New insights into the characteristic flavor components of traditional sour beers such as Lambic and Flanders Red Ale beers.

In recent years, the demand for beers with a variety of flavors has increased considerably owing to the diversification of consumer preferences. Sour beer is characterized by a sour taste unlike normal beer flavor, and previous studies on sour beer have been primarily focused on addressing issues, such as inconsistent product quality and long production time, and on the associated microorganisms. Scientific knowledge regarding the characteristic flavor of sour beer and flavor components is limited. Therefore, in this study, we aimed to clarify the characteristic sensory attributes of sour beer and the component profiles that explain these attributes. Component analysis was performed on 10 traditional sour beers (eight Flanders Red Ales and two Lambics), using untargeted gas chromatography-mass spectrometry with liquid-liquid extraction, liquid chromatography-mass spectrometry targeting amines and anionic compounds. Further, sensory evaluation was conducted by well-trained panelists via quantitative descriptive analysis. Orthogonal partial least squares regression analysis was also conducted to investigate candidate flavor components. Thus, 261 components were identified and our methods could explain the flavor attributes of the examined samples. Comprehensive component profiling data also showed that differences in fermentation method, barrel aging duration, and blending ratio affected beer flavor. Further, Lambics were found to be characterized by citrus and phenolic aroma, while Flanders Red Ales were characterized by solvent-like aroma, sourness complexity, full bodied, graininess, astringency, and bitterness. These findings may serve as a basis for addressing issues related to sour beer production and may facilitate process design for obtaining targeted sour beer flavors.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Characteristics of kopyor coconut (Cocos nucifera L.) using sensory analysis and metabolomics-based approach.

Kopyor is a coconut with unique characteristics from Indonesia, one of the largest coconut producers in the world. Kopyor is an edible mature coconut with soft endosperm. Although this fruit is one of the most popular coconuts in the world, there are limited studies on its properties, including its sensory attributes and metabolite profiles. This study investigates the characteristics of kopyor using sensory evaluation, a widely targeted metabolomics approach, and multivariate analysis. The liquid (water) and solid (flesh) endosperms were collected as the samples. The results showed that kopyor has characteristics that distinguish it from normal mature and young coconuts. Kopyor water has a milky, creamy, nutty, bitter, and astringent taste with an oily aftertaste and mouthfeel. Kopyor flesh is soft and moist and gives a sandy mouth feel. This study analyzed the sensory attributes of the kopyor endosperm for the first time and compared it with those of normal mature and young coconuts. A gas chromatography mass spectrometry analysis showed that kopyor contained wider variety of metabolites than normal coconuts of the same age. Based on the differential analysis and orthogonal projections to latent structures-regression, kopyor water was characterized by the accumulation of flavor-related metabolites, such as amino acids and organic acids, which contributed to its sensory complexity. This study solidified the effects of maturation and endosperm type on metabolite accumulation in kopyor endosperm. This pioneering information will lead to the future use of kopyor and other unique coconuts worldwide for food, contributing to the sustainability of the coconut industry.

Multidisciplinary approach combining food metabolomics and epidemiology identifies meglutol as an important bioactive metabolite in tempe, an Indonesian fermented food.

This study introduces a multidisciplinary approach to investigate bioactive food metabolites often overlooked due to their low concentrations. We integrated an in-house food metabolite library (n = 494), a human metabolite library (n = 891) from epidemiological studies, and metabolite pharmacological databases to screen for food metabolites with potential bioactivity. We identified six potential metabolites, including meglutol (3-hydroxy-3-methylglutarate), an understudied low-density lipoprotein (LDL)-lowering compound. We further focused on meglutol as a case study to showcase the range of characterizations achievable with this approach. Green pea tempe was identified to contain the highest meglutol concentration (21.8 ± 4.6 mg/100 g). Furthermore, we identified a significant cross-sectional association between plasma meglutol (per 1-standard deviation) and lower LDL cholesterol in two Hispanic adult cohorts (n = 1,628) (ß [standard error]: -5.5 (1.6) mg/dl, P = 0.0005). These findings highlight how multidisciplinary metabolomics can serve as a systematic tool for discovering and enhancing bioactive metabolites in food, such as meglutol, with potential applications in personalized dietary approaches for disease prevention.

