|Budget Amount *help
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2003: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2002: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥7,500,000 (Direct Cost: ¥7,500,000)
(1)Chemical composition of fish sauce produced in Tobishima Island are NaCl 20.7%, VBN 181mg/100g, lactic acid 1,620mg/100g, glutamic acid 1,322mg/100g, histamine 163mg/100mg and halophilic histamine-producing bacterium of Tetragenococus muriaticus appeared in the early stage of fermentation, while T.halophilus appeared throughout the period.
(2)Histamine formation in cultures of Tetragenococcus muriaticus was observed in low acidity (pH 5.8), O_2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively. Histamine formation could not be prevented even at 20% (w/v) NaCl.
(3)A histidine decarboxylase from Tetragenococcus muriaticus was purified to homogeneity, for the first time. The pure enzyme consisted of two polypeptide chains with molecular mass of 28.8 and 13.4kDa. The N-terminal amino acid sequences of these polypeptides highly correlated with those of the alpha-and beta-chains of other Gram-positive bacterial histidine decarboxylases
. The optimum and stable pH for the enzyme was 4.5-7.0 and 4.0-7.0, respectively. The enzyme activity decreased with the addition of NaCl.
(4)The gene co d ing histidine decarboxylase (HDC) has been cloned by EcoRI fragment of chromosomal DNA from Tetragenococcus muriaticus. Sequence analysis of this fragment identified two open reading frames (ORFs). The first ORF (hdcA) encoded 316 amino acid residue s and it was identical to N-terminal amino acid sequence of HDC from T.muriaticus. The deduced amino acid sequence was 98% identity with HDC of Oenococcus oeni. Enzyme activity was reconstituted by treatment with 8M urea solution. The specific activity of renatured HdcA was 3.6×10^<-4> unit/mg and pH optimum at 4.8. The transcript analysis revealed that the hdcA was expressed in stationary-phase cell under O_2-limiting and low pH condition.
(5)DGGE analysis of 16S rDNA fragments amplified directly from kusaya gravy revealed the existence of Bacteroides, Fusobacterium, Eggerthela lenta, and Clostridium, suggesting that unculturable organisms prevail in the gravy.
(6)Dominant microflora of funazushi crucian carp fermented with rice, were Lactobacillus kefir in the late stage of fermentation, and L.alimentarius in the early stage. Less