The growth and gas production test for Escherichia coli in the microbiological examination of food additives is stipulated in the ninth edition of Japan's Specifications and Standards for Food Additives (JSFA) and described as a part of the "Confirmation Test for Escherichia coli" in "Microbial Limit Tests" in the same manuscript. The growth and gas production test for E. coli indicated that the positive or negative of "gas production and/or turbidity" in EC broth should be confirmed after incubating at 45.5±0.2℃ for 24±2 h. If both gas production and turbidity are negative, the culture is additionally incubated up to 48±2 h to determine E. coli contamination. The internationally referenced Bacteriological Analytical Manual of the U.S. FDA had revised the incubation temperature in tests for coliforms and E. coli from 45.5±0.2℃ to 44.5±0.2℃ in 2017. Therefore, we conducted research in anticipation of this temperature change being reflected in the microbiological examination of the JSFA. We used seven EC broth products and six food additives across eight products that are available in Japan in order to compare the growth and gas production at temperatures of 45.5±0.2℃ and 44.5±0.2℃ of E. coli NBRC 3972, which is designated as the test strain in JSFA. Both with/without food additives, the number of EC broth products in which medium turbidity and gas production by the strain were positive in three out of three tubes at all test times was greater at 44.5±0.2℃ than at 45.5±0.2℃. These results suggest that the growth and gas production test for E. coli could be more appropriately conducted by incubation at 44.5±0.2℃ in the "Confirmation Test for Escherichia coli" for E. coli in the JSFA in comparison to 45.5±0.2℃. Furthermore, there were differences in the growth and gas production of E. coli NBRC 3972 depending on the EC broth product used. Therefore, the importance of "Media growth promotion test" and "Method suitability test" in the ninth edition of the JSFA should be emphasized.
Keywords: EC broth; Escherichia coli; culture condition; food additive; gas production; growth.