Satoru Tomita

A NMR-based, non-targeted multistep metabolic profiling revealed L-rhamnitol as a metabolite that characterised apples from different geographic origins.

This study utilises (1)H NMR-based metabolic profiling to characterise apples of five cultivars grown either in Japan (Fuji, Orin, and Jonagold) or New Zealand (Fuji, Jazz, and Envy). Principal component analysis (PCA) showed a clear separation between the Fuji-Orin-Jonagold class and the Jazz-Envy class, primarily corresponding to the differences in sugar signals, such as sucrose, glucose, and fructose. Multistep PCA removed the influence of dominant sugars and highlighted minor metabolites such as aspartic acid, 2-methylmalate, and an unidentified compound. These minor metabolites separated the apples into two classes according to different geographical areas. Subsequent partial least squares discriminant analysis (PLS-DA) indicated the importance of the unidentified metabolite. This metabolite was isolated using charcoal chromatography, and was identified as L-rhamnitol by 2D NMR and LC/MS analyses. The remarkable contribution of L-rhamnitol to geographic discrimination suggests that apples may be characterised according to various factors, including storage duration, cultivation method, and climate.

Characterization of Leaf Blade- and Leaf Sheath-Associated Bacterial Communities and Assessment of Their Responses to Environmental Changes in CO2, Temperature, and Nitrogen Levels under Field Conditions

Rice shoot-associated bacterial communities at the panicle initiation stage were characterized and their responses to elevated surface water-soil temperature (ET), low nitrogen (LN), and free-air CO2 enrichment (FACE) were assessed by clone library analyses of the 16S rRNA gene. Principal coordinate analyses combining all sequence data for leaf blade- and leaf sheath-associated bacteria revealed that each bacterial community had a distinct structure, as supported by PC1 (61.5%), that was mainly attributed to the high abundance of Planctomycetes in leaf sheaths. Our results also indicated that the community structures of leaf blade-associated bacteria were more sensitive than those of leaf sheath-associated bacteria to the environmental factors examined. Among these environmental factors, LN strongly affected the community structures of leaf blade-associated bacteria by increasing the relative abundance of Bacilli. The most significant effect of FACE was also observed on leaf blade-associated bacteria under the LN condition, which was explained by decreases and increases in Agrobacterium and Pantoea, respectively. The community structures of leaf blade-associated bacteria under the combination of FACE and ET were more similar to those of the control than to those under ET or FACE. Thus, the combined effects of environmental factors need to be considered in order to realistically assess the effects of environmental changes on microbial community structures.

A survey of metabolic changes in potato leaves by NMR-based metabolic profiling in relation to resistance to late blight disease under field conditions.

Non‐targeted nuclear magnetic resonance (NMR)‐based metabolic profiling was applied to potato leaves to survey metabolic changes associated with late blight resistance under field conditions. Potato plants were grown in an experimental field, and the compound leaves with no visible symptoms were collected from 20 cultivars/lines at two sampling time points: (i) the time of initial presentation of symptoms in susceptible cultivars and (ii) 12 days before this initiation. 1H NMR spectra of the foliar metabolites soluble in deuterium oxide‐ or methanol‐d4‐based buffers were measured and used for multivariate analysis. Principal component analysis for six cultivars at symptom initiation showed a class separation corresponding to their levels of late blight resistance. This separation was primarily explained by higher levels of malic acid, methanol, and rutin and a lower level of sucrose in the resistant cultivars than in the susceptible ones. Partial least squares regression revealed that the levels of these metabolites were strongly associated with the disease severity measured in this study under field conditions. These associations were observed only for the leaves harvested at the symptom initiation stage, but not for those collected 12 days beforehand. Subsequently, a simple, alternative enzymatic assay for l‐malic acid was used to estimate late blight resistance, as a model for applying the potential metabolic marker obtained. This study demonstrated the potential of metabolomics for field‐grown plants in combination with targeted methods for quantifying marker levels, moving towards marker‐assisted screening of new cultivars with durable late blight resistance. Copyright

Dietary intake of heat-killed Lactococcus lactis H61 delays age-related hearing loss in C57BL/6J mice

Age-related hearing loss (AHL) is a common disorder associated with aging. In this study, we investigated the effect of the intake of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) on AHL in C57BL/6J mice. Measurement of the auditory brainstem response (ABR) demonstrated that female mice at 9 months of age fed a diet containing 0.05% strain H61 for 6 months maintained a significantly lower ABR threshold than control mice. The age-related loss of neurons and hair cells in the cochlea was suppressed by the intake of strain H61. Faecal analysis of bacterial flora revealed that the intake of strain H61 increased the prevalence of Lactobacillales, which is positively correlated with hearing ability in mice. Furthermore, plasma fatty acid levels were negatively correlated with hearing ability. Overall, the results supported that the intake of heat-killed strain H61 for 6 months altered the intestinal flora, affected plasma metabolite levels, including fatty acid levels, and retarded AHL in mice.

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.