嗜盐四联球菌及其在发酵食品中的应用

嗜盐四联球菌是一类存在于酱油、鱼酱、豆制品等多种发酵食品及糖浆中的耐盐或耐高渗的乳酸菌。它们在食品发酵过程中可提高食品中有机酸、醛类和酯类等风味物质含量,具有广泛的应用价值。该文对嗜盐四联球菌的分类、生理生化特性、鉴定、胁迫环境下适应机制以及在发酵食品中的功能特性等几个方面的研究进展进行了综述。     

自然发酵豆酱中嗜盐四联球菌的分离筛选及生长特性研究

以采集自不同地区的14份自然发酵豆酱为试材,对其中的嗜盐四联球菌（Tetragenococcus halophilus）进行分离鉴定及益生特性研究,同时进一步探讨优良菌株的最佳生长条件,并通过单因素试验及响应面试验对其增殖培养基进行优化。结果表明,分离鉴定得到10株嗜盐四联球菌。其中,嗜盐四联球菌YSJ-5具有良好的耐酸、耐胆盐特性及抑菌特性,其最适培养条件为接种量3%、NaCl含量5%、培养温度33 ℃、初始pH值8.0。在此最适培养条件下,最适增殖培养基成分为葡萄糖2.70%,蛋白胨2.36%,黄豆浆4.41%,嗜盐四联球菌YSJ-5的活菌数为2.20×109 CFU/mL。     

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.     

臭鳜鱼中优良乳酸菌的分离筛选与鉴定

从臭鳜鱼中分离纯化得到50株疑似乳酸菌,按照肉制品发酵剂筛选标准,获得了4株优良乳酸菌L4、L12、L20和L36,其均具良好的耐盐性、发酵性和生长特性。经传统生理生化鉴定及16S rDNA序列分析,优良乳酸菌L4、L12、L20和L36分别为屎肠球菌(Enterococcus faecium)、弯曲乳杆菌(Lactobacillus curvatus)、坚强肠球菌(Enterococcus durans)、干酪乳杆菌(Lactobacillus casei)。     

甘氨酸甜菜碱和分子伴侣dnaK在嗜盐四联球菌耐盐机制中的作用

能耐高盐的嗜盐四联球菌CICC 10469是从传统的酱料中分离得到.为研究耐盐特性与相容性溶质积累和分子伴侣dnaK特性的关系,使用核磁共振来检测其主要的相容性溶质.不同盐浓度对耐盐的嗜盐四联球菌和不耐盐的乳酸乳球菌MG 1363的生长的影响通过测量OD600来获得.盐浓度对分子伴侣dnaK表达的影响通过实时荧光定量RT-PCR来检测.相容性溶质和可溶性蛋白随盐浓度的变化通过HPLC和酶标仪来检测.结果显示嗜盐四联球菌以甘氨酸甜菜碱作为主要的相容性溶质.适合的盐浓度能够促进菌体的生长,在一定盐浓度范围内,甘氨酸甜菜碱,可溶性蛋白和分子伴侣dnaK随盐浓度的增加而升高,然而超过一定盐浓度范围后,表达量将会减少.这表明细菌能够根据外部环境做出适当的调整以维持细胞的正常生理功能.而乳酸乳球菌MG1363很少有相容性溶质和分子伴侣dnaK以耐受外部盐浓度.这表明相容性溶质和分子伴侣dnak在嗜盐四联球菌生长中起重要的作用.总之,中度嗜盐菌的主要耐盐机制是通过快速合成和释放甘氨酸甜菜碱及分子伴侣dnaK高活性表达来完成.     

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.     

Immunomodulatory effect of halophilic lactic acid bacterium Tetragenococcus halophilus Th221 from soy sauce moromi grown in high-salt medium

A halophilic lactic acid bacterium, Tetragenococcus halophilus, was found to possess an immunomodulatory activity that promotes T helper type 1 (Th1) immunity in addition to its important roles in soy sauce brewing. Strain Th221 was selected from 151 strains isolated from soy sauce (shoyu) moromi, since it induced strong interleukin (IL)-12 production by mouse peritoneal macrophages in vitro. The relationship between the salt concentration in the medium and the IL-12 production-inducing activity of this strain was investigated, and the activity was found to be strong when the bacteria were grown in medium containing > or =10% (w/v) salt. The Th1-promoting activity was also manifested in an in vivo mouse study, since Th1-dependant contact sensitivity was augmented and Th2 immunity, as evaluated by specific immunoglobulin E production, was suppressed following oral ingestion of Th221. Based on these findings, Th221 administration may be useful for improving allergic symptoms.     

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.     

