Integration of bioassay and non-target metabolite analysis of tomato reveals that ß-carotene and lycopene activate the adiponectin signaling pathway, including AMPK phosphorylation.

Adiponectin, an adipokine, regulates glucose metabolism and insulin sensitivity through the adiponectin receptor (AdipoR). In this study, we searched for metabolites that activate the adiponectin signaling pathway from tomato (Solanum lycopersicu). Metabolites of mature tomato were separated into 55 fractions by liquid chromatography, and then each fraction was examined using the phosphorylation assay of AMP-protein kinase (AMPK) in C2C12 myotubes and in AdipoR-knockdown cells by small interfering RNA (siRNA). Several fractions showed AMPK phosphorylation in C2C12 myotubes and siRNA-mediated abrogation of the effect. Non-targeted metabolite analysis revealed the presence of 721 diverse metabolites in tomato. By integrating the activity of fractions on AMPK phosphorylation and the 721 metabolites based on their retention times of liquid chromatography, we performed a comprehensive screen for metabolites that possess adiponectin-like activity. As the screening suggested that the active fractions contained four carotenoids, we further analyzed ß-carotene and lycopene, the major carotenoids of food. They induced AMPK phosphorylation via the AdipoR, Ca2+/calmodulin-dependent protein kinase kinase and Ca2+ influx, in addition to activating glucose uptake via AdipoR in C2C12 myotubes. All these events were characteristic adiponectin actions. These results indicated that the food-derived carotenoids, ß-carotene and lycopene, activate the adiponectin signaling pathway, including AMPK phosphorylation.

Insights into the mechanism of diurnal variations in methane emission from the stem surfaces of Alnus japonica.

Recent studies have suggested that in certain environments, tree stems emit methane (CH4 ). This study explored the mechanism of CH4 emission from the stem surfaces of Alnus japonica in a riparian wetland. Stem CH4 emission rates and sap flux were monitored year-round, and fine-root anatomy was investigated. CH4 emission rates were estimated using a closed-chamber method. Sap flux was measured using Granier-type thermal dissipation probes. Root anatomy was studied using both optical and cryo-scanning electron microscopy. CH4 emissions during the leafy season exhibited a diurnally changing component superimposed upon an underlying continuum in which the diurnal variation was in phase with sap flux. We propose a model in which stem CH4 emission involves at least two processes: a sap flux-dependent component responsible for the diurnal changes, and a sap flux-independent component responsible for the background continuum. The contribution ratios of the two processes are season-dependent. The background continuum possibly resulted from the diffusive transport of gaseous CH4 from the roots to the upper trunk. Root anatomy analysis indicated that the intercellular space of the cortex and empty xylem cells in fine roots could serve as a passageway for transport of gaseous CH4 .

Development of a Method for Quantitation of Glyceraldehyde in Various Body Compartments of Rodents and Humans.

There is limited information available about the physiological content of glyceraldehyde, a precursor of toxic advanced glycation end products. The conventional derivatization method for aldoses using 1-phenyl-3-methyl-5-pyrazolone did not allow reproducible quantification of glyceraldehyde due to the instability of glyceraldehyde compared to other aldoses. We optimized the derivatization condition to achieve high and reproducible recovery of derivatives for liquid chromatography tandem mass spectrometry quantification. Based on the stability of glyceraldehyde during sample preparation and high recovery of spiked standard, the present method provides reproducible quantification of glyceraldehyde in the body. The glyceraldehyde contents in fasting conditions in the rodent liver (mice: 50.0 ± 3.9 nmol/g; rats: 35.5 ± 4.9 nmol/g) were higher than those in plasma (9.4 ± 1.7 and 7.2 ± 1.2 nmol/mL). The liver glyceraldehyde levels significantly increased after food consumption (p < 0.05) but remained constant in the plasma. High fat diet feeding significantly increased plasma glyceraldehyde levels in mice (p < 0.005). In healthy human volunteers, the plasma glyceraldehyde levels remained unchanged after the consumption of steamed rice. In patients with type 2 diabetes, the plasma glyceraldehyde level was positively correlated with the plasma glucose level (r = 0.84; p < 0.0001).

