Characterisation of the bacterial community structures of sunki, a traditional unsalted pickle of fermented turnip leaves.

The bacterial community structure in 29 naturally fermented samples of sunki, an unsalted lactic-fermented pickle in Japan, was determined by 16S rRNA gene-targeted metagenomic analysis. The data revealed that genus Lactobacillus was dominant in all samples and various bacterial species, related to Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus intestinalis, and Lactobacillus gasseri, showed a range of dominance depending on the samples. Comparative analysis of the bacterial composition by principal coordinate analysis and hierarchical clustering classified the varied bacterial composition of the 29 samples into three types of bacterial community structure. These types comprised lactobacilli belonging to different phylogenetic groups: type A had a certain ratio of Lactobacillus fermentum (70.3-22.1%, average 41.2%) in combination with several species belonging to Lactobacillus delbrueckii-phylogenetic group, type B comprised remarkably high levels of species Lactobacillus delbrueckii (average 89.5%), and type C had combinations of species belonging to Lactobacillus plantarum- and Lactobacillus buchneri-phylogenetic groups. Interestingly, these types differed in the compositional profiles of water-soluble and volatile compounds, and statistically significant differences were observed in the levels of acetic acid, succinic acid, ethanol, ethyl acetate, glutamic acid, γ-aminobutyric acid, and isovaleraldehyde. Furthermore, metabolomic analysis revealed a correlation of Lactobacillus fermentum dominance with pH value and lactic and acetic acid levels, with high R values of 0.643, -0.642, and 0.528, respectively. The data reported in this study showed the characteristics of the bacterial composition in the unsalted sunki pickle and its potential relationship with the compositional profile.

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation.

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.

NMR- and GC/MS-based metabolomic characterization of sunki, an unsalted fermented pickle of turnip leaves.

This study revealed the compositional characteristics of sunki, a traditional, unsalted, lactic acid-fermented pickle produced using turnip leaf in Kiso district, Japan. Comprehensive compositional analysis by two metabolomic approaches based on NMR and solid-phase microextraction-GC/MS methods was used to determine its chemical composition by annotating 54 water-soluble and 62 volatile compounds. Principal component analysis showed that samples had different compositions, depending on the agricultural processing factory and production year. This variation potentially resulted from the differences in the lactic acid bacterial community produced during the spontaneous fermentation of sunki and in the initial nutritional composition of the turnip leaf. Partial least squares regression revealed that the acetic acid level showed a strong positive correlation with pH (R = 0.810), in contrast to the negative correlations of lactic acid and ethanol levels (R =  -0.533 and -0.547). This indicated the crucial impact of acetic acid-related metabolism on acidification during sunki fermentation.

Nuclear magnetic resonance- and gas chromatography/mass spectrometry-based metabolomic characterization of water-soluble and volatile compound profiles in cabbage vinegar.

Non-targeted metabolomic analyses employing nuclear magnetic resonance- and gas chromatography/mass spectrometry-based techniques were applied for an in-depth characterization of cabbage vinegar, an original agricultural product made from cabbage harvested in Tsumagoi, Japan. Water-soluble and volatile metabolite profiles of cabbage vinegar were compared with those of various vinegars: rice vinegar, grain vinegar, apple vinegar, and black vinegar (Japanese kurozu made of brown rice). Principal component analysis (PCA) of the water-soluble metabolites indicated that cabbage vinegars belonged to an isolated class by the contributions of fructose, pyroglutamic acid, choline, and methiin (S-methylcysteine sulfoxide). Regarding the volatile compounds, the PCA data represented that rice, black, and apple vinegars were characterized by most of the dominant volatiles, such as acetate esters, alcohols, ketones, and acids. Cabbage and grain vinegars were included in the same class although these two vinegars have different flavors. Orthogonal partial least squares-discrimination analysis exhibited the differences in volatile compound profile between cabbage and grain vinegars, revealing that cabbage vinegars were characterized by the presence of sulfides (dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide), nitriles (allyl cyanide and 4-methylthio-butanenitrile), 3-hexene-1-ol, and crotonic acid. The time-course changes in these highlighted compounds during the acetic acid fermentation of cabbage vinegar suggested that pyroglutamic and crotonic acids were produced through fermentation, whereas choline, methiin, sulfides, nitriles, and 3-hexene-1-ol were derived from cabbage, suggesting the key role of these compounds in the unique taste and flavor of cabbage vinegar.

