Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce.

We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. T. muriaticus formed histamine in low acidity (pH 5.8), O2 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, indicating that NaCl could not prevent histamine formation by this bacterium. A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions. Studies of cell suspension experiments confirmed the results obtained from cultured cells.     

耐盐乳酸菌和酵母菌强化技术对高盐稀态发酵酱醪品质的影响

分别将耐盐乳酸菌Tetragenococcus halophilus CGMCC 3792、耐盐酵母菌Zygosaccharomyces rouxii CGM-CC 3791和Candida versatilis CGMCC 3790菌悬液接入酱醪中,利用顶空-固相微萃取偶联气质联用技术(HS-SPME/GC-MS)检测了发酵125 d酱醪中挥发性组分的变化。研究结果表明,T.halophilus CGMCC 3792强化的酱醪中酸类、醇类、酮类、呋喃类、吡嗪类和其他组分的相对含量降低,酯类、醛类和酚类组分的相对含量增加,其中苯乙醇、乙酸乙酯、辛酸乙酯、癸酸乙酯和4-乙基愈创木酚等的相对含量显著增加,而Z.rouxii CGMCC 3791、C.versatilis CGMCC 3790共培养强化的酱醪中醇类、酯类、醛类和其他组分的相对含量增加,酸类、酚类、呋喃类和吡嗪类组分的相对含量降低,其中苯乙醇、长链脂肪酸乙酯(月桂酸乙酯、棕榈酸乙酯、亚油酸乙酯等)的相对含量显著增加。此外,本研究中T.halophilus CGMCC 3792强化的酱醪中4-乙基愈创木酚的相对含量大幅增加,且在整个发酵过程中其含量也单调增加,对于T.halophilus CGMCC 3792是否具有分泌4-乙基愈创木酚的能力以及是否冷温工艺赋予该菌株合成4-乙基愈创木酚的能力需进一步探究。     

强化嗜盐四联球菌对模拟低盐酱油发酵的影响

通过模拟低盐酱油快速发酵,在添加鲁氏接合酵母（Zygosaccharomyces rouxii）的基础上,考察嗜盐四联球菌（Tetragenococcus halophilus）不同添加量对酱醪理化指标、氨基酸和有机酸含量、挥发性风味物质的影响,揭示嗜盐四联球菌在提高低盐酱油品质方面的应用潜力。结果表明,嗜盐四联球菌对酱油中酵母菌的生长有一定的抑制作用,会使酱醪pH略有下降,总酸和氨基酸态氮含量提高。最适宜提高酱油品质的菌株添加方式是发酵开始接入鲁氏接合酵母,发酵10 d后添加108 CFU/g嗜盐四联球菌,酱油样品中氨基酸和有机酸总量比单独添加酵母菌分别提高43.8%、55.6%,其中鲜味和甜味氨基酸分别提高33.7%、29.9%;挥发性风味物质种类增加43.2%,总量提高2.4倍,其中香气物质愈创木酚和1-辛烯-3-醇含量分别提高11.1倍、8.9倍。     

耐盐乳酸菌在高盐稀态酱油中的应用

该文研究耐盐乳酸菌的分离、培养、保藏及其在高盐稀态酱油中与酵母菌的协同发酵作用，确定了可应用于高盐稀态酱油中的乳酸菌种类及添加时间和添加量。通过乳酸菌的作用使酱油颜色更加鲜亮、有光泽，并增加了酱油的香气。     

Effect of Tetragenococcus halophilus,Zygosaccharomyces rouxii,and Torulopsis versatilis addition sequence on soy sauce fermentation

The aim of this study was to investigate the effect that the sequences of ingredient addition had on soy sauce fermentation. Five strategies were assessed for the inoculation of Tetragenococcus halophilus, Zygosaccharomyces rouxii, and Torulopsis versatilis. The results of physicochemical analysis and levels of flavor compounds demonstrated that the best strategy for soy sauce fermentation was to inoculate T. halophilus (2 × 10⁵ CFU/mL) on the 15th day, Z. rouxii (1.0 × 10⁶ CFU/mL) on the 30th day, and T. versatilis (1.0 × 10⁶ CFU/mL) on the 45th day; this enhanced its fruity, sauce, alcoholic, and caramel-like flavors by 64.3%, 22.7%, 43.1%, and 36.2%, respectively, and saline taste by 64.3%. Spearman correlation analysis revealed the content of ethanol positively correlated with acetic acid (r = 0.82) and succinic acid (r = 0.80), while the level of formic acid negatively correlated with ethyl phenylacetate (r = −0.88). In summary, these results provided fundamental evidence for strain addition sequence for producing flavor-enhanced soy sauce with potential application in the future.     