TOMATOMET: A metabolome database consists of 7118 accurate mass values detected in mature fruits of 25 tomato cultivars.

The total number of low-molecular-weight compounds in the plant kingdom, most of which are secondary metabolites, is hypothesized to be over one million, although only a limited number of plant compounds have been characterized. Untargeted analysis, especially using mass spectrometry (MS), has been useful for understanding the plant metabolome; however, due to the limited availability of authentic compounds for MS-based identification, the identities of most of the ion peaks detected by MS remain unknown. Accurate mass values of peaks obtained by high accuracy mass measurement and, if available, MS/MS fragmentation patterns provide abundant annotation for each peak. Here, we carried out an untargeted analysis of compounds in the mature fruit of 25 tomato cultivars using liquid chromatography-Orbitrap MS for accurate mass measurement, followed by manual curation to construct the metabolome database TOMATOMET (http://metabolites.in/tomato-fruits/). The database contains 7,118 peaks with accurate mass values, in which 1,577 ion peaks are annotated as members of a chemical group. Remarkably, 71% of the mass values are not found in the accurate masses detected previously in Arabidopsis thaliana, Medicago truncatula or Jatropha curcas, indicating significant chemical diversity among plant species that remains to be solved. Interestingly, substantial chemical diversity exists also among tomato cultivars, indicating that chemical profiling from distinct cultivars contributes towards understanding the metabolome, even in a single organ of a species, and can prioritize some desirable metabolic targets for further applications such as breeding.

Revealing 3D structure of gluten in wheat dough by optical clearing imaging.

Gluten, which makes up 85% of endosperm wheat protein, is considered a crucial quality determinant of wheat-based food products. During wheat dough manufacture, the molecular packing of gluten causes formation of large structures that exceed the millimetre scale. However, due to lack of imaging techniques for complex systems composed of giant macromolecules, the entire gluten structure remains unknown. Here, we develop an optical clearing reagent (termed SoROCS) that makes wheat-based products transparent. Combined with two-photon microscopy, we image the three-dimensional (3D) structure of gluten at the size in the millimetre scale and at submicron resolution. Further, we demonstrate how the 3D structure of gluten dramatically changes from a honeycomb-shaped network to sparse large clumps in wheat noodles, depending on the salt added during dough making, thereby reducing stress when compressing the noodle. Moreover, we show that SoROCS can be used for noodle imaging using confocal laser scanning microscopy.

Anti-Inflammatory and Antioxidative Properties of Isoflavones Provide Renal Protective Effects Distinct from Those of Dietary Soy Proteins against Diabetic Nephropathy.

SCOPE: Dietary soy reportedly protects from diabetic nephropathy (DN), but its active components and mechanism of action remain unknown. METHODS AND RESULTS: In this study, KKAy mice are fed three types of diet: Dietary soy isoflavones with soy protein (Soy-IP) diet, reduced isoflavones soy protein (RisoP), and oral administration of isoflavones aglycones (IsoAgc). Albuminuria and glycosuria are decreased only in the soy-IP group. The risoP group show reduced expansion of mesangial matrix and renal fibrosis, the IsoAgc group show renal anti-fibrotic and anti-inflammatory effects; however, these renal pathological changes are repressed in the soy-IP group, suggesting the distinct protective roles of soy protein or isoflavones in DN. The isoflavone genistein has a better inhibitory effect on the inflammatory response and cellular interactions in both mouse tubular cells and macrophages when exposed to high glucose and albumin (HGA). Genistein also represses HGA-induced activator protein 1 activation and reactive oxidases stress generation, accompanied by reduced NADPH oxidase (NOX) gene expression. Finally, diabetic mice show a decrease in lipid peroxidation levels in both plasma and urine, along with lower NOXs gene expression. CONCLUSION: The data elucidate the detailed mechanism by which isoflavones inhibit renal inflammation and provide a potential practical adjunct therapy to restrict DN progression.

Pyroglutamyl leucine, a peptide in fermented foods, attenuates dysbiosis by increasing host antimicrobial peptide.

