In recent years, individuals have increasingly pursued healthier lifestyles, making fish the preferred alternative to beef, pork, and poultry (Cortés-Sánchez et al., 2021). Tilapia, the second largest farmed species globally, is mainly distributed in Asia, Africa, and America. It offers various benefits such as rapid growth, and adaptability to different environments, and is a rich source of protein and nutrients (Chen et al., 2023). However, due to its high water content, tilapia is susceptible to spoilage during storage, which can lead to food safety risks and affect its nutritional value (Wong et al., 2020). Given these challenges, the food industry faces a crucial need to inhibit microbial growth effectively and prevent food spoilage.

Antimicrobial peptides (AMPs) are naturally occurring peptides with low molecular weights that exhibit antimicrobial activity. They can be synthesized by various organisms such as bacteria, fungi, higher plants, and higher animals (Hancock et al., 2016). Due to their positive charge, AMPs are attracted to the negatively charged lipid bilayer of bacteria, leading to an electrostatic interaction that enables them to adhere to the membrane, penetrate it, and disrupt bacterial growth (Wang et al., 2018). Meanwhile, certain AMPs can attach to ribosomes and hinder bacterial growth by interfering with protein synthesis (Graf et al., 2017). AMPs exhibit broad-spectrum antimicrobial properties against bacteria, fungi, and parasites, making them promising candidates for applications in the food industry (Zasloff et al., 2002).

The skin secretions of amphibians contain numerous bioactive compounds, including proteins, peptides, and steroids (Zhou et al., 2017). Frogs, being typical amphibians found worldwide, are a significant source of AMP production (Ladram et al., 2016). Odorranain-C1, isolated from the skin of Odorrana grahami (Li et al., 2007), demonstrates strong inhibitory effects against bacteria and fungi, along with potential anticarcinogenic properties (Zhao et al., 2021). Consequently, Odorranain-C1 holds promise as a novel antibacterial agent for food preservation. AMPs sourced directly from natural origins have a limited yield (Yazdi et al., 2019), while chemically synthesized AMPs are more complex and costly (Parachin et al., 2012). In contrast, recombinant DNA technology enables the cloning of codon-optimized foreign genes in specific vectors for expression in prokaryotic and/or eukaryotic host cells, which provides an opportunity for the mass production of AMPs (Kesidis et al., 2020). Compared to prokaryotic expression systems, Pichia pastoris (P. pastoris) (also known as Komagataella phaffii) is a commonly utilized host for expression (Karbalaei et al., 2020). It exhibits superior post-translational modification capabilities, which is crucial for maintaining the biological activity of recombinant proteins (Daly et al., 2005). By utilizing DNA recombination techniques, numerous AMPs, including Lunasin-4 (Zhu et al., 2018), Lactolisterin BU (Dong et al., 2021), and Hispidalin (Meng et al., 2019), have been successfully expressed in P. pastoris. These accomplishments have provided a theoretical foundation for the production and application of Odorranain-C1.

In this study, we utilized diverse bioinformatics tools to predict the structure of Odorranain-C1. Subsequently, we utilized DNA recombination technology and the P. pastoris expression system to generate recombinant Odorranain-C1 with enhanced antibacterial activity. To determine the quantity of Odorranain-C1 in the expression supernatant, we utilized an enzyme-linked immunosorbent assay (ELISA) assay. Following quantification, we comprehensively investigated Odorranain-C1's biological properties, including its antimicrobial spectrum, minimum inhibitory concentration (MIC), stability, and hemolytic activity. Additionally, we investigated the impact of Odorranain-C1 on bacterial membranes through membrane permeability alterations assay, total nucleotide leakage assay, and scanning electron microscopy to explore its antimicrobial mechanism. Finally, we investigated the effects of Odorranain-C1 on total bacterial counts, pH, total volatile basic nitrogen (TVB-N), and thiobarbituric acid (TBA) of tilapia fillets, and preliminarily evaluated the preservation effect of Odorranain-C1 on tilapia fillets.