Identification and quantitation of the rice aroma compound, 2-acetyl-1-pyrroline,in bread flowers (Vallaris glabra Ktze)

The aroma impact compound, 2-acetyl-1-pyrroline (2AP), has been identified for the first time in headspace of fresh bread flowers (Vallaris glabra Ktze) in which volatile components were extracted by solid-phase microextraction (SPME) at room temperature prior to analysis by gas chromatography-mass spectrometry. A total of 50 volatiles were detected. Among these, 23 volatiles were identified, predominantly in a group of terpenes. More volatiles were found in the extract of fresh bread flowers obtained by continuous steam distillation and solvent extraction (SDS). Of the 40 volatiles identified, the additional components were mainly straight-chain saturated hydrocarbons. 2AP was found in the extracts obtained by both SPME (0.37%) and SDS (2.71% relative proportion). Quantitative analyses of 2AP in bread flowers and other plant materials were performed by solvent extraction employing acidic solutions and capillary GC with flame ionization detection. The highest concentration of 2AP was found in dried flowers of V. glabra at 26.1 mg/kg. By comparison with other plant sources, fresh leaves of Pandanus amaryllifolius Roxb contain 2AP at 10.3 mg/kg and Thai fragrant rice, Khao Dawk Mali 105, at 3.0 mg/kg.     

Photodegradation of 1-(2-chlorobenzoyl)-3-(4-chlorophenyl) urea in different media and toxicity of its reaction products

The photodegradation of 1-(2-chlorobenzoyl)-3-(4-chlorophenyl) urea in simulated air, methanol, dioxane, hexane, and water, with a xenon lamp as light source, was studied. The rate constants and half-lives of this compound in various media under nitrogen or oxygen were determined. The photoreaction products were analyzed with HPLC-UV, GC-MS, and direct probe MS and found to have some differences in different cases. With (32)P postlabeling DNA adduct formation experiments, one of the main products, 2-chorobenzamide, was found to be able to form a DNA adduct.     

Metabolism of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and its isomer, N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), in rats. 1. Identification of metabolites in feces and urine

Rats were orally dosed with a 1:1 diastereomixture of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), both labeled with 14C, at 200 mg/kg/day for 5 consecutive days, and 16 metabolites in urine and feces were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Several of them were unique decyanated and/or cyclic compounds (lactone, imide, cyclic amide, cyclic imino ether forms). Major biotransformation reactions of the mixture of S-2900 and S-2900S in rats are proposed on the basis of the metabolites identified in this study.     

Simultaneous quantitation of 2-acetyl-4-tetrahydroxybutylimidazole, 2- and 4-methylimidazoles, and 5-hydroxymethylfurfural in beverages by ultrahigh-performance liquid chromatography-tandem mass spectrometry

An ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric (MS/MS) method was developed for the simultaneous quantification of 2-acetyl-4-tetrahydroxybutylimidazole (THI), 2- and 4-methylimidazoles (2-MI and 4-MI), and 5-hydroxymethylfurfural (HMF) in beverage samples. A C30 reversed-phase column was used in this method, providing sufficient retention and total resolution for all targeted analytes, with an MS/MS instrument operated in selected reaction monitoring (SRM) mode for sensitive and selective detection using isotope-labeled 4-methyl-d(3)-imidazole (4-MI-d(3)) as the internal standard (IS). This method demonstrates lower limit of quantification (LLOQ) at 1 ng/mL and coefficient of determination (r(2)) >0.999 for each analyte with a calibration range established from 1 to 500 ng/mL. This method also demonstrates excellent quantification accuracy (84.6-105% at 5 ng/mL, n = 7), precision (RSD < 7% at 5 ng/mL, n = 7), and recovery (88.8-99.5% at 10, 100, and 200 ng/mL, n = 3). Seventeen carbonated beverage samples were tested (n = 2) in this study including 13 dark-colored beverage samples with different flavors and varieties and 4 light-colored beverage samples. Three target analytes were quantified in these samples with concentrations in the range from 284 to 644 ng/mL for 4-MI and from 706 to 4940 ng/mL for HMF. THI was detected in only one sample at 6.35 ng/mL.     

