This study aims to detect Clostridium botulinum and its control using natural leaf extracts of Citrus limon, Citrus sinensis, and Citrus unshiu in Egyptian fish products, e.g., canned tuna, canned sardine, canned mackerel, fesikh, moloha, and renga, as well the application of C. limon in tuna. Moreover, the antibacterial activity of the C. limon leaf extract was also estimated. In the water extract, ascorbic acid, total flavonoid content (TFC), and total phenolic content (TPC) were determined by volumetric, aluminum chloride, and Folin-Ciocalteu approaches, respectively. The antioxidant ability of the extract was analyzed in vitro via free radical scavenging (DPPH) and Ferric reducing assays. The results showed variability in the distribution of the total number of positive C. botulinum in fish samples from three different governorates under study, which were (24) Alexandria, (16) Beheira, and (17) Gharbia, out of the 120 tested samples in each governorate. Additionally, the findings revealed that all three Citrus extracts contain an appropriate number of secondary metabolites, with a sustainable presence of saponin and tannins in the C. limon extract. Furthermore, all Citrus extracts inhibited bacterial growth by increasing the inhibition zone, with C. limon being the best extract (25 mm) compared to C. sinensis and C. unshiu. The overall results showed the high antioxidant and anti-Clostridium powers (p < 0.05) of C. limon leaf extract, indicating its preservative activity in fishery products during storage. Finally, C. limon leaf extract can fight off C. botulinum and is considered a promising natural preservation candidate in ensuring safe and fresh fishery products.
Keywords: Citrus; Clostridium botulinum; botulism sickness; fishery products; multiplex PCR; natural antimicrobials; natural antioxidants; natural extracts; shelf-life.