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Alzheimer's disease (AD) is the most common dementia characterized by the excessive accumulation of amyloid-beta (Aβ) and tau aggregates, as well as neuronal damage and neuroinflammation. Metabolic disruption in AD has been noticed because metabolite alterations closely correlate with Aβ neuropathology and behavioral phenotypes. Accordingly, controlling various neuropathological processes and metabolic disruption is an efficient therapeutic strategy for AD treatment. In this study, we evaluated the effects of a combination of Cuscuta seeds and Lactobacillus paracasei NK112 (CCL01) on AD neuropathology and altered metabolism in five familial AD (5xFAD) transgenic mice and neuronal cell cultures. First, we observed that CCL01 exerted neuroprotective effects in HT22 hippocampal neurons and primary cultured neurons. CCL01 ameliorated memory decline and protected synapses and neuronal survival in 5xFAD mice. These effects were related to the inhibition of tau phosphorylation. CCL01 also inhibited the activation of mitogen-activated protein kinase (MAPK) signaling and neuroinflammatory processes. Moreover, the metabolite profile-particularly characterized by altered phospholipid metabolism-was significantly changed in the 5xFAD group, while CCL01 partly restored the alteration. Lysophosphatidylcholine (lysoPC), the levels of which were higher in the brains of 5xFAD mice, exerted neurotoxicity in vitro, whereas CCL01 protected neurons from lysoPC-induced toxicity by regulating MAPK signaling. Additionally, CCL01 administration reduced gut inflammation in the 5xFAD mice. In summary, we demonstrated that CCL01 improved the memory function of 5xFAD mice by protecting neurons against Aβ- and lysoPC-induced toxicity through the regulation of MAPK signaling, neuroinflammation, tau phosphorylation, and gut inflammation, suggesting the potential of CCL01 as treatment for AD.

Parkinson's disease (PD) is the second most common neurodegenerative disease, after Alzheimer's disease, and becomes increasingly prevalent with age. α-Synuclein (α-syn) forms the major filamentous component of Lewy bodies, which are pathological hallmarks of α-synucleinopathies such as PD. We evaluated the neuroprotective effects of MT101-5, a standardized herbal formula that consists of an ethanolic extract of Genkwae Flos, Clematidis Radix, and Gastrodiae Rhizoma, against α-synuclein-induced cytotoxicity in vivo. MT101-5 protected against behavioral deficits and loss of dopaminergic neurons in human α-syn-overexpressing transgenic mice after treatment with 30 mg/kg/day for 5 months. We investigated transcriptomic changes within MT101-5 mechanisms of action (MOA) suppressing α-syn aggregation in an α-synuclein preformed fibril (α-syn PFF) mouse model of sporadic PD. We found that inhibition of α-syn fibril formation was associated with changes in transcripts in mitochondrial biogenesis, electron transport, chaperones, and proteasomes following treatment with MT101-5. These results suggest that the mixed herbal formula MT101-5 may be used as a pharmaceutical agent for preventing or improving PD.

In this study, we investigated the altered enzymatic activities and metabolite profiles of koji fermented using varying permutations of Aspergillus oryzae and/or Bacillus amyloliquefaciens. Notably, the protease and β-glucosidase activities were manifold increased in co-inoculated (CO) koji samples (co-inoculation of A. oryzae and B. amyloliquefaciens). Furthermore, gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling indicates that levels of amino acids, organic acids, sugars, sugar alcohols, fatty acids, nucleosides, and vitamins were distinctly higher in CO, SA (sequential inoculation of A. oryzae, followed by B. amyloliquefaciens), and SB (sequential inoculation of B. amyloliquefaciens, followed by A. oryzae). The multivariate principal component analysis (PCA) plot based on GC-MS datasets indicated a clustered pattern for MA and MB (koji samples inoculated either with A. oryzae or B. amyloliquefaciens) across PC2 (20.0%). In contrast, the CO, SA, and SB metabolite profiles displayed segregated patterns across PLS1 (22.2%) and PLS2 (21.1%) in the partial least square discriminant analysis (PLS-DA) model. Intriguingly, the observed disparity in the levels of primary metabolites was engendered largely by higher relative levels of sugars and sugar alcohols in MA, SA, and CO koji samples, which was commensurate with the relative amylase activities in respective samples. Collectively, the present study emphasizes the utility of integrated biochemical and metabolomic approaches for achieving the optimal permutation of fermentative inocula for industrial koji preparation.

Meju, a type of fermented soybean paste, is used as a starter in the preparation of various Korean traditional soybean-based foods. In this study, we performed Illumina-MiSeq paired-end sequencing for microbial communities and mass spectrometry analysis for metabolite profiling to investigate the differences between 11 traditional meju products from different regions across Korea. Even though the bacterial and fungal communities showed remarkable variety, major genera including Bacillus, Enterococcus, Variovorax, Pediococcus, Weissella, and Aspergillus were detected in every sample of meju. The metabolite profile patterns of the 11 samples were clustered into two main groups: group I (M1-5) and group II (M6-11). The metabolite analysis indicated a relatively higher amino acid content in group I, while group II exhibited higher isoflavone, soyasaponin, and lysophospholipid contents. The bioactivity analysis proved that the ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical-scavenging activity was higher in group II and the FRAP (ferric reducing antioxidant power) activity was higher in group I. The correlation analysis revealed that the ABTS activity was isoflavonoid, lipid, and soyasaponin related, whereas the FRAP activity was amino acid and flavonoid related. These results suggest that the antioxidant activities of meju are critically influenced by the microbiome and metabolite dynamics.

Production of good Koji primarily depends upon the selection of substrate materials and fermentative microflora, which together influence the characteristic flavor and aroma. Herein, we performed comparative metabolomic analyses of volatile organic compounds (VOCs) and primary metabolites for Koji samples fermented individually with Bacillus amyloliquefaciens and Aspergillus oryzae. The VOCs and primary metabolites were analyzed using headspace solid phase microextraction (HS-SPME) followed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). In particular, alcohols, ketones, and furans were mainly detected in Bacillus-fermented Koji (Bacillus Koji, BK), potentially due to the increased levels of lipid oxidation. A cheesy and rancid flavor was characteristic of Bacillus Koji, which is attributable to high content of typical 'off-flavor' compounds. Furthermore, the umami taste engendered by 2-methoxyphenol, (E,E)-2,4-decadienal, and glutamic acid was primarily detected in Bacillus Koji. Alternatively, malty flavor compounds (2-methylpropanal, 2-methylbutanal, 3-methylbutanal) and sweet flavor compounds (monosaccharides and maltol) were relatively abundant in Aspergillus-fermented Koji (Aspergillus Koji, AK). Hence, we argue that the VOC profile of Koji is largely determined by the rational choice of inocula, which modifies the primary metabolomes in Koji substrates, potentially shaping its volatolome as well as the aroma characteristics.

