Quantitation using a stable isotope dilution assay (SIDA) and thresholds of taste-active pyroglutamyl decapeptide ethyl esters (PGDPEs) in sake

A stable isotope dilution assay (SIDA) for two taste-active pyroglutamyl decapeptide ethyl esters (PGDPE1; (pGlu)LFGPNVNPWCOOC₂H₅, PGDPE2; (pGlu)LFNPSTNPWCOOC₂H₅) in sake was developed using deuterated isotopes and high-resolution mass spectrometry. Recognition thresholds of PGDPEs in sake were estimated as 3.8 μg/L for PGDPE1 and 8.1 μg/L for PGDPE2, evaluated using 11 student panelists aged in their twenties. Quantitated concentrations in 18 commercial sake samples ranged from 0 to 27 μg/L for PGDPE1 and from 0 to 202 μg/L for PGDPE2. The maximum levels of PGDPE1 and PGDPE2 in the sake samples were approximately 8 and 25 times higher than the estimated recognition thresholds, respectively. The results indicated that PGDPEs may play significant sensory roles in the sake. The level of PGDPEs in unpasteurized sake samples decreased during storage for 50 days at 6 °C, suggesting PGDPEs may be enzymatically decomposed.     

Taste-Guided Fractionation and Instrumental Analysis of Hydrophobic Compounds in Sake

The taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA). All of the high-TDA factor fractions showed a bitter or astringent taste in common, but their taste characters were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionation, using RP-HPLC and an instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of the peptides of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl ester of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably with the compounds of high hydrophobicity.     

Taste-guided fractionation and instrumental analysis of hydrophobic compounds in sake

The taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA). All of the high-TDA factor fractions showed a bitter or astringent taste in common, but their taste characters were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionation, using RP-HPLC and an instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of the peptides of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl ester of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably with the compounds of high hydrophobicity.     

Characteristics of Egyptian boza and a fermentable yeast strain isolated from the wheat bread

A sample of indigenous beer, boza was collected at Cairo, Egypt and analysed. Boza was an off-white porridge-like slurry containing 3.8% (v/v) ethanol. Volatile esters and higher alcohols such as ethyl acetate and isoamyl alcohol were detected in the boza by gas chromatography. The pH of the boza was 3.7. Organoleptically, this alcoholic beverage had an estery flavour and a sour taste. A fermentable yeast strain EG1 was isolated from the material wheat bread and identified, and was considered to resemble Candida krusei. The rice sake made with the yeast strain C. krusei EG1 at 30 °C contained 11.7% ethanol, 74.1 mg/l ethyl acetate and its pH value was 4.2.     

Characteristics of a rice beer (zutho) and a yeast isolated from the fermented product in Nagaland,India

Rice beer, known locally as zutho was collected in the Kohima district in Nagaland, India, and subjected to analytical and microbiological characterization. Zutho was a whitish porridge-like slurry containing 5.0% (v/v) ethanol. Volatile esters and higher alcohols, such as ethyl acetate and 3-methylbutanol, were detected in this indigenous alcoholic beverage by gas chromatography. The pH and acidity of zutho were 3.6 and 5.1, respectively. Zutho had a fruity aroma and sour taste and its unique aroma had characteristics similar to those of Japanese sake and sprouted rice sake. A fermentation yeast isolated from zutho was identified as being a strain of Saccharomyces cerevisiae and was found to be suitable as the brewing yeast for ethanol fermentation.     

Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1–7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with “Kimoto yeast starter” and “Yamahaimoto” had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with “Sokujo-moto”. When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50–5,000 μM dl-Ala did not effect on the aroma of the sakes at all.     

