A study of oxygen isotope scrambling in the enzymic and non-enzymic oxidation of linoleic acid.

Lipoxygenases-1 and -2 isolated from soybeans were incubated with linoleic acid in the presence of a mixture of 16O2 and 18O2. The formation of 16O/18O-molecules which is indicative for a head-to head reaction of peroxy radicals was determined and compared with that produced during autoxidation of a linoleic acid emulsion in the presence of ferric ions. Lipoxygenase-1 was much less active in scrambling than lipoxygenase-2 which was comparable to that found in the autoxidation reaction.

Photolysis of unsaturated fatty acid hydroperoxides. 4. Fatty acid products from the aerobic decomposition of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate dissolved in cyclohexane.

In the presence of oxygen, UV-irradiation of a solution of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) in cyclohexane leads to a broad pattern of reaction products of which a trihydroxyene, seven epoxyhydroxides, four hydroxydienes, four epoxyhydroperoxides, six oxodienes and an epoxycyclohexylene were identified as the main components. Two oxodienes having a (Z)-double bond adjacent to the carbonyl group and the epoxycyclohexylene are reported for the first time. In contrast to results published recently for the UV-degradation of the 13-HPOD in methanol, the decomposition of the 13-HPOD in cyclohexane results in the formation of the 9-HPOD by a rearrangement of the hydroperoxy group. Consequently the reaction products are formed as mixtures of positional isomers. The reaction pathways leading to the identified compounds are discussed.

Formation of aroma-active strecker-aldehydes by a direct oxidative degradation of Amadori compounds.

alpha-Dicarbonyls, generated by sugar degradation, catalyze the formation of the so-called Strecker aldehydes from alpha-amino acids. To check the effectiveness of Amadori compounds (suggested as important intermediates in alpha-dicarbonyl formation from carbohydrates) in Strecker aldehyde formation, the amounts of phenylacetaldehyde (PA) formed from either an aqueous solution of L-phenylalanine/glucose or the corresponding Amadori compound N-(1-deoxy-D-fructosyl-1-yl)-L-phenylalanine (ARP-Phe) were compared. The results revealed the ARP-Phe as a much more effective precursor in PA generation. On the contrary, a binary mixture of glucose/phenylalanine yielded preferentially phenylacetic acid, in particular, when reacted in the presence of oxygen and copper ions. Further model experiments gave evidence that a transition-metal-catalyzed oxidation of the ARP-Phe by air oxygen into the 2-hexosulose-(phenylalanine) imine is the key step responsible for the favored formation of phenylacetaldehyde from the Amadori compound. This mechanism might explain differences in the ratios of Strecker aldehydes and the corresponding acids depending on the structures of carbohydrate degradation products involved.

Comparison of the most odor-active compounds in fresh and dried hop cones (Humulus lupulus L. variety spalter select) based on GC-olfactometry and odor dilution techniques.

Application of aroma extract dilution analysis on the volatiles obtained from dried cones of Spalter Select hops grown in the German hop-growing area of Hallertau revealed 23 odorants in the flavor dilution (FD) factor range of 16-4096, 20 of which could be identified. On the basis of high FD factors, trans-4, 5-epoxy-(E)-2-decenal, linalool, and myrcene were identified as the most potent odorants, followed by ethyl 2-methylpropanoate, methyl 2-methylbutanoate, (Z)-1,5-octadien-3-one, nonanal, (E,Z)-1,3, 5-undecatriene, 1,3(E),5(Z),9-undecatetraene, propyl 2-methylbutanoate, 4-ethenyl-2-methoxyphenol, and 1-octen-3-one. Ten of the high-impact hop aroma compounds had previously not been identified as hop constituents and, in particular, 1,3(E),5(Z), 9-undecatetraene has not yet been reported as a food odorant. In an extract obtained from fresh hops, in addition to the odorants found in dry hops, (Z)-3-hexenal was characterized as a further key odorant rendering an additional green aroma note to the fresh material.

Evaluation of key aroma compounds in hand-squeezed grapefruit juice (Citrus paradisi Macfayden) by quantitation and flavor reconstitution experiments.

