Current Status, Challenges, and Future Trends in Mining and Functional Characterization of Novel Protease Genes in Traditional Fermented Foods

Traditional fermented foods are rich in microorganisms and serve as important sources of proteases that possess different biochemical and technological characteristics. During fermentation, microbial proteases help degrade proteins for the development of flavors, modification in texture, release of nutrients, and formation of bioactive peptides. New technologies in the fields of genome sequencing, metagenomics, transcriptomics, proteomics, and bioinformatics have paved the way for a shift from traditional culture-dependent screening to sequence- and function-guided protease discovery. Protease-producing Bacillus, Aspergillus, and Rhizopus are important in plant-based fermented foods, particularly fermented soybean foods, while animal-based fermented foods harbor diverse protease-producing microorganisms, including lactic acid bacteria, Staphylococcus, Tetragenococcus, Bacillus, yeasts, and molds. However, the identification of a putative protease gene does not imply its catalytic or technological role. This review summarizes the latest advances in protease gene mining, food applications, and functional characterization, identifies key methodological challenges, and outlines future prospects for next-generation food-grade proteases using integrated multi-omics, artificial intelligence, protein engineering, high-throughput screening, and synthetic biology. Special focus is placed on validation in real food matrices, salt tolerance, and substrate specificity.