Thermal generation of 3-amino-4,5-dimethylfuran-2(5H)-one, the postulated precursor of sotolone, from amino acid model systems containing glyoxylic and pyruvic acids

4,5-Dimethyl-3-hydroxy-2(5H)-furanone (sotolone), a naturally occurring flavor impact compound, can be isolated from various sources, especially fenugreek seeds. It can also be thermally produced from intermediates generated from the Maillard reaction such as pyruvic and ketoglutaric acids, glyoxal, and 2,3-butanedione. A naturally occurring precursor of sotolone, 3-amino-4,5-dimethyl-2(5H)-furanone, was thermally generated for the first time from pyruvic acid and glycine or from glyoxylic acid and alanine model systems. Isotope labeling studies have implicated 4,5-dimethylfuran-2,3-dione as an intermediate that can be converted into 3-amino-4,5-dimethyl-2(5H)-furanone through Strecker-like interaction with any amino acid. Furthermore, these studies have also indicated the presence of two pathways for the formation of 4,5-dimethylfuran-2,3-dione, one requiring pyruvic acid and a formaldehyde source and the other requiring glyoxylic acid and acetaldehyde. Self-aldol condensation of pyruvic acid followed by lactonization and further aldol reaction with formaldehyde can generate the same intermediate as the self-aldol addition product of acetaldehyde with glyoxylic acid followed by lactonization. The pyruvic acid pathway was found to be a more efficient route than the glyoxylic acid pathway. Furthermore, the pyruvic acid/glycine model system was able to generate sotolone in the presence of moisture, and in the presence of ammonia, commercial sotolone was converted back into 3-amino-4,5-dimethyl-2(5H)-furanone.     

Mousy off-flavor of wine: precursors and biosynthesis of the causative N-heterocycles 2-ethyltetrahydropyridine, 2-acetyltetrahydropyridine,and 2-acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176

The N-heterocyclic bases, 2-ethyltetrahydropyridine (1), 2-acetyl-1-pyrroline (2), and 2-acetyltetrahydropyridine (3) are associated with the occurrence of mousy off-flavor in wine. The biosynthesis of these N-heterocycles by the wine lactic acid bacterium, Lactobacillus hilgardii DSM 20176, was studied by high-cell-density incubation in combination with a minimal chemically defined N-heterocycle assay medium. The key components of the defined N-heterocycle assay medium included D-fructose, ethanol, L-lysine, L-ornithine, and mineral salts. N-heterocycle formation was quantitatively determined by gas chromatography-mass spectrometry. The formation of 2 and 3 required the concomitant availability of a fermentable carbohydrate (D-fructose), ethanol, and iron (Fe(2+)). In addition, L-ornithine stimulated the formation of 2 and repressed 3 formation, whereas L-lysine stimulated the formation of 3 and repressed 2 formation. Incorporation of d(6)-ethanol into the acetyl side chain of 2 and 3, and of d(4)-acetaldehyde into the acetyl side chain of 3, confirmed that ethanol and acetaldehyde could serve as major side chain precursors. A pathway for the formation of 2 and 3 by heterofermentative lactic acid bacteria is proposed involving the interaction of accumulated C-2 intermediates from the heterolactic pathway and N-heterocyclic intermediates derived from the metabolism of L-ornithine and L-lysine.     

Degradation studies on benzoxazinoids. Soil degradation dynamics of (2R)-2-O-beta-D-glucopyranosyl-4-hydroxy-(2H)- 1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and its degradation products, phytotoxic allelochemicals from Gramineae

Wheat (Triticum aestivum L.) has been found to possess allelopathic potential and studies have been conduced to apply wheat allelopathy for biological weed control. 2,4-Dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) is a common product found in wheat, corn, and rye exudates and it can be released to the environment by that way. In this report, the stability of DIBOA is studied in two soils from crop lands of wheat cv. Astron and cv. Ritmo. These varieties were selected by their concentrations of DIBOA and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) from aerial parts and by the bioactivities of their aqueous extracts in the growth of wheat coleoptile sections. The degradation rate of DIBOA in these soils was measured in laboratory tests during 90 h by high-pressure liquid chromatography methods. These analyses demonstrate that DIBOA was transformed primarily into 2-benzoxazolinone (BOA). This transformation was similar in both soil types with an average half-life of 43 h. The degradation studies for BOA show its biotransformation to 2-aminophenoxazin-3-one (APO) with a half-life of 2.5 days. Therefore, BOA is an intermediate product in the biotransformation from DIBOA to APO in these wheat crop soils and is consistent with previous findings. APO was not degraded after three months in soil, suggesting that its degradation rate in soil is very slow.     

