Superior high-efficiency and high-throughput volatile flavor extraction of Japanese fermented seasonings by solvent-assisted stir bar solid extraction with reverse extraction.

Fermented seasonings have pleasant flavors that stimulate our appetite. Their flavoring properties change depending on factors such as their materials and fermented conditions. Therefore, a comparative analysis of their flavor is important when evaluating their quality. However, seasonings contain high levels of various matrices such as sugars, proteins, lipids, and ethanol, making it difficult to extract aroma compounds efficiently from them. In this study, we verified a high-efficient and high-throughput volatile flavor analysis of fermented seasonings by solvent-assisted stir bar solid extraction (SA-SBSE) with reverse extraction. We applied SA-SBSE to Japanese fermented seasonings, soy sauce, miso (fermented beans), and mirin (sweet rice wine) and compared their profiles with those from other common extraction methods, headspace gas-solid-phase microextraction (HS-SPME), liquid extraction with solvent-assisted flavor evaporation (LE-SAFE), and conventional SBSE (C-SBSE). The aroma properties and profiles of extracts from SA-SBSE were close to those of the original sample, being similar to that of LE-SAFE. In addition, potent aroma compounds in each sample were extracted by SA-SBSE and LE-SAFE, which were far superior to those by C-SBSE. For quantification, SA-SBSE extracts showed a good standard curve by the standard addition method. We could quantify maltol, one of the most common potent aroma compounds in all samples, for various commercial samples by such high-throughput analysis.

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.

Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii.

Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

Efficient ammonia production from food by-products by engineered Escherichia coli.

Ammonia is used as a fertilizer for agriculture, chemical raw material, and carrier for transporting hydrogen, and with economic development, the demand for ammonia has increased. The Haber-Bosch process, which is the main method for producing ammonia, can produce ammonia with high efficiency. However, since it consumes a large amount of fossil energy, it is necessary to develop an alternative method for producing ammonia with less environmental impact. Ammonia production from food by-products is an appealing production process owing to unused resource usage, including waste, and mild reaction conditions. However, when food by-products and biomass are used as feedstocks, impurities often reduce productivity. Using metabolic profiling, glucose was identified as a potential inhibitor of ammonia production from impure food by-products. We constructed the recombinant Escherichia coli, in which glucose uptake was reduced by ptsG gene disruption and amino acid catabolism was promoted by glnA gene disruption. Ammonia production efficiency from okara, a food by-product, was improved in this strain; 35.4 mM ammonia was produced (47% yield). This study might provide a strategy for efficient ammonia production from food by-products.

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.

Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara).

Ammonia is an essential substance for agriculture and the chemical industry. The intracellular production of ammonia in yeast (Saccharomyces cerevisiae) by metabolic engineering is difficult because yeast strongly assimilates ammonia, and the knockout of genes enabling this assimilation is lethal. Therefore, we attempted to produce ammonia outside the yeast cells by displaying a glutaminase (YbaS) from Escherichia coli on the yeast cell surface. YbaS-displaying yeast successfully produced 3.34 g/L ammonia from 32.6 g/L glutamine (83.2% conversion rate), providing it at a higher yield than in previous studies. Next, using YbaS-displaying yeast, we also succeeded in producing ammonia from glutamine in soybean residues (okara) produced as food waste from tofu production. Therefore, ammonia production outside cells by displaying ammonia-lyase on the cell surface is a promising strategy for producing ammonia from food waste as a novel energy resource, thereby preventing food loss.

Effect of temperature on the stability of various peptidases during peptide-enriched soy sauce fermentation.

We previously developed a peptide-enriched soy sauce-like seasoning called Fermented Soybean Seasoning (FSS) with high-temperature fermentation, and we have reported the antihypertensive effects of FSS. Seryl-tyrosine (Ser-Tyr) and glycyl-tyrosine (Gly-Tyr) were identified from FSS as active constituents in the antihypertensive effects. They were found to be particularly enriched in FSS; more so than in regular soy sauce. In the present study, we clarified one of the mechanisms underlying the accumulation of these bioactive peptides during high temperature soy sauce brewing. Crude enzyme extracts were prepared from model soy sauce mash (moromi) fermented at various temperatures. Leucine aminopeptidase-I, II, and seryl-tyrosine hydrolytic activity were found to decrease in the moromi incubated at the fermentation temperature of FSS whereas almost no decrease was observed in that of regular soy sauce. The concentrations of ACE inhibitory peptides, Ser-Tyr and Gly-Tyr, in the moromi incubated at high temperature were revealed to be higher than those at low temperature through quantitative LC-MS/MS analysis. These results suggested that the peptidases responsible for degrading low molecular weight bioactive peptides were inactivated during the high temperature fermentation, thus, these peptides would be likely to remain in the high temperature fermentation.

Antihypertensive mechanism of a peptide-enriched soy sauce-like seasoning: the active constituents and its suppressive effect on renin-angiotensin-aldosterone system.

We previously reported that a peptide-enriched soy sauce-like seasoning called fermented soybean seasoning (FSS) demonstrated antihypertensive effects both in spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats. Angiotensin I-converting enzyme (ACE) inhibitory substances (9 kinds of dipeptides and a nicotianamine) were identified from FSS. In the present study, we clarified the mechanisms underlying the antihypertensive effects of FSS in SHR. FSS was divided into the nicotianamine fraction and the peptide fraction. The peptide fraction was found to exert a more prevalent antihypertensive effect than the nicotianamine fraction in SHR. Among the peptides, we identified Gly-Tyr and Ser-Tyr as the 2 primary substances in FSS that contributed to the antihypertensive effect in SHR. These peptides were neither degraded by acid nor gastrointestinal proteases, and were absorbed into the circulating blood. FSS (2000 mg/kg) exerted ACE inhibitory activity in the lung of rats and provided a decrease (P= 0.0067) in the level of serum aldosterone after a single oral administration in SHR, resulting in the antihypertensive effect. The antihypertensive mechanism was found to be similar to therapeutic ACE inhibitors and other food-derived ACE inhibitory peptides, which are in wide use and are recognized as safe.

Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identification of its angiotensin I-converting enzyme inhibitory substances.

We have developed a peptide-enriched soy sauce-like seasoning termed Fermented Soybean Seasoning (FSS), by modifying the process of soy sauce brewing. The FSS has a 2.7-fold higher concentration of total peptides than regular soy sauce. The angiotensin I-converting enzyme (ACE) inhibitory activity of FSS (IC(50) = 454 microg/mL) was greater than that of regular soy sauce (IC(50) = 1620 microg/mL). The FSS demonstrated antihypertensive effects both in spontaneously hypertensive rats and in Dahl salt-sensitive rats during continuous feeding. The ACE inhibitory substances were purified from FSS by reversed-phase chromatography. Ala-Trp IC(50) = 10 microM; Gly-Trp IC(50) = 30 microM; Ala-Tyr IC(50) = 48 microM; Ser-Tyr, IC(50) = (67 microM; Gly-Tyr, IC(50) = 97 microM; Ala-Phe, IC(50) = 190 microM; Val-Pro, IC(50) = (480 microM; Ala-Ile, IC(50) = 690 microM; Val-Gly, IC(50) = 1100 microM; and a nicotianamine, IC(50) = 0.26 microM. [corrected] The concentrations of these substances in the FSS were revealed to be higher than that of regular soy sauce through quantitative LC-MS/MS analysis.