[Effect of Incubation Temperature for Tetrathionate and Rappaport-Vassiliadis Broths on Salmonella Recovery from Food...

The microbial safety of food additives is regulated under Japan's Specifications and Standards for Food Additives (JSFA), which includes detection tests for Salmonella. The current standard procedure in JSFA requires selective enrichment of Salmonella using tetrathionate (TT) and Rappaport-Vassiliadis (RV) broths with differing incubation temperatures: 43 ± 0.2℃ for TT broth under high microbial load conditions, 35 ± 2℃ for TT broth under low bacterial load conditions, and 42 ± 0.2℃ for RV broth. These distinct incubation requirements necessitate separate temperature control, thereby increasing the complexity of laboratory procedures. In this study, we evaluated the temperature-dependent enrichment behavior of Salmonella under a limited set of conditions relevant to the JSFA method. Salmonella enterica subsp. enterica serovars Abony and Typhimurium were inoculated into food additive products in lactose broth with or without Escherichia coli at 10², 1.3×10⁵, and 1.3×10⁶ CFU/sample as a competing bacterium. The inoculated mixtures were incubated at 35 ± 1℃ for 22 hr, after which aliquots (TT: 1 mL/RV: 0.1 mL) were inoculated into TT and RV broths and incubated at 42 ± 0.2℃ and 43 ± 0.2℃ for RV broth under none/low-level E. coli co-inoculation, whereas RV broth was incubated at 42 ± 0.2℃ under high-level E. coli co-inoculation, and at 35 ± 2℃, 42 ± 0.2℃, and 43 ± 0.2℃ for TT broth. The cultures were plated on selective agar to evaluate Salmonella recovery. For TT broth, no clear differences in recovery scores were observed among 35 ± 2℃, 42 ± 0.2℃, and 43 ± 0.2℃, regardless of E. coli co-inoculation. Under none/low-level E. coli co-inoculation, recovery after RV enrichment at 42 ± 0.2℃ and 43 ± 0.2℃ was comparable. In some food additive products, colonies were not recovered on one or more selective media in some replicate experiments; however, when three selective agars were used in parallel, the incubation temperature for TT broth did not markedly affect the overall test interpretation. The recovery of S. Typhimurium on some selective agars was reduced following incubation in RV broth containing food additives, protease and isoamylase co-inoculated with E. coli at high microbial loads. This suggests that high levels of bacterial contamination in certain food additive enzyme products, including protease and isoamylase, may suppress Salmonella recovery in RV broth. Further investigation using a broader range of contaminating microorganisms and food additive types is required to validate these findings.