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LWT - Food Science and Technology 57 (2014) 656e662

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LWT - Food Science and Technology

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Characterisation of the key odorants in a squid broth (Illex argentinus)

Vanesa Carrascon, Ana Escudero, Vicente Ferreira, Ricardo Lopez*

Laboratory for Flavour Analysis and Enology, Aragón Institute of Engineering Research, I3A, Department of Analytical Chemistry, Faculty of Sciences,Universidad de Zaragoza, 50009 Zaragoza, Spain

a r t i c l e i n f o

Article history:Received 22 May 2013Received in revised form12 September 2013Accepted 11 February 2014

Keywords:Squid aromaGCeOGCeMSSPE2-Acetyl-1-pyrroline

* Corresponding author. Tel.: þ34 976761290; fax:E-mail address: riclopez@unizar.es (R. Lopez).

http://dx.doi.org/10.1016/j.lwt.2014.02.0100023-6438/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The characteristic flavour of cooked squid has been studied by gas chromatographyeolfactometry (GCeO) and gas chromatographyemass spectrometry (GCeMS) in order to identify and quantify the com-pounds responsible for this aroma. Squid meat (Illex argentinus) was boiled in water and the volatilecompounds of this soup were isolated with different sample preparation techniques. 2-Acetyl-1-pyrroline was identified for the first time in squid aroma and turned out to be the key odorant. Inaddition, 3-(methylthio)propanal, 2-furfurylthiol, 2-methyl-3-furanthiol, sotolon, furaneol and butyricacid were also found to be important odorants present in the cooked squid extract.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Squid is a popular food consumed in Europe, Asia and America.Being one of the most consumed cephalopods in the world, it isrelatively important within the fishing industry with around 1million tons caught in 2007 (FAO, 2010). Raw squid has a very faintodour, similar to other uncooked fish, but during cooking it gen-erates a strong and distinctive aroma that is completely differentfrom other fish or seafood and which is usually pleasant to con-sumers. Squid is cooked in different ways depending on the type ofdish or the country. The most common cooking techniques forsquid include baking, boiling, pan-frying, and tempura (Kubota,Matsukage, Sekiwa, & Kobayashi, 1996).

Scientific literature on the specific subject of squid aroma isscarce and to the best of our knowledge begins in 1987. Yakush et al.found heterocyclic compounds among the volatiles of canned squidand reported 4,5-dimethylthiazole as one of the main componentsof squid flavour (Yakush, Misharina, & Golovnya, 1987). In 1989Korean researchers published an analysis of the volatile fraction ofthe Todarodes pacificus species of squid, reporting 38 compoundsidentified by GCeMS (Lee, Choi, Lee, & Ryu, 1989). The volatilecompounds produced by heating dried squid (sp. Loligo edulisedulis) were analysed by Kawai et al. (Kawai & Ishida, 1989; Kawaiet al., 1991). The authors isolated a concentrate with a strongroasted odour but although many heterocycles containing N and S

þ34 976761292.

were found, no clear attribution of the compounds causing theodour was reported. In 1991, Ohshima et al. found that the mostimportant precursors for the characteristic squid odour wereribose, basic amino acids and sulphur-containing amino acids, andthat these precursors were in the white mantle muscle and not inthe skin (Ohshima, Yokoyama, Wada, Lee, & Koizumi, 1991). How-ever, the authors did not analyse the volatile compounds in thesesamples.