PyroGlu-Leu is present in certain food protein hydrolysates and traditional Japanese fermented foods. Our previous study demonstrated that the oral administration of pyroGlu-Leu (0.1 mg/kg body weight) attenuates dysbiosis in mice with experimental colitis. The objective of this study was to elucidate why such a low dose of pyroGlu-Leu attenuates dysbiosis in different animal models. High fat diet extensively increased the ratio of Firmicutes/Bacteroidetes in feces of rats compared to control diet. Oral administration of pyroGlu-Leu (1 mg/kg body weight) significantly attenuated high fat diet-induced dysbiosis. By focusing on the production of intestinal antimicrobial peptides, we found that pyroGlu-Leu significantly increased the level of 4962 Da peptides, which identified as the propeptide of rattusin or defensin alpha 9, in ileum. We also observed increased tryptic fragment peptides from rattusin in the lumen. Here, we report that orally administered pyroGlu-Leu attenuates dysbiosis by increasing in the host antimicrobial peptide, rattusin.

Amount of Collagen in the Meat Contained in Japanese Daily Dishes and the Collagen Peptide Content in Human Blood after Ingestion of Cooked Fish Meat.

Objectives of the present study were to evaluate amounts of collagen in Japanese daily dishes and contents of food-derived collagen peptides in human blood. The meat in one serving of most Japanese daily dishes contains 0.2-2.5 g of collagen, except for beef tendon, eel with skin, and skinned shark tail (7.6-13.3 g). After ingestion of cooked shark meat, nine collagen di- and tripeptides were detected in plasma and the area under the curve of most peptides, except for Hyp-Gly and Pro-Hyp-Gly, was ∼30% of that after ingestion of collagen hydrolysate containing an equivalent amount of collagen. Likewise, only ∼30% of the total collagen in the meat was liberated into solution by pepsin and pancreatin digestion. Thus, ingestion of collagen-rich meat increases the collagen peptides in blood, which depends on not only the collagen content in the meat but also the susceptibility of the collagen/gelatin to digestive endoproteinases.

Effects of heat treatment under low moisture conditions on the protein and oil in soybean seeds.

The effects of autoclave and microwave heating on the protein and oil in soybean seeds were investigated under low moisture conditions. The nitrogen solubility index (NSI) decreased on heating. The reduction in the NSI was accompanied by an increase in the size and deformation of the oil bodies in the cellular tissue of soybean seeds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that lipoxygenase was susceptible to heat denaturation, but 7S and 11S globulins were only partially denatured. The partial denaturation of the proteins was confirmed by Fourier transform infrared spectroscopy measurements. The ratio of oil to protein peaks increased with increasing heating, suggesting the exudation of oil to the surface or outside of oil bodies. Microwave heating is more efficient in changing the oil distribution in soybean seeds than autoclave heating. On the other hand, the degree of protein denaturation is lower after microwave heating.

Tolerance of bitter stimuli and attenuation/accumulation of their bitterness in humans.

Some components of bitterness make key flavor contributions to promote the palatability of foods, whereas other components are recognized as aversive signals to avoid consuming harmful substances. These contradictory behaviors suggest that humans tolerate tastes of bitterants based on certain criteria. Here, we investigated human taste tolerance and sensory cues leading to diverse taste tolerance of bitter compounds. Tolerance of eight bitter compounds, which are typically contained in foods, was evaluated by measuring detection and rejection thresholds. The results revealed that the level of tolerance of each compound was variable, and some compounds showed an acceptable concentration regarding the suprathreshold intensity. Tolerance did not depend on the nutritive value or attenuation and accumulation characteristics of bitterness and bitter taste receptors. These results suggest that the criteria controlling tolerance of bitter compounds may be derived from a complex relationship between the taste quality and cognitive process.

Diversity of γ- glutamyl peptides and oligosaccharides, the "kokumi" taste enhancers, in seeds from soybean mini core collections.

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the "kokumi" taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the "kokumi" taste components in soybean by performing a genetic analysis.

Wide-range screening of anti-inflammatory compounds in tomato using LC-MS and elucidating the mechanism of their functions.

