Histological and microbiological alterations in sardines subjected to repeated freezethaw cycles: Implications for muscle integrity and food safety

Abstract

A total of thirty freshly caught sardines were subjected to one to five freeze–thaw cycles, while unfrozen samples served as controls. Muscle structure was assessed by histological and histomorphometric analyses, including measurements of vacuole area, vacuolated fiber area, vacuolated-to-fiber area ratio, and ice crystal size. Microbiological analyses included aerobic mesophilic bacteria, total coliforms, coagulase-positive Staphylococcus aureus, and Salmonella spp. Histological observations revealed a progressive deterioration of muscle architecture with increasing numbers of freeze–thaw cycles, characterized by fiber disorganization, extensive vacuolation, and widening of inter-fiber spaces. Histomorphometric analysis showed significant increases (P < 0.05) in ice crystal area, from 1050 ± 300 µm² at one freeze–thaw cycles to 2300 ± 150 µm² at five freeze–thaw cycles, as well as in the vacuolated area ratio, which increased from 25 ± 7 % to 65 ± 6 %. Microbial counts showed a slight but consistent decrease during the first three freeze–thaw cycles and remained well below the spoilage threshold of 7 log CFU/g. A significant negative correlation (r = −0.78; P < 0.05) was observed between vacuole area and total aerobic mesophilic counts, indicating that structural muscle damage did not promote microbial proliferation under controlled cold storage conditions. In conclusion, repeated freeze–thaw cycles notably impair sardine muscle microstructure and may slightly reduce microbial counts, without favoring bacterial growth when the cold chain is properly maintained. These findings highlight the importance of strict temperature control to preserve both the quality and safety of sardine products.