Polysaccharide intercellular adhesin and proper phospholipid composition are important for aggregation in Tetragenococcus halophilus SL10.

Aggregating strains of Tetragenococcus halophilus tend to be trapped during soy sauce mash-pressing process and are, therefore, critical for clear soy sauce production. However, the precise molecular mechanism involved in T. halophilus aggregation remains elusive. In previous studies, we isolated a number of aggregating strains, including T. halophilus AB4 and AL1, and showed that a cell surface proteinaceous aggregation factor is responsible for their aggregation phenotype. In the present study, we explored the role of polysaccharide intercellular adhesin (PIA) in aggregate formation in T. halophilus SL10, isolated from soy sauce. SL10 exhibited similar aggregation to AB4 and AL1 but formed a non-uniform precipitate with distinctive wrinkles at the bottom of the test tube, unlike AB4 and AL1. Insertion sequence mutations in each gene of the ica operon diminished aggregation and PIA production, highlighting the critical role of IcaADBC-mediated PIA production in T. halophilus aggregation. Furthermore, two non-aggregating cardiolipin synthase (cls) gene mutants with intact ica operon did not produce detectable PIA. Phospholipid composition analysis in cls mutants revealed a decrease in cardiolipin and an increase in phosphatidylglycerol levels, highlighting the association between phospholipid composition and PIA production. These findings provide evidence for the pivotal role of cls in PIA-mediated aggregation and lay the foundation for future studies to understand the intricate networks of the multiple aggregation factors governing microbial aggregation.IMPORTANCEAggregation, commonly observed in various microbes, triggers biofilm formation in pathogenic variants and plays a beneficial role in efficient food production in those used for food production. Here, we showed that Tetragenococcus halophilus, a microorganism used in soy sauce fermentation, forms aggregates in a polysaccharide intercellular adhesin (PIA)-mediated manner. Additionally, we unveiled the relationship between phospholipid composition and PIA production. This study provides evidence for the presence of aggregation factors in T. halophilus other than the proteinaceous aggregation factor and suggests that further understanding of the coordinated action of these factors may improve clarified soy sauce production.

Sucrose contributed to the biofilm formation of Tetragenococcus halophilus and changed the biofilm structure.

Based on the previous research results that the addition of sucrose in the medium improved the biofilm formation of Tetragenococcus halophilus, the influence of sucrose on biofilm formation was explored. Moreover, the influence of exogenous expression of related genes sacA and galE from T. halophilus on the biofilm formation of L. lactis NZ9000 was investigated. The results showed that the addition of sucrose in the medium improved the biofilm formation, the resistance of biofilm cells to freeze-drying stress, and the contents of exopolysaccharides (EPS) and eDNA in the T. halophilus biofilms. Meanwhile, the addition of sucrose in the medium changed the monosaccharide composition of EPS and increased the proportion of glucose and galactose in the monosaccharide composition. Under 2.5% (m/v) salt stress condition, the expression of gene sacA promoted the biofilm formation and the EPS production of L. lactis NZ9000 with the sucrose addition in the medium and changed the EPS monosaccharide composition. The expression of gene galE up-regulated the proportion of rhamnose, galactose, and arabinose in the monosaccharide composition of EPS, and down-regulated the proportion of glucose and mannose. This study will provide a theoretical basis for regulating the biofilm formation of T. halophilus, and provide a reference for the subsequent research on lactic acid bacteria biofilms.

Transcriptomic profiling reveals differences in the adaptation of two Tetragenococcus halophilus strains to a lupine moromi model medium.

BACKGROUND: Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. RESULTS: The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. CONCLUSIONS: Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.

Arginine deiminase pathway of Tetragenococcus halophilus contributes to improve the acid tolerance of lactic acid bacteria.

Arginine deiminase pathway, controlled by arginine deiminase, ornithine carbamoyltransferase and carbamate kinase, could affect and modulate the intracellular pH homeostasis of lactic acid bacteria under acid stress. Herein, strategy based on exogenous addition of arginine had been proposed to improve the robustness of Tetragenococcus halophilus during acid stressed condition. Results indicated cells cultured in the presence of arginine acquired high tolerance to acid stress mainly through maintaining the homeostasis of intracellular microenvironment. Additionally, metabolomic analysis and q-PCR showed the content of intracellular metabolites and expression levels of genes involved in ADI pathway significantly increased when cells encountered acid stress with the presence of exogenous arginine. Furthermore, Lactococcus lactis NZ9000 with heterologous overexpression of arcA and arcC from T. halophilus exhibited high stress tolerance to acidic condition. This study may provide an insight into the systematical understanding about the mechanism underlying acid tolerance and improve the fermentation performance of LAB during harsh condition.