Obesity-induced chronic inflammation is a key factor in type 2 diabetes. A vicious cycle involving pro-inflammatory mediators between adipocytes and macrophages is a common cause of chronic inflammation in the adipose tissue. Tomato is one of the most popular vegetables and is associated with a reduced risk of diabetes. However, the molecular mechanism underlying the effect of tomato on diabetes is unclear. In this study, we focused on anti-inflammatory compounds in tomato. We found that the extract of tomato reduced plasma glucose and inflammatory markers in mice. We screened anti-inflammatory fractions in tomato using lipopolysaccharide-stimulated RAW264.7 macrophages, and active compounds were estimated by liquid chromatography-mass spectrometry over a wide range. Surprisingly, a large number of compounds including oxylipin and coumarin derivatives were estimated as anti-inflammatory compounds. Especially, 9-oxo-octadecadienoic acid and daphnetin suppressed pro-inflammatory cytokines in RAW264.7 macrophages inhibiting mitogen-activated protein kinase phosphorylation and inhibitor of kappa B α protein degradation. These findings suggest that tomato containing diverse anti-inflammatory compounds ameliorates chronic inflammation in obese adipose tissue.

Microgelation imparts emulsifying ability to surface-inactive polysaccharides-bottom-up vs top-down approaches.

In order to impart emulsifying ability to gel-forming polysaccharides that have not been used as emulsifying agents, three kinds of polysaccharides, agar, curdlan, and gellan gum were converted to microgels by different gelation methods via the bottom-up and top-down approaches. We clearly demonstrated that agar and curdlan acquired the ability to emulsify an edible oil by microgel formation. Among the colloidal properties of microgel suspensions such as microstructure, particle size, zeta-potential, viscosity, and surface hydrophobicity, we pointed out the importance of particle size on the emulsifying ability of polysaccharide-based microgels. The creaming behavior of the microgel-stabilized emulsions depended on the polysaccharide types and microgel preparation methods. The emulsion stability against oil droplet coalescence was extremely high for agar and curdlan microgel-stabilized emulsions during storage in the static condition, whereas different stability was observed for both the emulsions, that is, the curdlan microgel-based ones were more resistant to dynamic forcible destabilization by centrifugation than the agar ones, which can be attributed to the surface hydrophobicity of the microgels.

Isolation and characterization of key contributors to the "kokumi" taste in soybean seeds.

The water extract of soybean seeds (Glycine max (L.) Merr.) is nearly tasteless, but "kokumi" taste sensation was confirmed upon addition of a basic umami solution containing glutamic acid, inosine monophosphate, and sodium chloride. To identify the key contributors to the "kokumi" taste sensation in soybean seeds, sensory-guided fractionation, taste sensory analyses, and LC-MS/MS analyses were utilized. γ-glutamyl-tyrosine and γ-glutamyl-phenylalanine were identified as contributors to "kokumi taste"; specifically, these γ-glutamyl peptides imparted the "kokumi" taste sensation at a low taste threshold in a basic umami solution. Raffinose and stachyose, which are sufficiently present in soybean seeds, exhibited a synergistic effect in regard to the enhanced "kokumi" taste sensation of γ-glutamyl peptides. This is the first report that the combined use of γ-glutamyl peptides and oligosaccharides can increase the "kokumi" intensity, which suggests that soybean extracts or soymilk can be used to enhance the "kokumi" taste sensation in food products.

Solubility of soy lipophilic proteins: comparison with other soy protein fractions.

Solubility of soy lipophilic proteins (LP) was studied as compared with that of other soy protein fractions. LP, ß-conglycinin, glycinin, and soy protein isolate (N-SPI) were prepared under the condition to avoid heat denaturation. Solubility of LP was lower than that of other soy protein fractions under all the tested conditions varying in pH values and ionic strength. The solubility of LP was increased constantly by elevating temperature until 90 °C, whereas that of ß-conglycinin and glycinin dropped at high temperature. Temperature-dependent change in solubility of N-SPI might reflect the balance among that of glycinin, ß-conglycinin and LP. Based on the results of SDS-PAGE, determination of phospholipid content and Fourier Transform Infrared spectroscopy, we discussed the solubilization behavior of LP relating to its origin and composition.

Protein-surfactant interactions between bovine lactoferrin and sophorolipids under neutral and acidic conditions.