The stochasticity of Aspergillus oryzae (Trivially: the koji mold) pan-metabolomes commensurate with its ubiquitously distributed landscapes, i.e. growth matrices have been seemed uncharted since its food fermentative systems are mostly being investigated. In this review, we explicitly have discussed the likely tendencies of A. oryzae metabolomes pertaining to its growth milieu formulated with substrate matrices of varying nature, composition, texture, and associated physicochemical parameters. We envisaged typical food matrices, namely, meju, koji, and moromi as the semi-natural cultivation models toward delineating the metabolomic patterns of the koji mold, which synergistically influences the organoleptic and functional properties of the end products. Further, we highlighted how tailored conditions in sub-natural growth matrices, i.e. synthetic cultivation media blends, inducers, and growth surfaces, may influence A. oryzae metabolomes and targeted phenotypes. In general, the sequential or synchronous growth of A. oryzae on formulated matrices results in a number of metabolic tradeoffs with its immediate microenvironment influencing its adaptive and regulatory metabolomes. In broader context, evaluating the metabolic plasticity of A. oryzae relative to the tractable variables in formulated growth matrices might help approximate its growth and metabolism in the more complex natural matrices and environs. These approaches may considerably help in the design and manipulation of hybrid cultivation systems towards the efficient harnessing of commercial molds.

Volatile organic compounds (VOCs) are increasingly been recognized as the chemical mediators of mold interactions, shaping their community dynamics, growth, and metabolism. Herein, we selectively examined the time-correlated (0 D-11 D, where D = incubation days) effects of intraspecies VOC-mediated interactions (VMI) on Aspergillus oryzae KCCM 60345 (S1), following co-cultivation with partner strain A. oryzae KACC 44967 (S2), in a specially designed twin plate assembly. The comparative evaluation of S1VMI (S1 subjected to VMI with S2) and its control (S1Con) showed a notable disparity in their radial growth (S1VMI S1Con) at 3-5 D, amylase activity (S1VMI S1Con) at 3 D. Furthermore, we observed a distinct clustering pattern for gas chromatography-time of flight-mass spectrometry datasets from 5 D extracts of S1VMI and S1Con in principle component analysis (PC1: 30.85%; PC2: 10.31%) and partial least squares discriminant analysis (PLS-DA) (PLS1: 30.77; PLS2: 10.15%). Overall, 43 significantly discriminant metabolites were determined for engendering the metabolic variance based on the PLS-DA model (VIP > 0.7, p S1Con) at 5 D, organic acids (S1VMI > S1Con) at 5 D, and kojic acid (S1VMI < S1Con) at 5-7 D were observed. Examining the headspace VOCs shared between S1 and S2 in the twin plate for 5 D incubated samples, we observed the relatively higher abundance of C-8 VOCs (1-octen-3-ol, (5Z)-octa-1,5-dien-3-ol, 3-octanone, 1-octen-3-ol acetate) having known semiochemical functions. The present study potentially illuminates the effects of VMI on commercially important A. oryzae’s growth and biochemical phenotypes with subtle details of altered metabolomes.

Two different doenjang manufacturing processes, the industrial process (IP) and the modified industrial process (mIP) with specific microbial assortments, were subjected to metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The multivariate analyses indicated that both primary and secondary metabolites exhibited distinct patterns according to the fermentation processes (IP and mIP). Microbial community analysis for doenjang using denaturing gradient gel electrophoresis (DGGE), exhibited that both bacteria and fungi contributed proportionally for each step in the process viz., soybean, steaming, drying, meju fermentation, cooling, brining, and aging. Further, correlation analysis indicated that Aspergillus population was linked to sugar metabolism, Bacillus spp. with that of fatty acids, whereas Tetragenococcus and Zygosaccharomyces were found associated with amino acids. These results suggest that the components and quality of doenjang are critically influenced by the microbial assortments in each process.

Short-term abiotic stress treatment before harvest can enhance the quality of horticultural crops cultivated in controlled environments. Here, we investigated the effects of combined chilling and UV-A treatment on the accumulation of phenolic compounds in kale (Brassica oleracea var. acephala). Five-week-old plants were subjected to combined treatments (10 &deg, C plus UV-A LED radiation at 30.3 W/m2) for 3-days, as well as single treatments (4 &deg, C, 10 &deg, C, or UV-A LED radiation). The growth parameters and photosynthetic rates of plants under the combined treatment were similar to those of the control, whereas UV-A treatment alone significantly increased these parameters. Maximum quantum yield (Fv/Fm) decreased and H2O2 increased in response to UV-A and combined treatments, implying that these treatments induced stress in kale. The total phenolic contents after 2- and 3-days of combined treatment and 1-day of recovery were 40%, 60%, and 50% higher than those of the control, respectively, and the phenylalanine ammonia-lyase activity also increased. Principal component analysis suggested that stress type and period determine the changes in secondary metabolites. Three days of combined stress treatment followed by 2-days of recovery increased the contents of quercetin derivatives. Therefore, combined chilling and UV-A treatment could improve the phenolic contents of leafy vegetables such as kale, without growth inhibition.

Aim To identify the active compounds from the Streptomyces sp. MJM4426 that can protect rice from bacterial blight disease (BB), and to evaluate the potential of this Streptomyces strains and the compound for biocontrol of rice bacterial blight disease. Methods and results The ethyl acetate extract of Streptomyces sp. MJM4426 can significantly protect rice leaf explants from the infection of Xanthomonas oryzae pv. oryzaeKACC 10331 (Xoo), the pathogen which cause BB. To identify the active compounds, the ethyl acetate extract of Streptomyces sp. MJM4426 was fractionated through a Sephadex LH-20 column chromatography, and further purified by preparative HPLC guided by the inhibitory activity against BB in rice leaf explants. UPLC-Q-TOF/MS analysis showed the active compound displayed its m/z values at [M+H](+) 467·2086 and [M+FA-H](-) 511·1963, and the molecular formula was estimated as C28 H26 N4 O3 which is identical to commercial standard staurosporine. In this study, the isolated staurosporine dramatically suppressed bacterial blight in rice leaf explants with the lowest concentration at 12·5 μmol l(-1) , however, it exhibited low inhibitory activity against Xoo with the MIC value at 256 μg ml(-1) . In addition, greenhouse study showed both crude extract and purified staurosporine can suppress the bacterial blight at the concentration of 5000 and 200 μg ml(-1) respectively. Conclusion Streptomyces sp. MJM4426 can protect rice leaf explants from the infection of Xoo by producing staurosporine, but not by direct inhibitory activity against Xoo. Significance and impact of the study This is the first report that staurosporine can protect rice leaf against bacterial blight disease and showed the potential of Streptomyces sp. MJM4426 as an alternative to chemical bactericide for bacterial blight disease in rice.