Selective extracellular secretion of small double-stranded RNA by Tetragenococcus halophilus.

Small double-stranded RNAs (dsRNAs) abundantly produced by lactic acid bacteria demonstrate immunomodulatory activity and antiviral protective immunity. However, the extracellular secretion of dsRNA from lactic acid bacteria and their compositional and functional differences compared to the intracellular dsRNA is unknown. In this study, we compared the intracellular and secreted extracellular dsRNA of the lactic acid bacteria, Tetragenococcus halophilus, commonly present in fermented foods, by growing in RNA-free and RNase-free media. We used RNA deep sequencing and in-silico analysis to annotate potential regulatory functions for the comparison. A time series sampling of T. halophilus culture demonstrated growth phase-dependent dynamics in extracellular dsRNA secretion with no major change in the intracellular dsRNA profile. The RNA deep sequencing resulted in thousands of diverse dsRNA fragments with 14-21 nucleotides in size from T. halophilus culture. Over 70% of the secreted extracellular dsRNAs were unique in their sequences compared to the intracellular dsRNAs. Furthermore, the extracellular dsRNA abundantly contains sequences that are not T. halophilus genome encoded, not detected intracellularly and showed higher hits on human transcriptome during in-silico analysis, which suggests the presence of extrachromosomal mobile regulatory elements. Further analysis showed significant enrichment of dsRNA target genes of human transcriptome on cancer pathways and transcription process, indicating the extracellular dsRNA of T. halophilus is different not only at the sequence level but also in function. Studying the bacterial extracellular dsRNA is a promising area of future research, particularly for developing postbiotic fermented functional foods and understanding the impact of commensal gut bacteria on human health.

The diversity among the species Tetragenococcus halophilus including new isolates from a lupine seed fermentation.

BACKGROUND: Tetragenococcus (T.) halophilus can be isolated from a variety of fermented foods, such as soy sauce, different soy pastes, salted fish sauce and from cheese brine or degraded sugar beet thick juice. This species contributes by the formation of short chain acids to the flavor of the product. Recently, T. halophilus has been identified as a dominant species in a seasoning sauce fermentation based on koji made with lupine seeds. RESULTS: In this study we characterized six strains of T. halophilus isolated from lupine moromi fermentations in terms of their adaptation towards this fermentation environment, salt tolerance and production of biogenic amines. Phylogenic and genomic analysis revealed three distinctive lineages within the species T. halophilus with no relation to their isolation source, besides the lineage of T. halophilus subsp. flandriensis. All isolated strains from lupine moromi belong to one lineage in that any of the type strains are absent. The strains form lupine moromi could not convincingly be assigned to one of the current subspecies. Taken together with strain specific differences in the carbohydrate metabolism (arabinose, mannitol, melibiose, gluconate, galactonate) and amino acid degradation pathways such as arginine deiminase pathway (ADI) and the agmatine deiminase pathway (AgDI) the biodiversity in the species of T. halophilus is greater than expected. Among the new strains, some strains have a favorable combination of traits wanted in a starter culture. CONCLUSIONS: Our study characterized T. halophilus strains that were isolated from lupine fermentation. The lupine moromi environment appears to select strains with specific traits as all of the strains are phylogenetically closely related, which potentially can be used as a starter culture for lupine moromi. We also found that the strains can be clearly distinguished phylogenetically and phenotypically from the type strains of both subspecies T. halophilus subsp. halophilus and T. halophilus subsp. flandriensis.

Co-culture with Tetragenococcus halophilus improved the ethanol tolerance of Zygosaccharomyces rouxii by maintaining cell surface properties.

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.

Characterization of the two nonidentical ArgR regulators of Tetragenococcus halophilus and their regulatory effects on arginine metabolism.

