Volatile Flavor Components of Dried Bonito (Katsuobushi) II. From Neutral Fraction

The aqueous extract of dried bonito (Katsuobushi) was distilled under reduced pressure. The resulting distillate with diethyl ether and the extract was separated into acidic, phenolic, basic and neutral fractions. The neutral fraction was further fractionated into ten sub-fractions by silica gel column chromatography. All these sub-fractions were analyzed by gas chromatography and gas chromatography-mass spectrometry.

One hundred and sixty-five compounds were identified and 12 compounds were tentatively identified from the neutral fraction. Among them, 111 compounds were newly identified as flavor components of Katsuobushi.     

Volatile Flavor Compounds of Boiled Buckwheat Flour

Volatile flavor compounds of boiled buckwheat flour were collected using a Likens and Nickerson apparatus type and were fractionated into acidic, weak acidic, basic and neutral fractions. The neutral fraction was further fractionated by silica gel column chromatography. All fractions were analyzed by gas chromatography and gas chromatography-mass spectrometry.

Two hundred and nine compounds were thus identified. Among them, 168 were newly identified as volatile flavor compounds of buckwheat flour; special note is made of 2-(1′-ethoxyethyl)pyrazine and 2-(1′-ethoxyethyl)-5-methylpyrazine being found which are compounds previously unreported in any literature.     

Volatile Components of Roasted Shrimp

The components of the strong and favorable aroma obtained from roasted shrimp were investigated. The aroma concentrate of roasted shrimp was isolated by combining the dichloromethane extract and carbon dioxide distillation methods. The concentrate was fractionated into acidic, basic and neutral fractions. Each fraction was analyzed by combined gas chromatography-mass spectrometry. Seventy-seven compounds were identified, among which isovaleric acid, alkyl pyrazines, isovaleramide, ketones and some sulfur-containing compounds were the main and characteristic constituents of the roasted shrimp aroma.

These constituents were compared with those of the volatiles of boiled shrimp, which was prepared by using simultaneous distillation and extraction methods in a modified Likens and Nickerson’s apparatus.     

Fractionation and Identification of Volatile Compounds in Patis,a Philippine Fish Sauce

Commercial Philippine fish sauce (Patis) was steam-distilled and the distillate was fractionated into four fractions, neutral, basic, acidic and phenolic and each fraction was analyzed using gas chromatography and gas chromatography-mass spectrometry.

As a result of this study, a total of 66 compounds were identified in Patis, 14 of which were only a tentative identification. Out of these 66 identified compounds, 40 identified compounds have not been reported in previous studies on fish sauces. These identified compounds include 19 acids, 14 alcohols, 12 nitrogen containing compounds, 5 esters, 3 sulfur containing compounds, 1 phenol, 3 carbonyls, 7 hydrocarbons and 2 others. In the acidic fraction, 5 acids were considered major constituents which accounted for about 98% of the total acids. n-Butanoic acid was found to be the most abundant accounting for about 50% of the total acids.     

Volatile Acidic Compounds in Heat-Degradated Pork Fat

Pork fat was heated at 160~170°C for 3 hr under bubbling with air, and the volatile acidic compounds were extracted with 3 % aqueous sodium carbonate solution from the total volatile compounds collected during heating. Gas chromatographic identification of the acidic compounds was carried out on PEG-20M and Apiezon L columns. Some of the isolated compounds were identified by infrared and mass spectrometry. The compounds identified include; saturated acids, trans-2, trans-3, cis-3 alkenoic acids, hydroxy acids, keto acids, and dicarboxylic acids. Each of the unsaturated acid and γ-keto acid might be one of the precursors of γ- and δ-lactones reported previously.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

Volatile components obtained by the extraction of “Katsuobushi” with 80% ethanol and by the subsequent steam distillation of the extract were fractionated by the usual methods, and the resulting hydrocarbon fraction was investigated. Gas chromatographic study on this fraction originated from “Katsuobushi” of bonito (Katsuwonus pelamis) revealed 9 hydrocarbons, including n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, n-eicosane, n-heneicosane and n-docosane, which were tentatively identified by the retention times with the aid of authentic hydrocarbons. n-Pentadecane and n-heptadecane that were main components among these hydrocarbons were identified further by NMR and IR spectrometry. “Katsuobushi” of frigate mackerel (Auxis thazard), mackerel (Scomber Japonicus Houttuyn) or muroaji (Decapterus muroadsi) also contained n-penta-decane and n-heptadecane in large amounts, but did other hydrocarbons in negligible amounts.