A new type of sulfation reaction: C-sulfonation for α,ß-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1.

Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,ß-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate to the α-carbon of α,ß-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,ß-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,ß-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.

Rice grain structural characteristics of sake rice cultivar Hakutsurunishiki for daiginjo-shu brewing.

Hakutsurunishiki is a sake rice cultivar bred using Yamadabo (seed parent) and Wataribune 2 (pollen parent), equivalent to a Yamadanishiki sibling. This study evaluated the structural characteristics of the Hakutsurunishiki rice grain that contribute to the brewing characteristics of daiginjo-shu, via a comparison with Yamadanishiki. Hakutsurunishiki brown rice was a little heavy and had a large white core. Observing a cross-section of white rice after soaking revealed that the rice grain structure of Hakutsurunishiki was different from that of Yamadanishiki. Hakutsurunishiki white rice showed fewer voids than Yamadanishiki, promoting a slower water absorption rate. Glucose distribution in rice koji obtained by mass spectrometry imaging showed that Hakutsurunishiki rice koji, like Yamadanishiki, is tsuki-haze type, suggesting that its grain structure is suitable for making rice koji for daiginjo-shu. With these observations, we were able to clarify the structural characteristics of Hakutsurunishiki rice grain.

Effects of n-butanol production on metabolism and the photosystem in Synecococcus elongatus PCC 7942 based on metabolic flux and target proteome analyses.

Although n-butanol (BuOH) is an ideal fuel because of its superior physical properties, it has toxicity to microbes. Previously, a Synechococcus elongatus PCC 7942 derivative strain that produces BuOH from CO2 was developed by introducing six heterologous genes (BUOH-SE strain). To identify the bottleneck in BuOH production, the effects of BuOH production and its toxicity on central metabolism and the photosystem were investigated. Parental (WT) and BUOH-SE strains were cultured under autotrophic conditions. Consistent with the results of a previous study, BuOH production was observed only in the BUOH-SE strain. Isotopically non-stationary 13C-metabolic flux analysis revealed that the CO2 fixation rate was much larger than the BuOH production rate in the BUOH-SE strain (1.70 vs 0.03 mmol gDCW-1 h-1), implying that the carbon flow for BuOH biosynthesis was less affected by the entire flux distribution. No large difference was observed in the flux of metabolism between the WT and BUOH-SE strains. Contrastingly, in the photosystem, the chlorophyll content and maximum O2 evolution rate per dry cell weight of the BUOH-SE strain were decreased to 81% and 43% of the WT strain, respectively. Target proteome analysis revealed that the amounts of some proteins related to antennae (ApcA, ApcD, ApcE, and CpcC), photosystem II (PsbB, PsbU, and Psb28-2), and cytochrome b6f complex (PetB and PetC) in photosystems decreased in the BUOH-SE strain. The activation of photosynthesis would be a novel approach for further enhancing BuOH production in S. elongatus PCC 7942.

Correlation between plasma glutamate and adiponectin in patients with type 2 diabetes.

Visceral fat accumulation is a major determinant of type 2 diabetes mellitus and cardiovascular diseases. Recent studies have reported that glutamate is the most elevated amino acid in the plasma amino acid profile in patients with obesity and/or visceral fat accumulation. Here, we show the relationship between plasma glutamate and the clinical features of patients with type 2 diabetes. The study subjects were 62 (28 men and 34 women) Japanese patients with type 2 diabetes. Blood profiles, including glutamate and adiponectin (APN) levels and estimated visceral fat area (eVFA), were measured. We also evaluated the plasma amino acid levels in mice with or without obesity by GC/MS analysis. In patients with type 2 diabetes, plasma glutamate was positively correlated with BMI, eVFA, and fasting insulin but negatively correlated with APN and duration of diabetes. Additionally, multiple regression analysis revealed that plasma glutamate was a significant determinant of APN. The plasma glutamate level was most significantly increased in obese mice compared to control mice, and it was negatively correlated with APN. These results suggest that the level of plasma glutamate could be a strong indicator of adipocyte dysfunction in patients with type 2 diabetes.