The halophilic lactic acid bacterium Tetragenococcus halophilus has been widely used in high-salinity fermentation processes of food. Previous studies have indicated that the catabolism of arginine may contribute to the osmotic stress adaptation of T. halophilus. Unusually, in the chromosome of T. halophilus, preceding the arginine deiminase (ADI) operon, locate two co-transcribed genes, both encoding an ArgR regulator; similar structure was rarely found and the roles of the regulators have not been demonstrated. In the current study, regulatory roles of these two nonidentical ArgR regulators on the arginine metabolism of T. halophilus were investigated. The results show that these two regulators play different roles in arginine metabolism, ArgR1 acts as a negative regulator of the ADI pathway by binding to the promoter sequences and repressing the transcription of genes, and the addition of arginine or hyper-osmotic stress conditions can abolish the ArgR1 repression, whereas ArgR2 negatively regulates the genes involved in arginine biosynthesis. Our study found that despite the commonly known roles of the ArgR regulators as the activator of arginine catabolism and the repressor of arginine biosynthesis, which are found in most studied bacteria possessed one ArgR regulator, the two nonidentical ArgR regulators of T. halophilus both act as repressors, and the repression by which is regulated when sensing changes of environments. By revealing the regulation of arginine metabolism, the current study provides molecular insights and potential tools for future applications of halophiles in biotechnology. KEY POINTS: • The expression of the ADI pathway of T. halophilus is regulated by carbon sources and osmotic stress. • The arginine metabolism process of T. halophilus is fine-tuned by the two ArgR regulators. • The ADI pathway may contribute to the osmotic stress adaptation by generating more energy and accumulating citrulline which acts as compatible solute.

The role of microorganisms in soy sauce production.

Soy sauce is a salty condiment commonly used in Eastern Asia that is made from soy beans with varying amounts of wheat or no wheat at all. It is known as shoyu in Japan, chiang-yu (or -yi) in China, kecup in Indonesia, kunjang in Korea, toyo in The Philippines, and see-ieu in Thailand (Beuchat, 1985; Djien, 1982; Fukushima, 1989). It provides flavor in an otherwise bland diet, and nutritionally it provides salt (NaCl) and predigested proteins in a diet that is traditionally protein poor. It has been made for centuries on a small scale in many towns and villages in Asia, but since 1950, particularly in Japan, the manufacturing process has been studied and modernized so that its manufacture is now concentrated in large factories using modern, controlled methods of production (Sasaki & Nunomura, 1993). In Japan, soy sauce fermentation is a major food manufacturing activity. More than 1.1 million kiloliters of soy sauce was produced in 1986 by 3000 producers, and the Kikkoman Company supplied 30% of the market (Fukushima, 1989). By 1990 there were 2871 manufacturers, 5 of which produced about 50% of the total production (Sasaki & Nunomura, 1993). While modern methods are used for most of the soy sauce produced in Japan, and factory production in other Asian countries is growing, soy sauce is still produced by methods involving no modern technological inputs (Röling, Prasetyo, Timotius, Stouthamer, & van Verseveld, 1994).

Identification of a GntR family regulator BusRTha and its regulatory mechanism in the glycine betaine ABC transport system of Tetragenococcus halophilus.

Glycine betaine is one of the most effective compatible solutes of the halophilic lactic acid bacterium Tetragenococcus halophilus, the transportation of which is essential for its survival under salinity stress condition. In the current study, we attempted to define a glycine betaine ABC transporter system of T. halophilus, busATha, which plays an important role in adapting to salinity condition. The expression of busATha enhanced the growth of the recombinant strain under high salinity. BusRTha, a transcription regulator that represses the expression of busATha, was characterized, and the repression was abrogated under high salinity. The binding of the regulator was demonstrated through electrophoretic mobility shift assays, and the binding sites were characterized as 5'-AAA(T/G)TGAC(C/A)(G/A)T(C/A)C-3'. This is the first studied transcription regulator of T. halophilus, and our findings provide insights into the molecular mechanism of halophilic life and tools for further application of halophiles as chassis in industrial biotechnology.

Genomic and metabolic features of Tetragenococcus halophilus as revealed by pan-genome and transcriptome analyses.