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。     

Paenibacillus tyraminigenes sp. nov. isolated from Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy

A bacterial strain H3029, a gram-positive, rod-shaped, oxidase-negative, endospore-forming bacterium that characteristically produces tyramine from tyrosine, was isolated from a Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy (Engraulis japonicus). The H3029 strain showed a high ability to produce 4140 microg/ml of tyramine from the culture broth containing 5000 microg/ml tyrosine. On the other hand, the strain produced a relatively low level of other putrefactive amines, at 973 microg/ml of putrescine and 147 microg/ml of cadaverine from the media, with each 5000 microg/ml of ornithine hydrochloride and lysine hydrochloride. The H3029 strain produced no detectable level of histamine (detection limit of 4 microg/ml) from the media containing 5000 microg/ml of histidine hydrochloride. Meanwhile, tyramine, the main product of the strain, showed the antimicrobial activity at the level of over 1 mg/disk against Staphylococcus aureus by agar diffusion test, and the mutagenicity in Ames test at 0.1 mg/plate using Salmonella typhimurium TA98 and TA1535. On the basis of the polyphasic taxonomic study, the H3029(T) strain was assigned a novel species of the genus Paenibacillus as Paenibacillus tyraminigenes sp. nov. The type strain of which is strain H3029(T) (=KCTC 10694BP(T)).     

Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce

Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006^T were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated > 99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements.     

Differentiation of Tetragenococcus populations occurring in products and manufacturing processes of puffer fish ovaries fermented with rice-bran

Tetragenococcus strains isolated from the manufacturing process of Japanese puffer fish ovaries fermented with rice-bran were characterized and differentiated phenotypically and genotypically. A total of 413 Tetragenococcus isolates were evaluated. On the basis of five representative substrates, the isolates were grouped into seven groups. An RFLP (restriction fragment length polymorphisms) analysis of the 16S rRNA gene of representative strains of major groups revealed that they could be grouped into two groups: one was identified as the most prominent halophilic lactic acid coccus, Tetragenococcus halophilus, and the other as T. muriaticus, which has recently been added to the genus Tetragenococcus as a new species. Physiologically, the major differences between the two groups were found in the ability to grow in medium not supplemented with NaCl and the fermentation of L-arabinose, sucrose and D-mannitol, and several other carbohydrates.     

Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli

Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli.     

多重胁迫对嗜盐四联球菌CGMCC 3792存活率及细胞成分的影响

本研究以嗜盐四联球菌（Tetragenococcus halophilus CGMCC 3792）为实验菌株,考察了单因素（酸、热、盐）胁迫和交互保护对其存活的影响。结果表明,T.halophilus CGMCC 3792在p H4.0和45℃预适应处理60 min后细胞对酸致死胁迫（p H2.5,60 min）的存活率分别提高了10.6倍和8.8倍。此外,还考察了酸胁迫对细胞膜不饱和脂肪酸和胞内氨基酸含量的影响,结果表明,酸预适应后再经酸致死胁迫导致细胞膜不饱和脂肪酸18∶1n-9和18∶2n-6,9含量增加,分别是直接经酸致死胁迫的2.3倍和2.0倍;同时,分析胞内氨基酸含量,胞内谷氨酸、天冬氨酸、亮氨酸和异亮氨酸浓度分别是直接经酸致死胁迫的2.5倍、2.4倍、2.2倍和2.7倍。研究结果将有助于进一步解析嗜盐四联球菌环境胁迫抗性的生理机制。      

Formation of cadaverine, histamine, putrescine and tyramine by bacteria isolated from meat, fermented sausages and cheeses

Enterobacteriaceae (EB, n = 149), Lactobacillus (LB, n = 162) and Leuconostoc sp. (LC, n = 89) and enterococci (EC, n = 137), isolated from raw meat (n = 65), fermented sausages (n = 50) and cheese (n = 55) samples, were cultivated in a broth containing precursor amino acids (each 3 g/l). After incubation, the liquid culture was chemically analysed for cadaverine (CAD), putrescine (PUT), histamine (HIS) and tyramine (TYR) formation at pH 5.2 and at pH 6.7. The majority of EB isolates (147 of 149) was capable of forming >100 mg/l of either CAD or PUT. Among the most frequently isolated species Hafnia alvei and Serratia liquefaciens, formation of >100 mg/l HIS occurred, but with low prevalence (1.6 and 6.5%, respectively). Twelve of 149 isolates (8%) were able to produce more than 10 mg/l HIS. One hundred forty-two isolates (95.3%) produced less than 10 mg/l TYR, and 7 isolates (4.7%) 10 mg/l to a maximum of 35.3 mg/l TYR. For LB + LC, one isolate (Leuconostoc mesenteroides ssp. mesenteroides) formed >100 mg/l PUT and one >100 mg/l CAD (of all 251 LB + LC isolates 0.4% each). Formation of >100 mg/l HIS and TYR was detected in 3.6 and 19% of the LB + LC isolates, respectively. For the EC isolates, maximum levels for PUT, CAD and HIS were 25.4 mg/l, 6.0 mg/l and 15.7 mg/l, respectively. TYR was formed in quantities of 100–1000 mg/l by 47.9% of EC faecalis (n = 75), and 59.7% of EC faecium (n = 62) isolates. More than 1000 mg/l TYR were formed by 50.7 and 35.5% of the isolates, respectively. A low initial pH of 5.2 compared to the initial pH of 6.7 favoured tyramine production by lactic acid bacteria, but was associated with lower CAD yield by EB. A considerable intra-species variability in amine formation was observed.     

