Profiling volatile compounds from culture supernatants of periodontal bacteria using gas chromatography/mass spectrometry/olfactometry analysis with a monolithic silica gel adsorption device.

Halitosis is formed mainly by the volatile compounds produced by periodontal bacteria. Three volatile sulfur compounds (VSCs), hydrogen sulfide, methanethiol, and dimethyl sulfide, have attracted attention as major components of halitosis. However, these compounds cannot account for all odors. In this study, we profiled volatile compounds from the culture supernatants of periodontal bacteria using gas chromatography/mass spectrometry/olfactometry analysis with a monolithic silica gel adsorption device to investigate the potential odorous compounds. Periodontal bacteria have been found to produce volatile compounds belonging to various classes, such as alcohols, ketones, fatty acids, and aromatic compounds, in addition to VSCs. In addition, VSCs different from hydrogen sulfide and methanethiol, which are considered important causative compounds, may also influence to halitosis.

Metabolomic investigation of differences in components and taste between hon-mirin and mirin-like-seasoning.

Hon-mirin (HM) is a traditional Japanese brewed seasoning used to confer sweetness and koku. Mirin-like-seasoning (MLS) is a less-expensive alternative to HM because it is not subjected to liquor tax in Japan. In this study, components and taste qualities of HM and MLS were compared by gas chromatography/mass spectrometry (GC/MS)-based metabolomics and a sensory evaluation. GC/MS analyses of foods with high sugar content are limited by contamination of the ion source and difficulty in detecting other compounds. To resolve this issue, solid-phase analytical derivatization (SPAD), in which the extraction and derivatization of analytes can be conducted in a single step, was applied as a novel sample preparation method in this study. The effect of sugar was removed by the specific absorption, derivatization, and elution of ionic compounds, such as amino acids and organic acids, with ion-exchange solid-phases. The SPAD method application enabled the detection of 15 amino acids and 14 organic acids using ion-exchange solid-phases by performing GC/MS analysis twice. These ionic compounds were not detected in mirin using conventional sample preparation. HM samples had a higher amino acid content and a lower sugar content than those of MLS samples. Furthermore, differences in sweetness and koku between HM and MLS were observed in a sensory evaluation. This is the first GC/MS-based metabolomics analysis of mirin using the SPAD method; our results provide insight into the differences between HM and MLS.

Profiling of volatile compounds in Japanese sake stored in sherry casks using solid phase microextraction/gas chromatography/mass spectrometry analysis.

The combination of solid phase microextraction (SPME) and gas chromatograph-mass spectrometer (GC-MS) is frequently used for comprehensive analysis of aroma components in foods because it can be used to easily analyze volatile components, allowing saving of the amount of solvent used. In this study, SPME-GC-MS analysis of sake samples before and after sherry cask storage was performed to investigate the special flavor derived from sherry cask storage. A GC column with polyethylene glycol as the stationary phase, which is the first choice for volatile component analysis, was used. However, the peak of the acid having a carbonyl group was tailed due to its bond with the hydroxyl group of the stationary phase. In the analysis of sake samples, a large and tailing peak derived from the large amount of fatty acids in Japanese sake was observed. Additionally, it was not possible to analyze other co-eluting components. To overcome this problem, a novel extraction condition was examined using SPME and tris (hydroxymethyl) aminomethane (Tris). By adding Tris solution to sake, the fatty acid peak was removed successfully, thereby facilitating analysis of the peaks of compounds co-eluting with fatty acids and comprehensive analysis of the aroma components in sake. Furthermore, a comparative analysis of sake before and after storage in sherry cask showed that levels of fatty alcohols, organic acid esters, fatty acid esters, and terpenes increased significantly after storage in sherry cask, suggesting that these ingredients might constitute the special flavor of sherry cask-stored sake.

Serine racemase enhances growth of colorectal cancer by producing pyruvate from serine.