Biosynthetic mechanism of 2-acetyl-1-pyrroline and its relationship with Delta1-pyrroline-5-carboxylic acid and methylglyoxal in aromatic rice (Oryza sativa L.) callus

2-Acetyl-1-pyrroline (2-AP) was identified as the major flavor compound in aromatic rice varieties Tainung 71 and 72. In order to understand the mechanism of 2-AP biosynthesis in aromatic rice, we studied the formation of putative precursors, Delta(1)-pyrroline-5-carboxylic acid and methylglyoxal. The endogenous Delta(1)-pyrroline-5-carboxylic acid contents of Tainung 71 and 72 calli reached 191 to 276%, compared to nonaromatic rice Tainung 67. In addition, calli of Tainung 71 and 72 contained 1.30- and 1.36-fold, respectively, higher methylglyoxal levels than that of Tainung 67. Specific enzyme activities of Delta(1)-pyrroline-5-carboxylic acid-synthetic enzyme including Delta(1)-pyrolline-5-carboxylic acid synthetase (P5CS) and ornithine aminotransferase (OAT) increased significantly in aromatic rice varieties. The expression levels of P5CS1 and P5CS2 genes were found to be significantly higher in aromatic rice than nonaromatic rice. Results of a tracer experiment with (15)N-labeled glutamic acid revealed that the nitrogen atom of 2-acetyl-1-pyrroline was derived from glutamic acid. Upregulation of P5CS in aromatic rice Tainung 72 may contribute to the increase of Delta(1)-pyrroline-5-carboxylic acid level and thus leads to the accumulation of an extra amount of 2-acetyl-1-pyrroline.     

Liquid chromatography-tandem mass spectrometry analysis of 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine in cooked meats

Several cooked meats such as beef (fried, coated-fried), pork (fried, coated-fried), and chicken (fried, griddled, coated-fried, roasted) were analyzed for the heterocyclic amine 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5- b]pyridine (4'-OH-PhIP) not commonly determined in food and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). The highest content of 4'-OH-PhIP was found in fried and griddled chicken breast, the concentration being 43.7 and 13.4 ng/g, respectively, whereas the corresponding PhIP concentrations were 19.2 and 5.8 ng/g. The estimated concentration of both pyridines in fried pork loin, in fried pork sausages, and in coated-fried chicken was below 2.5 ng/g. In the rest of the samples, 4'-OH-PhIP was not detected. The analyses were performed by solid-phase extraction and LC-MS/MS. The fragmentation of 4'-OH-PhIP in an ion trap mass analyzer was studied in order to provide information for the identification of 4'-OH-PhIP. Additionally, the effect of red wine marinades on the formation of 4'-OH-PhIP in fried chicken was examined, finding a notable reduction (69%) in the amine's occurrence.     

Improved synthesis and deployment of (2S,3R)-2-(2Z,5Z-octadienyl)-3-nonyloxirane, a pheromone of the pink moth, Lymantria mathura

We report two new syntheses of (2S,3R)-2-(2Z,5Z-octadienyl)-3-nonyloxirane, the main sex pheromone component of the pink moth, Lymantria mathura. The key step in the first route was the construction of (Z,Z)-1-bromo-1,4-heptadiene (6), which was coupled in the final step with 2-iodomethyl-3-nonyloxirane 4 via a Grignard reaction. The second approach employed alkylation of 1,4-heptadiynyllithium with epoxy triflates 7 in ether/hexane and provided the pheromone in >/=37% overall yield from alcohol 2. The 4:1 ratio of pheromone enantiomers, reportedly the most attractive to pink moth males, can be directly crafted from appropriately selected Sharpless asymmetric epoxidation conditions.     