Synthesis of DHA/EPA-rich phosphatidylcholine by immobilized phospholipase A<Subscript>1:</Subscript> effect of water addition and vacuum condition

DHA/EPA-rich phosphatidylcholine (PC) was successfully synthesized by immobilized phospholipase A₁ (PLA₁)-catalyzed transesterification of PC and DHA/EPA-rich ethyl esters in a solvent-free system. Effects of reaction temperature, water addition and substrate mass ratio on the incorporation of DHA/EPA were evaluated using response surface methods (RSM). Water addition had most significant effect on the incorporation. Reaction temperature and substrate mass ratio, however, had no significant effect on the incorporation. The maximal incorporation was 19.09 % (24 h) under the following conditions: temperature 55.7 °C, water addition 1.1 wt % and substrate mass ratio (ethyl esters/PC) 6.8:1. Furthermore, effects of water addition (from 0 to 1.25 wt %) on DHA/EPA incorporation and the composition of products were further investigated. The immobilized PLA₁ was more active when water addition was above 0.5 wt %. By monitoring the reaction processes with different water addition, a possible reaction scheme was proposed for transesterification of PC with DHA/EPA-rich ethyl esters. In summary, PC and sn2-lysophosphatidylocholine (LPC) were predominant in the mixtures at early stages of reaction, whereas sn1-LPC and glycerophosphocholine (GPC) predominant at later stages. The vacuum employed after 24 h significantly increased the incorporation of DHA/EPA and the composition of PC, and the highest incorporation (30.31 %) of DHA/EPA was obtained at 72 h and the yield of PC was 47.2 %.     

Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu(1) conversion is favored at pH 6.0 while Gln(1) conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.     

Flow of toxic metals in food-web components of tropical mangrove ecosystem,Southern India

The concentration of heavy metals in water, sediment, and various food-web components like plankton, shrimp, bivalve, and fishes were collected from Muthupet mangrove ecosystem. Heavy metal concentration in water samples was relatively lesser than the biological and sediment samples. Among the heavy metals studied, zinc showed highest concentration ranged from 1.81 to 81.5 mg/kg or mg/L, whereas Cd (0–26.06 mg/kg or mg/L) was found to be lesser in all the samples except a few organisms viz. Anadara sp. (26.06 mg/kg), Coilia sp. (10.09 mg/kg), Anguila sp. (9.14 mg/kg), and Tachysurus maculates (6.95 mg/kg) observed during this study. Pb and Cu were ranged from 10.29 to 14.99 mg/kg and 0.59 to 16.06 mg/kg, respectively. The reported values of heavy metals were several folds higher than permissible levels of international regulatory agencies like WHO, FAO, and USEPA. The order of accumulation of heavy metals in biological samples are as follows: Pb > Cu > Zn > Cd. All the biota showed a higher degree of bioconcentration factor for Zn, in the range of 3.90–34.39. Principal component analysis concluded that Muthupet was contaminated by lithogenic as well as anthropogenic activities.Therefore, field observation and sample analysis clearly indicated that sampling sites were polluted with both point and nonpoint source of pollution.     

Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1

The fruity odor of Chinese liquor is largely derived from ester formation. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is one of the most important esters in the strong aromatic Chinese liquor (or Luzhou-flavor liquor), which is the most popular and best-selling liquor in China. In the traditional fermentation process, ethyl caproate in strong aromatic liquor is mainly produced by aroma-producing yeast, bacteria, and mold with high esterification abilities in a mud pit at later fermentation stages at the expense of both fermentation time and grains rather than by the ethanol-fermenting yeast Saccharomyces cerevisiae. To increase the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae AY15) and shorten the fermentation period, we constructed a recombinant strain EY15 by overexpressing EHT1 (encoding ethanol hexanoyl transferase), in which FAA1 (encoding acyl-CoA synthetases) was deleted. In liquid fermentation of corn hydrolysate and solid fermentation of sorghum, ethyl caproate production by EY15 was remarkably increased to 2.23 and 2.83 mg/L, respectively, which were 2.97- and 2.80-fold higher than those of the parental strain AY15. Furthermore, an increase in ethyl octanoate (52 and 43 %) and ethyl decanoate (61 and 40 %) production was observed. The differences in fermentation performance between EY15 and AY15 were negligible. This study resulted in the creation of a promising recombinant yeast strain and introduced a method that can be used for the clean production of strong aromatic Chinese liquor by ester-producing S. cerevisiae without the need for a mud pit.     

