Impact of Various Slaughter Techniques on Microbial Succession and Spoilage Dynamics in Grouper ( Epinephelus fuscoguttatus ) During Cold Storage at 4°C

Introduction Slaughtering methods can influence microbial growth and spoilage in fish, thereby affecting meat quality. This study investigated the effects of different slaughtering techniques on the microbial community and spoilage potential of grouper (Epinephelus fuscoguttatus) during cold storage at 4°C.Methods The grouper was subjected to five slaughtering methods: head striking (M1), carbon dioxide/nitrogen/oxygen asphyxiation (M2), clove oil anesthesia followed by ice slurry treatment (M3), ice water immersion (M4), and gradient cooling (M5). Microbial analysis included total viable cell count (TVC) and 16S rRNA sequencing, conducted at 0, 4, and 8 days. Additionally, inoculation experiments with Pseudomonas fluorescens and Pseudomonas poae were performed to assess spoilage potential.Results Gradient cooling (M5) provided the best inhibitory effect on microbial growth, with the lowest TVC. 16S rRNA analysis revealed that Pseudomonas spp. was the dominant microbial community, with P. fluorescens and P. poae being the predominant species. Inoculation experiments indicated that P. fluorescens was responsible for higher spoilage potential, evidenced by elevated levels of TVC, TVB-N, TMA, lipid oxidation, and protein hydrolysis.Discussion This study identified Pseudomonas spp. as key spoilage organisms in refrigerated grouper and showed that gradient cooling most effectively delays spoilage.