Identification of potential quality markers in Indonesia's Arabica specialty coffee using GC/MS-based metabolomics approach.

INTRODUCTION: The cupping test is a widely used method for quality assessment of Arabica coffee. However, the cupping test is limited by the low number of certified panelists and the low throughput. Therefore, an analytical-based quality assessment may be a promising tool to complement the cupping test. A present, there is no report investigating quality marker candidates, focusing only on "specialty" grade Arabica coffee from Indonesia. OBJECTIVE: This study identified the potential quality marker(s) in Arabica Specialty coffee at different stages (green beans, roasted beans, and brewed coffee. METHODS: The metabolite profiles of ten different Arabica specialty-grade coffees were analyzed with different cup scores using gas chromatography-mass spectrometry (GC/MS). From the ten samples, green coffee beans, roasted coffee beans, and brewed coffee were selected. In addition, an orthogonal projection to latent structure (OPLS) regression analysis was conducted to obtain a potential quality marker based on the variable importance in projection (VIP). The potential quality marker(s) were validated by GC/MS metabolome profiling and OPLS analysis of different sets of samples consisting of 35 Arabica specialty-grade coffee samples. RESULTS: In Arabica coffee samples, the OPLS model of the three stages showed galactinol to have a high VIP score. Galactinol showed a consistent positive correlation with cup scores at all stages of coffee production (green beans, roasted beans, and brewed coffee). The correlation suggests galactinol is a potential quality marker after further validation using different samples. CONCLUSION: GC/MS combined with OPLS regression analysis suggested galactinol as a quality marker and provide an early screening method for Arabica coffee quality that complements the cupping test performed by certified panelists.

Chitosan-based coating enriched with melezitose alters primary metabolites in fresh-cut pineapple during storage.

Demand for minimally processed fresh fruit is increasing due to its convenience. However, the distribution of fresh-cut fruits is limited because of their short shelf life. Pineapple, a popular tropical fruit, sold in fresh-cut form has a shelf life of approximately 5-7 days at 4 °C. Chitosan, an edible coating, is commonly used to prolong the shelf life of food products. Similarly, the sugar melezitose has been reported to change during pineapple ripening and may play a role in regulating the shelf life of pineapple. However, the direct effects of this sugar have yet to be studied. The objective of this study was to investigate the effect of chitosan coating with melezitose to prolong the shelf life of fresh-cut pineapple. Full-ripe Bogor pineapples from Okinawa, Japan, were cut into cubes and soaked in either chitosan 1.25%, melezitose 5 mg/L, or chitosan+melezitose and stored for 5 days under dark conditions (23.6 ± 0.5 °C; relative humidity, 40.0 ± 10.4%). None of the treatments significantly altered the weight loss or color changes in the fresh-cut fruit. However, treatment significantly altered the primary metabolites, namely quinic acid, sucrose, and xylitol based on orthogonal projection to latent structures data with the screening from p-value score. Moreover, cell-wall metabolism is possibly affected in pineapple cut fruit treated by chitosan-melezitose as shown from metabolite sets enrichment analysis. This study showed that chitosan added with melezitose might have potential to prolong the shelf-life of fresh-cut pineapple, providing a basis for further post-harvest studies of the whole pineapple fruit.

Improvement of the functional value of green soybean (edamame) using germination and tempe fermentation: A comparative metabolomics study.