Rapid discrimination of strain-dependent fermentation characteristics among Lactobacillus strains by NMR-based metabolomics of fermented vegetable juice.

In this study, we investigated the applicability of NMR-based metabolomics to discriminate strain-dependent fermentation characteristics of lactic acid bacteria (LAB), which are important microorganisms for fermented food production. To evaluate the discrimination capability, six type strains of Lactobacillus species and six additional L. brevis strains were used focusing on i) the difference between homo- and hetero-lactic fermentative species and ii) strain-dependent characteristics within L. brevis. Based on the differences in the metabolite profiles of fermented vegetable juices, non-targeted principal component analysis (PCA) clearly separated the samples into those inoculated with homo- and hetero-lactic fermentative species. The separation was primarily explained by the different levels of dominant metabolites (lactic acid, acetic acid, ethanol, and mannitol). Orthogonal partial least squares discrimination analysis, based on a regions-of-interest (ROIs) approach, revealed the contribution of low-abundance metabolites: acetoin, phenyllactic acid, p-hydroxyphenyllactic acid, glycerophosphocholine, and succinic acid for homolactic fermentation; and ornithine, tyramine, and γ-aminobutyric acid (GABA) for heterolactic fermentation. Furthermore, ROIs-based PCA of seven L. brevis strains separated their strain-dependent fermentation characteristics primarily based on their ability to utilize sucrose and citric acid, and convert glutamic acid and tyrosine into GABA and tyramine, respectively. In conclusion, NMR metabolomics successfully discriminated the fermentation characteristics of the tested strains and provided further information on metabolites responsible for these characteristics, which may impact the taste, aroma, and functional properties of fermented foods.

Prevention of GABA reduction during dough fermentation using a baker's yeast dal81 mutant.

γ-Aminobutyric acid (GABA) is consumed by yeasts during fermentation. To prevent GABA reduction in bread dough, a baker's yeast mutant AY77 deficient in GABA assimilation was characterized and utilized for wheat dough fermentation. An amber mutation in the DAL81 gene, which codes for a positive regulator of multiple nitrogen degradation pathways, was found in the AY77 strain. The qPCR analyses of genes involved in nitrogen utilization showed that transcriptional levels of the UGA1 and DUR3 genes encoding GABA transaminase and urea transporter, respectively, are severely decreased in the AY77 cells. The AY77 strain cultivated by fed-batch culture using cane molasses exhibited inferior gas production during dough fermentation compared to that of wild-type strain AY13. However, when fed with molasses containing 0.5% ammonium sulfate, the mutant strain exhibited gas production comparable to that of the AY13 strain. In contrast to the AY13 strain, which completely consumed GABA in dough within 5 h, the AY77 strain consumed no GABA under either culture condition. Dough fermentation with the dal81 mutant strain should be useful for suppression of GABA reduction in breads.

Identification of a gene, FMP21, whose expression levels are involved in thermotolerance in Saccharomyces cerevisiae.

Elucidation of the mechanism of high temperature tolerance in yeasts is important for the molecular breeding of high temperature-tolerant yeasts that can be used in bioethanol production. We identified genes whose expression is correlated with the degree of thermotolerance in Saccharomyces cerevisiae by DNA microarray analysis. Gene expression profiles of three S. cerevisiae strains showing different levels of thermotolerance were compared, and we chose three of them as candidate genes. Among these genes, FMP21 was investigated as a thermotolerance-related gene in S. cerevisiae by comparing the growth at high temperature with the gene expression in eight strains. The expression ratio of FMP21 at 37°C was correlated with the doubling time ratio at a coefficient of determination of 0.787. The potential involvement of the Fmp21 in the thermotolerance of yeasts was evaluated. The FMP21 deletion variant showed a decreased respiratory growth rate and increased thermosensitivity. Furthermore, the overexpression of FMP21 improved thermotolerance in yeasts. In conclusion, the function of Fmp21 is important for thermotolerance in yeasts.

