# Impact of Chitooligosaccharide Conjugated Epigallocatechin Gallate and Non-Thermal High-Voltage Atmospheric Cold Plasma on Vibrio parahaemolyticus: An In Vitro Study and the Use in Blood Clam Meat

## TL;DR

This study shows that combining chitooligosaccharide conjugated with epigallocatechin gallate and high-voltage atmospheric cold plasma can effectively kill Vibrio parahaemolyticus in blood clams and extend their shelf life.

## Contribution

The novel contribution is the combined use of COS-EGCG and HVACP to inhibit Vibrio parahaemolyticus in shellfish.

## Key findings

- ACP-CE400 treatment completely killed Vibrio parahaemolyticus after 6 hours of incubation.
- ACP-CE400 extended the shelf life of contaminated blood clam meat to nine days.
- SEM and confocal microscopy showed cell damage and disrupted biofilm formation after treatment.

## Abstract

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.