Pull apart a piece of cooked chicken, and the meat separates into long, fibrous strands. Replicating this texture using plant proteins remains difficult in developing convincing meat alternatives. During high-moisture extrusion, proteins are exposed to heat, moisture, shear and pressure, causing them to unfold, align and form structured networks. Protease enzymes can further modify this process by partially cutting protein chains. If the proteins are broken down too little, the product can remain rubbery and difficult to chew. Too much hydrolysis, however, can weaken the protein network and cause it to collapse into a soft paste.
“Different proteases cut within the protein chain, while others trim from the ends,” said Jie Hong Chiang, a Senior Scientist at the A*STAR Singapore Institute of Food and Biotechnology Innovation (A*STAR SIFBI). “That changes whether the protein network becomes more fibrous and gel-like or too broken and weak.” Their enzymatic activity also affects in-vitro digestibility, as breaking down proteins into smaller peptides, amino acids and soluble fragments may improve their accessibility during digestion.
To better understand these variations, the A*STAR SIFBI team, in collaboration with A*STAR Singapore Institute of Manufacturing Technology (A*STAR SIMTech), compared three commercial proteases—Alcalase® (AL), Flavourzyme® (FL) and Protana Prime (PP). AL cuts anywhere along the protein chain; PP trims from the ends; and FL does both.
The researchers used high-moisture extrusion to develop soy-pea meat analogues, testing the three enzymes at various concentrations up to five percent by weight relative to the protein blend. They found that both enzyme type and concentration determined the extent of protein breakdown and texturisation.
At low-to-moderate concentrations, FL and PP improved fibrous structure formation and slightly reduced hardness and chewiness. Meanwhile, AL induced extensive breakdown at higher concentrations. As the protein chains could no longer form a continuous network, this reduced chewiness by nearly 75 percent and led the structure to collapse into fragmented pieces, confirmed through X-ray tomography imaging.
“The key factor is not only the type of enzyme used, but also whether the remaining protein fragments are still long and reactive enough to form a stable fibrous matrix,” said Caleb Ong, a Research Officer at A*STAR SIFBI.
When the team simulated the stomach’s digestive processes, all enzyme-treated samples released more soluble protein and free amino acids than untreated counterparts. However, these differences eventually normalised during intestinal digestion, suggesting that protease selection is particularly important for product structure, early digestibility and stomach-specific protein release.
The researchers next aim to examine how extrusion features and enzyme concentration can be tuned together to create processing conditions that improve texture and digestibility without damaging the fibrous structure.
“By understanding proteolytic mechanisms, we can use enzymes more like precision tools rather than general additives,” said Grace Ng, former Scientist at A*STAR SIFBI. “We see this as part of a platform for designing plant-based meats with targeted texture and nutritional quality tailored for different consumers.”
The A*STAR-affiliated researchers contributing to this research are from the A*STAR Singapore Institute of Food and Biotechnology Innovation (A*STAR SIFBI) and A*STAR Singapore Institute of Manufacturing Technology (A*STAR SIMTech).