Green soybean, also known as edamame, is a legume with high nutritional and functional value. Despite its growing popularity and potential health benefits, the functionality of green soybean has not been thoroughly studied. Previous research on the functionality of green soybean has largely focused on a limited number of specific, well-studied, bioactive metabolites, without comprehensively investigating the metabolome of this legume. Additionally, very few studies have explored the improvement of the functional value of green soybean. This study aimed to investigate the metabolome profile of green soybean, identify bioactive metabolites, and to further explore the potential improvement of the identified bioactive metabolites using germination and tempe fermentation. A total of 80 metabolites were annotated from green soybean using GC-MS and HPLC-PDA-MS. Among them, 16 important bioactive metabolites were identified: soy isoflavones daidzin, glycitin, genistin, malonyl daidzin, malonyl genistin, malonyl glycitin, acetyl daidzin, acetyl genistin, acetyl glycitin, daidzein, glycitein, and genistein, as well as other metabolites including 3,4-dihydroxybenzoic acid, 3-hydroxyanthranillic acid, 3-hydroxy-3-methylglutaric acid (meglutol), and 4-aminobutyric acid (GABA). Germination and tempe fermentation techniques were employed to potentially improve the concentrations of these bioactive metabolites. While showing improvements in amino acid contents, germination process did not improve bioactive metabolites significantly. In contrast, tempe fermentation was found to significantly increase the concentrations of daidzein, genistein, glycitein, acetyl genistin, acetyl daidzin, 3-hydroxyanthranillic acid, and meglutol (>2-fold increase with p < 0.05) while also improving amino acid levels. This study highlights the potentials of germination and fermentation to improve the functionality of legumes, particularly green soybean.

Visualization of azoxystrobin penetration in wheat leaves using mass microscopy imaging.

Fungicides must penetrate the internal tissues of plants to kill pathogenic fungi. Mass spectrometers have been used to confirm this penetration, but conventional mass spectrometric methods cannot distinguish the fungicides in different internal tissues owing to the extraction steps. However, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect the penetration of fungicides into leaf sections through direct analysis of the sample surfaces. Therefore, the objective of this study was to establish a method for visualizing fungicide penetration in wheat leaf cross sections using MALDI-MSI. The penetration of azoxystrobin from the epidermal to the internal tissue of the leaves was observed. Moreover, azoxystrobin accumulates in the cells around the vascular bundle. This study suggests that MSI can be useful for the evaluation of fungicide penetration in plant leaves.

Metabolite Changes in Indonesian Tempe Production from Raw Soybeans to Over-Fermented Tempe.

Tempe is fermented soybean from Java, Indonesia, that can serve as a functional food due to its high nutritional content and positive impact on health. Although the tempe fermentation process is known to affect its nutrient content, changes in the metabolite profile during tempe production have not been comprehensively examined. Thus, this research applied a metabolomics approach to investigate the metabolite profile in each step of tempe production, from soybean soaking to over-fermentation. Fourteen samples of raw soybeans, i.e., soaked soybeans (24 h), steamed soybeans, fungal fermented soybeans, and over-fermented soybeans (up to 72 h), were collected. Untargeted metabolomics by gas chromatography/mass spectrometry (GC-MS) was used to determine soybean transformations from various fermentation times and identify disparity-related metabolites. The results showed that soybeans samples clustered together on the basis of the different fermentation steps. The results also showed that sugar, sugar alcohol, organic acids, and amino acids, as well as fermentation time, contributed to the soybean metabolite profile transformations. During the fermentation of tempe, sugars and sugar alcohols accumulated at the beginning of the process before gradually decreasing as fermentation progressed. Specifically, at the beginning of the fermentation, gentiobiose, galactinol, and glucarate were accumulated, and several metabolites such as glutamine, 4-hydroxyphenylacetic acid, and homocysteine increased along with the progression of fermentation. In addition, notable isoflavones daidzein and genistein increased from 24 h of fermentation until 72 h. This is the first report that provides a complete description of the metabolic profile of the tempe production from soybean soaking to over-fermentation. Through this study, the dynamic changes at each step of tempe production were revealed. This information can be beneficial to the tempe industry for the improvement of product quality based on metabolite profiling.

Online measurement of the viscosity in shake flasks enables monitoring of γ-PGA production in depolymerase knockout mutants of Bacillus subtilis with the phosphate-starvation inducible promoter Ppst.

Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (Viscosity Monitoring Online System) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the Ppst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The Ppst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in gγ-PGA /gglucose of 190% could be achieved with the triple knockout mutant compared to the Ppst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production.

