Flavor and Lipid Chemistry of Seafoods - American Chemical Society

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Chapter 4

Determination of Potent Odorants in Ripened Anchovy (Engraulis encrasicholus L.) by Aroma Extract Dilution Analysis and by Gas Chromatography-Olfactometry of Headspace Samples 1

Réda Triqui and Helmut Guth

2

1

Institut Agronomique et Vétérinaire Hassan II, B.P. 6202-Instituts 10101, Rabat, Morocco Deutsche Forschungsanstalt für Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany

2

The potent odorants of ripened anchovy were evaluated by aroma extract dilution analysis (AEDA) and gas chromatography-olfactometry of headspace samples (GCO-H). For AEDA, volatiles were isolated by direct solvent extraction followed by sublimation in vacuo to recover solvent plus volatile constituents. Methional and (Z)-1,5-octadien-3one were the most potent odorants among eighteen odorants in the flavor dilution range of 10-1000. Eleven odorants were detected by GCO-H of 20 mL headspace samples of ripened anchovy equilibrated at 35°C. Results of GCO-H were in good agreement with those of AEDA in that methional and (Z)-1,5-octadien-3-one were perceived at the small headspace samples of ripened anchovy (1 mL). Based on results of GCO-H, acetaldehyde, 3-methyl-butanal and 3-methylropanal were the most intense odorants among the highly volatile constituents of ripened anchovy. Recently, we reported on volatile compounds of importance to the aroma of anchovy and on changes in flavor profiles associated with the ripening process (1-2). In these investigations, the volatiles were isolated from anchovy by distillation of an aqueous slurry under vacuum at temperatures of 57-58°C and by subsequent solvent extraction of the distillate. Due to the relatively high temperatures used for isolation, the potential for generating thermally induced artifacts was not dismissed. Therefore, in the present study the volatiles were isolated by sublimation in vacuo (3). Aroma extract dilution analysis is limited to odorants boiling higher than the solvent used for the extraction and dilution steps. Furthermore, odorants boiling in the same range as the extraction solvent are partially lost during the concentration of the extract by distilling off the solvent (4). To evaluate the contribution of highly volatile compounds to the flavor of ripened anchovy, A E D A was complimented by gas chromatography-olfactometry of headspace samples (GCO-H) (5-6). © 1997 American Chemical Society Shahidi and Cadwallader; Flavor and Lipid Chemistry of Seafoods ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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FLAVOR AND LIPID CHEMISTRY OF SEAFOODS

Table I. Volatile compounds identified in ripened anchovy RI

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No 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

a

Compound

Odor Description

2,3-pentanedione hexanal (E)-2-hexenal methional 2-acetyl-1 -pyrroline l-octen-3-one (Z)-1,5 -octadien-3 -one 2,4-heptadienal (E,E)-2,4-heptadienal 5-ethyl(5H)-dihydro-furan-2-one (agelicalactone) phenylacetaldehyde (E)-2-octenal (E,Z)-3,5-octadien-2-one (E,E)-2,4-octadienal (E,Z)-2,6-nonadienal (E)-2-nonenal (E,E)-2,4-nonadienal 2,4,6-nonatrienal 2,4,6-nonatrienal (E,Z)-2,4-decadienal (E,E)-2,4-decadienal 4,5-epoxy-(E)-2-decenal

butter-like green, fresh grass green potato-like roasty, popcorn-like mushroom-like geranium-like fatty fatty

e

DB-5 FFAP

e

floral fatty fatty-fruity fatty cucumber-like cucumber-like, green fried fat-like sweat, anise-like sweat, anise-like fatty, green fried fat-like metallic

1053 1076 1207 1445 1323 1289 1365

ID

C

1457 1588

(1) (1) (1) (1) (2) (2) (2) (2) (1) (4)

1050 1619 1060 1415 1095 1110 1576 1152 1571 1160 1521 1212 1683 1270 1913 1278 1296 1734 1316 1790 1387 1989

(1) (1) (1) (1) (1) (1) (1) (3) (3) (1) (1) (2)

