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

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.

Keywords: COS-EGCG conjugate; HVACP; V. parahaemolyticus; cell membrane damage; non-thermal technology.

Figure 1

Experimental outline for preparation of challenged blood clam meat.

Figure 2

Antibacterial effect of the COS-EGCG conjugate combined with and without HVACP treatment against V. parahaemolyticus after every 3 h for 48 h. CON: control without any treatment; CE200: COS-EGCG conjugate at 200 ppm; CE400: COS-EGCG conjugate at 400 ppm; ACP: high-voltage atmospheric cold plasma (HVACP); ACP-CE200: HVACP combined with COS-EGCG conjugate at 200 ppm and ACP-CE400: HVACP combined with COS-EGCG conjugate at 400 ppm. HVACP treatment was performed using a gas mixture ratio of 70% Ar: 30% O₂ for 60 s.

Figure 3

Cell membrane permeability of V. parahaemolyticus cells treated with COS-EGCG conjugate in combination with and without HVACP. (A) CON, (B) ACP, (C) CE200, (D) ACP-CE200, (E) CE400, and (F) ACP-CE400. For abbreviations see Figure 2 caption.

Figure 4

FTIR spectra of freeze-dried V. parahaemolyticus cells after treatment with COS-EGCG conjugate in combination with and without HVACP. For abbreviations see Figure 2 caption.

Figure 5

CLSM analysis of biofilm formation by V. parahaemolyticus cells treated with COS-EGCG conjugate in combination with and without HVACP. For abbreviations see Figure 2 caption.

Figure 6

Scanning electron microscopy (SEM) (A,C,E,G) and transmission electron microscopy (TEM) (B,D,F,H) analyses of V. parahaemolyticus cells treated with COS-EGCG conjugate in combination with and without HVACP. (A,B) CON, (C,D) CE400, (E,F) ACP, and (G,H) ACP-CE400. For abbreviations see Figure 2 caption.

Figure 7

Changes in V. parahaemolyticus count in inoculated blood clam meat treated with COS-EGCG conjugate in combination with and without HVACP during storage of 9 days at 4 °C. The bars represent the standard deviation (n = 3). Different lowercase letters on the bars within the same storage time indicate significant differences (p < 0.05). For abbreviations see Figure 2 caption.

Figure 8

Morphological changes of V. parahaemolyticus cells adhered to the surface of blood clam meat in control group (A,B) and treated with COS-EGCG conjugate 400 ppm in combination with HVACP (C,D) on day nine of the shelf-life study. The yellow arrows indicated the pores on V. parahaemolyticus cells.