Visualization of Glutamate Decarboxylase Activity in Barley Seeds under Salinity Stress Using Mass Microscope.

γ-Aminobutyric acid (GABA) accumulates in plants in response to environmental stresses. The activity levels of glutamate decarboxylase (GAD), an enzyme involved in GABA biosynthesis, are reported to increase during germination under salinity stress. However, it is not clear which tissues of the plant seeds are affected by GAD activity in response to salinity stress. In this study, the effects of salinity stress on the distribution of barley seeds GAD activity during germination were investigated. The mass spectrometry imaging (MSI) method was optimized, and the distribution of GAD activity in germinated seeds exposed to salinity stress at different germination stages from 12 to 48 h after imbibition was investigated. In this study, MSI was successfully applied to enzyme histochemistry to visualize the relative GAD activity in germinating barley seeds for the first time. The salinity stress increased the GAD activity, mostly due to the increase in relative GAD activity in the embryo. Higher GAD activity was detected in seeds exposed to salinity stress in the scutellum or aleurone layer, which are difficult to separate for extraction. This method can be used to clarify the role of GABA shunts, including GAD enzyme responses, in barley seeds under stress.

MALDI Mass Spectrometry Imaging Reveals the Existence of an N-Acyl-homoserine Lactone Quorum Sensing System in Pseudomonas putida Biofilms.

Quorum sensing (QS) is generally used to describe the process involving the release and recognition of signaling molecules, such as N-acyl-homoserine lactones, by bacteria to coordinate their response to population density and biofilm development. However, detailed information on the heterogeneity of QS metabolites in biofilms remains largely unknown. Here, we describe the utilization of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to follow the production of specific metabolites, including QS metabolites, during Pseudomonas putida biofilm development. To do so, a method to grow an agar-based biofilm was first established, and MALDI-MSI was used to detect and visualize the distribution of QS metabolites in biofilms at different cultivation times. This study demonstrated that N-acyl-homoserine lactones are homogeneously produced in the early stages of P. putida biofilm formation. In contrast, the spatial distribution of quinolones and pyochelin correlated with the swarming motility of P. putida in mature biofilms. These two metabolites are involved in the production of extracellular polymeric substances and iron chelators. Our study thus contributes to establishing the specific temporal regulation and spatial distribution of N-acyl-homoserine lactone-related metabolites and quinolone and pyochelin in P. putida biofilms.

Metabolomics-driven strain improvement: A mini review.

In recent years, mass spectrometry-based metabolomics has been established as a powerful and versatile technique for studying cellular metabolism by comprehensive analysis of metabolites in the cell. Although there are many scientific reports on the use of metabolomics for the elucidation of mechanism and physiological changes occurring in the cell, there are surprisingly very few reports on its use for the identification of rate-limiting steps in a synthetic biological system that can lead to the actual improvement of the host organism. In this mini review, we discuss different strategies for improving strain performance using metabolomics data and compare the application of metabolomics-driven strain improvement techniques in different host microorganisms. Finally, we highlight several success stories on the use of metabolomics-driven strain improvement strategies, which led to significant bioproductivity improvements.

Mass spectrometry imaging enables visualization of the localization of glutamate decarboxylase activity in germinating legume seeds.

Visualizing the distribution of enzymes is vital for understanding physiological phenomena. Enzyme histochemistry is a technique used to investigate the localization of enzyme activity. However, the target is restricted to enzymes with easy-to-design artificial substrates that can develop color through reactions. Mass spectrometry imaging (MSI)-based enzyme histochemistry has been developed as a novel method to visualize enzyme localization. It can be applied to enzyme histochemistry as it detects products from the supplied substrate using enzymes present on the tissue sections. However, enzyme histochemistry using MSI has not been applied to plant tissue samples yet. Glutamate decarboxylase (GAD, EC: 4.1.1.15) is an enzyme that catalyzes the decarboxylation reaction of l-glutamic acid to produce γ-aminobutyric acid (GABA). GABA biosynthesis is important both in the field of food chemistry and plant physiology. This study focused on GAD during the legume germination process and successfully visualized GAD activity in legume seeds using MSI for the first time. Furthermore, the localization of GAD activity in the embryonic axis of germinated soybean seeds and alfalfa seeds could be visualized.