Cristazine (1), a new class of dioxopiperazine alkaloid, along with previously isolated chetomin (2), neoechinulin A (3), and golmaenone (4), were isolated from the mudflat-sediment-derived fungus Chaetomium cristatum. The structure and absolute stereochemistry of 1 was assigned on the basis of NMR, electron impact (EI)-MS, tandem FAB-MS/MS, and circular dichroism (CD) experiments. Compounds 1-4 displayed potent radical-scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), with IC50 values of 19, 15, 24, and 20 µM, respectively, which were similar to that of the positive control, ascorbic acid (IC50, 20 µM). Compound 1 also displayed cytotoxic activity against human cervical carcinoma (HeLa) cells, with an IC50 value of 0.5 µM.

Ultraviolet B (UV-B) light, as a physical elicitor, can promote the secondary metabolites biosynthesis in plants. We investigated effects of different energy levels of UV-B radiation on growth and bioactive compounds of Crepidiastrum denticulatum. Three-week-old seedlings were grown in a plant factory for 5 weeks. Plants were subjected to different levels of UV-B (0, 0.1, 0.25, 0.5, 1.0, and 1.25 W m&minus, 2), 6 h a day for 6 days. All UV-B treatments had no negative effect on the shoot dry weight, however, relatively high energy treatments (1.0 and 1.25 W m&minus, 2) inhibited the shoot fresh weight. UV-B light of 0.1, 0.25, and 0.5 W m&minus, 2 did not affect total chlorophyll and H2O2 contents, however, they increased total carotenoid content. On 4 days, 0.25 W m&minus, 2 treatment increased antioxidant capacity, total hydroxycinnamic acids (HCAs) content, and several sesquiterpenes. Treatments with 1.0 and 1.25 W m&minus, 2 increased total carotenoid, total HCAs, and H2O2 contents, and destroyed chlorophyll pigments, reducing maximum quantum yield of photosystem II and causing visible damage to leaves. Partial least squares discrimination analysis (PLS-DA) showed that secondary metabolites were distinguishably changed according to energy levels of UV-B. The potential of 0.25 W m&minus, 2 UV-B for the efficient production of bioactive compounds without growth inhibition in C. denticulatum was identified.

Red drupelet is a postharvest disorder of blackberries with several drupelets turning back to red. This affects visual quality and thus marketability and consumers’ acceptance. However, the cause of this disorder as well as metabolite changes during color reversion have not been fully understood. Anthocyanins, cyanidin 3-glucoside, cyanidin 3-malonylglucoside, cyanidin 3-dioxalylglucoside, and total anthocyanin, were significantly lower in red drupelets than in black drupelets after 7 days of storage. Sugars and organic acids, lipids, and free amino acids also changed with storage and by color reversion. The untargeted metabolomics analyses indicated that red drupelets were generally differentiated from berries at harvest or black drupelets at metabolite level. The results of this study help better understand the red drupelet disorder. To our knowledge, this is the first study investigating red drupelet disorder by comparing black and red drupelets at metabolite level.

Untargeted metabolomics unraveled the effects of varying substrates (soybean, wheat, and rice) and inocula (Aspergillus oryzae and Bacillus amyloliquefaciens) on metabolite compositions of koji, a starter ingredient in various Asian fermented foods. Multivariate analyses of the hyphenated mass spectrometry datasets for different koji extracts highlighted 61 significantly discriminant primary metabolites (sugars and sugar alcohols, organic acids, amino acids, fatty acids, nucleosides, phenolic acids, and vitamins) according to varying substrates and inocula combinations. However, 59 significantly discriminant secondary metabolites were evident for koji-types with varying substrates only, viz., soybean (flavonoids, soyasaponins, and lysophospholipids), wheat (flavones and lysophospholipids), and rice (flavonoids, fatty acids derivatives, and lysophospholipids). Independently, the substrates influenced primary metabolite compositions in koji (soybean > wheat, rice). The inocula choice of A. oryzae engendered higher carbohydrates, organic acids, and lipid derivative levels commensurate with high α-amylase and β-glucosidase activities, while B. amyloliquefaciens affected higher amino acids levels, in respective koji types.

In this study, a comprehensive metabolite profile analysis of doenjang, a fermented soybean paste, at various steps of its industrial 5-step production process was conducted, by combining gas and liquid chromatography-mass spectrometry techniques with multivariate analysis. From the partial least squares discriminant analysis of primary and secondary metabolites, the patterns were clearly distinguishable between the various processing steps (step 1: steaming, step 2: drying, step 3: meju fermentation, step 4: brining, step 5: doenjang aging). Of the primary metabolites, most of the monosaccharides, amino acids, and fatty acids increased in steps 3-5. Isoflavone and soyasaponin derivatives were major secondary metabolites identified during the processing of doenjang. Isoflavone glycosides gradually decreased after step 1, while isoflavone aglycones distinctly increased in steps 4-5. Soyasaponins generally decreased during processing after step 2. Increased isoflavone aglycones, such as daidzein, glycitein, and genistein, were observed in steps 4-5 showed the strongest positive correlation with doenjang's antioxidant potential and total phenolic content.

Glucosinolates, their hydrolysis products and primary metabolites were analyzed in five pak choi cultivars to determine the effect of methyl jasmonate (MeJA) on metabolite flux from primary metabolites to glucosinolates and their hydrolysis products. Among detected glucosinolates (total 14 glucosinolates; 9 aliphatic, 4 indole and 1 aromatic glucosinolates), indole glucosinolate concentrations (153–229%) and their hydrolysis products increased with MeJA treatment. Changes in the total isothiocyanates by MeJA were associated with epithiospecifier protein activity estimated as nitrile formation. Goitrin, a goitrogenic compound, significantly decreased by MeJA treatment in all cultivars. Changes in glucosinolates, especially aliphatic, significantly differed among cultivars. Primary metabolites including amino acids, organic acids and sugars also changed with MeJA treatment in a cultivar-specific manner. A decreased sugar level suggests that they might be a carbon source for secondary metabolite biosynthesis in MeJA-treated pak choi. The result of the present study suggests that MeJA can be an effective agent to elevate indole glucosinolates and their hydrolysis products and to reduce a goitrogenic compound in pak choi. The total glucosinolate concentration was the highest in “Chinese cabbage” in the control group (32.5 µmol/g DW), but indole glucosinolates increased the greatest in “Asian” when treated with MeJA.