The information derived from non-target volatile analysis isimportant but incomplete given that it is necessary to identify andevaluate which of the compounds are in fact important odorants.One attempt to relate chemical composition with sensory percep-tion in squid was carried out by Morita et al. (Morita, Kubota, &Aishima, 2002). The authors searched for relationships betweenvolatile components and sensory attributes obtained by quantita-tive descriptive analysis bymeans of applying a partial least squaresregression. With this statistical method they found positive con-tributions of different N and S-containing compounds to the cookedsquid aroma. Among these were 4,5-dimethylthiazole, 3-methylbutylpropylsulfide and 2-pyrrolidinone. Olfactometricstudies are frequently used to identify and rank the importance ofpotential odorants. As far as we know, the only study of this kindrelating to squid was conducted by Kubota et al. (1996). They car-ried out a GC-O study to determine the odorants in boiled squid(Todarodes pacificus) applying the technique of aroma extractdilution analysis (AEDA). The result of their GC-O analysis was a listof 10 potent odorants of cooked squid. Among the identifiedodorants, the most important were 4,5-dimethylthiazole, 2-acetyl-1-thiazoline, 2,5-dimethylpyrazine, 3-(methylthio)propanal and

Fig. 1. HSeSPE system used to obtain a representative extract of cooked squid.

V. Carrascon et al. / LWT - Food Science and Technology 57 (2014) 656e662 657

2,5-dimethyl-4-hydroxy-3(2H)-furanone (furaneol) with dilutionvalues of 16. The authors estimated the concentration of volatilecompounds in their extract from the peak area percentage. How-ever, no estimation of the odour activity values of these compoundswas reported.

From all the studies mentioned above, it can be concluded thatthe characterization of cooked squid flavour is not complete.Therefore, the main aim of this study is to examine the GCeO ar-omatic profile of cooked squid, to identify and determine theconcentration of the most important odorants and, with this data,to find the key odorant compounds in the squid sample.

2. Material and methods

2.1. Samples and reagents

Samples of frozen squid (Illex argentinus) were supplied by theAnfaco-Cecopesca company. The specimens were between 15 and18 cm long and were frozen on board at the time of their captureand stored at�18 �C. For preparation of the samples, squid portionsof approximately 300 g were thawed at 4 �C for 24 h.

Dichloromethane (GC, � 99.8%), methanol (GC, � 99.8%),ethanol (GC, � 99.8%), n-hexane (GC, � 98.0%), diethyl ether (GC, �99.7%), phosphoric acid (85%), LiChrolut EN resins and poly-propylene cartridges (3 mL internal volume and 0.8 cm internaldiameter) were supplied by Merck (Darmstadt, Germany). BondElut-ENV cartridges (50 mg) were purchased from Varian (WalnutCreek, CA, USA). Water was purified in a Milli-Q system from Mil-lipore (Bedford, MA). Reagents 2-acetylpyrazine (�99%), 2-furfurylthiol (98%), 2-methyl-3-furanthiol (95%), 3-(methylthio)propanal (�97%), 4,5-dimethylthiazole (�97%), 4,5-dimethyl-3-hydroxy-2 (5H)-furanone (sotolon) (�97%), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (furaneol) (�98%), butyric acid (�99%)and 3-methylbutyric acid (99%), 5-chloro-2-pentanone (85%), eth-ylenediaminetetracetic acid disodium salt 2- hydrate (EDTA), L-cysteine chlorohydrate (99%), octafluoronaphtalene 96% (OFN) and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were supplied bySigmaeAldrich (Madrid, Spain). The standard solution of alkanes(C8eC20) 40 mg L�1 in hexane, docosane (�98%), tetracosane(�99%), hexacosane (�99%), octacosane (�98%), 2-phenylethanethiol, 4-methoxy-a-toluenothiol, mercaptoglycerol,and pentafluorobenzyl bromide (PFBBr) were purchased from Fluka(Madrid, Spain). Anhydrous sodium sulphate (99%) and sodiumchloride (99%) were obtained from Panreac (Barcelona, Spain). Thereagent 2-acetyl-1-pyrroline (10% in triacetin) was supplied byChemstep (Carbon Blanc, France).

2.2. Sample preparation

Only the white parts of the squid muscle were selected. The skinwas removed and the muscle was washed with Milli-Q water andcut into pieces of approximately 5� 5mm. Two hundred and fifty gof squid were boiled with 250 mL of Milli-Q water in a 1 L roundbottom flask. The cooking process was carried out in a reflux sys-tem at 100 �C for 30 min. The broth was cooled in a water bathmaintaining the reflux system to prevent volatile compounds fromescaping. Once room temperature was reached, the broth wasfiltered.