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria.

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.

Tomato FRUITFULL homologs regulate fruit ripening via ethylene biosynthesis.

Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes.

Ethanol production by repeated-batch simultaneous saccharification and fermentation (SSF) of alkali-treated rice straw using immobilized Saccharomyces cerevisiae cells.

Repeated-batch simultaneous saccharification and fermentation (SSF) of alkali-treated rice straw using immobilized yeast was developed to produce ethanol. Saccharomyces cerevisiae cells were immobilized by entrapping in photocrosslinkable resin beads, and we evaluated the possibility of its reuse and ethanol production ability. In batch SSF of 20% (w/w) rice straw, the ethanol yields based on the glucan content of the immobilized cells were slightly low (76.9% of the theoretical yield) compared to free cells (85.2% of the theoretical yield). In repeated-batch SSF of 20% (w/w) rice straw, stable ethanol production of approx. 38gL(-1) and an ethanol yield of 84.7% were obtained. The immobilizing carrier could be reused without disintegration or any negative effect on ethanol production ability.

Identification of an acetate-tolerant strain of Saccharomyces cerevisiae and characterization by gene expression analysis.

A massive screening was performed to identify an acetate-tolerant strain of Saccharomyces cerevisiae. We found that S. cerevisiae ATCC 38555 is acetate-tolerant, with a fermentation profile indicating that it has a high level of acetate adaptation. The global gene expression analysis indicated that AFT1- and HAA1-regulated genes are clearly up-regulated.

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast.

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (D-xylose and L-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ D-xylose and L-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ D-xylose, and 10 g l⁻¹ L-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both D-xylose and L-arabinose.

Isolation of a novel strain of Candida shehatae for ethanol production at elevated temperature.

Considering the cost-effectiveness of bioethanol production, there is a need for a yeast strain which can convert glucose and xylose into ethanol at elevated temperatures. We succeeded in isolating a yeast strain, designated strain ATY839, which was capable of ethanolic fermentation at temperatures above those previously reported for yeasts able to ferment both glucose and xylose. Strain ATY839 was capable of producing a substantial amount of ethanol at up to 37°C from 2% glucose or 2% xylose. The results of a phylogenetic analysis suggest that strain ATY839 belongs to Candida shehatae. In additional, ethanol production from rice straw by strain ATY839 was examined. Compared with the control strains (Saccharomyces cerevisiae NBRC 0224, Scheffersomyces stipitis NBRC 10063, and C. shehatae ATCC 22984), strain ATY839 produced more ethanol in SSF even at 37°C. The theoretical maximum yield of strain ATY839 was 71.6% at 24 h. Thus, strain ATY839 is considered to be the most tolerant to high temperature of the C. shehatae strains.

A UV-induced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance.

In the fermentation process of lignocellulosic biomass (such as wood and rice straw), efficient conversion of pentose (mainly xylose) into ethanol is important. Mutants of Pichia stipitis NBRC1687 were obtained after UV mutagenesis and selection of large colonies on ethanol-containing medium. One mutant, PXF58, produced 4.3% ethanol from 11.4% xylose while the parent strain only produced 3.1%. The ethanol productivities of PXF58 from glucose and fructose were about were about 1.4-fold higher than those of the parent strain. After continuous cultivation of PXF58 in YNB (yeast nitrogen base) medium containing 2% xylose and 5-7% ethanol, an ethanol-tolerant mutant, PET41, was obtained. Strain PET41 was able to produce 4.4% ethanol when first supplied with xylose then with glucose. This isolate might be thus useful for two-phase fermentation in which xylan is saccharified by xylanase to produce xylose, and glucan is saccharified later by cellulase and ß-glucosidase to produce glucose.

Selection of stress-tolerant yeasts for simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash to ethanol.