A time-resolved non-targeted metabolomic study towards evaluating the effects of three rice substrate types viz., white rice (WR), giant embryo rice (GER), and brown rice (BR), on Koji fermentation was performed. Notwithstanding the relatively higher proportions of metabolites in unfermented BR (BR 0>WR 0, GER 0), the relative levels of metabolic repertoire in BR Koji were marginally enhanced during 36h fermentation except those for phenolic acids, fatty acids, and vitamins. The WR Koji showed higher levels of lysophospholipids (LysoPC's) and protease-released amino acids (WR 36>GER 36>BR 36). The higher β-glucosidase activity in GER Koji effected the increased levels of sugars and flavonoid aglycons complementing its higher antioxidant activity (GER Koji>BR Koji∼WR Koji). The present study holistically underpins the dynamic metabolomes and enzymatic states during rice Koji fermentation with varying substrate types. The present study finds applications in optimization of commercial Koji production.

Key message Using Illumina sequencing technology, we have generated the large-scale transcriptome sequencing data containing abundant information on genes involved in the metabolic pathways in R. idaeus cv. Nova fruits. Abstract Rubus idaeus (Red raspberry) is one of the important economical crops that possess numerous nutri- ents, micronutrients and phytochemicals with essential health benefits to human. The molecular mechanism underlying the ripening process and phytochemical bio- synthesis in red raspberry is attributed to the changes in gene expression, but very limited transcriptomic and genomic information in public databases is available. To address this issue, we generated more than 51 million sequencing reads from R. idaeus cv. Nova fruit using Illumina RNA-Seq technology. After de novo assembly, we obtained 42,604 unigenes with an average length of 812 bp. At the protein level, Nova fruit transcriptome showed 77 and 68 % sequence similarities with Rubus coreanus and Fragaria versa, respectively, indicating the evolutionary relationship between them. In addition, 69 % of assembled unigenes were annotated using public dat- abases including NCBI non-redundant, Cluster of Orthol- ogous Groups and Gene ontology database, suggesting that our transcriptome dataset provides a valuable resource for investigating metabolic processes in red raspberry. To analyze the relationship between several novel transcripts and the amounts of metabolites such as c-aminobutyric acid and anthocyanins, real-time PCR and target metabolite analysis were performed on two different ripening stages of Nova. This is the first attempt using Illumina sequencing platform for RNA sequencing and de novo assembly of Nova fruit without reference genome. Our data provide the most comprehensive transcriptome resource available for Rubus fruits, and will be useful for understanding the rip- ening process and for breeding R. idaeus cultivars with improved fruit quality.

Plant abiotic stress tolerance has been modulated by engineering the trehalose synthesis pathway. However, many stress-tolerant plants that have been genetically engineered for the trehalose synthesis pathway also show abnormal development. The metabolic intermediate trehalose 6-phosphate has the potential to cause aberrations in growth. To avoid growth inhibition by trehalose 6-phosphate, we used a gene that encodes a bifunctional in-frame fusion (BvMTSH) of maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH) from the nonpathogenic bacterium Brevibacterium helvolum. BvMTS converts maltooligosaccharides into maltooligosyltrehalose and BvMTH releases trehalose. Transgenic rice plants that over-express BvMTSH under the control of the constitutive rice cytochrome c promoter (101MTSH) or the ABA-inducible Ai promoter (105MTSH) show enhanced drought tolerance without growth inhibition. Moreover, 101MTSH and 105MTSH showed an ABA-hyposensitive phenotype in the roots. Our results suggest that over-expression of BvMTSH enhances drought-stress tolerance without any abnormal growth and showes ABA hyposensitive phenotype in the roots. [BMB Reports 2014; 47(1): 27-32]

This study aimed to investigate the biotransformation of soybean isoflavones to hydroxyisoflavones, and the primary and secondary metabolite change during Aspergillus oryzae KACC40247-mediated fermentation by gas chromatography–time of flight-mass spectrometry and LC-MS with multivariate analysis. The mass spectrometric analysis revealed that acetylglycosides and glycosides decreased during the first 12 h of fermentation, while the aglycones increased up to that time point. This was followed by a decrease in aglycone levels due to the formation of hydroxyisoflavones. The hydroxyflavones, 8-hydroxydaidzein, hydroxygenistein, and hydroxyglycitein, resulting from the biotransformation of the corresponding aglycones, increased up to 24 h, and then subsequently decreased. During fermentation, the levels of monosaccharides, aspartic acid, pyroglutamic acid, gamma-aminobutyric acid, and organic acids gradually decreased, whereas the levels of threonine, serine, and glycine increased. Hydroxyisoflavone was more strongly correlated with antioxidant activity than the other metabolites. Our results suggest that biotransformation has the potential to improve the nutritional properties of soy-based food.

The microbial community and the metabolites of barley nuruk were studied to determine the time-dependent correlation between the fermentation of microbes and metabolites. Samples were analyzed by a polyphasic approach based on culture-dependent, culture-independent (PCR-DGGE and qPCR analysis), and metabolite analysis using GC-MS. Barley nuruk consists of varying amounts of bacteria, yeasts, and molds. The PCR-DGGE results showed that only one phylotype, Aspergillus oryzae, was predominant throughout fermentation, reaching a maximum on day 9. The bacterial load was higher on day 6 of fermentation, and then gradually decreased because of increased fungal activity. The shift in fungal and bacterial diversity observed by DGGE was further confirmed by qPCR analysis. In addition, microbes closely related to Pantoea agglomerans and Saccharomycopsis fibuligera appeared to play key roles in the fermentation of barley nuruk. GC-MS analysis combined with multivariate analysis, including PCA, PLS-DA, and OPLS-DA, showed fermentation time-dependent metabolite patterns. A total of 21 metabolites, including organic acids, amino acids, sugars, and sugar alcohols, were identified. In particular, glycerol, malic acid, fructose, glucose, sucrose, and maltose were produced at the early fermentation stages (0-6 d), whereas glutamine, aspartic acid, glutamic acid, mannitol, and xylitol were produced during the latter stages of fermentation (9-18 d). Mixed culture fermentation was found throughout the natural fermentation of barley nuruk starter. Most likely, A. oryzae had a major role in saccharification, along with other mixed cultures.

