INTEGRATION OF NUTRITIONAL, GENETIC AND PHYSIOLOGICAL APPROACHES TO IMPROVE PRODUCTION EFFICIENCY OF RAINBOW TROUT
Location: Small Grains and Potato Germplasm Research
Title: Protein-Protein Interactions during High-Moisture Extrusion for Fibrous Meat Analogues and Comparison of Protein Solubility Methods Using Different Solvent Systems
Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 7, 2008
Publication Date: March 1, 2008
Citation: Liu, K.S. and F.-H. Hsieh. 2008. Protein-Protein Interactions during High-Moisture Extrusion for Fibrous Meat Analogues and Comparison of Protein Solubility Methods Using Different Solvent Systems J. Agric. Food Chem. 56: 2681-2687.
Interpretive Summary: This report related to a non-ARS funded project. The work dealt with extrusion of plant proteins, mainly soy proteins and gluten, into fibrous meat analogs under a high moisture and high temperature condition, using a twin screw extruder. High moisture extrusion is a promising and emerging technology in producing meat alternatives having structure similar to muscle meat. Yet, the mechanism of protein-protein interactions during extrusion has not been well understood, and controversy exists in the literature. On the practical aspect, by knowing the mechanism, we can improve production efficiency and end product quality. Therefore, the objectives of the study were: 1) to determine the relative importance between non-covalent bonding in stabilizing extruded products and/or creating fiber structure, and 2) to determine when these bonds form or change during extrusion. To achieve the objectives, we used 6 types of selective reagents to design 11 extraction solutions through addition, subtraction or multiple combinations of the reagents. Instead of just comparing between raw mix and extrudates, we compared protein solubility among samples collected at different zones of the extruder barrel after a dead-stop run and among extrudates made at different moisture contents. We also used two-dimensional gel electrophoresis to map out protein changes before and after high moisture extrusion.
The significant findings of this study reflect in the following three aspects. First, a key reason for poor understanding of protein-protein interactions during extrusion is inherent difficulty for studying the interactions. The most common approach is based on protein resolubilization by selective reagents with known mechanisms of protein solubilization. Yet, with regard to how to conduct such protein solubility study, all the previous investigators used extracting solutions containing single or dual reagents and compared solubility values with that of a basic buffer solution for basically two types of samples: raw material and final extruded product. This study was the first one to point out the pitfalls of this common method, and suggested a correct procedure in designing a protein solubility study, which was based on an isoelectric focus buffer with subtraction of one or more reagents from it. Second, using the new correct procedure, this study was the first one to conclude that although non-covalent bonding has important roles in forming secondary and tertiary structures of proteins, it is disulfide bonding that plays a primary role in not only holding the rigid structure but also forming the fibrous texture of extrudates. Third, because this study used a set of samples collected from different zones of the extruder after a dead-stop run, it was the first one to show that the sharpest changes in protein solubility occurred when the mix passed through the intermediate zones of the extruder barrel and indicated formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die the chemical nature of the product did not change significantly.
Soy protein, mixed with gluten and starch, was extruded into fibrous meat analogs under high moisture and high temperature conditions. The protein solubility of samples collected at different extruder zones and extrudates made with different moistures was determined by 11 extraction solutions consisting of 6 selective reagents and their combinations: phosphate salts, urea, DTT, thiourea, Triton X-100, and CHAPS. Protein solubility by most extractants showed decreasing patterns as the material passed through the extruder, but the solution containing all the 6 reagents, known as isoelectric focus (IEF) buffer, solubilized the highest levels and equal amounts of proteins in all samples, indicating that there are no other covalent bonds involved beside disulfide bonds. This was confirmed by comparing 2D gel electrophoresis of the raw and extrudate samples. Yet, regarding relative importance between disulfide bonds and non-covalent interactions, different conclusions could be made from protein solubility patterns, depending on the type of extracting systems and baseline for comparison used. Our observations addressed common pitfalls of protein solubility studies and explained why controversy exists in the literature. We believe that the IEF buffer system with omission of one or more selective reagents was the right one to conduct protein solubility study. Results obtained with this system indicates that disulfide bonding plays a more important role in not only holding the rigid structure of extrudates but also forming fibrous texture than non-covalent bonds. The sharpest changes in protein solubility occurred when the mix passed through the intermediate section of the extruder barrel, indicating formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die the chemical nature of the product did not change significantly.