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To characterize the histamine-producing bacteria isolated from a marinade broth accumulating approximately 25 mg/100 mL of histamine in shimesaba production, taxonomic and physiological features of the isolates were studied. Nineteen strains capable of producing histamine were isolated from a marinade broth by using AC plate agar with the anaerobic culture method and a member of lactobacilli was identiˆed based on the phenotypic characteristics. Moreover, the closest phylogenetic neighbor of the strains was Lactobacillus otakiensis sharing 100  similarity based on 16S rRNA gene sequence. The strains harbored a pyruvoyl-dependent histidine decarboxylase gene, which is widespread in histamine-producing gram positive bacteria. Growth of the strains was inhibited by low pH (below 3.6), low water activity (below 0.939), high salt concentration (above 10), and supplementation of 2 acetic acid (ˆnal concentration). However, all of the strains were able to grow in MRS broth with pH 5.7 at temperatures below 8°C and produce histamine within 4 weeks of cultivation. To prevent histamine accumulation in shimesaba production, it is necessary to use some bacteriostatic conditions, low pH (around 4.0), low temperature (below 8°C), and an appropriate concentration of vinegar, to preserve marinade broth based on the hurdle technology theory.

A total of 12 strains of histamine-producing bacteria harboring a pyruvoyl-dependent histidine decarboxylase gene, identiˆed as Tetragenococcus halophilus, were isolated from aˆsh sauce mash accumulating over 40 mg/100 g of histamine. To conˆrm that the strain can produce histamine inˆsh sauce mashes, the representative histamine-producing strain, FS-4, was inoculated into aˆsh sauce mash. The histamine content of theˆsh sauce mash inoculated with FS-4 accumulated over 100 mg/100 g of histamine after 7 days at 30°C. In contrast, histamine accumulation was not observed in sterileˆsh sauce mashes irradiated with over 10.0 kGy of gamma-rays, indicating that the causative agent of histamine accumulation inˆsh sauce mashes was microorganisms. The addition of starter culture consisting of T. halophilus NBRC12172 (isolated from soy sauce mash), which does not harbor the histidine decarboxylase gene, to theˆsh sauce mashes eŠectively suppressed histamine accumulation. According to the result of a competitive growth test between strain FS-4 and starter culture inˆsh sauce mashes, it is necessary to suppress histamine accumulation duringˆsh sauce fermentation by adding toˆsh sauce mashes a starter culture with a total biomass more than one thousand times that of the histamine-producing bacterial cells.