Granulomorphometry: A suitable tool for identifying hydrophobic and disulfide bonds in beta-lactoglobulin aggregates. Application to the study of beta-lactoglobulin aggregation mechanism between 70 and 95 degrees C

• Published in: Journal of dairy science Vol. 95; no. 8; pp. 4188 - 4202
• Main Authors: Petit, Jérémy, Herbig, Anna-Lena, Moreau, Anne, Le Page, Jean-Francois, Six, Thierry, Delaplace, Guillaume
• Format: Journal Article
• Language: English
• Published: American Dairy Science Association 01.08.2012
• Subjects: Chemical and Process Engineering; Engineering Sciences; Food engineering; Life Sciences; beta-lactoglobulin; granulomorphometry; disulfide bond; hydrophobic interaction
• ISSN: 0022-0302
• DOI: 10.3168/jds.2011-4146

This work deals with the investigation of beta-lactoglobulin (beta-LG) aggregation by granulomorphometry. In the first part of this study, we showed that the binding interactions involved in aggregate structure could be identified by their appearance in granulomorphometric pictures. The reliability of this analytical approach was demonstrated by comparing the appearance of beta-LG aggregates in the presence and absence of a thiol-blocking agent (N-ethylmaleimide). The translucency of the aggregates was associated with hydrophobic interactions and their opacity was associated with disulfide bonds. We state, based on the morphology of the aggregates, along with the color of protein aggregates and insoluble materials, that hydrophobic interactions had a better water-holding capacity than disulfide bonds. Additionally, our results suggest that disulfide and hydrophobic bonds compete for beta-LG aggregate shaping. In the second part of this work, interesting features of granulomorphometry useful for identifying aggregate binding interactions were highlighted to clarify the effect of temperature on the aggregation mechanisms occurring in a beta-LG concentrate with a moderate calcium content (6.6 mmol.L-1). Heat treatment experiments were performed between 70 and 95 degrees C, and granulomorphometric measurements (aggregate size, aggregate number, and gray level of the picture) were conducted at different sampling times up to 4 h. Results, which were interpreted in light of calculated beta-LC denaturation levels, revealed that the aggregation mechanism could be split into 2 steps. Initially, beta-LG denatured quickly, leading to fast beta-LG aggregation by disulfide bonds. The denaturation rate then declined, which drastically slowed the disulfide aggregation mechanism. From that point on, a second aggregation path became preponderant. It consisted of the agglomeration of small aggregates by hydrophobic interactions and resulted in the formation of large aggregates containing both interaction types. This second aggregation mechanism was clearly favored at high temperatures because it was not detected in our experiments at temperatures below 85 degrees C.