Analysis of volatile aroma constituents of wine produced from Indian mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) by GC-MS

Volatile aroma compounds are synthesized by wine yeast during wine fermentation. In this study the volatile aroma composition of two varieties of mango wine were determined to differentiate and characterize the wines. The wine was produced from the fruits of two varieties of mango cultivars namely Banginapalli and Alphonso. The volatile compounds formed in mango wine were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Thirty-two volatile compounds in wines were determined of which four were new and unidentified present in lower concentration. Apart from the ethanol (8.5 ± 0.28 and 7.2 ± 0.28% v/v), 1-propanol (54.11 ± 0.33 and 42.32 ± 0.57 mg/l), isobutyl alcohol (102 ± 1.57 and 115.14 ± 2.88 mg/l) and isoamyl alcohol (123 ± 2.88 and 108.40 ± 0.23 mg/1) were found to be the major flavouring higher alcohols in the mango wines produced from the fruits of Banginapalli and Alphonso respectively. Ethyl acetate (35 ± 0.57 and 30.42 ±1.15 mg/l) was the major ester component in both wines produced. Besides, other esters like ethyl octonoate, ethyl hexanoate and ethyl decanoate were also present in the wines. Cyclohexane methanol (1.45 ± 0.11 mg/l) was present only in wine made from Banginapalli and β-phenylethyl butanoate (0.62 ± 0.01 mg/1) was found only in Alphonso wine. The results demonstrate that the wine prepared from Banginapalli variety had better aroma composition and good taste than that from the Alphonso variety.     

Yeast strain effect on the concentration of major volatile compounds and sensory profile of wines from Vitis vinifera var. Treixadura

The fermentative ability of five Saccharomyces cerevisiae strains and their influence on the aroma and sensory properties of wine from Treixadura were evaluated to determine the most suitable yeast that produces a high quality wine from this grapevine variety. The results indicated that all strains, except T2, were able to lead the vinification process and have good fermentative powers. The chemical composition of wines obtained with resident cellar yeasts consisted of a significant amount of glycerol, a compound that contributes to the structure and smoothness in taste of the wine. In addition, these strains were good producers of acetates, ethyl esters and fatty acids, compounds that positively influence wine aroma. Compounds with a direct contribution to the aroma of Treixadura wines (Odour Activity Value >1) included 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isoamyl and ethyl acetate and the ethyl esters, ethyl butyrate, ethyl hexanoate, ethyl octanoate and ethyl decanoate. Sensory analysis supported the fact that cellar strains produced more fruity and floral wines than commercial or spontaneous fermentations. We conclude that resident cellar yeasts enhanced sensory quality of wines from Treixadura.     

Identification of five new bradykinin potentiating peptides (BPPs) from Bothrops jararaca crude venom by using electrospray ionization tandem mass spectrometry after a two-step liquid chromatography

Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme displaying strong anti-hypertensive effects in human subjects. The BPPs can be recognized by their typical pyroglutamyl proline-rich oligopeptide sequences presenting invariably a proline residue at the C-terminus. In the present study, we identified 18 BPPs, most of them already described for the B. jararaca venom. We isolated and sequenced new peptides ranging from 5 to 14 amino acid residues exhibiting similar amino acid sequence features. The applied methodology consisted of a strait two-step liquid chromatography, followed by mass spectrometry analysis. Besides the amino acid sequence homology, the corresponding synthetic peptides were able to potentiate bradykinin on the isolated guinea-pig ileum.     

Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation

This work investigates the continuous production of alkyl esters from soybean fatty acid (FA) charges using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of FA charge to alcohol (methanol and ethanol) molar ratio, from 1:1 to 1:6, substrate flow rate in the range of 0.5–2.5 mL/min and output irradiation power up to 154 W, at fixed temperature of 65 °C, on the reaction conversion. Results showed that almost complete conversions to fatty acids ethyl esters were achieved at mild ultrasonic power (61.6 W), FA to ethanol molar ratio of 1:6, operating temperature (65 °C) and remained nearly constant for long-term reactions without negligible enzyme activity losses.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.     