Bacterial blight, an important and potentially destructive bacterial disease in rice caused by Xanthomonas oryzae pv. oryzae (Xoo), has recently developed resistance to the available antibiotics. In this study, mass spectrometry (MS)-based metabolite profiling and multivariate analysis were employed to investigate the correlation between time-dependent metabolite changes and antimicrobial activities against Xoo over the course of Phomopsis longicolla S1B4 fermentation. Metabolites were clearly differentiated based on fermentation time into phase 1 (days 4-8) and phase 2 (days 10-20) in the principal component analysis (PCA) plot. The multivariate statistical analysis showed that the metabolites contributing significantly for phases 1 and 2 were deacetylphomoxanthone B, monodeacetylphomoxanthone B, fusaristatin A, and dicerandrols A, B, and C as identified by liquid chromatography-mass spectrometry (LC-MS), and dimethylglycine, isobutyric acid, pyruvic acid, ribofuranose, galactofuranose, fructose, arabinose, hexitol, myristic acid, and propylstearic acid were identified by gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling. The most significantly different secondary metabolites, especially deacetylphomoxanthone B, monodeacetylphomoxanthone B, and dicerandrol A, B and C, were positively correlated with antibacterial activity against Xoo during fermentation.

The metabolite profile of meju during fermentation was analyzed using mass spectrometry techniques, including GC-MS and LC-MS, and the bacterial diversity was characterized. The relative proportions of bacterial strains indicated that lactic acid bacteria, such as Enterococcus faecium and Leuconostoc lactis, were the dominant species. In partial least-squares discriminate analysis (PLS-DA), the componential changes, which depended on fermentation, proceeded gradually in both the GC-MS and LC-MS data sets. During fermentation, lactic acid, amino acids, monosaccharides, sugar alcohols, and isoflavonoid aglycones (daidzein and genistein) increased, whereas citric acid, glucosides, and disaccharides decreased. MS-based metabolite profiling and bacterial diversity characterization of meju demonstrated the changes in metabolites according to the fermentation period and provided a better understanding of the correlation between metabolites and bacterial diversity.

The metabolic perplexes for gochujang (GCJ) fermentative bioprocess, a traditional Korean pepper paste, has largely remain equivocal for preparative conditions and raw material (RM) additives exacerbating its commercial standardization. Herein, we outlined a differential non-targeted metabolite profiling for three GCJ (white rice-WR; brown rice-BR; wheat-WT) under varying processing steps (P1 - fermentation; P2 - meju addition; P3 - ripening; and P4 - red pepper addition). We correlated the process specific metabolomes with corresponding physicochemical factors, enzymatic phenotypes, and bioactivities for GCJ-types. The P1 was characterized by a uniform increase in the levels of RM-derived lysoPCs. In contrast, P2 was observed with proportionally higher levels of meju-released isoflavones and soyasaponins in WR-GCJ, followed by BR and WT-GCJ. The P3 involved a cumulative increase in primary metabolites in all GCJ samples except lower organic acid contents in WT-GCJ. The pepper derived flavonoids and alkaloids were selectively increased while P4 in all GCJ-types.

The structure and absolute stereochemistry of cristazine (I), a new class of dioxopiperazine alkaloid, is presented.

A multi-parallel approach gauging the mass spectrometry-based metabolite fingerprinting coupled with bioactivity and pathway evaluations could serve as an efficacious tool for inferring plant taxonomic orders. Thirty-four species from three plant families, namely Cornaceae (7), Fabaceae (9), and Rosaceae (18) were subjected to metabolite profiling using gas chromatography–time-of-flight-mass spectrometry (GC–TOF-MS) and ultrahigh performance liquid chromatography–linear trap quadrupole-ion trap-mass spectrometry (UHPLC–LTQ-IT-MS/MS), followed by multivariate analyses to determine the metabolites characteristic of these families. The partial least squares discriminant analysis (PLS-DA) revealed the distinct clustering pattern of metabolites for each family. The pathway analysis further highlighted the relatively higher proportions of flavonols and ellagitannins in the Cornaceae family than in the other two families. Higher levels of phenolic acids and flavan-3-ols were observed among species from the Rosaceae family, while amino acids, flavones, and isoflavones were more abundant among the Fabaceae family members. The antioxidant activities of plant extracts were measured using ABTS, DPPH, and FRAP assays, and indicated that extracts from the Rosaceae family had the highest activity, followed by those from Cornaceae and Fabaceae. The correlation map analysis positively links the proportional concentration of metabolites with their relative antioxidant activities, particularly in Cornaceae and Rosaceae. This work highlights the pre-eminence of the multi-parallel approach involving metabolite profiling and bioactivity evaluations coupled with metabolic pathways as an efficient methodology for the evaluation of plant phylogenies.

Consumption of fruits and vegetables is correlated with a lower incidence of cancer. Here, we identified hyperoside as an active compound from Acanthopanax sessiliflorus , and investigated the effect of hyperoside on UVB-induced transactivation of activator protein 1 (AP-1) and on the mitogen-activated protein kinase signalling pathway in JB6 P + cells. Hyperoside inhibited UVB-induced AP-1 transactivation. It inhibited the UVB-induced phosphorylation of p90 RSK . Kinase assays revealed that hyperoside significantly inhibited ERK1/2 activity. Furthermore, hyperoside bound to ERK1/2 to suppress its activity. In addition, phosphorylation of cAMP response element binding protein (CREB) and signal transducers and activators of transcription (STAT) 3 were suppressed by hyperoside. Overall, these results indicate that hyperoside may be a promising chemopreventive agent that acts by suppressing the transactivation of AP-1 and the phosphorylation of p90 RSK , CREB, and STAT3 through the binding and inhibition of ERK1/2.

In this study, the metabolite profiles of three Aspergillus strains during rice koji fermentation were compared. In the partial least squares discriminant analysis-based gas chromatography-mass spectrometry data sets, the metabolite patterns of A. oryzae (KCCM 60345) were clearly distinguished from A. kawachii (KCCM 60552) and only marginal differences were observed for A. oryzae (KCCM 60551) fermentation. In the 2 days fermentation samples, the overall metabolite levels of A. oryzae (KCCM 60345) were similar to the A. oryzae (KCCM 60551) levels and lower than the A. kawachii (KCCM 60552) levels. In addition, we identified discriminators that were mainly contributing tyrosinase inhibition (kojic acid) and antioxidant activities (pyranonigrin A) in A. oryzae (KCCM 60345) and A. kawachii (KCCM 60552) inoculated rice koji, respectively. In this study, we demonstrated that the optimal inoculant Aspergillus strains and fermentation time for functional rice koji could be determined through a metabolomics approach with bioactivity correlations.

Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating pathogen to Oryza sativa and has been shown to cause bacterial blight. Two bioactive compounds showing antimicrobial activities against Xoo strain KACC 10331 were isolated from a Streptomyces bottropensis strain. The ethyl acetate extract was fractionated on a Sephadex LH-20 column, and then purified by preparative HPLC. The purified compounds were identified as bottromycin A2 and dunaimycin D3S by HR/MS and 1H NMR analyses. The MIC value against Xoo and the lowest concentration still capable of suppressing rice bacterial blight were 2 microgram/ml and 16 microgram/ml for bottromycin A2, and 64 microgram/ml and 0.06 microgram/ml for dunaimycin D3S, respectively. These two compounds were shown to exert different bioactivities in vitro and in rice leaf explants.

Chana series are new chalcone derivatives. To evaluate the possibility of Chana series as therapeutic agents of type 2 diabetes, the inhibitory effects of Chana series on the activities of α-glucosidase and DPP-4 were investigated using in vitro enzyme assays, and their effects on adipocyte differentiation were investigated in C3H10T1/2 cells. Chana 1 and Chana 7 among the Chana series showed significant inhibition of α-glucosidase activity. In DPP-4 enzyme assay, Chana 1 exhibited the highest inhibitory activity while Chana 7 did not. In MTT assay, Chana 1 did not show significant cytotoxicity up to a concentration of 250 μM, whereas cytotoxicity was observed with Chana 7 at a concentration of 300 μM. In addition, Chana 1 induced adipocyte differentiation. Therefore, Chana 1 showed inhibitory effects on α-glucosidase and DPP-4 as well as a stimulatory effect on adipocyte differentiation, suggesting that Chana 1 may be a potential beneficial agent for the treatment of type 2 diabetes.

Liquid chromatography mass spectrometry and multivariate analysis were employed to investigate the correlation between fermentation time-dependent metabolite changes in cheonggukjang, a traditional fermented soybean product, and changes in its antioxidant activity over 72 h. The metabolite patterns were clearly distinguished not by strains but by fermentation time, into patterns I (0-12 h), II (12-24 h), and III (24-72 h), which appeared as distinct clusters on principal component analysis. The compounds that significantly contributed to patterns I, II, and III were soyasaponins, isoflavonoid derivatives, and isoflavonoid aglycons respectively. Partial least square analysis for metabolite to antioxidant effects showed correlations between the ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay during 24-36 h, and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) test and total phenol content (TPC) during 36-72 h. Compared with the strong negative correlations of glucosylated-isoflavonoids with DPPH, ABTS and TPC during fermentation, the isoflavonoid aglycon displayed strong positive correlations with these compounds during fermentation.

Liquid chromatography tandem mass spectrometry (LCMS/MS)-based metabolite profiling was applied to elucidate the correlation between metabolite release and antioxidant activity during water blanching of Chrysanthemum coronarium L. (CC). Some major metabolites showing differences between fresh CC and blanched CC (BCC) were selected by principal component analysis (PCA) and partial least-square discriminate analysis (PLS-DA) loading plots, and were identified as dicaffeoylquinic acid (DCQA), succinoyl-DCQA, and acetylmycosinol. By PLS regression analysis of the correlation between antioxidant components and effects, candidate antioxidative metabolites were predicted due to strong positive correlations with DCQA and succinoyl-DCQA, and by a relatively weak positive correlation with acetylmycosinol.

In this study, seven Trichoderma species (33 strains) were classified using secondary metabolite profile-based chemotaxonomy. Secondary metabolites were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) and multivariate statistical methods. T. longibrachiatum and T. virens were independently clustered based on both internal transcribed spacer (ITS) sequence and secondary metabolite analyses. T. harzianum formed three subclusters in the ITS-based phylogenetic tree and two subclusters in the metabolitebased dendrogram. In contrast, T. koningii and T. atroviride strains were mixed in one cluster in the phylogenetic tree, whereas T. koningii was grouped in a different subcluster from T. atroviride and T. hamatum in the chemotaxonomic tree. Partial least-squares discriminant analysis (PLS-DA) was applied to determine which metabolites were responsible for the clustering patterns observed for the different Trichoderma strains. The metabolites were hetelidic acid, sorbicillinol, trichodermanone C, giocladic acid, bisorbicillinol, and three unidentified compounds in the comparison of T. virens and T. longibrachiatum; harzianic acid, demethylharzianic acid, homoharzianic acid, and three unidentified compounds in T. harzianum I and II; and koninginin B, E, and D, and six unidentified compounds in T. koningii and T. atroviride. The results of this study demonstrate that secondary metabolite profiling-based chemotaxonomy has distinct advantages relative to ITSbased classification, since it identified new Trichoderma clusters that were not found using the latter approach.

Previously, we prepared a hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) containing undetectable amounts of glycyrrhizin, which is known to induce hypertension. This study assessed the effect of HEGU on inflammatory responses in lipopolysaccharide (LPS)-treated Raw264.7 macrophages and in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate. In the LPS-stimulated macrophages, HEGU (0–2 μg/ml) reduced nitric oxide (NO) release and the protein expression and transcriptional activity of inducible NO synthase (iNOS). HEGU reduced prostaglandin E 2 release and phospholipase A 2 transcripts. HEGU reduced the secretion and mRNA levels of tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. HEGU prevented IκBα degradation, p65 nuclear translocation, NFκB DNA binding and transcriptional activities. Additionally, dehydroglyasperin C, isolated from HEGU, reduced NO production, iNOS expression, and NFκB transcriptional activity. In the mouse inflammation model, HEGU suppressed skin swelling and iNOS and cyclooxygenase-2 expression. These results indicate that HEGU exerts powerful anti-inflammatory effects, probably mediated via the inhibition of NFκB signalling.

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography-mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between alpha6 and alpha7 helices of the active site and alpha9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the alpha7 and the alpha9 helices play an important role in positioning m-xylene, respectively.