Twenty-five odor-active compounds were quantified in the fresh, hand-squeezed juice of White Marsh seedless grapefruits using stable isotope dilution assays. By calculation of the odor activity values of the odorants (ratio of their concentrations in the juice to their odor thresholds in water) it was shown that the fruity esters ethyl 2-methylpropanoate, ethyl butanoate, and (S)-ethyl 2-methylbutanoate, and the fruity, sweet winelactone, as well as the grassy smelling (Z)-hex-3-enal, and trans-4,5-epoxy-(E)-dec-2-enal with metallic odor, were among the most potent odorants of the fresh grapefruit juice. The typical sulfurous, grapefruit-like odor quality was mainly due to the catty, blackcurrant-like 4-mercapto-4-methylpentan-2-one and the grapefruit-like smelling 1-p-menthene-8-thiol. These findings were confirmed by reconstitution experiments to simulate the aroma of the fresh grapefruit juice.

Evaluation of aroma differences between hand-squeezed juices from Valencia late and Navel oranges by quantitation of key odorants and flavor reconstitution experiments.

Twenty-five odor-active compounds were quantified in hand-squeezed juices of Valencia late and Navel oranges using stable isotope dilution assays. Odor activity values (OAVs, ratio of the concentration to odor thresholds) based on odor thresholds in water were calculated for the entire set of aroma compounds in both varieties. It was shown that due to their high OAVs, the fruity-smelling esters ethyl 2-methylpropanoate, ethyl butanoate, (S)-ethyl 2-methylbutanoate, and 3a,4,5,7a-tetrahydro-3,6-dimethyl-2(3H)-benzofuranone (wine lactone), the grassy smelling (Z)-hex-3-enal, and the citrus-like decanal were the most potent odorants in both juices. The weaker fruity note in the Navel oranges was clearly correlated with significantly lower OAVs of all fruity-smelling esters but a higher OAV of (Z)-3-hexenal compared to Valencia late. Model solutions simulating the odor of both orange varieties confirmed the findings of the quantitation studies.

Aroma properties of a homologous series of 2,3-epoxyalkanals and trans-4,5-epoxyalk-2-enals.

A few odor-active epoxyaldehydes, formed during lipid peroxidation, have recently been reported as intense aroma compounds in foods. However, very little is known about their flavor properties in general. Syntheses of homologous trans-2,3-epoxyalkanals (C(6)-C(12)) and trans-4,5-epoxy-(E)-2-alkenals (C(7)-C(12)) followed by structural characterization using mass spectrometry (MS/EI; MS/CI) and (1)H NMR measurements were performed. An evaluation of their odor qualities and odor thresholds by gas chromatography-olfactometry revealed the following: within the trans-2,3-epoxyalkanals, the odor quality changed from grassy for the compounds with six and seven carbon atoms to citrus-like or soapy for aldehydes with eight and more carbon atoms. The odor thresholds lay in the range of 3-15 ng/L (in air) and were nearly identical within the series; however, a slight minimum was measured for trans-2,3-epoxyoctanal to trans-2,3-epoxydecanal. In the series of the trans-4,5-epoxyalk-(E)-2-enals the C(10) compound was characterized by the lowest odor threshold of 0.6-2.5 pg/L of air. However, all trans-4,5-epoxy-alk-(E)-2-enals smelled intensely metallic.

Determination of key aroma compounds in the crumb of a three-stage sourdough rye bread by stable isotope dilution assays and sensory studies.

An investigation of the volatile fraction of a freshly prepared sourdough rye bread crumb by means of the aroma extract dilution analysis (AEDA), followed by identification experiments, revealed 22 flavor compounds in the flavor dilution (FD) factor range of 128 to 2048. Quantitations performed by stable isotope dilution assays (SIDA) and a calculation of odor activity values (OAV; ratio of concentration to odor threshold) revealed the following as contributors to the overall crumb flavor: 3-methylbutanal (malty), (E)-2-nonenal (green, fatty), (E,E)-2,4-decadienal (fatty, waxy), hexanal (green), acetic acid (sour, pungent), phenylacetaldehyde (honey-like), methional (boiled potato-like), vanillin (vanilla-like), 2,3-butandione (buttery), 3-hydroxy-4,5-dimethyl-2(5H)-furanone (spicy), and 2- and 3-methylbutanoic acid (sweaty). Using either citrate buffer, starch, or deodorized crumb as model matrixes, the typical malty and sour rye bread crumb flavor was reproduced by adding a mixture of 20 reference odorants in the "natural" concentrations as quantitatively determined in the fresh crumb.