2.3. Head spaceesolid phase extraction (HSeSPE)

A system designed to obtain representative extracts for olfac-tometry analysis was used to capture the aroma of the sample (San-Juan, Pet’ka, Cacho, Ferreira, & Escudero, 2010). The device con-sisted of a glass flask with two outlets: one in the upper part in

which the SPE cartridge is placed to retain the volatiles, andanother outlet at the liquid surface to introduce a stream of nitro-gen to purge the headspace. The system is represented in Fig. 1.

To retain the volatiles, a LiChrolut EN resin cartridge was used.The cartridge was prepared with 400 mg of resin and conditionedwith 20 mL of dichloromethane. Then it was dried under vacuum at0.6 bar for 10 min. Eighty mL of the sample were purged with anitrogen stream at a flow rate of 500 mL min�1 for 100 min. Nosquid aromawas detected at the cartridge end, so it can be assumedthat all the aroma was retained. After the retention process, thecartridge was dried for about 5 min with a nitrogen stream toremove water traces. The retained volatiles were eluted at0.1 mL min�1 with 3.2 mL of dichloromethane-5% methanol. Theextract was concentrated to 200 mL with a stream of pure nitrogen.This extract was used to obtain the olfactometric profile.

2.4. Solid phase extraction (SPE): identification and quantification

An adaptation of the method described by Lopez et al. was usedto identify and quantify the flavour volatiles (Lopez, Aznar, Cacho, &Ferreira, 2002). Commercial cartridges of 200 mg of resin LiChrolutEN were used. The resin was washed with 4 mL of dichloro-methane, 4 mL of methanol and 4 mL of Milli-Q water and driedunder vacuum at 0.6 bar. The squid broth was previously centri-fuged at 4000 rpm (1646� g) for 10 min to prevent solids fromclogging the cartridge frits.

For identifying the compounds, 50 mL of centrifuged broth werepassed through the cartridge slowly by gravity. The cartridge wasthen washed with 10 mL of Milli-Q water and dried under vacuumfor 1 h to remove most of the water. The retained molecules wereeluted with 1.6 mL of dichloromethane containing 1% methanol.Anhydrous sodium sulphate was added to the dichloromethane

V. Carrascon et al. / LWT - Food Science and Technology 57 (2014) 656e662658

eluate to remove traces of water. This extract was used to identifythe compounds by GCeMS and GCeOeGCeOeMS.

For quantitative analysis, a squid broth sample was spiked withpure compounds at expected levels according to the literature.Quantification was carried out using specific response factors (RF)for each compound. These RFs were calculated as:

RFanalyte ¼ Added concetrationanalyteAreaðspikedÞanalyteAreaðspikedÞIS � Areaðnon�spikedÞanalyte

Areaðnon�spikedÞIS

And analytes concentration was calculated as:

concetrationanalyte ¼ RFanalyte �Areaðnon� spikedÞanalyteAreaðnon� spikedÞIS

Spiked and non-spiked samples were extracted in the same wayas described above for identification: 10 mL were passed throughthe cartridge which was then washed with 10 mL of Milli-Q waterand dried under vacuum. The compounds were then eluted with1.6 mL of dichloromethane-1% methanol and finally anhydroussodium sulphate was added. Seventy mL of 5-chloro-2-pentanone80 mL L�1 in dichloromethane was added as a chromatographicinternal standard. Both samples (spiked and non spiked squidbroths) were prepared in duplicate and analysed by GCeMS.