Combined effects of fermentation temperature and pH on kinetic changes of chemical constituents of durian wine fermented with Saccharomyces cerevisiae

This study investigated the effects of temperature (20 and 30 °C) and pH (pH 3.1, 3.9) on kinetic changes of chemical constituents of the durian wine fermented with Saccharomyces cerevisiae. Temperature significantly affected growth of S. cerevisiae EC-1118 regardless of pH with a higher temperature leading to a faster cell death. The pH had a more significant effect on ethanol production than temperature with higher production at 20 °C (5.95%, v/v) and 30 °C (5.56%, v/v) at pH 3.9, relative to that at pH 3.1 (5.25 and 5.01%, v/v). However, relatively higher levels of isobutyl alcohol and isoamyl alcohol up to 64.52 ± 6.39 and 56.27 ± 3.00 mg/L, respectively, were produced at pH 3.1 than at pH 3.9 regardless of temperature. In contrast, production of esters was more affected by temperature than pH, where levels of ethyl esters (ethyl esters of octanoate, nonanoate, and decanoate) and acetate esters (ethyl acetate and isoamyl acetate) were significantly higher up to 2.13 ± 0.23 and 4.61 ± 0.22 mg/L, respectively, at 20 °C than at 30 °C. On the other hand, higher temperature improved the reduction of volatile sulfur compounds. This study illustrated that temperature control would be a more effective tool than pH in modulating the resulting aroma compound profile of durian wine.

     

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.     

Reconstruction of peptide using p-dimethylsulfoniophenol ester in aqueous solution

Summary p-Dimethylsulfoniophenol (DSP) ester was applied on the reconstruction of peptide, because its hydrophilicity was favorable for the reaction performed in aqueous media. As an example, the biological response of an enzymatically digested peptide hormone, Substance P (SP), was recovered by the reaction with pyroglutamyl (pGlu)-Phe-DSP. This result showed that pGlu-Phe-DSP restored the active site of SP destroyed by the enzyme.     

2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-glucoside Suppresses Expression of Adhesion Molecules in Aortic Wall of Dietary Atherosclerotic Rats and Promonocytic U937 Cells

We sought to investigate whether TSG suppressed the ICAM-1/VCAM-1 expression in dietary atherosclerotic rats and in Ox-LDL-induced U937 cells. For this purpose, 60 male Sprague–Dawley rats were randomly-and-equally divided into six groups. Atherosclerosis was induced by feeding rats a hyperlipidemic diet. TSG (120, 60 or 30 mg/kg/day) was administered by oral gavage. Simvastatin (2 mg/kg/day) was administered as positive control whereas physiological saline (0.9 % NaCl) served as untreated control. After 12 weeks, rats were euthanized by ethyl carbonate (1,200 mg/kg) and aortic wall samples were collected. Besides, U937 cells were stimulated for 48 h by Ox-LDL (80 μg/mL) with and without TSG (120, 60, 30 μg/L) or simvastatin (100 μg/L). ICAM-1/VCAM-1 mRNA expression was determined by RT-PCR and protein expression was detected by immunohistochemistry and/or western blotting. The data show that ICAM-1/VCAM-1 mRNA/protein expression was significantly enhanced in atherosclerotic aortas compared with normal diet group. Ox-LDL-induced ICAM-1/VCAM-1 mRNA/protein expression in U937 cells. Importantly, TSG significantly inhibited ICAM-1/VCAM-1 expression in atherosclerotic aortas in a dose-dependent manner. TSG-pretreatment also inhibited ICAM-1/VCAM-1 expression in Ox-LDL-induced U937 cells. Therefore, we concluded that TSG suppressed the expression of adhesion (ICAM-1/VCAM-1) molecules both in vivo (in aortic wall of dietary atherosclerotic rats) and in vitro (U937 cells).