Possible mechanisms of the hydrocarbon formation during the processing of “Katsuobushi” were discussed.     

Studies on the Flavor of Green Tea

The essential oil from manufactured green tea was separated into carboxylic, phenolic, carbonyl and alcoholic fractions and analysed by gas chromatography.

Seventeen alchols, two carbonyls, seven acids and two phenolic compounds were identified on the basis of their relative retention times and aroma of effluents by comparing with authentic compounds.

The quantities of these compounds were also determined by gas chromatography.

Four alcohols (present in rather high amounts), two caronyls, three esters and two phenols have remained unidentified.     

Antimicrobial Activity of Kumazasa (Sasa albo-marginata)

The organic solvent extract of Kumazasa leaves (Sasa albo-marginata) showed antimicrobial activity against bacteria, fungi and yeast. Kumazasa at a concentration of 0.2-1.0% showed stronger antimicrobial activity than potassium sorbate or sodium benzoate at the same concentration. Both acidic and phenolic fractions of the extract showed strong antimicrobial activity. Thirty acidic and phenolic compounds were identified by GC and GC-MS analysis. Acetic, propionic, benzoic, phenylacetic, salicylic, 3-hydroxybenzoic and o-anisic acids, and guaiacol, phenol, 4-ethylphenol, xylenol and 4-vinylphenol were the main components. It was estimated that these components play an important role in the formation of the antimicrobial activity of Kumazasa extract.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

The flavor concentrate obtained by the extraction of “Katsuobushi” of bonito (Katsuwonus pelamis) with 80% ethanol and by the subsequent steam distillation of the extract was fractionated by the usual method and the resulting neutral, non-carbonyl oxygenated fraction was investigated by gas chromatography. The following components were tentatively identified: 2-pentanol and 2-methyl-1-heptanol as free alcohols, and 4 alcohols of n- and isobutanol, n-pentanol and n-dodecanol and 9 carboxylic acids of propanoic, n-butanoic, n-pentanoic, n-octanoic, n-nonanoic n-decanoic, n-dodecanoic, n-tetradecanoic and n-hexadecanoic acid as the constituents of esters. A constituent alcohol existing in the largest amount was isolated by gas chromatography and identified as 2-methyl-1-heptanol by elemental analysis, NMR, IR, and MS. A constituent acid existing in large amount was also isolated and investigated similarly, and the structure was partially estimated. 2-Methyl-1-heptanol holds a fresh woody aroma and seems to have a major effect on “Katsuobushi” flavor.     

Shoyu (Soy Sauce) Flavor Components: Neutral Fraction

From the vacuum distilled volatiles of shoyu, a neutral fraction was obtained. Shoyu was also directly extracted with dichloromethane and then the extract was separated into ten (A~J) fractions. The J fraction was a neutral one. The J fraction was further separated into twelve fractions by liquid column chromatography. All fractions obtained were analyzed by gas chromatography and combined gas chromatography-mass spectrometry. Consequently, 142 components were identified, 82 of which have not been reported previously as volatile constituents of shoyu. The identified compounds were 37 hydrocarbons, 22 alcohols, 22 carbonyls, 22 esters, 12 furans, 6 sulfurous compounds, 1 pyrone, 5 phenols, 1 furanone, 1 acid, 1 lactone and 12 other compounds. From the results of quantitative analysis and organoleptic evaluation, phenylacetal-dehyde is considered to be most important in the neutral fraction.     

Volatile Compounds in Pyroligneous Liquids from Karamatsu and Chishima-sasa

Volatile components in pyroligneous liquids obtained from Karamatsu and Chishima-sasa. were extracted, fractionated into neutral/basic and acidic fractions, and identified by gas chromatography-mass spectrometry. In the neutral/basic fraction, 118 components were identified. Cyclic and acyclic ketones, pyridine derivatives, pyrazine derivatives and furan derivatives were important odorous components. Sixty-nine compounds were identified in the acidic fraction. The main groups were carboxylic acids and phenol compounds. The concentrations of alkyl phenols were almost the same between Karamatsu and Chishima-sasa, but there were large differences in the contents of 2-methoxyphenol derivatives and 2,6-dimethoxyphenol derivatives between them.     