2-Furfurylthiol and 2-methyl-3-furanthiol were quantifiedfollowing an adaptation of the procedure described by Mateo-Vivaracho et al. (Mateo-Vivaracho, Cacho, & Ferreira, 2008) bySPE and GC-negative chemical ionization-MS analysis. Twenty fivemL of squid broth were added with 0.2 g of EDTA and 0.6 g of L-cysteine chlorohydrate. This mixture was transferred to a 20 mLvolumetric flask with 15 mL of an ethanolic solution of 2-phenylethanethiol 1400 mg L�1 as an internal standard. Six milli-litres of this sample were loaded onto a 50 mg Bond Elut-ENV SPEcartridge (Varian, Walnut Creek, California, USA). Major volatileswere removed by rinsing with 4 mL of a 40% methanolewater so-lution 0.2 M in phosphate buffer at pH 7.7. A second InternalStandard was added to the cartridge (20 mL of 4-methoxy-a-tol-uenothiol 150 mg L�1 in ethanol mixed with 200 mL of water). Theretained mercaptans were derivatised in the cartridge with 1 mL ofan aqueous solution of DBU (6.7%) and 50 mL of a 2000 mg L�1

solution of PFBBr in hexane, allowing the cartridge to imbibe thereagent for 20 min at room temperature (25 �C). The remainingderivatising agent was removed with 100 mL of 2000 mg L�1

Table 1LRIs, odour description, % MF and identification of the 13 most important odour zones d

LRI DB-WAXa LRI VF-5MSb Odour descriptor

1349 903 Popcorn, roasted1466 908 Baked potato1447 1003 Roasted1447 1040 Coffee1447 1053 Roasted1323 937 Barbecue sauce1642 1022 Roasted, burnt hair1392 931 Molt, sweat2245 1110 Spicy2072 1065 Candy, burnt sugar1252 1078 Fish1547 e Green, earth1688 877 Rancid cheese1796 e Plastic1647 820 Cheese

a Linear Retention Index in a DB-WAX column.b Linear Retention Index in a VF-5MS column.c Modified frequency.d %MF in DB-WAS column with LRI ¼ 1447 which turned out to be three compoundse n.i. not identified.

mercaptoglycerol in an aqueous solution containing 6.7% DBU,allowing it to react for 20 min at room temperature. After that, thecartridge was rinsed with 4 mL of 0.2 M H3PO4 in water e 40%methanol (v/v) and 1 mL of water. Derivatised analytes were elutedwith 600 mL of hexane 25% in diethylether, spiked with 10 mL ofchromatographic internal standard OFN 22.5 mL L�1 in hexane. Theextract was finally washed 5 times with 1 mL of brine (200 g L�1

NaCl in water). Four mL of this sample were directly injected in coldsplitless mode into the GCeNCIeMS system (under the conditionsdescribed by (Mateo-Vivaracho et al., 2008). Concentration datawere obtained by applying the whole methodology in duplicate tothe spiked and non spiked squid broth.

2.5. Gas chromatographyeolfactometry (GCeO)

The instrument used to rank the odour zones was a Trace GC gaschromatograph (ThermoQuest, Milan, Italy) with a flame ionizationdetector (FID) and a sniffing port ODO-I from SGE (Ringwood,Australia). The capillary column used was a DB-WAX supplied byAgilent (Santa Clara, CA), 30 m � 0.32 mm i.d. �0.5 mm filmthickness, and a deactivated precolumn (3 m � 0.32 mm i.d.) fromSupelco (Bellefonte, PA). Hydrogen was used as carrier gas at aconstant flow rate of 3.5 mL min�1. The injection was conducted insplitless mode (60 s splitless time). The injection volume was 1 mL.The injector temperature was 250 �C and the detector temperaturewas 250 �C. The sniffing port was heated using a thermostat madein the laboratory to prevent the condensation of high boiling pointcompounds, and it was equipped with a humidifier of deionizedwater.

The temperature program used for analysis of the sample was40 �C for 5 min, increased by 4 �C min�1 to 100 �C and then6 �C min�1 to 220 �C, maintaining this temperature for 10 min. Thetotal run time was 50 min.