Quantification of the mycotoxin patulin by a stable isotope dilution assay.

Two stable isotope dilution assays for the quantification of patulin [4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one] in foods were developed using (13)C-labeled patulin as the internal standard. One method was performed by means of LC/MS in negative electrospray ionization mode without derivatization; the other used HRGC/HRMS after trimethylsilylation of the patulin isotopomers. In comparison with previously reported methods based on high-performance liquid chromatography with UV detection, HRGC/HRMS of the derivatized samples showed better repeatability, higher recovery rates (96% at a spike level of 200 ng/L), and a 100 times lower detection limit (12 ng/L). In contrast, LC/MS showed a much lower performance as compared to HPLC/UV or HRGC/HRMS. Using HRGC/HRMS, the mycotoxin was quantified in many different fruit products and in molded wheat bread.

Characterization of the most odor-active volatiles in fresh, hand-squeezed juice of grapefruit (Citrus paradisi Macfayden).

By application of the aroma extract dilution analysis on an extract prepared from fresh grapefruit juice, 37 odor-active compounds were detected in the flavor dilution (FD) factor range of 4-256 and subsequently identified. Among them the highest odor activities (FD factors) were determined for ethyl butanoate, p-1-menthene-8-thiol, (Z)-3-hexenal, 4,5-epoxy-(E)-2-decenal, 4-mercapto-4-methylpentane-2-one, 1-heptene-3-one, and wine lactone. Besides the 5 last mentioned compounds, a total of 13 further odorants were identified for the first time as flavor constituents of grapefruit. The data confirmed results of the literature on the significant contribution of 1-p-menthene-8-thiol in grapefruit aroma but clearly showed that a certain number of further odorants are necessary to elicit the typical grapefruit flavor.

Quantitative model studies on the formation of aroma-active aldehydes and acids by strecker-type reactions.

Application of aroma extract dilution analysis on the volatiles formed by reacting glucose and L-phenylalanine (30 min, 100 degrees C) revealed the Strecker aldehyde, phenylacetaldehyde (PA), and, in addition, phenylacetic acid (PAA) as the two key odorants among the volatiles formed. Quantitative measurements on alpha-dicarbonyl formation revealed that the 3-deoxyosone and glyoxal were formed as the first prominent sugar degradation products, whereas 2-oxopropanal became predominant after approximately 4 h at 100 degrees C. Among the four alpha-dicarbonyls analyzed, 2-oxopropanal proved to be the most effective in generating PA as well as PAA from phenylalanine, but the reaction parameters significantly influenced the ratio of both odorants; for example, at pH 3.0 the ratio of PA to PAA was 3:1, whereas at pH 9.0 the ratio was 1:5. Furthermore, in the presence of oxygen and copper ions the formation of the acid was further increased. 3-Deoxyosone and glucosone were found to be effective precursors of phenylacetaldehyde, but neither was very effective in acid generation. On the basis of the results, a new oxygen-dependent formation pathway of the Strecker reaction is proposed.

Differences in key odorants of handmade juice of yellow-flesh peaches (Prunus persica L.) induced by the workup procedure.