853 907 923 980 985 997 1012 1037

d

6

b

a

Compound number in Figure 1. Calculated retention index on capillaries DB-5 and FFAP. Compound identified on the basis of the following criteria: (1) Comparison with the reference compound on the basis of odor quality perceived at the sniffing port, the RI values on capillary DB-5 and/or FFAP and the mass spectra obtained by MS (EI) and MS (CI); (2) The MS signals were too weak for interpretation. The compound was identified on the basis of the remaining criteria listed in footnote (1).; (3) Identified from Ulrich (75) and by comparing the RI values on both capillaries and the mass spectra of the synthesized compound.; (4) Compound tentatively identified by comparison with data from the library of mass spectra. Odorless at the sniffing port. Configuration of isomer not determined. c

d

Shahidi and Cadwallader; Flavor and Lipid Chemistry of Seafoods ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

4. TRIQUI &GUTH

Odorants of Ripened Anchovy by AEDA and GCO-H

33

The aim of the present study was to employ A E D A and GCO-H procedures to evaluate potent odorants of ripened anchovy and to recommend indicator odorants for the objective assessment of ripening and of the quality of the final product.

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Materials and Methods Chemicals. Pure samples of standard compounds (Table I, footnote 1) were provided by the Deutsche Forchungsanstalt fur Lebensmittelchemie, Garching, Germany. Synthesis of 2,4,6-nonatrienal was done by the aldol condensation of (E,E)-2,4heptadienal and acetaldehyde (7). Acetaldehyde (1.3 g) was added to a test tube containing (E,E)-2,4-heptadienal (1.1 g in 1 ml ether). After addition of 2 drops of a 50% aqueous solution of potassium hydroxide, the mixture was allowed to react under stirring for 24 hr at room temperature. The yield by this procedure was low (about 5%). Anchovy. Two samples of whole (not filleted) fully ripened anchovy (12 weeks of ripening) were obtained from a local processor in the Rabat area, Morocco. Anchovy were immersed in a salt-brine and stored in closed cans at 4°C for later analysis. Isolation of the volatiles. Fish (100 g) were cut in small pieces, soaked in methylene chloride (100 ml) and then homogenized using an ultra-turrax for two min. To the suspension, 100 ml of methylene chloride was added and the solvent was filtered off under low vacuum. The remaining material was further washed with 50 ml of methylene chloride and filtered. The extract was then dried over anhydrous sodium sulfate and concentrated to about 100 ml by distilling off the methylene chloride on a Vigreux column (50 x 1 cm) at 40°C. The volatile compounds were distilled off from the nonvolatile material under high vacuum in the apparatus previously described (5), but with the following modifications: two traps cooled with liquid nitrogen were used and the water jacketed tube was held at a temperature of 35°C. The sample was poured into a 250 ml distillation flask and frozen for 30 min in liquid nitrogen. Following sublimation of the volatiles and of the solvent in vacuo (10 mbar), the temperature of the water bath was increased to 35°C and the sublimation was continued for a further 2 h. The condensate of the first cooling trap was dried over anhydrous sodium sulfate and then concentrated to about 2 ml by distilling off the solvent on a Vigreux column (50 x 1 cm) and then by microdistillation (8). -4

High Resolution Gas Chromatography (HRGC). HRGC was performed by means of a Carlo Erba GC (Carlo Erba, Hofheim, Germany) using DB-5 and DB-FFAP fused silica capillaries (each 30 m x 0.32 mm, film thickness 0.25 /mi; J & W Scientific, Folsom, USA). The samples were applied by the on-column injection technique at 35°C. After 1 min, the temperature of the GC oven was raised by 40°C/min to 60°C, held 5 min isothermally, then raised at 4°C/min to 250°C (230°C for DB-FFAP). The flow rate of the carrier gas (helium) was 2 ml/min. At the end of the capillary, the effluent was split 1:1 (by volume) into an FID detector and a sniffing port using deactivated uncoated fused silica capillaries (40 x 0.3 mm). The FID and sniffing port were held at a temperature of 200°C. The splitter was flushed with helium to accelerate the split flow to 10 ml/min. Nitrogen (20 ml/min) was used as makeup gas

Shahidi and Cadwallader; Flavor and Lipid Chemistry of Seafoods ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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FLAVOR AND LIPID CHEMISTRY OF SEAFOODS

for the FID . Retention data of the compounds are presented as retention indices (RI)

(9).