The olfactometric analysis was carried out by a panel of 8 trainedjudges (sniffers) belonging to the laboratory staff. Each olfac-tometry was performed in two sessions to avoid fatigue. Thesniffers indicated the time, description and odour intensity whenan aroma was detected. The scale used for intensity was 1e3 (1weak odour, 2 clear odour, 3 extremely strong odour), allowingintermediate values. The data were processed taking into accountthe frequency of citation (F) and the intensity of each odour zone (I),obtaining the modified frequency percentage (% MF) from the for-mula given by Dravnieks (Dravnieks & American Society for Testing

etected in an HSeSPE extract of squid broth by GCeO.

% MFc Identification

90 2-Acetyl-1-pyrroline70 3-(Methylthio)propanal

n.i.e

69d 2-Furfurylthioln.i.e

63 2-Methyl-3-furanthiol60 2-Acetylpyrazine43 4,5-Dimethylthiazole43 4,5-Dimethyl-3-hydroxy-2 (5H)-furanone (sotolon)41 2,5-Dimethyl-4-hydroxy-3(2H)-furanone (furaneol)40 n.i.e

38 n.i.e

37 3-Methylbutyric acid37 n.i.e

31 Butyric acid

in VF-5MS column.

Table 2Results of the quantitative analyses.

Compound m/za Concentration in squid broth (mg L�1)b Odour threshold in water (mg L�1)c Odour Activity Value (OAV)

2-Acetyl-1-pyrroline 83 97.3 (10.1) 0.1e 9733-(Methylthio)propanal 104 50.5 (8.8) 1.8e 28.1Butyric acid 73 1410 (152) 200f 7.12-Furfurylthiol 274d 0.049 (0.003) 0.01e 4.92-Methyl-3-furanthiol 274d 0.023 (0.005) 0.007e 3.34,5-Dimethyl-3-hydroxy-2 (5H)-furanone

(sotolon)128 0.93 (0.12) 0.3e 3.1

2,5-Dimethyl-4-hydroxy-3(2H)-furanone(furaneol)

128 22.5 (3.1) 25e <1

2-Acetylpyrazine 122 0.84 (0.10) 62g <13-Methylbutyric acid 87 18.2 (2.8) 750e <14,5-Dimethylthiazole 113 0.474 (0.07) 470h <1

a m/z used in the SIM analyses for quantification.b Average of two samples (standard deviation in brackets).c References are shown in superscripts.d m/z of the derivatised compound.e Munch and Schieberle (1998).f Boatright and Crum (1997).g Majcher and Jelen (2011).h Buttery, Guadagni, and Lundin (1976).

V. Carrascon et al. / LWT - Food Science and Technology 57 (2014) 656e662 659

and Materials, 1985): % MF ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

% F$% Ip

. A linear alkane solution(C8eC28) of 5 mg L�1 in dichloromethane was injected with thesame temperature program to calculate the linear retention index(LRI) of the odour zones.

Olfactometric analyses were also performed on a blank. Thisblankwas prepared by boiling 250mL ofMilli Qwater in the systemdescribed in the sample preparation section and the headspaceextraction was performed in the same way as for the samples.

2.6. Gas chromatographyemass spectrometry (GCeMS)

The instrument used was a 7890A gas chromatograph fromAgilent (Santa Clara, CA) with a mass spectrometry detector Agilent5975C. The capillary column used was a DB-WAX Agilent (SantaClara, CA), 60 m length, 0.25 mm inner diameter, 0.25 mm thickphase, and a precolumn (3� 0.32 mm ID) from Supelco (Bellefonte,PA). Heliumwas used as carrier gas at a flow rate of 1mL min�1. Theinjection was carried out in pulsed splitless mode (splitless time2 min) and a pulse pressure of 30 psi for 2 min. The injection vol-ume was 2 mL. The injector temperature was 250 �C. The detectorwas a mass spectrometer with an electron impact ionization sourcewhich was maintained at 230 �C. The quadrupole analyser was keptat 150 �C. The acquisition time was 13.5 mine50 min and the massacquisition range from 40 to 300 m/z. The temperature programused for the analysis was 40 �C for 5 min, after which the oventemperature was increased by 4 �C min�1 to 100 �C and then6 �C min�1 to 220 �C. This temperature was maintained for 15 min.The MSD ChemStation software version E.02.01.1177 was used.. Forquantitative analyses, each molecule was quantified with specificm/z ions to prevent interference and to increase the sensitivity ofthe method. The ions used for each compound appear in table 2.

