Vibrio parahaemolyticus is the leading cause of bacterial diarrhea in humans from shellfish consumption. In Thailand, blood clam is a popular shellfish, but homemade cooking often results in insufficient heating. Therefore, consumers may suffer from food poisoning due to Vibrio infection. This study aimed to determine the effect of chitooligosaccharide conjugated with epigallocatechin gallate (COS-EGCG) at different concentrations (200 and 400 ppm) combined with high-voltage atmospheric cold plasma (HVACP) on inhibiting V. parahaemolyticus in vitro and in challenged blood clam meat. Firstly, HVACP conditions were optimized for gas composition and treatment time (20 and 60 s); a 70% Ar and 30% O₂ gas mixture resulted in the highest ozone formation and a treatment time of 60 s was used for further study. COS-EGCG conjugate at 400 ppm with HVACP (ACP-CE400) completely killed V. parahaemolyticus after incubation at 37 °C for 6 h. Furthermore, an antibacterial ability of ACP-CE400 treatment against bacterial cells was advocated due to the increased cell membrane damage, permeability, and leakage of proteins and nucleic acids. Scanning electron microscopy (SEM) showed cell elongation and pore formation, while confocal microscopy revealed disrupted biofilm formation. Additionally, the shelf life of challenged blood clam meat treated with ACP-CE400 was extended to nine days. SEM analysis revealed damaged bacterial cells on the meat surface after ACP-CE400 treatment, indicating the antibacterial activity of the combined treatment. Thus, HVACP combined with COS-EGCG conjugate, especially at a highest concentration (400 ppm), effectively inhibited microbial growth and extended the shelf life of contaminated blood clam meat.

Natural compounds from microorganisms are gaining importance as food preservation agents to maintain seafood quality and ensure safety. Endophytic fungi from medicinal plants are a crucial microbial resource for producing bioactive compounds. This study is aimed at isolating and identifying the endophytic Fusarium spp. from Piper spp. plants and determining the antimicrobial activity of their crude extract against spoilage and pathogenic bacteria. The ethyl acetate extract of the endophytic fungus strain P2NS8 (EAE‐P2NS8) exhibited antibacterial activity against Shewanella sp. TBRC 5775, Listeria monocytogenes ATCC 15313, and Staphylococcus aureus ATCC 25923, with MIC and MBC values in the range of 0.128–0.512 mg/mL, 0.256–0.512 mg/mL, and 0.064–0.256 mg/mL, respectively. Scanning electron microscopic (SEM) study revealed significantly damaged cells with wrinkled surfaces and breakage in all three bacterial strains treated with EAE‐P2NS8 (4 × MIC). Additionally, the antioxidant activity of the EAE‐P2NS8 was determined using DPPH‐RSA, ABTS‐RSA, and FRAP assays. The EAE‐P2NS8 at 0.039 mg/mL showed no significant cytotoxicity on Caco‐2 cells. LC‐QTOF‐MS analysis identified kaempferol 3‐(3 ^″ ‐acetyl‐6 ^″ ‐p‐coumaroylglucoside) and catechin‐4beta‐ol as the most abundant flavonoids in positive and negative modes, respectively. The alkaloid (2E)‐piperamide‐C5:1 was also detected in EAE‐P2NS8 in negative mode. The endophytic fungus strain P2NS8 was identified as Fusarium concentricum through morphological and molecular analyses. (2E)‐Piperamide‐C5:1 was first reported to be produced by the endophytic fungus F. concentricum isolated from P. betel . These findings highlight the potential of secondary metabolites from F. concentricum as alternative antimicrobial and antioxidant agents.