耐盐乳酸菌和酵母菌对高盐稀态发酵酱油品质的影响

研究了添加耐盐乳酸菌(Tetragenococcus halophilus)和酵母菌(Zygosaccharomyces rouxii和Candida versatilis)对酱醪理化指标及挥发性组分的影响.研究结果表明,添加耐盐微生物的酱醪发酵125d后氨态氮(FN)均略高于对照样品(未添加耐盐微生物);添加T.halophilus的酱醪总酸(TA)高于其他样品,而采用T.halophilus、Z.rouxii和C.versatilis 共培养的酱醪TA和对照样品没有显著区别;分析酱醪还原糖(RS)含量结果表明,添加耐盐微生物后还原糖含量显著低于对照样品.此外,通过GC-MS分析了添加耐盐乳酸菌和酵母菌对酱醪挥发性组分的影响.发酵125d后,醇、酯类组分含量增加,酸类及其他类组分含量减少.所检出的各酱醪挥发性组分中,添加T.halophilus的酱醪中2-甲基丁酸、2-甲基丁醇、乙酸异戊酯含量分别较对照组高53.4％、337.3％、388.2％,添加Z.rouxii和C.versatilis的酱醪中乙醇和乙酸乙酯含量分别较对照组高64.2％和56.3％,采用T.halophilus、Z.rouxii和C.versatilis共培养的酱醪中1-辛烯-3-醇和苯甲醛含量高于对照样品56.7％和26.3％,显著改善了酱醪风味.     

耐盐乳酸菌和酵母菌对高盐稀态发酵酱油品质的影响

研究了添加耐盐乳酸菌（Tetragenococcus halophilus）和酵母菌（Zygosac charomyces rouxii和Candida versatilis）对酱醪理化指标及挥发性组分的影响。研究结果表明,添加耐盐微生物的酱醪发酵125d后氨态氮（FN）均略高于对照样品（未添加耐盐微生物）;添加T.halophilus的酱醪总酸（TA）高于其他样品,而采用T.halophilus、Z.rouxii和C.versatilis共培养的酱醪TA和对照样品没有显著区别;分析酱醪还原糖（RS）含量结果表明,添加耐盐微生物后还原糖含量显著低于对照样品。此外,通过GC-MS分析了添加耐盐乳酸菌和酵母菌对酱醪挥发性组分的影响。发酵125d后,醇、酯类组分含量增加,酸类及其他类组分含量减少。所检出的各酱醪挥发性组分中,添加T.halophilus的酱醪中2-甲基丁酸、2-甲基丁醇、乙酸异戊酯含量分别较对照组高53.4%、337.3%、388.2%,添加Z.rouxii和C.versatilis的酱醪中乙醇和乙酸乙酯含量分别较对照组高64.2%和56.3%,采用T.halophilus、Z.rouxii和C.versatilis共培养的酱醪中1-辛烯-3-醇和苯甲醛含量高于对照样品56.7%和26.3%,显著改善了酱醪风味。      

高盐辣椒坯中一株耐盐乳杆菌的分离及鉴定

目的采用选择性的MRS培养基,从湖南地区特色的盐坯辣椒中分离筛选出耐盐优势菌。方法得到一株能在18%NaCl培养基中生长的耐盐菌进行了生理生化实验,通过菌落PCR扩增其16S rDNA基因序列并测序,经过序列比对构建系统发育树。结果这株菌有较好的产乳酸、耐盐的能力,经过形态观察和生理生化试验验证,属于乳酸杆菌。结论经过16S rDNA进一步鉴定为植物乳杆菌。     

酱油发酵过程中的耐盐乳酸菌筛选及对低盐固态酱油品质的影响

本文通过对酱醪中耐盐乳酸菌的筛选,成功分离出一株耐18%盐度的乳酸菌,经过16S r DNA测序鉴定,确定为一株植物乳杆菌。在酱油酿造过程中添加该耐盐植物乳杆菌,无盐固形物、氨基酸态氮、还原糖和总酸含量等理化指标都有所上升。经过液相测定,发现酱油中乳酸含量比空白增加2倍左右。挥发性风味物质中酮类和酚类物质增加2倍左右,醇类物质稍有增加。本研究为进一步安全有效提高酱油风味物质奠定理论依据。      