2.7. Two-dimensional gas chromatographyeolfactometryemassspectrometry (GCeOeGCeOeMS)

The dual chromatography system comprised two separatechromatographs connected by a transfer line at 200 �C. The firstchromatograph (GC-1) was a Varian CP-3800 (Walnut Creek, CA)with a 1079 programmed temperature vaporizing injector. A glassliner of 3.4 mm internal diameter with a carbofrit (Restek, Belle-fonte, PA) was placed inside the PTV injector. The column used wasthe same as in the GCeO analysis, a DB-WAX Agilent (Santa Clara,CA), 30 m � 0.32 mm i.d. �0.5 mm film thickness. This first

chromatograph was equipped with a sniffing port (ODO-II fromSGE, Ringwood, Australia) and a FID detector, which was kept at300 �C, both connected to the column by a flow splitter which al-lows olfactometric detection simultaneously with FID signalacquisition. The carrier gas was helium maintained at a constantpressure of 30 psi. The injection conditions had been optimizedpreviously (Campo, Cacho, & Ferreira, 2007) for injecting largevolumes (10 mL). The injector was at 40 �C keeping the split valveopen to allow the evaporation of the solvent up to 0.4min. Then thevalve was closed and the split injector was heated to 250 �C. Theoven temperature program was 40 �C for 5 min, increasing by4 �C min�1 to 100 �C and then 6 �C min�1 to 220 �C.

This temperaturewasmaintained for 15min. The end of the firstcolumn was connected to a Deans valve (Valco Instruments,Houston, TX) for transferring the flow from the first to the secondGC. The second chromatograph (GC-2) was a Varian CP-3800 with aFactor Four VF-5MS column, 30 m � 0.32 mm i.d. �1 mm filmthickness, also from Varian. The column was connected directly tothe heated transfer line (200 �C) attached to the Deans valve of theGC-1. The column was then passed through a cryofocusing unitwith liquid CO2 to concentrate the analytes captured before startingthe chromatography in the second oven. This GC-2 had anothersniffing port (ODO-II of SGE, Ringwood, Australia) and a VarianSaturn 240-MS ion-trap mass spectrometric detector. The temper-ature of the GC-2 was 50 �C until the cryogenic trap of CO2 wasturned off. Then there was an increase of 4 �C min�1 to 200 �Cfollowed by a cleaning stage of 100 �C min�1 to 300 �C. This tem-perature was maintained for 10 min. The ion-trap was kept at150 �C. The acquisition mass range was 35e250 m/z, and 2 scanswere performed per second.

For identifying the compounds responsible for the odour zone,heart-cuttings were employed for the selective isolation of eachzone. In a first monitoring analysis, the retention time (tr, in mi-nutes) was found. Then a second analysis was carried out to capturethe unidentified compound (or compounds). The systemwas set upto cut the gas flow from GC-1 in the interval time (tr e

0.9) � 0.2 min, �0.9 min being the time delay between the Deansvalve and the sniffing port in which it was detected, and 0.2 minbeing the standard range for quantitatively transferring the odourzone to GC-2. The cryogenic trap was cooled from 2 min before thestart of the cutting interval to 2 min after the end. The ramp of theGC-2 and the acquisition in the mass spectrometry detector startedwhen the cryo-focalization was finished.