传统发酵豆酱中嗜盐四联球菌的分离鉴定及增鲜菌株筛选

为了获得高产鲜味肽嗜盐四联球菌（Tetragenococcus halophilus）优良菌株,从辽宁省7个不同地区的96份自然发酵豆酱样品中分离出嗜盐四联球菌疑似菌株83株,通过提取各菌株DNA、16S rDNA序列测定和同源性对比分析,其中47株菌为嗜盐四联球菌,分别测定47株菌的产多肽能力、产γ-谷氨酰转肽酶能力、产蛋白酶能力、电子舌风味值分析等指标,结合因子分析以及因子得分综合评价,筛选得到LY9-1产鲜味肽潜力最佳,通过电子舌结果也证明,LY9-1发酵液鲜味值最高,为15.05±0.02,产蛋白酶活力高达（85.45±0.03）U/mL,产γ-谷氨酰转肽酶能力（44.23±0.03）U/m L,产多肽（17.55±0.13）mg/mL,推测该菌株具有较强的增鲜潜力,可为进一步研究增鲜机理以及在发酵食品增鲜中的产业化应用提供理论支持。     

亚硝酸盐还原酶对咸鱼中亚硝酸盐的降解条件与效果研究

为了探明亚硝酸盐还原酶(Nitrite Reductase,NiRs)对咸鱼中亚硝酸盐的降解作用及应用条件,实验以亚硝酸盐降解量为评价指标,通过单因素实验和Design-expert响应面实验分析咸鱼加工过程中加入亚硝酸盐还原酶(NiRs)的酶浓度、温度、时间的最适条件,并通过测定色差值和感官指标分析NiRs对咸鱼品质的影响。结果表明:NiRs在咸鱼加工中的最佳应用条件是酶浓度4mg/m L、温度34℃、时间5h,亚硝酸盐降解率可达到32.7%。加入NiRs不影响咸鱼的品质,对咸鱼风味有提升作用。研究结果表明在咸鱼生产中添加NiRs可有效降解亚硝酸盐,在保证咸鱼的食用安全性的同时,为腌制水产品的加工提供理论依据和技术支撑。     

亚硝酸盐还原酶对咸鱼中亚硝酸盐的降解条件与效果研究

为了探明亚硝酸盐还原酶（Nitrite Reductase,NiRs）对咸鱼中亚硝酸盐的降解作用及应用条件,实验以亚硝酸盐降解量为评价指标,通过单因素实验和Design-expert响应面实验分析咸鱼加工过程中加入亚硝酸盐还原酶（NiRs）的酶浓度、温度、时间的最适条件,并通过测定色差值和感官指标分析NiRs对咸鱼品质的影响。结果表明:NiRs在咸鱼加工中的最佳应用条件是酶浓度4mg/m L、温度34℃、时间5h,亚硝酸盐降解率可达到32.7%。加入NiRs不影响咸鱼的品质,对咸鱼风味有提升作用。研究结果表明在咸鱼生产中添加NiRs可有效降解亚硝酸盐,在保证咸鱼的食用安全性的同时,为腌制水产品的加工提供理论依据和技术支撑。      

Characterization of anti-listerial lactic acid bacteria isolated from Thai fermented fish products

Thai fermented fish products were screened for lactic acid bacteria capable of inhibitingListeriasp. (Listeria innocua). Of 4150 assumed lactic acid bacteria colonies from MRS agar plates that were screened by an agar-overlay method 58 (1.4%) were positive. Forty four of these strains were further characterized and 43 strains were inhibitory againstListeria monocytogenes. The strains were inhibitory to other Gram-positive (lactic acid) bacteria probably because of production of bacteriocins. All 44 strains inhibited bothVibrio choleraeandVibrio parahaemolyticusand 37 were inhibitory to a mesophilic fish spoilage bacterium (anAeromonassp.). Inhibition of Gram-negative bacteria was attributed to production of lactic acid. Most strains were identified asLactobacillusspp., and all grew well at ambient temperatures (25–37°C) and tolerated up to 6.5% NaCl. Glucose was fermented rapidly in laboratory media whereas pH decreased only very slowly in fish juice supplemented with 4% glucose and 3.5% NaCl or in a rice–fish mixture. Only four of 44 isolates could degrade and ferment complex carbohydrates such as rice, potatoes and maize starch. This indicates that other types of bacteria may be responsible for the rapid spontaneous fermentation of the products or that other yet-unknown factors ensure rapid fermentation. Overall anti-listerial lactic acid bacteria do occur in fermented fish products and the antibacterial activity against pathogenic bacteria indicates that they may be important in product safety.