Application of aroma extract dilution analysis (AEDA) on a flavor extract isolated from a freshly prepared, enzyme-inactivated peach juice using solvent extraction and high-vacuum distillation (extract I) revealed 24 odor-active regions in the gas chromatogram. Flavor dilution (FD) factors ranged from 4 to 512. The highest FD factors were determined for beta-damascenone (cooked-apple-like) and gamma-decalactone (peach-like). Cooking of peaches for 2 h in an apparatus equipped for simultaneous steam distillation/extraction (extract II) yielded an overall more intense aroma extract (extract II). By AEDA, 30 odorants were detected in the FD-factor region of 4-16384 and were subsequently identified. The results revealed that in extract II, besides the two above-mentioned aroma compounds, both had FD factors of 16 384; delta-decalactone, gamma-dodecalactone additionally, and 6-dodeceno-gamma-lactone contributed with very high FD factors (FD 8192) to the overall aroma. In general, the thermal treatment led to the formation of 15 new odorants which were not detected in I. Furthermore, the lactones and beta-damascenone were significantly increased in II, thereby indicating their generation from precursors in the fresh juice.

Studies on the key odorants formed by roasting of wild mango seeds (Irvingia gabonensis).

Application of the aroma extract dilution analysis on a concentrate of volatiles obtained by solvent extraction and high vacuum distillation from roasted seeds (180 degrees C; 15 min) of wild mango (Irvingia gabonensis) revealed 32 odor-active compounds with flavor dilution (FD) factors ranging from 8 (low odor activity) to 2048 (high odor activity). The identification experiments based on the use of reference odorants revealed methional (cooked potato-like) followed by 2-acetyl-1-pyrroline (roasty, popcorn-like), butan-2,3-dione, pentan-2,3-dione, 2-ethyl-3,5-dimethylpyrazine, and 2,3-diethyl-5-methylpyrazine as the key aroma compounds among the 27 odorants identified. All odorants are reported for the first time as components of roasted wild mango seeds.

Model studies on the influence of coffee melanoidins on flavor volatiles of coffee beverages.

Addition of the total melanoidin fraction isolated by water extraction from medium-roasted coffee powder to a model solution containing a set of 25 aroma compounds mimicking the aroma of a coffee brew reduced, in particular, the intensity of the roasty, sulfury aroma quality. Model studies performed by static headspace analysis revealed that especially three well-known coffee odorants, that is, 2-furfurylthiol (FFT), 3-methyl-2-butene-1-thiol, and 3-mercapto-3-methylbutyl formate, were significantly reduced in the headspace above an aqueous model solution when melanoidins were added. In particular, the low molecular weight melanoidins (1500-3000 Da) led to the most significant decrease in FFT. In contrast, for example, aldehydes remained unaffected by melanoidin addition.

Characterization of the key aroma compounds in dried fruits of the West African peppertree Xylopia aethiopica (Dunal) A. Rich (Annonaceae) using aroma extract dilution analysis.

Application of aroma extract dilution analysis on an extract of the dried fruits of the West African peppertree Xylopia aethiopica obtained by extraction with diethyl ether followed by sublimation in vacuo revealed 28 odor-active compounds in the flavor dilution (FD) factor range of 4-8192, all of which could be identified. The highest FD factor was found for linalol (floral), followed by (E)-beta-ocimene (flowery), alpha-farnesene (sweet, flowery), beta-pinene (terpeny), alpha-pinene (pine needle-like), myrtenol (flowery), and beta-phellandrene (terpeny). Vanillin (vanilla-like) and 3-ethylphenol (smoky, phenolic) showing somewhat lower FD factors (FD = 128) were detected for the first time as constituents of the dried fruit.

Development of a stable isotope dilution assay for an accurate quantification of protein-bound N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine using a (13)C-labeled internal standard.

Syntheses of the labeled Amadori compound [(13)C(6)]-N(epsilon)-(1-deoxy-D-fructos-1-yl)-L-lysine ([(13)C(6)]-DFLys) and the labeled glycated tetrapeptide Ala-[(13)C(6)]-DFLys-Leu-Gly are presented. The compounds were used in the development of stable isotope dilution assays for the quantification of the degree of glycosylation of bovine serum albumin treated for 20 min at 95 degrees C in the presence of glucose. The experiments revealed that the use of the labeled standards in combination with LC/MS allowed the exact quantification of protein-bound DFLys with the high recovery rate of 95% (at a spike level of 150 nmol/mg of protein) and a low detection limit of 5 nmol/mg of protein. The data revealed, however, that DFLys is significantly degraded during the enzymic hydrolysis of the protein backbone generally needed in the quantification procedure and, furthermore, incomplete digestion of the protein was observed. Both sources of errors were clearly overcome by using in particular the labeled peptide as the internal standard.