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HRGC-Eluate Sniffing. The aroma concentrates were analyzed by HRGC and by eluate sniffing of the following dilution series: The original extract (100 ml) was stepwise diluted (1+9, v/v) by addition of methylene chloride. The potent odorants were located in the capillary gas chromatograms by A E D A (10). Mass Spectrometry (MS). MS analyses were performed with a Finnigan M S 8230 (Bremen, Germany) mass spectrometer using the aforementioned DB-5 and DB-FFAP capillary columns. Mass spectra in the electronic impact mode M S (EI) were generated at 70 eV and in the chemical ionization mode MS (CI) at 90 eV with isobutane as reagent gas. Gas chromatography-Olfactometry of Headspace Samples (GCO-H). G C O - H was performed with a CP-9001 GC connected to a purge and trap system TCT/PTI 4001 (Chrompack, Frankfurt, Germany) as recently reported (6-11), but with the following modifications: The GC was equipped with a DB-5 fused silica capillary as previously described. The sample of ripened anchovy (10 g) was put into the vessel (volume: 250 ml), sealed with a septum, and then held for 30 min in a 35°C water-bath. The headspace volumes detailed in Table I were drawn into a gastight syringe and then injected at a flow rate 10 ml/min into the purge system operating in the desorption mode for 10 min at a temperature of 250°C. The conditions used for headspace analysis were the same as previously reported ((5), except the temperature program for the capillary DB-5 was modified as follows: Before the start of each run the capillary was cooled to 0°C. After injection of the sample the temperature was held at 0°C for 1 min, then raised by 8°C/min to 250°C and held for 10 min. Results The volatile fraction of ripened anchovy was isolated from 100 g of fish by solvent extraction at room temperature followed by sublimation in high vacuo. The resulting concentrated isolate had an odor similar to the characteristic smell of ripened anchovy. The volatile compounds which were identified in the samples investigated are reported in Table I. With the exception of compound no. 10 and isomers of 2,4,6-nonatrienal (no. 18 and 19), all the remaining volatiles were identified earlier (7). Two potent volatiles previously reported were not detected in the present study: (Z)-4-heptenal and 3 -methyl-nonan-2,4-dione. The screening of the volatiles by A E D A indicated 18 potent odorants in the flavor dilution range of 10-1000 (Figure 1). Methional and (Z)-l,5-octadien-3-one showed the highest FD-factors. The aromagram (Figure 1) also shows that 7 additional odorants contribute with relatively high FD factors to the flavor of ripened anchovy. It is worth mentioning that compound no. 19, identified as 2,4,6nonatrienal, had a low FD factor in the present study, while previously it showed the highest FD value (7). The results of G C O - H are shown in Table II. Direct sniffing of the air drawn by a gas tight syringe showed that the characteristic odor of ripened anchovy was

Shahidi and Cadwallader; Flavor and Lipid Chemistry of Seafoods ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

4. TRIQUI &GUTH

Odorants of Ripened Anchovy by AEDA and GCO-H

35

O O (M

COCM

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o

CM

CO

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COCM

CMCM

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Shahidi and Cadwallader; Flavor and Lipid Chemistry of Seafoods ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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FLAVOR AND LIPID CHEMISTRY OF SEAFOODS

perceived in a headspace volume of 20 ml. This volume was used for starting the GCO-H. Eleven odorants were detected in the 20 ml samples of the air above ripened anchovy. In addition to the compounds identified by A E D A , some highly volatile odorants (no. 1, 2, 3, and 5) were identified by comparing their RI values and odor qualities with those of the corresponding reference compounds. Compounds no. 6, 9, 11, 12, 20 and 26 were readily identified on the basis of the results of the A E D A . The results of GCO-H are in good agreement with those of A E D A since methional and (Z)-l,5-octadien-3-one were perceived in the low headspace volume of 1 ml. On the basis of their low headspace volume, acetaldehyde, 3-methylbutanal and 2methylpropanal are the most potent of the highly volatile odorants of ripened anchovy.

Table II. Volatile compounds identified in ripened anchovy by static headspace gas chromatography-olfactometry 8

No

a

1 2 3 4 5 6 9 11 12 20 26

Compound

RI

b

acetaldehyde