solubility. Further investigations were made to determine if a specific type of aggregate was possible. Under the conditions tested, α-lactalbumin did not form the specific aggregates (spherulites), which helps explain why these blockers were ineffective in an α-lactalbumin protein system. The use of sodium dodecly sulfate (SDS) was used to control aggregation by binding to exposed hydrophobic patches on the protein's surface. SDS was not effective in controlling aggregation in β-lactalbumin systems, as monitored through turbidity development and protein solubility, but was effective in systems that contained β-lactoglobulin. The difference in response may be attributed to the structural differences between these proteins. Blocking of hydrophobic amino acid residues with hydroxyproply- β-cyclodextrin had the most potential for success in suppressing aggregation of α-lactalbumin. However, further work is needed to determine where they are binding, strength of binding, and the effect of polar components on the core cyclodextrin molecule.
Date: 2006-12-28
Degree: MS
Discipline: Food Science
URI: http://www.lib.ncsu.edu/resolver/1840.16/2129


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