Many studies have been carried out on bioactivities of individual herbs, however, no collective study on their comparative antioxidant and cytoprotective activities against oxidative damage has been reported. We selected 17 common commercial herbs and studied their relative phenolic contents, antioxidant activities, and cytoprotective activities on gap–junction intercellular communication and antioxidative enzymes in vitro under the same conditions. Total polyphenol content ranged from 464 to 870 gallic acid equivalents (GAE) mg/100 g and total flavonoid content from 212 to 494 catechin equivalents (CE) mg/100 g. Among the samples, chamomile, rosehip, hawthorn, lemon verbena, and green tea contained relatively high total phenolics (769–844 mg GAE/100 g) and flavonoids (400–4 mg CE/100 g). Chamomile also showed the highest antioxidant activity with 960 mg/100 g of vitamin C equivalent (VCE), followed by hawthorn (929 mg VCE/100 g) and black tea (916 mg VCE/100 g). Total phenolic and total flavonoids showed a higher correlation with antioxidant activity. Most of herbs enhanced cell viability and showed protective effects against oxidative stress induced by hydrogen peroxide in Chinese hamster lung fibroblast (V79-4) cells. Furthermore, herbs used in this study showed higher protective effect on gap–junction intercellular communication (GJIC) as compared to gallic acid and catechin, and also enhanced activity of the antioxidative enzymes such as superoxide dismutase (SOD) and catalase (CAT) in a dose-dependent manner.

Methanol extracts of Cheonggukjang, fermented for various times, were evaluated for their free-radical-scavenging and tyrosinase-inhibitory activities and the underlying mechanisms were elucidated. The free-radical-scavenging activity was highest for Cheonggukjang extracts fermented for 40 h and this decreased as the fermentation time shortened. The tyrosinase-inhibition activity increased with the length of fermentation. While levels of total phenolic compounds were highest in the 40-h-fermented Cheonggukjang samples, the total flavonoid content was lower compared to samples fermented for shorter times. Therefore, the antioxidative and tyrosinase-inhibition activities exhibited by methanol-extracted Cheonggukjang samples may be attributable to the contents of total phenolic compounds.

Secondary metabolite-based chemotaxonomic classification of Streptomyces (8 species, 14 strains) was performed using ultraperformance liquid chromatography-quadrupole-time-offlight- mass spectrometry with multivariate statistical analysis. Most strains were generally well separated by grouping under each species. In particular, S. rimosus was discriminated from t he r emaining s ev en s pecies ( S. coelicolor, S. griseus, S. indigoferus, S. peucetius, S. rubrolavendulae, S. scabiei, and S. virginiae) in partial least squares discriminant analysis, and oxytetracycline and rimocidin were identified as S. rimosus-specific metabolites. S. rimosus also showed high antibacterial activity against Xanthomonas oryzae pv. oryzae , the pathogen responsible for rice bacterial blight. This study demonstrated that metabolite-based chemotaxonomic classification is an effective tool for distinguishing Streptomyces spp. and for determining their species-specific metabolites.

A Gram-negative, non-motile, non-spore-forming and rod-shaped bacterial strain, DSW-6T, was isolated from sea water and subjected to a polyphasic study. Strain DSW-6T grew optimally at 25 degrees C, in the presence of 2 % (w/v) NaCl and at pH 7.0-8.0. It was characterized chemotaxonomically as having MK-6 as the predominant menaquinone and iso-C(15 : 0), anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and C(15 : 0) as the major fatty acids (10 % of total fatty acids). Major polar lipids were phosphatidylethanolamine, unidentified phospholipids and amino-group-containing lipids that are ninhydrin-positive. The DNA G+C content was 36.9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DSW-6(T) forms an independent line of descent within the family Flavobacteriaceae. The 16S rRNA gene sequence of strain DSW-6T exhibited similarity values of less than 94.7 % to the sequences of other members of the family Flavobacteriaceae. Strain DSW-6T could be distinguished from other phylogenetically related genera by differences in several phenotypic properties. Therefore, in view of the combined phenotypic and phylogenetic data, it is proposed that strain DSW-6T (= KCTC 12402T = DSM 17205T) represents a novel genus and species, Donghaeana dokdonensis gen. nov., sp. nov.

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein (2 μg/mouse) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.

A Gram-positive or Gram-variable, motile, endospore-forming, halophilic bacterial strain, MSS-402T, was isolated from a marine solar saltern in Korea, and subjected to a polyphasic taxonomic study. Some cells of strain MSS-402T were long filamentous rods. The isolate grew optimally at 37 °C and in the presence of 3–5 % (w/v) NaCl. Strain MSS-402T had cell-wall peptidoglycan based on l-orn–d-Asp, MK-7 as the predominant menaquinone and anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0 as major fatty acids. The DNA G+C content was 42·9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain MSS-402T falls within the evolutionary radiation of species of the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between strain MSS-402T and the type strains of recognized Halobacillus species ranged from 98·0 % (with Halobacillus halophilus) to 99·2 % (with Halobacillus litoralis and Halobacillus trueperi). Levels of DNA–DNA binding indicated that strain MSS-402T represents a genomic species that is distinct from recognized Halobacillus species. Strain MSS-402T was differentiated from Halobacillus species by means of several phenotypic characteristics. On the basis of its phenotypic properties and phylogenetic and genetic distinctiveness, strain MSS-402T (=KCTC 3957T=DSM 17110T) should be classified as the type strain of a novel Halobacillus species, for which the name Halobacillus yeomjeoni sp. nov. is proposed.

Two Gram-negative, non-motile, non-spore-forming, slightly halophilic bacterial strains, DSW-1T and DSW-21, were isolated from sea water and subjected to a polyphasic taxonomic study. They grew optimally at 30 °C and in the presence of 2 % (w/v) NaCl. Strains DSW-1T and DSW-21 were characterized chemotaxonomically as containing MK-6 as the predominant menaquinone and iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 as the major fatty acids. Their DNA G+C content was 38 mol%. Strains DSW-1T and DSW-21 exhibited four nucleotide differences in their 16S rRNA gene sequences and possessed a mean DNA–DNA relatedness level of 78 %. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strains DSW-1T and DSW-21 formed a distinct lineage within the family Flavobacteriaceae. The 16S rRNA gene sequences of strains DSW-1T and DSW-21 had similarity levels of less than 92·2 % to the sequences of other members of the family Flavobacteriaceae. There were some differences in phenotypic properties between the two strains and Cellulophaga species, the nearest phylogenetic neighbours. On the basis of phenotypic, phylogenetic and genetic data, strains DSW-1T (=KCTC 12391T=DSM 17200T) and DSW-21 were classified in a novel genus and species, Dokdonia donghaensis gen. nov., sp. nov.