Isolation and Characterization of Acidic Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. The acidic subfractions with various acidities were further fractionated, using a preparative amino acid analyzer and by paper chromatography to separate the acidic peptide components.

Four dipeptides and sugar derivatives of ten dipeptides and two tripeptides were isolated and characterized as the major acidic peptides in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their contents and taste intensities.     

Flavor Components of Roasted Almond

Roasted almond volatiles were separated into basic, carbonyl and non-carbonyl fractions. Each fraction was analyzed by combination gas chromatography-mass spectrometry. Twenty-five compounds, in addition to eighteen known components, were identified. Many of the components identified were considered to contribute to the overall flavor. 2,5-Dimethyl-4- hydroxy-3(2H)-furanone, which was identified from methanol extract of roasted almond, seemed to make the largest contribution to the sweet aroma of roasted almond.     

Collection and Identification of Allelopathic Compounds from the Undisturbed Root System of Bigalta Limpograss (Hemarthria altissima)

Collection of allelopathic chemicals from the undisturbed plant root system is difficult because of their low concentrations and the high level of contaminants in growth media such as soil. A new approach for the continuous trapping of quantities of extracellular chemicals from donor plants is described. Bigalta limpograss (Hemarthria altissima), a tropical forage with allelopathic activities, was established in sand culture. Nutrient solution was circulated continuously through the root system and a column containing XAD-4 resin. Extracellular hydrophobic metabolites were selectively adsorbed by the resin, while inorganic nutrients were recycled to sustain plant growth. Columns were eluted with methanol and the eluate separated into neutral, acidic, and basic fractions. Bioassays of trapped root exudates using lettuce seed combined with paper and thin layer chromatography showed that the inhibitors were mainly phenolic compounds. The active neutral fraction was methylated and analyzed by gas chromatography-mass spectrometry. Twelve compounds were identified, with two additional compounds tentatively identified. 3-Hydroxyhydrocinnamic, benzoic, phenylacetic, and hydrocinnamic acids were the major rhizospheric compounds with known growth regulatory activities.     

Reaction between Nitrite and Low Salt-soluble Diffusible Fraction of Meat. Some Compounds Influencing Nitrite Depletion and Producing Unidentified-N Compounds

A low salt-soluble, diffusible fraction of meat was separated into basic, neutral and acidic fractions. Investigations were then conducted on the reaction of each fraction with nitrite and on the recovery of the added nitrite.

The basic fraction shared the lowest ability to decompose nitrite and had 88% recovery of added nitrite-N. Hypoxanthine was one of the main components that affected the recovery in the basic fraction. In the neutral fraction, 42% of nitrite was decomposed, and the recovery of the nitrite was 107%. In the acidic fraction, more than 80% of nitrite was decomposed and 30% was converted to unidentified-N compounds. Among endogenous acidic substances tested, cysteic acid showed the highest ability to decompose nitrite, accompanying the production of unidentified-N compounds.     

Volatile Flavor Components of Watermelon (Citrullus vulgaris)

Volatile flavor components of watermelon fruit (Citrullus vulgaris) were obtained by distilling juice under reduced pressure and extracting the resulting distillate with Freon-11. The volatiles were separated into acidic and neutral fractions and then analyzed by gas chromatography and gas chromatography-mass spectrometry.

Fifty-two compounds were found for the first time as flavor components of watermelon. Among them, the following compounds have not been previously reported as naturally occurring flavor components: 4-oxononanal and 2-hydroxy-, 2-pentyloxy-, 2-hexyloxy-, 2-octyloxy-, 2-nonyloxy-, 2-[(Z)-3-noneyloxy]-, 2-[(Z)-2-nonenyloxy]-, 2-[(Z,Z)-3,6-nonadienyloxy]-, 2-benzyl-oxy-, and 2-phenetyloxy-5-pentyltetrahydrofurans.     