The genomic and metabolic diversity and features of Tetragenococcus halophilus, a moderately halophilic lactic acid bacterium, were investigated by pan-genome, transcriptome, and metabolite analyses. Phylogenetic analyses based on the 16S rRNA gene and genome sequences of 15 T. halophilus strains revealed their phylogenetic distinctness from other Tetragenococcus species. Pan-genome analysis of the T. halophilus strains showed that their carbohydrate metabolic capabilities were diverse and strain dependent. Aside from one histidine decarboxylase gene in one strain, no decarboxylase gene associated with biogenic amine production was identified from the genomes. However, T. halophilus DSM 20339T produced tyramine without a biogenic amine-producing decarboxylase gene, suggesting the presence of an unidentified tyramine-producing gene. Our reconstruction of the metabolic pathways of these strains showed that T. halophilus harbors a facultative lactic acid fermentation pathway to produce l-lactate, ethanol, acetate, and CO2 from various carbohydrates. The transcriptomic analysis of strain DSM 20339T suggested that T. halophilus may produce more acetate via the heterolactic pathway (including d-ribose metabolism) at high salt conditions. Although genes associated with the metabolism of glycine betaine, proline, glutamate, glutamine, choline, and citrulline were identified from the T. halophilus genomes, the transcriptome and metabolite analyses suggested that glycine betaine was the main compatible solute responding to high salt concentration and that citrulline may play an important role in the coping mechanism against high salinity-induced osmotic stresses. Our results will provide a better understanding of the genome and metabolic features of T. halophilus, which has implications for the food fermentation industry.

Tetragenococcus halophilus MJ4 as a starter culture for repressing biogenic amine (cadaverine) formation during saeu-jeot (salted shrimp) fermentation.

Biogenic amines (BAs) are frequently present in traditionally fermented salted foods. In this study, a Tetragenococcus halophilus strain (MJ4) with no BA-producing ability was isolated from a fish (anchovy) sauce. Strain MJ4 did not produce BAs from supplied precursors and no BA-producing genes were identified in its genome. Bacterial community analysis showed that in non-inoculated saeu-jeot (shrimp sauce) fermentation, Tetragenococcus predominated after 82 days, while in strain MJ4-inoculated saeu-jeot, Tetragenococcus predominated during the entire fermentation. Strain MJ4 repressed the growth of T. muriaticus, a known BA producer, during fermentation, but metabolite analysis demonstrated that metabolite profiles, including amino acids, were similar regardless of MJ4 inoculation. The metabolite analysis also showed that strain MJ4 clearly repressed the formation of cadaverine during fermentation. This study suggests that the use of strain MJ4 as a starter culture in salted fish fermentation may be a good strategy for the reduction of BA formation.

Effect of raw material and starters on the metabolite constituents and microbial community diversity of fermented soy sauce.

BACKGROUND: The quality of soy sauce is strongly affected by microorganisms and raw materials (defatted soybean or whole soybean). The present study investigated the effect of two types of fortified pattern, including inoculation with starters (Tetragenococcus halophilus combined with Zygosaccharomyces rouxii and Candida versatilis), and adding culture medium (saccharified rice flour solution), on the metabolite profiles and microbial community of soy sauce produced from defatted soybean (DP) and whole soybean (HD). Relationships between microbes and volatiles, and their interactions, were shown. RESULTS: The dominant metabolites differed in the soy sauce samples except for isoflavones. Alcohols and phenols were higher in DP moromi. Two classes of dominant esters, long-chain fatty acid esters (LFAE) and unsaturated-short-chain fatty acid esters (USFAE), were higher in HD moromi than DP. Weissella, Leuconostoc, and Aspergillus were the dominant microbes. Leuconostoc, and Aspergillus increased, and Weissella decreased in moromi inoculated with starters compared with a control. Similar changes to Leuconostoc were observed in moromi added culture medium. CONCLUSIONS: The microbes were responsible for the formation of volatiles. The intergeneric interactions with microbes were affected by fortified pattern. The effect of starters or culture medium on microbial community and metabolites of soy sauce depended on the raw material. © 2019 Society of Chemical Industry.

Formation of Sulforaphane and Iberin Products from Thai Cabbage Fermented by Myrosinase-Positive Bacteria.

