Studies on lactic acid producing bacteria in fermented seafoods

发酵海产品中产乳酸菌的研究

• 批准号: 13460090
• 负责人: FUJII Tateo
• 金额: $ 8.51万
• 依托单位: 東京水産大学
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13460090/
• 关键词: lactic acid bacteria; fish sauce; kusaya; funazushi; Tetragenococcus; histamine; histidine decarboxylase; Lactobacillus; 塩辛; Staphylococcus; 好塩性乳酸菌

(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 … More . 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

（1）飞岛产酱料的化学成分为：NaCl 20.7%，VBN 181 mg/100 g，乳酸1, 620 mg/100 g，谷氨酸1, 322 mg/100 g，组胺163 mg/100 g，嗜盐组胺产生菌Tetragenococus muriaticus出现在发酵初期，而嗜盐嗜盐菌T.halophilus则出现在整个发酵期。（2）在低酸度（pH5.8）、限氧条件下，盐酸四联球菌（Tetragenococcus muriaticus）培养物中组胺的生成量最大，NaCl浓度为5-7%（w/v），葡萄糖浓度大于1%。即使在20%（w/v）NaCl下也不能阻止组胺形成。(3)A首次从Tetragenococcus muriaticus中纯化了组氨酸脱羧酶。纯化的酶由两条多肽链组成，分子量分别为28.8和13.4kDa。这些多肽的N-末端氨基酸序列与其它革兰氏阳性细菌组氨酸脱羧酶的α-和β-链高度相关 ...更多信息 .该酶的最适和稳定pH分别为4.5-7.0和4.0-7.0。随着氯化钠的加入，酶活性下降。（4）利用大肠杆菌染色体DNA的EcoR Ⅰ片段克隆了组氨酸脱羧酶（HDC）基因。对该片段的序列分析鉴定出两个开放阅读框（ORF）。第一个ORF（hdcA）编码316个氨基酸残基，与鼠毛癣菌HDC的N端氨基酸序列完全相同。推导的氨基酸序列与酒酒球菌HDC的同源性为98%。通过用8 M尿素溶液处理来重建酶活性。复性后的HdcA比活力为3.6×10^<-4>unit/mg，最适pH为4.8。转录本分析表明，hdcA基因在稳定期细胞中表达，且在O_2限制和低pH条件下表达。（5）DGGE分析从草屋肉汁中直接扩增出的16 SrDNA片段，结果表明，草屋肉汁中存在拟杆菌属、梭杆菌属、迟缓蛋杆菌属和梭菌属，表明草屋肉汁中存在不可培养的微生物。（6）发酵后期的优势菌群为开菲尔乳杆菌，发酵前期的优势菌群为食物乳杆菌。少

项目成果

Y.Konagaya: "Purification and properties of a histidine decarboxylase from Tetragenococcus muriaticus, a halophilicus, lactic acid bacterium"J.Appl.Microbiol.. 92・6. 1136-1142 (2002)

Y.Konagaya：“来自嗜盐乳酸菌四联球菌的组氨酸脱羧酶的纯化和特性”J.Appl.Microbiol.. 1136-1142 (2002)。

H.Takahashi: "Cloning and sequencing of the histidine decarboxylase gene of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish."Appl.Environ.Microbiol.. 69. 2568-2579 (2003)

H.Takahashi：“革兰氏阴性、产生组胺的细菌的组氨酸脱羧酶基因的克隆和测序及其在鱼类中这些生物体的检测和鉴定中的应用。”Appl.Environ.Microbiol.. 69. 2568-2579 (2003

H.Takahashi: "Cloning and sequencing of the histidine decarboxylase gene of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish"Appl.Environ.Microbiol.. 69・5. 2568-2579 (2003)

H.Takahashi：“革兰氏阴性、产生组胺的细菌的组氨酸脱羧酶基因的克隆和测序及其在鱼类中这些生物体的检测和鉴定中的应用”Appl.Environ.Microbiol.. 69・5。 2003）

藤井建夫: "増補 塩辛・くさや・かつお節-水産発酵食品の製法と旨味"恒星社厚生閣. 121 (2001)

藤井猛夫：“膨化咸鱼、草鱼和鲣鱼片——发酵海鲜食品的制造方法和风味”《Seiseisha Koukaku》121（2001）。

M.Satomi: "Marinospilillum insulare sp.nov., a novel halophilic helical bacterium isolated from kusaya gravy."Int.J.Syst.Evol.Microbiol.. 54. 163-167 (2004)

M.Satomi：“Marinospilillum insulare sp.nov.，一种从 kusaya 肉汁中分离出来的新型嗜盐螺旋细菌。”Int.J.Syst.Evol.Microbiol.. 54. 163-167 (2004)