Serine racemase (SRR) catalyses not only the racemization but also the dehydration of L-serine and D-serine, resulting in the formation of pyruvate and ammonia. Although SRR activity is important in the central nervous system, SRR has not been linked to cancer metabolism before. Here we show that SRR supports proliferation of colorectal-cancer cells. We find that SRR expression is upregulated in colorectal adenoma and adenocarcinoma lesions compared with non-neoplastic mucosa in human colorectal-cancer specimens. SRR-mediated dehydration of serine contributes to the pyruvate pool in colon-cancer cells, enhances proliferation, maintains mitochondrial mass and increases basal reactive oxygen species production, which has anti-apoptotic effects. Moreover, SRR promotes acetylation of histone H3 by maintaining intracellular acetyl-CoA levels. Inhibition of SRR suppresses growth of colorectal tumours in mice and augments the efficacy of 5-fluorouracil treatment. Our findings highlight a previously unknown mechanism through which a racemase supports cancer-cell growth and suggest that SRR might be a molecular target for colorectal-cancer therapy.

Component Profiling of Soy-Sauce-Like Seasoning Produced from Different Raw Materials.

Soy sauce is a traditional Japanese umami seasoning commonly made from soybeans, wheat, and salt water. Soy-sauce-like seasoning, made from other raw materials, such as rice and peas, has recently been developed. However, differences in the taste of soy-sauce-like seasoning, depending on the raw materials, have not been evaluated. Component profiling based on GC/MS combined with a paired comparison test were used to investigate the effect of raw materials on seasoning components and umami taste in five grain-based and four bean-based soy-sauce-like seasonings. In a principal component (PC) analysis, grain-based samples and bean-based samples were separated along the PC1 axis (explaining 48.1% of the total variance). Grain-based samples had a higher saccharide content, and bean-based samples had a higher amino acid content. Furthermore, differences in the umami intensity were also observed among sample types. This is the first detailed metabolomics study of the characteristic compounds and umami of a variety of soy-sauce-like seasonings made from different raw materials.

Profiling of taste-related compounds during the fermentation of Japanese sake brewed with or without a traditional seed mash (kimoto).

Japanese sake production involves three processes: rice koji fermentation, seed mash fermentation, and main mash fermentation. Traditional seed mash (kimoto) production utilizes natural lactic acid produced by lactic acid bacteria for pure cultures of only sake yeast, preventing the growth of wild yeast and other unwanted bacteria. Recently, because kimoto production requires substantial time and labor, sake yeast mass-cultured in usual liquid medium has been used as a seed mash alternative. Sake quality is highly similar to that of kimoto, suggesting that they share similar component profiles. However, comparative component analyses of sake brewed with kimoto and sake brewed with cultured yeast are lacking. In this study, a time-course analysis of hydrophilic compounds in the main mash brewed with kimoto and with cultured yeast as well as a sensory evaluation of the products were performed. As a result, differences in various compounds and in umami taste level between sake brewed with kimoto and cultured yeast were detected. This is the first comparative analysis of changes in the component profile during sake main mash brewing using kimoto seed mash and cultured sake yeast; our results clarify the effects of kimoto seed mash on main mash brewing and sake quality.

High-Throughput LC-MS/MS Method for Chiral Amino Acid Analysis Without Derivatization.

D-Amino acids have recently attracted much attention in various research fields including medical, clinical, and food industry due to their important biological functions that differ from L-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. This chapter describes a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method allows the simultaneous analysis of 18 D-amino acids with high sensitivity and reproducibility. Additionally, this chapter also focuses on the application of the method to real samples for the quantification of targeted amino acids.

High-sensitive liquid chromatography-tandem mass spectrometry-based chiral metabolic profiling focusing on amino acids and related metabolites.

Metabolomics has been an evolving science with a wide range of applications in various fields. However, previous studies have rarely focused on metabolite chirality. In this study, to achieve metabolic profiling of chiral amino acids and related metabolites, we developed a high-throughput method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The combination of two types of chiral columns (with binaphthyl-based crown ether and cinchona alkaloid-derived zwitterionic stationary phases) enabled the analysis of 115 chiral and non-chiral metabolites. By finely optimizing MS/MS parameters, the method allowed the highly sensitive (0.001-50 nmol/mL) and wide dynamic range detection of targeted analytes in a standard solution without derivatization. We applied the method to food samples (cheese), and successfully quantified trace levels of metabolites such as d-amino acids in samples. Additionally, we performed principal component analysis on the metabolome data and obtained unique profiles that reflected metabolite chirality. These results demonstrated the applicability and feasibility of the LC-MS/MS method as an effective tool for wide-targeted chiral metabolome analysis.