Myrosinase-positive bacteria from local fermented foods and beverages in Thailand with the capacity to metabolize glucosinolate and produce isothiocyanates (ITCs) were isolated and used as selected strains for Thai cabbage fermentation. Enterobacter xiangfangensis 4A-2A3.1 (EX) from fermented fish and Enterococcus casseliflavus SB2X2 (EC) from fermented cabbage were the two highest ITC producers among seventeen strains identified by 16S rRNA technique. EC and EX were used to ferment Thai cabbage (Brassica oleracea L. var. capitata) containing glucoiberin, glucoraphanin and 4-hydroxyglucobrassicin at 430.5, 615.1 and 108.5 µmol/100 g DW, respectively for 3 days at 25 °C. Different amounts of iberin nitrile, iberin, sulforaphane and indole 3-acetonitrile were produced by spontaneous, EX- and EC-induced cabbage fermentations, and significantly higher ITCs were detected (p < 0.01) with increased antioxidant activities. Iberin and sulforaphane production in EX-induced treatment peaked on day 2 at 117.4 and 294.1 µmol/100 g DW, respectively, significantly higher than iberin at 51.7 µmol/100 g DW but not significantly higher than sulforaphane at 242.6 µmol/100 g DW in EC-induced treatment at day 2. Maximum health-promoting benefits from this functional food can be obtained from consumption of a liquid portion of the fermented cabbage with higher ITC level along with a solid portion.

Spoilage potential of Vagococcus salmoninarum in preservative-free, MAP-stored brown shrimp and differentiation from Brochothrix thermosphacta on streptomycin thallous acetate actidione agar.

AIMS: During a previous study concerning brown shrimp (Crangon crangon), selective streptomycin thallous acetate actidione (STAA) agar was used to determine the growth of Brochothrix thermosphacta. However, the growth of Vagococcus salmoninarum on this medium was also noticed. This study explores the spoilage potential of this organism when inoculated on sterile shrimp. METHODS AND RESULTS: Isolates growing on STAA were identified using (GTG)5 clustering followed by partial 16S rRNA gene sequence analysis. Their biochemical spoilage potential was analysed for H2 S production and enzymatic activities were tested using an APIZYM test. Headspace solid phase micro-extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were used to analyse the volatile organic compounds (VOCs) produced during storage of inoculated shrimp. CONCLUSION: Fifty-five per cent of isolates taken from STAA could be identified as V. salmoninarum, while no apparent morphological difference with B. thermosphacta isolates was identified upon the prescribed incubation conditions. For isolates identified as V. salmoninarum, production of 2-heptanone, 2-nonanone, 2-undecanone was found, as was the possibility to form H2 S. SIGNIFICANCE AND IMPACT OF THE STUDY: When using the STAA medium for detecting B. thermosphacta, one should consider the possible abundant presence of V. salmoninarum as well. Based on this study, V. salmoninarum does not exhibit great spoilage potential, although it can produce H2 S and formed VOCs which are also found in other spoiled seafood products.

Quantification of viable bacterial starter cultures of Virgibacillus sp. and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR.

Real-time quantitative polymerase chain reaction (qPCR) methods were developed for the quantification of Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33, which were added as starter cultures in fish sauce fermentation. The PCR assays were coupled with propidium monoazide (PMA) treatment of samples to selectively quantify viable cells and integrated with exogenous recombinant Escherichia coli cells to control variabilities in analysis procedures. The qPCR methods showed species-specificity for both Virgibacillus halodenitrificans and T. halophilus as evaluated using 6 reference strains and 28 strains of bacteria isolated from fish sauce fermentation. The qPCR efficiencies were 101.1% for V. halodenitrificans and 90.2% for T. halophilus. The quantification limits of the assays were 10(3) CFU/mL and 10(2) CFU/mL in fish sauce samples with linear correlations over 4 Logs for V. halodenitrificans and T. halophilus, respectively. The matrix effect was not observed when evaluated using fish sauce samples fermented for 1-6 months. The developed PMA-qPCR methods were successfully applied to monitor changes of Virgibacillus sp. SK37 and T. halophilus MS33 in a mackerel fish sauce fermentation model where culture-dependent techniques failed to quantify the starter cultures. The results demonstrated the usability of the methods as practical tools for monitoring the starter cultures in fish sauce fermentation.

Microbiology of sugar-rich environments: diversity, ecology and system constraints.