V. Carrascon et al. / LWT - Food Science and Technology 57 (2014) 656e662660

The software used was Varian WorkStation version 6.30. NISTand Wiley spectral libraries were used for identification purposesbefore the injection of reference compounds.

3. Results and discussion

3.1. Considerations for sample preparation

The squid species Illex argentinus was chosen for this studybecause it represents ca. 22% of the total squids captured in 2007(FAO, 2010), and once cooked it has a good and intense flavour.Boiling has been used to prepare the raw squid samples in order tocharacterize and generate their aroma (Kubota et al., 1996),although other preparations such as canned squid (Yakush et al.,1987) or dried squid (Kawai et al., 1991) have also been studied.Boiling was chosen for the present study after ascertaining that thecharacteristic squid flavour was generated in the process and that itwas a reproducible process for preparation of the sample.

The previous published work on squid aroma characterizationby GCeO (Kubota et al., 1996) had some limitations. The process toobtain the GCeO extract was to pass the squid soup through aTenax TA column followed by elution with ethyl ether. The limitedability of Tenax to retain volatiles has been proved when comparedwith newgeneration sorbents such as LiChrolut EN that can providea trapping capacity up to 1000-fold higher (Lopez, Batlle, Nerin,Cacho, & Ferreira, 2007; Lopez, Ferreira, Cullere, Grasa, & Cacho,2003). Another limitation is the likely loss of representativity ofthe extract. By trapping all the volatiles present in the liquid phaseand not only those that are really able to reach the headspace abovethe sample, some low boiling compounds will be positivelydiscriminated (Ferreira & Cacho, 2009). To overcome these limita-tions, in the present work the GCeO extract was obtained by col-lecting the volatiles in the headspace of the soup using a purge andtrap system with a LiChrolut EN cartridge. The performance of thissystem for obtaining representative extracts for olfactometry hasbeen validated previously for other foodstuffs (Barata et al., 2011;San-Juan et al., 2010).

3.2. Ranking and identification of odorants

Table 1 shows the ranking of the olfactometric odour zonesfound. For the sake of simplicity, those odorants not reaching ascore of 30% (maximum¼ 100%) were eliminated, being consideredas noise. After this operation the number of odour zones to beidentified was 13. It is worth mentioning that none of the detectedodour zones was described as “cooked squid”, which means thatthe characteristic aroma of squid is most likely the consequence ofthe combination of all the odorants.

For identification purposes, the olfactometric extract was ana-lysed by GCeMS and GCeOeGCeOeMS. With these techniques,most of the odour zones were identified as shown in Table 1. Theidentity of the odorants was determined through their massspectra and their LRIs in DB-WAX and VF-5MS columns, andconfirmed by the injection of the pure reference compound. Sixodour zones were attributed to 2-acetyl-1-pyrroline, 3-(methyl-thio)propanal, 2-acetylpyrazine, 4,5-dimethylthiazole, 3-methylbutyric acid and butyric acid. It was also found that thethird odour zone in the ranking (LRI 1447 in DB-WAX column) wassplit into three different odour zones in the two-dimensional sys-tem (LRIs 1003, 1040 and 1053 in the VF-5MS column), of whichonly 2-furfurylthiol (LRI 1040 in the VF-5MS column)was identifiedby its mass spectrum and a distinctive coffee odour. The remainingtwo compounds had roasted descriptors (“popcorn”, “fried”), butno conclusive mass spectra were found for either of them. Theidentity of compounds 2-methyl-3-furanthiol, sotolon and

furaneol, was also confirmed in the GCeOeGCeOeMS systemalthough no mass spectrum was obtained in the olfactometricextract. 2-methyl-3-furanthiol has an extremely low odourthreshold (0.007 mg L�1)(Munch & Schieberle, 1998), but its lowconcentration in the sample and its poor chromatographic prop-erties prevented the acquisition of its mass spectrum. The confir-mation of its identity was achieved by applying a specific methodfor the analysis of thiols (see Section 2.4). Furaneol and sotolon arehighly polar molecules which tend to tail in chromatographic col-umns (Ferreira, Jarauta, Lopez, & Cacho, 2003), therefore it wasnecessary to inject a more concentrated SPE extract to obtain theconfirmation by mass spectrometry. The odour zones with LRIs1252, 1547 and 1796 in DB-WAX remain unidentified so far.

