Riichiro Uchida

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.

Analysis of the correlation between dipeptides and taste differences among soy sauces by using metabolomics-based component profiling

Characterizing the relationships between the components and taste differences among soy sauces can help evaluate and improve the quality of soy sauces. Although previous studies have reported that certain taste-active dipeptides, the relationships between taste differences and dipeptides of soy sauces are unknown. Therefore, our objective in this study was to investigate the correlations between the dipeptides and the taste differences among soy sauces. To analyze the dipeptides, we constructed an analytical method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode. Based on this method, we detected 237 dipeptides, the largest number ever detected in soy sauce research. Next, orthogonal projections to latent structures regressions were performed. The data matrix of components, including dipeptides and other low-molecular-weight hydrophilic components obtained from gas chromatography/mass spectrometry (GC/MS), served as explanatory variables (366 in total), whereas a sensory data matrix obtained using quantitative descriptive analysis served as the response variable. The accuracy of models for the sweetness and saltiness differences constructed using the LC/MS/MS and GC/MS data matrix were higher than did models constructed using only the GC/MS data matrix. As a result of investigation of the correlation between the dipeptides and taste differences among soy sauces by using variable importance in the projection (VIP) score, many dipeptides showed the high correlation with taste differences. Specifically, Ile-Gln, Pro-Lys, Ile-Glu, Thr-Phe, and Leu-Gln showed the high VIP score on sweet differences. This study is the first report that reveals the correlations between the dipeptides and taste differences among soy sauces.

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.

A convenient synthesis of adenosien 3',5'-cyclic phosphate (cAMP) benzyl and methyl triesters

The benzyl and methyl triesters of cAMP were synthesized from cAMP tri-n-butylammonium salt by alkylation with alkyl halides in the presence of Na 2 CO 3 in dimethylacetamide (DMA)

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.

Syntheses of modified 2-chloro-4-nitrophenyl β-maltopentaosides as useful substrates for assay of human alpha amylase

Twenty-three novel 2-chloro-4-nitrophenyl beta-D-maltopentaosides modified at the 6(5) and/or 4(5) position were synthesized as substrates for human alpha amylase. Two human alpha amylases hydrolyzed 6(5)-deoxy-6(5)-, 6(5)-O-, and 4(5),6(5)-di-O-substituted derivatives at essentially a single D-glucosidic linkage, but 4(5),6(5)-O-bridged and 4(5)-O-substituted derivatives were hydrolyzed at two or more linkages. The amylases displayed smaller Km values for the compounds having hydrophobic modifications. In these derivatives, 2-chloro-4-nitrophenyl O-(6-bromo-6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-tris[O-alpha-D- glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (10), 2-chloro-4-nitrophenyl O-(6-azido-6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (19), and 2-chloro-4-nitrophenyl O-[6-O-(N-isopropyl)carbamoyl-alpha-D-glucopyranosyl]-(1-->4)- tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (30), which were rapidly hydrolyzed by the two amylases at a limited position at an approximately equal rate, were shown to be very useful blocked-type substrates for assay of human alpha amylase.

Determination of nicotianamine in soy sauce and other plant-based foods by LC-MS/MS.

Nicotianamine is a nonproteinogenic amino acid, known to be an important metal chelator in plants. Recently, the antihypertensive effect of nicotianamine was discovered. In this study, a simple method to determine nicotianamine was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a multimode ODS column. This method does not need derivatizing or ion-pairing reagents to retain nicotianamine, which is known for its poor retention on reversed-phase columns because of its high polarity. Moreover, this method showed a sufficient limit of detection (0.5 ng/mL), so it was found to be suitable for the analysis of nicotianamine in soy sauce and other foods, without cleanup. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of nicotianamine in soy sauce ranged from <0.25 to 71 μg/g. Nicotianamine was also determined in other foods, including soy milk, vegetable juice, fruit juice, and bottled tea.

New enzymatic synthesis of 63-modified maltooligosaccharides and their inhibitory activities for human α-amylases

Ten new 6(3)-modified maltopentaoses and tetraoses were synthesized by enzymatic reactions utilizing cyclodextrin glycosyltransferase (EC 2.4.1.19) and subsequent human salivary alpha-amylase (HSA) (EC 3.2.1.1). Among these compounds, alpha-D-glucopyranosyl-(1-->4)- alpha-D-glucopyranosyl-(1-->4)-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (11) and alpha-D-glucopyranosyl-(1-->4)-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (12) showed strong inhibitory activities for human pancreatic alpha-amylase (HPA) and HSA. The IC50 of 6(3)-deoxymaltopentaose 11 (8.0 x 10(-5) M for HPA, 1.0 x 10(-4) M for HSA) and 6(3)-deoxymaltotetraose 12 (2.0 x 10(-3) M for HPA, 2.0 x 10(-3) M for HSA) were lower than that of 6(3)-deoxymaltotriose [(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-alpha-D- glucopyranosyl-(1-->4)-D-glucopyranose 13; 2.0 x 10(-3) M for HPA, 4.2 x 10(-2) M for HSA].

A new enzyme, maltobionate α-d-glucohydrolase, from alkalophilic Bacillus sp. N-1053

A new enzyme, maltobionate alpha-D-glucohydrolase, was purified to apparent homogeneity from a cell-free extract of alkalophilic Bacillus sp. N-1053 about 930-fold with a yield of 18% and some of its properties were investigated. The enzyme showed optimum activity at about pH 7.0, and was stable over the range of pH 6.0-9.5. The molecular weight was estimated to be 152,000 and 71,000 by HPLC gel filtration on TSKgel G3000SWXL and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme hydrolyzed maltobionate more effectively than disaccharides such as maltose and maltitol or trisaccharides such as maltotrionate, maltotriose and maltotriitol, but showed no activity toward polysaccharides such as amylose, amylopectin and soluble starch. The reaction products from 1 mol of maltobionate were found to be 1 mol of beta-D-glucose and 1 mol of D-gluconate. The Km value for maltobionate was 1.63 mM and the Vmax/Km value for maltobionate was the largest among the substrates tested. The enzyme activity was almost completely inhibited by Hg2+, Ag+, iodine and N-bromosuccinimide, and also inhibited by p-nitrophenyl alpha-D-glucoside, maltose and maltitol.

Positive/Negative Allosteric Modulation Switching in an Umami Taste Receptor (T1R1/T1R3) by a Natural Flavor Compound, Methional

Taste is a vital sensation for vertebrates, enabling the detection of nutritionally important substances or potential toxins. A heteromeric complex of two class C GPCRs, T1R1 and T1R3, was identified as the umami (savory) taste receptor. Amino acids and 5′-ribonucleotides are well known to be natural ligands for human T1R1/T1R3. In this study, we reveal that methional, which is a familiar flavor component in foods, is an allosteric modulator of T1R1/T1R3. Receptor expression experiments showed that methional served as a positive allosteric modulator (PAM) of human T1R1/T1R3 and functioned as a negative allosteric modulator (NAM) of mouse T1R1/T1R3. Although amino acids and 5′-ribonucleotides bound to the extracellular domain of T1R1, the use of interspecies chimeric receptors demonstrated that methional interacted with the transmembrane domain of T1R1. Site-directed mutagenesis and molecular modeling showed that methional could potentially bind at two distinct sites in the transmembrane domain of T1R1 and that the amino acid residues in the bottom of the allosteric pocket engendered the switch between the PAM and NAM modes, which could contribute to switching the binding position of methional. These results may be applicable for elucidating the molecular mechanisms underlying ligand recognition by other class C GPCRs.