Microbial habitats that contain an excess of carbohydrate in the form of sugar are widespread in the microbial biosphere. Depending on the type of sugar, prevailing water activity and other substances present, sugar-rich environments can be highly dynamic or relatively stable, osmotically stressful, and/or destabilizing for macromolecular systems, and can thereby strongly impact the microbial ecology. Here, we review the microbiology of different high-sugar habitats, including their microbial diversity and physicochemical parameters, which act to impact microbial community assembly and constrain the ecosystem. Saturated sugar beet juice and floral nectar are used as case studies to explore the differences between the microbial ecologies of low and higher water-activity habitats respectively. Nectar is a paradigm of an open, dynamic and biodiverse habitat populated by many microbial taxa, often yeasts and bacteria such as, amongst many others, Metschnikowia spp. and Acinetobacter spp., respectively. By contrast, thick juice is a relatively stable, species-poor habitat and is typically dominated by a single, xerotolerant bacterium (Tetragenococcus halophilus). A number of high-sugar habitats contain chaotropic solutes (e.g. ethyl acetate, phenols, ethanol, fructose and glycerol) and hydrophobic stressors (e.g. ethyl octanoate, hexane, octanol and isoamyl acetate), all of which can induce chaotropicity-mediated stresses that inhibit or prevent multiplication of microbes. Additionally, temperature, pH, nutrition, microbial dispersion and habitat history can determine or constrain the microbiology of high-sugar milieux. Findings are discussed in relation to a number of unanswered scientific questions.

Comparative transcriptomic analysis reveals novel genes and regulatory mechanisms of Tetragenococcus halophilus in response to salt stress.

Tetragenococcus halophilus, a moderately halophilic Gram-positive bacterium, was isolated from Chinese style soy sauce. This species is a valuable resource for investigating salt tolerance mechanisms and improving salinity resistance in microorganisms. RNA-seq was used to sequence T. halophilus samples treated with 0 M (T1), 1 M (T2), and 3.5 M NaCl (T3). Comparative transcriptomic analyses of the different treatments were performed using gene ontology and Kyoto encyclopedia of genes and genome. The comparison of T1 and T2 by RNA-seq revealed that genes involved in transcription, translation, membrane system, and division were highly up-regulated under optimum salt condition. The comparison of T2 and T3 showed that genes related to heat shock proteins or the ATP-binding cassette transport systems were significantly up-regulated under maximum-salt condition. In addition, a considerable proportion of the significantly differently expressed genes identified in this study are novel. These data provide a crucial resource that may determine specific responses to salt stress in T. halophilus.

Tetragenococcus koreensis is part of the microbiota in a traditional Italian raw fermented sausage.

This study reports the isolation of Tetragenococcus koreensis, a bacterial species currently represented only by the type strain isolated from kimchi, from a raw fermented and ripened Italian sausage, Ventricina Vastese, all over the ripening period of five months. Rep-PCR genotyping showed that different T. koreensis strains, identified by sequencing of the 16S rRNA gene, were present in the same production batch. Tests on representative isolates showed intra-species physiological variability and the possession of phenotypic traits relevant for the production of fermented sausages, i.e. ability to grow at high salt concentrations, to induce some changes in the peptide profile of the culture medium and inability to produce histamine and tyramine, confirmed by the absence of the respective decarboxylase genes. Therefore the opportunity to further investigate the suitability of T. koreensis as a starter for fermented meat products was suggested.

Isolation and characterization of halophilic lactic acid bacteria acting as a starter culture for sauce fermentation of the red alga Nori (Porphyra yezoensis).

AIMS: A screening test was conducted for environmental samples to isolate halophilic lactic acid bacteria (HLAB) that can act as a starter in a Nori (Porphyra yezoensis)-sauce culture. METHOD AND RESULTS: After 9 months of incubation of enrichment cultures added with 25 kinds of environmental samples, growth of HLAB-like microorganisms was observed in six cultures salted at a 15% w/w level, including culture samples originally from mesopelagic water taken from 321 m-depth and from mountain snow taken at 2450 m-height. Ten strains were isolated and characterized as Tetragenococcus halophilus based on sequence analysis of the 16S rRNA gene. The isolates were inoculated into a newly prepared Nori-sauce culture and were confirmed to be able to act as a starter culture while three reference strains of T. halophilus obtained from a culture collection could not grow in the same culture. CONCLUSIONS: Halophilic lactic acid bacteria strains that can make growth in a highly salted Nori-sauce culture were isolated from environmental samples for the first time. All the isolates were identified as T. halophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated strains are expected to be utilized as a starter culture for manufacturing fermented seaweed-sauce, which will be the first fermented food products obtained from algae.