Occurrence of 12-methyltridecanal in microorganisms and physiological samples isolated from beef.

12-Methyltridecanal (MT) smelling tallowy, beef-like was formed from plasmalogens when beef was boiled. To clarify the origin of MT, its concentration was determined by a stable isotope dilution assay in bacteria and protozoa isolated from the rumen of bovine animals as well as in the plasma, erythrocytes, and other physiological samples. The highest amounts of MT were found in bacteria followed by protozoa. The MT content of the erythrocytes was small. The results support the hypothesis that microorganisms are the main source of MT of which a small amount is resorbed by the animal and transported to the muscular tissue where MT is incorporated into plasmalogens.

Development of a stable isotope dilution assay for the quantification of 5-methyl-(E)-2-hepten-4-one: application to hazelnut oils and hazelnuts.

A stable isotope dilution assay was developed for the quantitation of the hazelnut odorant 5-methyl-(E)-2-hepten-4-one by mass chromatography using synthesized [(2)H](2)-5-methyl-(E)-2-hepten-4-one as the internal standard. Application of the method on two batches of commercial hazelnut oils, processed from either roasted or unroasted nuts, revealed 6.4 microg 5-methyl-(E)-2-hepten-4-one per kg of unroasted oil whereas 315.8 microg per kg was determined in the roasted nut oil. The about 50-fold higher amount of 5-methyl-(E)-2-hepten-4-one in roasted hazelnut oil suggested the necessity of a thermal treatment to generate the flavor compound. Pan frying of raw hazelnuts (9 to 15 min) or boiling of the crushed nut material for 1 h in water led to an increase of 5-methyl-(E)-2-hepten-4-one by factors of 600 and 800, respectively, thereby corroborating that the major part of the nut flavorant is formed during heat treatment from a yet unknown precursor in hazelnuts.

Quantification of the mycotoxins patulin and ochratoxin A by stable isotope dilution assays.

For analysis of trace compounds, stable isotope dilution assays (SIDAs) have gained increasing importance in the past years. This methodology is based on the use of stable isotopically labelled analogues of the analytes as internal standards (IS). To take the mycotoxins patulin and ochratoxin A as examples, the benefits of SIDAs were demonstrated both for foods and for clinical analyses.Regarding PAT, an isotopomer labelled with(13)C was used as IS and enabled quantitation of the mycotoxin in tissues and blood. By applying this technology, a fast passive diffusion into tissue was proven with the model of the perfused rat stomach. Furthermore, rapid degradation of PAT was observed when it was reacted with blood, which was attributed to the formation of PAT-GSH adducts detected by LC-MS/MS.For OTA, a SIDA was based on the use of [(2)H5]-OTA as the IS and proved to be more accurate when compared to alternative methods such as HPLC-FD or ELISA. In contrast to PAT, OTA was detectable in human blood and urine samples. Under the assumption that the majority of OTA is circulating in blood, an urinary excretion rate of about 1% of the whole body content per day was calculated.

Carry over (transfer) of feed-borne acrylamide into eggs, muscle, serum, and faeces - a pilot study with Japanese quails (Coturnix coturnix japonica).

Two groups of 10 quails each were fed for 30 days on a diet containing either 663 microg feed-borne acrylamide (AA)/kg feed (group B) or 2472 microg AA/kg feed (group C) respectively. The concentrations of AA present in excreta and eggs were compared with those measured in eggs and excreta obtained from a control group (group A) fed on a diet low in AA (<10 microg/kg). The results clearly showed that the eggs of quails in group C contained approximately 53-112 microg AA/kg based on dry weight, while eggs collected from group A did not contain AA (limit of detection in eggs 3.5 microg/kg). Nearly 5% of the AA fed was recovered in the excreta. The AA concentrations in liver, muscle and serum were detectable, but below the limit of quantitation.