Fusobacterium nucleatum Metabolically Integrates Commensals and Pathogens in Oral Biofilms.

Fusobacterium nucleatum is a common constituent of the oral microbiota in both periodontal health and disease. Previously, we discovered ornithine cross-feeding between F. nucleatum and Streptococcus gordonii, where S. gordonii secretes ornithine via an arginine-ornithine antiporter (ArcD), which in turn supports the growth and biofilm development of F. nucleatum; however, broader metabolic aspects of F. nucleatum within polymicrobial communities and their impact on periodontal pathogenesis have not been addressed. Here, we show that when cocultured with S. gordonii, F. nucleatum increased amino acid availability to enhance the production of butyrate and putrescine, a polyamine produced by ornithine decarboxylation. Coculture with Veillonella parvula, another common inhabitant of the oral microbiota, also increased lysine availability, promoting cadaverine production by F. nucleatum. We confirmed that ArcD-dependent S. gordonii-excreted ornithine induces synergistic putrescine production, and mass spectrometry imaging revealed that this metabolic capability creates a putrescine-rich microenvironment on the surface of F. nucleatum biofilms. We further demonstrated that polyamines caused significant changes in the biofilm phenotype of a periodontal pathogen, Porphyromonas gingivalis, with putrescine accelerating the biofilm life cycle of maturation and dispersal. This phenomenon was also observed with putrescine derived from S. gordonii-F. nucleatum coculture. Lastly, analysis of plaque samples revealed cooccurrence of P. gingivalis with genetic modules for putrescine production by S. gordonii and F. nucleatum. Overall, our results highlight the ability of F. nucleatum to induce synergistic polyamine production within multispecies consortia and provide insight into how the trophic web in oral biofilm ecosystems can eventually shape disease-associated communities. IMPORTANCE Periodontitis is caused by a pathogenic shift in subgingival biofilm ecosystems, which is accompanied by alterations in microbiome composition and function, including changes in the metabolic activity of the biofilm, which comprises multiple commensals and pathogens. While Fusobacterium nucleatum is a common constituent of the supra- and subgingival biofilms, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Here, we report that amino acids supplied by other commensal bacteria induce polyamine production by F. nucleatum, creating polyamine-rich microenvironments. Polyamines reportedly have diverse functions in bacterial physiology and possible involvement in periodontal pathogenesis. We show that the F. nucleatum-integrated trophic network yielding putrescine from arginine through ornithine accelerates the biofilm life cycle of Porphyromonas gingivalis, a periodontal pathogen, from the planktonic state through biofilm formation to dispersal. This work provides insight into how cooperative metabolism within oral biofilms can tip the balance toward periodontitis.

Visualization of dipeptidyl peptidase B enzymatic reaction in rice koji using mass spectrometry imaging.

Enzyme histochemistry via mass spectrometry imaging (MSI) has garnered attention as a straightforward approach for visualizing enzymatic reactions. While several studies in the medical and physiological fields have shown its promising application potential, its applicability to agricultural or food studies has not yet been demonstrated. Rice koji, known as an enzyme source for various fermented products, is a suitable model for demonstrating the applicability of this method to food-related materials. In this study, the enzymatic reaction of dipeptidyl peptidase B (DppB) in rice koji was visualized using MSI for the first time. The method was optimized and applied to investigate the effects of rice variety, polishing ratio, and cultivation time on the location of the DppB reaction. The DppB enzymatic reaction was found to occur in different locations in each of the two rice varieties, Yamadanishiki and Hakutsurunishiki. The polishing ratio also affected the distribution of the DppB enzymatic reactions. Furthermore, a time-course investigation of rice koji cultivation revealed that while the location of the reaction was largely associated with mycelial penetration, the structure and features of the rice grain may also affect the location of the enzymatic reaction. In summary, these results demonstrate the applicability of enzyme histochemistry by MSI to food-related materials.