A Gram-negative, non-motile, non-spore-forming, slightly halophilic bacterial strain, SW-238T, was isolated from sea water collected from the South Sea in Korea, and subjected to a polyphasic taxonomic study. The organism grew optimally at 25–30 °C and in the presence of 2–3 % (w/v) NaCl. Strain SW-238T was characterized chemotaxonomically as containing ubiquinone (Q-8) as the predominant respiratory lipoquinone and C18 : 1 ω9c as the major fatty acid. Its DNA G+C content was 47·6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SW-238T fell within the radiation of the cluster comprising Psychrobacter species. The 16S rRNA gene sequence of strain SW-238T had similarity levels of 94·8–97·9 % to sequences of the type strains of recognized Psychrobacter species. Levels of DNA–DNA relatedness between strain SW-238T and the type strains of 10 phylogenetically related Psychrobacter species were below 70 %. On the basis of phenotypic and phylogenetic data and genetic distinctiveness, strain SW-238T (=KCTC 12313T=JCM 12601T) was placed in the genus Psychrobacter as the type strain of a novel species, for which the name Psychrobacter celer sp. nov. is proposed.

A Gram-positive, rod- or coccus-shaped bacterial strain, KSL-104T, was isolated from an alkaline soil from Korea and its taxonomic position was investigated by a polyphasic approach. Strain KSL-104Tgrew optimally at pH 7·0–8·0 and 30 °C. It was characterized chemotaxonomically as having a cell-wall peptidoglycan type based onll-2,6-diaminopimelic acid with MK-8(H4) as the predominant menaquinone. The major fatty acid was iso-C16 : 0and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The DNA G+C content was 70·6 mol%. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain KSL-104Tjoined the cluster comprisingNocardioides jenseniiandMarmoricola aurantiacus. The cellular fatty acid profile of strain KSL-104Twas different from that ofM. aurantiacus. Strain KSL-104Tand the type strain ofN. jenseniiexhibited a 16S rRNA gene sequence similarity value of 97·1 % and a mean DNA–DNA relatedness value of 13 %. Levels of 16S rRNA gene sequence similarity between strain KSL-104Tand the type strains of otherNocardioidesspecies were in the range 94·0–96·5 %. On the basis of phenotypic, phylogenetic and genetic data, strain KSL-104T(=KCTC 9992T=JCM 13008T) represents a novel species of the genusNocardioides, for which the nameNocardioides dubiussp. nov. is proposed.

A Gram-negative, motile, slightly halophilic bacterial strain, SW-150T, was isolated from sea water of the Yellow Sea, Korea, and was characterized by a polyphasic taxonomic approach. Strain SW-150T grew optimally at 25–30 °C and in the presence of 2 % (w/v) NaCl. The isolate could be distinguished from other Sphingopyxis species in producing an orange pigment. It contained ubiquinone-10 as the predominant respiratory lipoquinone and C18 : 1 ω7c and C17 : 1 ω6c as the major fatty acids. No 3-hydroxy fatty acids were detected. Major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63 mol%. Comparative 16S rRNA gene sequence analyses showed that strain SW-150T was phylogenetically affiliated to the genus Sphingopyxis of the family Sphingomonadaceae. Similarity values between the 16S rRNA gene sequences of strain SW-150T and the type strains of Sphingopyxis species ranged from 91·6 to 94·2 %, making it possible to categorize strain SW-150T as a species that is separate from previously described Sphingopyxis species. On the basis of phenotypic properties and phylogenetic distinctiveness, SW-150T (=KCTC 12231T=DSM 16222T) should be classified as the type strain of a novel Sphingopyxis species, for which the name Sphingopyxis baekryungensis sp. nov. is proposed.

Two Gram-negative, non-motile, non-spore-forming, slightly halophilic bacterial strains, SW-210T and SW-242T, were isolated from sea water of the South Sea in Korea, and were characterized taxonomically by means of a polyphasic approach. The two isolates grew optimally at 25–30 °C and in the presence of 2–3 % (w/v) NaCl. Strains SW-210T and SW-242T contained Q-8 as the predominant respiratory lipoquinone and C18 : 1 ω9c as the major fatty acid. The DNA G+C contents of strains SW-210T and SW-242T were 43·2 and 45·3 mol%, respectively. The 16S rRNA gene sequences of strains SW-210T and SW-242T were 98·9 % similar, and the mean DNA–DNA relatedness value between them was 24 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains SW-210T and SW-242T form distinct phylogenetic lineages within the radiation of the cluster comprising Psychrobacter species, having 16S rRNA gene sequence similarity levels of 95·9–99·2 % with respect to the type strains of Psychrobacter species. The levels of DNA–DNA relatedness between the two isolates and the type strains of 15 phylogenetically related Psychrobacter species were well below 70 %. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains SW-210T and SW-242T were classified in the genus Psychrobacter as representing two distinct novel species, for which the names Psychrobacter aquimaris sp. nov. (type strain, SW-210T=KCTC 12254T=DSM 16329T) and Psychrobacter namhaensis sp. nov. (type strain, SW-242T=KCTC 12255T=DSM 16330T) are proposed, respectively.

Four Gram-positive, rod- or coccus-shaped bacterial strains, KSL-1T, KSL-9, KSL-10 and KSL-12, were isolated from an alkaline serpentinite soil in Korea, and their taxonomic positions were investigated in a polyphasic study. The four strains exhibited no difference in their 16S rRNA gene sequences. Phylogenetic analyses based on 16S rRNA gene sequences showed that the four strains were phylogenetically affiliated to the genus Nocardioides. The four strains had cell-wall peptidoglycan based on ll-diaminopimelic acid as the diamino acid, indicating wall chemotype I. The predominant menaquinone detected in the four strains was MK-8(H4). The major fatty acid components were iso-C16 : 0, 10-methyl-C18 : 0, C18 : 1 ω9c and C17 : 1 ω6c. The DNA G+C contents were 72·4–73·6 mol%. The four strains exhibited 16S rRNA gene sequence similarity levels of 94·0–96·3 % to the type strains of Nocardioides species with validly published names. DNA–DNA relatedness levels between the four strains were 85–91 %. On the basis of phenotypic properties, phylogenetic distinctiveness and genotypic relatedness, strains KSL-1T, KSL-9, KSL-10 and KSL-12 were classified in the genus Nocardioides as members of a novel species, Nocardioides alkalitolerans sp. nov. The type strain is strain KSL-1T (=KCTC 19037T=DSM 16699T).