The effect of adding different proportions of transglutaminase (TG) on the structure and digestive characteristics of high-moisture pea protein extrudates prepared with a twin-screw extruder was investigated. The macrostructure and microstructure of the extrudates were characterized by measuring texture and texturization degree as well as using a scanning electron microscope (SEM). The structural properties and nutritional value were evaluated by measuring rheological properties, protein solubility and digestive properties. These results showed that addition of appropriate amounts of TG (0.25%–1.00%) promoted the cross-linking between pea protein molecules, forming a dense network structure, increasing the digestibility of extrudates and promoting the release of free amino acids. However, addition of excess TG (2%) inhibited the fiber formation and digestive properties of extrudates. The extrudate with 1.00% of TG had higher texturization degree and nutritional value. This study demonstrated that high-moisture extrusion combined with addition of TG can improve the structure and digestive characteristics of plant protein-based meat analogs.

Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (P_aprE, P₄₃, and P_glv) were compared and applied. The results indicated that the strain RIK1285-pBE-P_glv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.