Thermal decomposition of 5-(hydroxymethyl)-2-furaldehyde (HMF) and its further transformations in the presence of glycine

Thermal decomposition of HMF has been so far studied indirectly through carbohydrate degradation reactions assuming HMF as the main product. Such studies, however, do not necessarily generate relevant information on HMF decomposition because many other products are generated simultaneously. Direct thermal decomposition using different concentrations of HMF in silica gel was studied using pyrolysis-GC-MS. Undiluted HMF generated four peaks corresponding to 5-methylfurfural, 2,5-furandicarboxaldehdye, HMF, and a major unknown peak at retention time of 20.73 min. The diluted HMF in silica gel (15-fold) generated only the first three peaks. The generation of the unknown peak was dependent on the concentration of HMF, indicating the possibility of a dimeric structure; furthermore, when HMF was generated from [U-13C6]glucose in the reaction mixture, the highest mass in the spectrum of the unknown peak showed the incorporation of 11 carbon atoms from the glucose. Thermal decomposition studies of HMF have also indicated that in the absence of amino acids it can mainly dimerize and the initially formed dimer can degrade to generate 5-methylfurfural and 2,5-furandicarboxaldehyde. On the other hand, thermal degradation of HMF in the presence of glycine generated Schiff base adducts of HMF, 5-methylfurfural, and 2,5-furandicarboxaldehdye in addition to 2-acetyl-5-methylfuran and a newly discovered adduct, 5-[(dimethylamino)methyl]-2-furanmethanol.     

Analysis of a model reaction system containing cysteine and (E)-2-methyl-2-butenal, (E)-2-hexenal, or mesityl oxide

Cysteine conjugates, resulting from the addition of cysteine to alpha,beta-unsaturated carbonyl compounds, are important precursors of odorant sulfur compounds in food flavors. The aim of this work was to better understand this chemistry in the light of the unexpected double addition of cysteine to two unsaturated aldehydes. These reactions were studied as a function of pH. When (E)-2-methyl-2-butenal (tiglic aldehyde, 4) was treated with cysteine in water at pH 8, the major product formed was the new compound (4R)-2-(2-[[(2R)-2-amino-2-carboxyethyl]thio]methylpropyl)-1,3-thiazolidine-4-carboxylic acid (6). Under acidic conditions (pH 1), we also observed a double addition, but the second cysteine was linked by a vinylic sulfide bond to form the previously unreported major product, (2R,2'R,E)-S,S'-(2,3-dimethyl-1-propene-1,3-diyl)bis-cysteine (7). When (E)-2-hexenal (12) was treated with cysteine under acidic conditions, the major product was the novel (4R,2' 'R)-2-[2'-(2' '-amino-2' '-carboxyethylthio)pentyl]-1,3-thiazolidine-4-carboxylic acid (13), and the formation of an vinylic sulfide compound analogous to 7 was not observed. Reduction of the acidic crude reaction mixture with NaBH(4) afforded 13 and the cysteine derivative (R)-S-[1-(2-hydroxyethyl)butyl]cysteine (14) in 14% yield. Treating (E)-2-hexenal with cysteine at pH 8 followed by NaBH(4) reduction yielded the new product (3R)-7-propylhexahydro-1,4-thiazepine-3-carboxylic acid (15). Addition of cysteine to mesityl oxide (16), at pH 8, followed by reduction with NaBH(4) furnished (R)-S-(3-hydroxy-1,1-dimethylbutyl)cysteine (3) and the new compound (3R)-hexahydro-5,7,7-trimethyl-1,4-thiazepine-3-carboxylic acid (18).     

Studies on the aroma of five fresh tomato cultivars and the precursors of cis- and trans-4,5-epoxy-(E)-2-decenals and methional

Three tasty (BR-139, FA-624, and FA-612) and two less tasty (R-144 and R-175) fresh greenhouse tomato cultivars, which significantly differ in their flavor profiles, were screened for potent odorants using aroma extract dilution analysis (AEDA). On the basis of AEDA results, 19 volatiles were selected for quantification in those 5 cultivars using gas chromatography-mass spectrometry (GC-MS). Compounds such as 1-penten-3-one, ( E, E)- and ( E, Z)-2,4-decadienal, and 4-hydroxy-2,5-dimethyl-3(2 H)-furanone (Furaneol) had higher odor units in the more preferred cultivars, whereas methional, phenylacetaldehyde, 2-phenylethanol, or 2-isobutylthiazole had higher odor units in the less preferred cultivars. Simulation of the odor of the selected tomato cultivars by preparation of aroma models and comparison with the corresponding real samples confirmed that all important fresh tomato odorants were identified, that their concentrations were determined correctly in all five cultivars, and that differences in concentration, especially of the compounds mentioned above, make it possible to distinguish between them and are responsible for the differential preference. To help elucidate formation pathways of key odorants, labeled precursors were added to tomatoes. Biogenesis of cis- and trans-4,5-epoxy-( E)-2-decenals from linoleic acid and methional from methionine was confirmed.     