Mechanistic study on the high-selectivity enantioseparation of amino acids using a chiral crown ether-bonded stationary phase and acidic, highly organic mobile phase by liquid chromatography/time-of-flight mass spectrometry.

The separation mechanism of amino acid enantiomers using a chiral crown ether-bonded stationary phase, CROWNPAK CR-I(+), and acetonitrile (ACN)-rich mobile phases (MPs) was studied. The retention factors of proteinogenic l-amino acids (except proline) formed U-shaped plots against the ACN content in the MP with a sharp increase at a high ACN content, while d-amino acids showed much smaller increases or monotonous decreases in retention within the same range. The use of an acidic, highly organic MP with trifluoroacetic acid (TFA) provided a high enantioselectivity with a short separation time from the contribution of the increased binding of the ammonium group of the analytes to the crown ether functionality of the stationary phase and electrostatic repulsion counteracting the hydrophilic partition mechanism. Optimizing the sample diluent and MP alleviated the peak distortion caused by a moving water band that accompanied the hydrophilic interaction liquid chromatography-like elution conditions. The liquid chromatography/time-of-flight mass spectrometry method with the optimized MP - ACN/ethanol/water/TFA = 80/15/5/0.5 (v/v/v/v) - enabled the determination of eighteen pairs of proteinogenic amino acid enantiomers within 10 min. The conditions also provided the following advantages: (i) fast and highly reproducible separations under isocratic conditions, (ii) high sensitivity and low backpressure using the MP with a high organic content, and (iii) highly reliable peak identification by combining two columns (CR-I(+) and CR-I(-)), reversing the elution orders of the enantiomers.

Investigation of storage time-dependent alterations of enantioselective amino acid profiles in kimchi using liquid chromatography-time of flight mass spectrometry.

Although naturally abundant amino acids are represented mainly by l-enantiomers, fermented foods are known to contain various d-amino acids. Enantiospecific profiles of food products can vary due to fermentation by bacteria, and such alterations may contribute to changes in food properties that would not be dependent exclusively on l-amino acids. Therefore, more attention should be paid to the study of temporal alterations of d-amino acid profiles during fermentation process. However, there have been very few studies reporting time-dependent profiling of d-amino acids because enantioseparation of widely targeted d-amino acids is technically difficult. This study aimed to achieve high throughput profiling of amino acids enantiomers. Enantioselective profiling of amino acids using CROWNPAK CR-I(+) column, liquid chromatography, time of flight mass spectrometry, and principle component analysis was performed to investigate time-dependent alterations in concentrations of free d- and l-amino acids in kimchi stored at 4°C or 25°C. We demonstrated significant changes in d- and l-amino acid profiles in kimchi stored at 25°C. In particular, concentrations of the amino acids d-Ala, d-Ser, d-allo-Ile, d-Leu, d-Asp, d-Glu, and d-Met became higher in kimchi with storage time. This is the first report of time-dependent alterations of d- and l-amino acid contents in kimchi. This study showed that our analytical method of enantioselective detection of amino acids using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) with CROWNPAK CR-I(+) enables high throughput food screening and can be recommended for advanced studies of the relationship between d-amino acid content and food properties.

Novel high-throughput and widely-targeted liquid chromatography-time of flight mass spectrometry method for d-amino acids in foods.

Recently, the demand for d-amino acid profiling has been drastically increasing because the significance of d-amino acid in various biological events is suggested. However, the present methodologies for d-amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for d-amino acid profiling must be developed. In this paper, we developed a novel method for d-amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOFMS). To our knowledge, our approach has the best performance for d-amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the d-amino acid research in the future.

Development of a liquid chromatography-tandem mass spectrometry method for quantitative analysis of trace d-amino acids.

d-Amino acids have recently attracted much attention in various research fields including medical, clinical and food industry due to their important biological functions that differ from l-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. Thus, the aim of this research is to develop a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By optimizing MS/MS parameters, we established a quantification method that allowed the simultaneous analysis of 18 d-amino acids with high sensitivity and reproducibility. Additionally, we applied the method to food sample (vinegar) for the validation, and successfully quantified trace levels of d-amino acids in samples. These results demonstrated the applicability and feasibility of the LC-MS/MS method as a novel, effective tool for d-amino acid measurement in various biological samples.