Highly concentrated bioethanol production requires less volume in fermentation tanks and conserves distillery energy. We screened osmotolerant yeasts from a collection of 1699 yeast strains at our institute and found that three strains, NFRI3062, NFRI3213, and NFRI3225, were candidates for use in bioethanol production. All of these strains belonged to Saccharomyces cerevisiae. NFRI3062 produced 15.0% (w/v) of ethanol from YPD medium containing 35% glucose cultivated at 30 degrees C for 60 h, while S. cerevisiae NBRC0224, which has previously been reported suitable for ethanol production, only produced 13.0% (w/v). The thermotolerances of NFRI3213 and NFRI3225 were also superior to those of NBRC0224 and NFRI3062. We also demonstrated the simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash and sweet-potato mash. NFRI3225 produced ethanol from potato mash at the fastest rate and in the highest volume (13.7% (w/v)) among the tested strains. The maximum productivity and ethanol yields were 9.1g/L/h and 92.3%, respectively. Although the potato mash was not sterilized, bacterial contamination was not observed. This may have been due to the growth inhibition of bacteria by the rapid glucose consumption and ethanol production of NFRI3225 during the VHG-SSF process.

Strategy for simultaneous saccharification and fermentation using a respiratory-deficient mutant of Candida glabrata for bioethanol production.

High temperature and agitation are advantageous for ethanol production from insoluble feedstock when using the simultaneous saccharification and fermentation (SSF) technique. To construct an effective SSF system, a respiratory-deficient mutant was isolated from the thermotolerant yeast Candida glabrata. Our results suggest that this respiratory-deficient mutant has higher ethanol production abilities in SSF.

Multicopy suppression of oxidant-sensitive eos1 mutation by IZH2 in Saccharomyces cerevisiae and the involvement of Eos1 in zinc homeostasis.

EOS1 is required for tolerance to oxidative stress in Saccharomyces cerevisiae; mutants are defective in the gene sensitive to hydrogen peroxide and tolerant to tunicamycin. To clarify the function of Eos1, we screened yeast genomic DNA libraries for heterologous genes that, when overexpressed from a plasmid, can suppress the hydrogen peroxide-sensitive eos1 mutation. We identified one such gene, IZH2, which has previously been reported to be a Zap1-regulated gene. However, the EOS1 and IZH2 genes do not themselves appear to be functionally interchangeable. Double disruption of the EOS1 and IZH2 genes yielded a slow-growth phenotype, suggesting that the two proteins are involved in related cellular processes. DNA microarray analysis revealed decreased expression of Zap1-regulated genes in the eos1-deletion mutant (Deltaeos1). Thus, it is likely that Eos1 is involved in zinc homeostasis.

Prevention of bacterial contamination using acetate-tolerant Schizosaccharomyces pombe during bioethanol production from molasses.

Bacterial contamination causes yield reduction during ethanol production from molasses. To prevent contamination, construction of a fermentation system using acetate-tolerant yeast under an acetate-containing condition was attempted. Schizosaccharomyces pombe was screened as an acetate-tolerant strain. Bacterial contamination was significantly prevented by the combined use of Sc. pombe and acetate.

Involvement of ergosterol in tolerance to vanillin, a potential inhibitor of bioethanol fermentation, in Saccharomyces cerevisiae.

A vanillin-tolerant strain of Saccharomyces cerevisiae was screened and its intracellular ergosterol levels were compared with several laboratory yeast strains to study the potential relationship between ergosterol content and vanillin tolerance. Saccharomyces cerevisiae NBRC1950 was selected as a vanillin-tolerant strain. Its ergosterol content was higher than those of the laboratory strains. The results of DNA microarray and quantitative reverse transcriptase-polymerase chain reaction analysis showed that five genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5, and ERG7) were upregulated in NBRC 1950 compared with strain X2180, suggesting that high expression of genes involved in ergosterol biosynthesis may cause high ergosterol content in strain NBRC 1950. The S. cerevisiae HX strain, which was a high-ergosterol-containing strain derived from X2180, was more tolerant to vanillin than the parental strain, suggesting that high ergosterol content may, in part, be responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin.

Characterization of a spontaneous flocculation mutant derived from Candida glabrata: a useful strain for bioethanol production.

A spontaneous flocculation mutant (Cgflo1) was obtained from Candida glabrata. Flocculation of Cgflo1 was dependent on divalent cations such as Ca(2+) and Mg(2+) and was inhibited by galactose. Cgflo1 is believed to be a useful strain for bioethanol production based on its flocculation ability at higher temperatures.