Generating Animal-Free Protein Scaffolds using Antisolvent Precipitation under Couette Flow

A common feature of animal-based proteins is their ability to self-assemble into continuous protein fibers, as seen in muscle and connective tissues. Their fibrous nature has inspired research and development in the tissue engineering, textile, and synthetic meat industries, to find methods of replicating this aligned fiber structure in nonanimal-based proteins. Zein is a nitrogen storage protein that is primarily found in corn, that has become popular due to its status of Generally Regarded As Safe (GRAS), its biocompatibility, its fabrication flexibility and its unique solubility, solvating in 60-90% mixtures of ethanol and water. This makes it a prime candidate for antisolvent precipitation and extrusion methods to produce zein fibers for food industry applications. However, extrusions methods produce a rapid but random generation of fibers in terms of diameter and alignment when utilizing this antisolvent precipitation, and the conclusion has been drawn that further antisolvent zein fiber formation should focus on fiber orientation as a next step in development. In this work we aimed to produce aligned fibrous microstructures on zein scaffolds like those found in animal-based proteins. We investigate how salt concentration and identity influence the aggregation patterns of zein as it precipitates into nanoparticles through its addition to an aqueous antisolvent. We then use the conclusions drawn from these findings in combination with a custom-built device to induce anti-solvent precipitation under Couette-flow to produce continuous fibrous scaffolds, demonstrating their use as scaffolds for future tissue engineering applications.