To understand the protein-surfactant interactions between naturally derived sophorolipids (SLs) and bovine lactoferrin (bLf), we carried out spectroscopic, microscopic, and biochemical experiments under weakly acidic and neutral pH conditions. Particle size analysis, microscopy, and enzymatic digestion indicated that bLf and SLs interact with each other to form sheet-like and small aggregated structures reflecting the original self-organization of SLs at pH 5.0 and 7.0, respectively. Circular dichroism (CD) showed that SLs did not significantly affect the secondary structure of bLf.

Influence of processing factors on the stability of model mayonnaise with whole egg during long-term storage.

Mayonnaise-like oil-in-water emulsions with different stabilities-evaluated from the degree of macroscopic defects, e.g., syneresis-were prepared by different formulations and processing conditions (egg yolk weight, homogenizer speed, and vegetable oil temperature). Emulsions prepared with lower egg yolk content were destabilized for shorter periods. The long-term stability of emulsions was weakly related to initial properties, e.g., oil droplet distribution and protein coverage at the interface. Protein aggregation between oil droplets was observed and would be responsible for the instability of emulsions exhibited by the appearance defects. SDS-PAGE results for adsorbed and unadsorbed proteins at the O/W interface suggested that predominant constituents adsorbed onto the interface were egg white proteins as compared with egg yolk components when the amount of added egg yolk was low. In present condition, egg white proteins adsorbed at the O/W interface could be a bridge of neighboring oil droplets thereby causing flocculation in emulsions.

Dietary low-fat soy milk powder retards diabetic nephropathy progression via inhibition of renal fibrosis and renal inflammation.

SCOPE: Diabetic nephropathy (DN) is a major cause of end-stage renal disease. Here, we examined the effect of long-term consumption of a low-fat soy milk powder (LFSMP) on the diabetic kidney structure and function. METHODS AND RESULTS: KKAy mice were fed a casein-, LFSMP-, or high-fat soy mixture powder (HFSMP)-based diet for 4 months. Plasma and urine were subjected to a biochemical assay every 2-4 wk. Renal morphology and protein expression were evaluated by histochemical staining and western blots. Although HFSMP-based diet showed no protective effect on DN. LFSMP-fed mice exhibited lower water intake, urine output, and urinary albumin, and glucose excretion. Furthermore, strong preservation of renal structural proteins and low urinary N-acetyl-beta-d-glucosaminidase activity were observed in LFSMP-fed mice, indicating alleviation of renal injury. LFSMP-fed mice showed a lesser degree of mesangial matrix expansion, of tubulointerstitial fibrosis, and of myofibroblast differentiation. Finally, milder renal inflammation was found in LFSMP-fed mice, as evidenced by a decrease in urinary monocyte chemoattractant protein- 1 excretion and lesser macrophage infiltration into the tubulointerstitium. CONCLUSION: The present data suggests that long-term consumption of LFSMP but not HFSMP retards DN progression via suppressing renal injury, myofibroblast differentiation, and renal macrophage infiltration in diabetic condition.

Enantioselective GC-MS analysis of volatile components from rosemary (Rosmarinus officinalis L.) essential oils and hydrosols.

Essential oils and hydrosols were extracted from rosemary harvested in different seasons, and the chemical compositions of volatile components in the two fractions were analyzed by gas chromatography-mass spectrometry (GC-MS). Enantiomers of some volatile components were also analyzed by enantioselective GC-MS. Classification of aroma components based on chemical groups revealed that essential oils contained high levels of monoterpene hydrocarbons but hydrosols did not. Furthermore, the enantiomeric ratios within some volatile components were different from each other; for example, only the (S)-form was observed for limonene and the (R)-form was dominant for verbenone. These indicate the importance of determining the enantiomer composition of volatile components for investigating the physiological and psychological effects on humans. Overall, enantiomeric ratios were determined by volatile components, with no difference between essential oils and hydrosols or between seasons.

Localization of 9- and 13-oxo-octadecadienoic acids in tomato fruit.

We previously reported that the two peroxisome proliferator-activated receptor-α agonists, 9- and 13-oxo-octadecadienoic acids (oxo-ODAs), were found in the tomato fruit. However, their localization remains unknown. Herein, we showed that oxo-ODAs localize primarily in the fruit peel and their amount increases after the homogenization of the tomato fruit.