Quantitative analysis, occurrence, and stability of (E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene in wine

A stable isotope dilution assay has been developed for quantification of (E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene (4) in wine using a [(2)H(6)]-analogue. Using this method, 4 was found in 96 out of 97 white wines, but in none of 12 red wines analyzed. 4 was found to be most prevalent in Semillon wines, followed by Chardonnay, with Riesling showing the least amount of 4 among these three varieties. 4, like 1,1,6-trimethyldihydronaphthalene (TDN, 3), appears to be formed during the aging process. 4 was found to be unstable in model wine, and in both white and red wine, with the order of stability being model > white > red. In a PVPP-stripped red wine, the rate of degradation of 4 was substantially lessened, with the final concentrations very close to those observed in model wine. When treated with either grape or wine tannin extracts in model wine, the concentration of 4 was found to decrease to levels very close to those observed with an untreated red wine. When white wine was heated at 45 degrees C, 4 was formed, indicating the presence of precursor forms. The amounts formed were much higher than those found in a commercial white wine. 4 was also observed in red wine heated to 45 degrees C, but the concentration produced was much less than that with white wine.     

Influence of human saliva on odorant concentrations. 2. aldehydes,alcohols, 3-alkyl-2-methoxypyrazines,methoxyphenols, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone

The influence of human whole saliva on selected alcohols, aldehydes, 3-alkyl-2-methoxypyrazines, and phenols in food-relevant concentrations was investigated. At pH 7.5-8 it was found that the alcohols, methoxyphenols, methoxypyrazines, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone remained unmodified by saliva, whereas aldehydes were reduced to their corresponding alcohols. Generally, the processes were found to be dependent on the salivary activity of the panelists as well as on the concentration of the applied odorants. Reduction of the aldehydes did not occur after thermal treatment of the saliva. These investigations are aimed at finding an explanation for longer lasting aftertaste in humans, as it is induced by some odor-active compounds after the consumption of food materials.     

Early post-mortem AMP-activated protein kinase (AMPK) activation leads to phosphofructokinase-2 and -1 (PFK-2 and PFK-1) phosphorylation and the development of pale, soft, and exudative (PSE) conditions in porcine longissimus muscle

Pale, soft, and exudative (PSE) meat has been recognized for decades. Fast glycolysis during early post-mortem stage while the muscle temperature is still high is the cause of PSE meat. To elucidate the molecular mechanism underlying this fast glycolysis in muscle to become PSE meat, post-mortem ATP metabolism, fructose-2,6-diphosphate content, and the activities of AMPK, glycogen phosphorylase, and pyruvate kinase were examined in post-mortem muscle. Earlier and faster post-mortem AMPK activation was responsible for the significantly lower pH and higher lactic acid accumulation (p<0.05) seen in PSE muscle, which resulted in the occurrence of PSE meat. In muscle that became PSE meat, AMPK was activated at 0 h post-mortem and reached maximal activation at 0.5 h post-mortem, whereas AMPK reached maximal activation at 1 h post-mortem in the normal pork loin. Higher fructose-2,6-diphosphate content (p<0.05) was detected in PSE muscle compared to normal muscle at early post-mortem stage. However, no difference in the activities of glycogen phosphorylase and pyruvate kinase, rate-controlling enzymes in glycogenolysis and glycolysis, respectively, was detected between PSE and normal pork loins. Because fructose-2,6-diphosphate is a product of phosphofructokinase-2 (PFK-2), these data suggest that AMPK regulates post-mortem glycolysis through its phosphorylation and activation of PFK-2, which then up-regulates the activity of phosphofructokinase-1 (PFK-1), a key rate-controlling enzyme in glycolysis. Early AMPK activation in PSE muscle is associated with early consumption of ATP, because higher AMP and IMP contents and lower ATP content were detected in PSE meat compared to normal meat. Other mechanisms causing early AMPK activation in PSE meat may exist, which warrants further investigation.