Effect of different halophilic microbial fermentation patterns on the volatile compound profiles and sensory properties of soy sauce moromi

The effect of inoculated or co-cultured halophilic lactic bacteria (Tetragenococcus halophilus) and yeasts (Zygosaccharomyces rouxii and Candida versatilis) on the quality of soy sauce moromi was investigated. Results showed no significant change in the major physicochemical properties of soy sauce moromi such as amino nitrogen, total acid, and reducing sugar at the end of fermentation, whereas the composition of free amino acids slightly changed with different fermentation patterns. In the sample inoculated with T. halophilus, the concentration of proline and tryptophan decreased by 18.83 and 50.87 % compared with the blank sample, whereas tyrosine and histidine increased by 32.41 and 27.04 %. In the sample inoculated with Z. rouxii and C. versatilis, the concentration of bitter-tasting lysine, tyrosine, and phenylalanine increased by more than 20 %, and the concentration of tryptophan decreased by 56.71 %. The proportion of amino acids with sweet taste increased by 0.8 % through the co-culture of the three strains. Meanwhile, the total amount of volatile compounds increased by 117.66, 23.28, and 216.88 % in the T. halophilus inoculated sample, two yeast strain inoculated sample, and three strain inoculated sample, respectively. Increased odour activity values of the nine compounds that mainly contributed to the flavour of moromi ranged from 1 to 7.87 times in the three strain inoculated sample. These findings revealed that the co-culture of the three strains during moromi fermentation was beneficial to the improvement of flavour of soy sauce moromi.     

酱醅中乳酸菌的分离与鉴定

对豆酱和酱油发酵酱醅中的乳酸菌进行了分离鉴定.通过溶钙圈实验、乳酸纸层析、形态特征和培养特征观察,从中筛选出8株菌.经API系统鉴定,JY-1和JY-6为戊糖片球菌,JY-2、JY-4和JY-7为短乳杆菌,JY-3为肠膜明串珠菌葡聚糖亚种,JY-5和JY-8为植物乳杆菌,并对菌株JY-1、JY-2、JY-3和JY-5的耐盐性进行了测试.     

Analysis of a 30 kbp plasmid encoding histidine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to analyze the genes related to the histamine production, a strain of histamine producing halophilic bacteria, referred to as strain H, was isolated using enrichment culture and dilution-to-extinction methods with histidine broth inoculated from the fish sauce mashes. The two Japanese fish sauce mashes used, accumulate over 1000 mg/l of histamine. Phenotypic and 16 S rRNA gene sequence analyses identified strain H as Tetragenococcus halophilus, the predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR and Southern blot) of the histamine producing strain confirmed that the strain harbored a 30 kbp plasmid (pHDC) encoding a single copy of the pyruvoyl dependent histidine decarboxylase gene (hdc). A comparison of hdcA that is a structural gene of histidine decarboxylase among strain H, Lactobacillus hilgardii 0006, L. sakei LTH2076, Oenococcus oeni 9204, T. halophilus and T. muriaticus JCM10006 (T) indicated >99% sequence similarity. The hdc gene cluster consisted of 4 ORFs, hdcP, hdcA, hdcB, and hdcRS, and were almost identical to that of L. hilgardii 0006 with 99% sequence similarity including the structural hdc spacer region. However, the approximately 500 bp regions upstream and downstream of the hdc gene were different between that of strain H and L. hilgardii 0006. The complete sequence of pHDC revealed 29,924 nucleotides including 28 ORFs, two pairs of IR (inverted repeat), similar sequence of plasmid conjugative elements, and a theta-type replicon. These results suggested that hdc could be encoded on transformable elements among lactic acid bacteria.     