Genotoxicity and PAC analysis of particulate and vapour phases of environmental tobacco smoke

Samples of indoor air were collected from an office room (88 m3) both before smoking and during experimental smoking of 96 cigarettes by 10 persons within 6 h. The particulates were collected on glass-fibre filters and the vapour-phase compounds on XAD-2 resin. The samples were extracted with acetone and analysed quantitatively for polycyclic aromatic compounds and qualitatively with GC-MS. The extracts of filters and XAD-2 resins were fractionated into neutral/acidic and 2 basic (strong and weak bases) fractions; all these fractions were tested with the sister-chromatid exchange (SCE) assay in Chinese hamster ovary (CHO) cells and with the Salmonella/microsome test (strain TA98). Total concentrations of PAC were 205 ng/m3 in the background sample and 1207 ng/m3 after contamination by cigarette smoking. The total PAC concentrations were 4-6 times higher in the vapour phase than in the particulate phase. The fractions of the particulate samples collected before smoking showed mainly marginal genotoxic activity, whereas after smoking their genotoxicity increased dramatically. The fractions of the vapour phase samples were not genotoxic before smoking, but after smoking the neutral/acidic and strong basic fractions induced responses in both assays. The SCE assay was more sensitive towards the vapour-phase mutagens of environmental tobacco smoke (ETS). The relative responses of the two basic fractions, whereas the fraction containing neutral and acidic compounds was the most potent in the SCE assay. In the Salmonella test, the mutagenic activity was mainly detected with metabolic activation, while the induction of SCE in CHO cells was also seen without an exogenous metabolic activation system.     

Smith Degradation and Methylation of an Acidic Polysaccharide from Soy Sauce

APS–I, an acidic polysaccharide isolated from soy sauce, was subjected to Smith degradation and methylation, and each product obtained was examined by paper chromatography, gas-liquid chromatography and mass spectrometry. Some compounds, such as ethylene glycol, propylene glycol, glycerol, D-threitol, D-galactose, L-rhamnose, D-galacturonic acid and D-threonic acid, were identified as Smith degradation products, and their molar ratios were estimated. Several kinds of methylated sugars were detected in the hydrolysate of methylated APS–I and identified by gas chromatograph-mass spectrometry. On the basis of those findings and previous discussion, the constitution of APS–I was discussed.     

Comparison of Phenols in Smokes of Lamina and Midrib of Flue-cured Tobacco

In order to reveal the difference of the main stream smoke composition between lamina and midrib of flue-cured tobacco, the yields of neutral, basic, acidic and phenolic fractions of the smoke condensates of lamina and midrib cigarettes were compared. The composition of the phenolic fractions were also compared by glass capillary gas chromatography. Neutral and basic fractions were dominant in lamina smoke condensate, while ether insoluble fraction was dominant and formed about a half in midrib smoke condensate. For the semi-quantitative analysis of phenols with gas chromatography mono- and dihydroxybenzenes were extracted from the smoke condensate and missing of these compounds by oxidation was prevented by addition of dl-ascorbic acid. After trimethylsilylation they were simultaneously examined with gas chromatography. It was found that 4-methyl, 4-ethyl and 4-vinylcatechol in lamina smoke were much more rich than those in midrib smoke, while coniferylalcohol (a compound found first in cigarettes smoke condensates) was much more rich in midrib smoke.     

Isolation and Identification of α-Ketoaldehydes in Calf and Rabbit Livers

Carbonyl compounds in calf liver stored at 4°C for 5~6 hr after slaughter were investigated, with emphasis on α-ketoaldehydes. After conversion of carbonyls into their 2,4-dinitrophenylhydrazones (2,4-DNPs), they were fractionated by preparative thin-layer chromatography (TLC); at least, twenty-three fractions were separated, among which eighteen fractions contained 2,4-DNPs of α-dicarbonyls. From some of the fractions, crystalline 2,4-DNPs were isolated, and identified by TLC and infrared spectra: pyruvaldehyde, 3-deoxypentosulose, xylosulose, 3-deoxyglucosulose, 2,3-diketogulonic acid or/and dehydroascorbic acid, and formaldehyde were identified, and glucosulose was tentatively identified.

The carbonyl pattern, obtained from rabbit liver frozen in liquid nitrogen immediately after slaughter, was also similar as that from calf liver, and further, the amount of each α-ketoaldehydes remained at the almost same level during storage at 4°C for 24 hr. These results indicate that the identified α-ketoaldehydes are constituents or metabolic intermediates in calf and rabbit livers.

The formation mechanisms of the carbonyls in tissues are discussed.