Production of tyramine in "moromi" mash during soy sauce fermentation

The concentrations of 7 non-volatile amines, tyramine (Tym), histamine (Him), phenethylamine (Phm), putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine (Spm) in the liquid part of "moromi" mash during soy sauce fermentation were studied. These amines, except for him and Cad, were detected during fermentation by the conventional production method in the laboratory. Put and Spd were detected at the beginning, and Tym, Phm and Spm appeared later; these 5 amines increased gradually during the fermentation. Put, Spd, Spm and Cad were present in the raw starting material for soy sauce; thus, Tym and Phm were produced by the fermentation. When "moromi" mash was added to liquid medium and cultivated, Tym was detected in some "moromi" mash and the other amines were not detected. Tym-producing bacterial strains were isolated from the liquid culture media of Tym-positive "moromi" mash. The Tym-producing strain was a gram-positive coccus. The conditions for production of amines by Tym-producing bacterial strains were examined. These strains grew and produced tyramine under various conditions, which may occur during soy sauce fermentation. Namely, Tym was produced at pH 5-10, at salt concentrations of less than 8%, under either aerobic or anaerobic conditions. During soy sauce fermentation, it is assumed that Tym would be produced by these strains during the early stages of soy sauce aging within a short period when the salt concentration and pH conditions are optimal for growth. Based on the bacteriological properties, the strains were identified as Enterococcus faecium. With the exception of Phm and Him, which did not exist in the starting raw material, non-volatile amines (including Put, Cad, Spd and Spm) were not produced and microorganisms producing them are not believed to be present during "moromi" fermentation.     

Structural characteristics of oligosaccharides from soy sauce lees and their potential prebiotic effect on lactic acid bacteria

Oligosaccharides have attracted much attention due to the beneficial health effect on human beings. Oligosaccharides were extracted from soy sauce lees (SSLO) and were purified in this work. It was a mixture of oligosaccharides, which accounted for 80.5% of the total weight, and free monosaccharides. Only two monosaccharides, xylose and mannose, were detected in the molecular chain of SSLO. Their relative molar percentages were 53.2% and 46.8% for the oligosaccharide and monosaccharide fractions, respectively. A dose-dependent behaviour was observed for the growth promoting effect of SSLO on Lactobacillus bulgaricus and Streptococcus thermophilus. When 200 μg/ml of SSLO was applied, the L. bulgaricus concentration was 15.7-fold higher than the control, whilst the S. thermophilus concentration was 34.7-fold higher than the control. The analysis of DPPH radical scavenging activity indicated poor antioxidant activity of SSLO. It also suggested that the prebiotic effect was not correlated with antioxidant activity.     

Effect of a halophilic aromatic yeast together with Aspergillus oryzae in koji making on the volatile compounds and quality of soy sauce moromi

Koji making is an essential step in the production of high‐quality soy sauce. In this study, we inoculated koji with a halophilic aromatic yeast (Zygosaccharomyces rouxii) and Aspergillus oryzae and investigated the effects on the volatile compounds and quality of soy sauce moromi. Our results suggest that the optimal yeast inoculation was approximately 1.5 × 10⁶ yeast per gram of koji, and excessive yeast greatly decreased A. oryzae enzyme activity. In comparison with the control group, there were no significant changes in the concentrations of amino nitrogen, total titratable acids, total soluble nitrogen and reducing sugar by extending the fermentation time during the first fermentation month, whereas the concentration of glutamate in the yeast‐inoculated sample increased by 46.16% and the arginine concentration decreased by 61.07%. After the second fermentation month, 17, 38 and 38 volatile components were identified by GC–MS in C1, C2 and C5, respectively. Moreover, a large number of volatile components, such as 2‐Methoxy‐4‐vinylphenol, ethyl acetate and 1‐Octen‐3‐ol were found in C2 and C5. And the total contents of volatile compounds in C2 were obviously higher than C5. This work furthers our understanding of the traditional multistrain koji making and provides a new method for enhancing the quality and flavour of soy sauce.     

乳酸菌在酱油酿造工业及蔬菜加工中的应用

该文扼要地介绍了乳酸菌的分类、生理特性和保藏方法，较详细地介绍了乳酸菌在酱油酿造、蔬菜加工中的应用概况。指出了乳酸菌参与酱油酿造有不同程度的增香效果；应用于蔬菜加工中，不仅改善了产品的风味，而且提高了营养价值。     

耐盐乳酸菌与酵母菌在酱醪汁中协同作用的研究

采用透析培养的方法,研究了添加耐盐乳酸菌与酱醪中酵母菌的协同作用以及发酵工艺的控制和对酱油主要风味的影响.结果表明:在酱醪发酵过程中,添加耐盐乳酸菌明显促进T酵母哮菌的生长,而与S酵母菌存在着相互促进的关系.在酱醪发酵前期,同时添加104个/mL耐盐乳酸菌与T酵母菌时对发酵酱醪中的pH值和总酸起到稳定作用,提高了还原糖的产生速度,对中后期酱油主体风味的形成和提高具有良好的效果.