In summary, 13 odour areas were found to be relevant(MF > 30%) in the aroma profile of cooked squid. Ten of themwereproduced by compounds that have been identified, but three odourzones were not assigned to any compound. It is worth mentioningthat although one odour area had the descriptor “fish”, which couldbe of interest for this product, it had a modified frequency of 40%and therefore its impact on the aroma is of limited importance.Most of the molecules identified are compounds in which hetero-cyclic or carbonyl groups are present, as could be expected fromMaillard reactions (Varlet, Prost, & Serot, 2007).

3.3. Quantitative results

The results of the quantitative analysis of the aroma compoundsreleased by the squid broth can be seen in Table 2. Only compoundsidentified in Table 1 were quantified. A specific method wasrequired for the determination of 2-furfurylthiol and 2-methyl-3-furanthiol due to their low concentrations and poor chromato-graphic properties. The compounds shown in Table 1 represent themost potent odorants in the GCeO experiment, but their real in-fluence on the squid aroma can be better assessed by means of theso-called odour activity values (OAVs) which represent the ratio ofthe concentration of a given substance in the sample to the sensorydetection threshold. To our knowledge, Table 2 is the first publisheddata of the OAVs of components in cooked squid.

According to its %MF, the most important compound in the GCeO experiment was 2-acetyl-1-pyrroline with a “roasted” descriptor.Its overwhelming importance for the aroma of this product waslater confirmed by its huge OAV of 973. To the best of our knowl-edge, this is the first time that 2-acetyl-1-pyrroline has been re-ported in squid aroma, although it has been found in other seafoodsuch as shrimp (Rochat, Egger, & Chaintreau, 2009), lobster (Lee,Suriyaphan, & Cadwallader, 2001) or crab (Yu & Chen, 2010), andin other oven-cooked food such as lamb (Bueno et al., 2011),hazelnut paste (Burdack-Freitag & Schieberle, 2012) or bread(Rychlik & Grosch, 1996). It is not surprising that 2-acetyl-1-pyrroline was ranked first among our odorants as it is consideredto be one of the Maillard reaction products with the highest aro-matic impact (Adams & De Kimpe, 2006). 2-acetyl-1-pyrroline wasthe compound with by far the highest OAV, one order of magnitudeabove the second compound, and consequently it should beconsidered the key odorant in the characteristic aroma of cookedsquid.

The second compound, as shown in Table 2, was 3-(methylthio)propanal. With an OAV of 28.1 and a “baked potato” descriptor, itcan be considered an important contributor to squid aroma. Thiscompound has been reported previously as an important aroma incooked squid by Kubota et al. (1996), and also in other seafood suchas cooked tail meat of lobster (Lee et al., 2001). The origin of 3-(methylthio)propanal in the sample is likely to be a degradation ofthe amino acid methionine (Escudero, Hernandez-Orte, Cacho, &Ferreira, 2000). Two other sulphur compounds were found in

V. Carrascon et al. / LWT - Food Science and Technology 57 (2014) 656e662 661

concentrations above their odour threshold in water: 2-furfurylthiol and 2-methyl-3-furanthiol with OAVs of 4.9 and 3.3,respectively. Both compounds have been previously found as keyodorants in roasted foods (Burdack-Freitag & Schieberle, 2012;Sabine Rochat, de Saint Laumer, & Chaintreau, 2007; Semmelroch& Grosch, 1995). It is likely that in our squid broth both com-pounds shared a common origin in ribose and cysteine Maillardreactions (Hofmann & Schieberle, 1995). Furthermore, Ohshimaet al. (Ohshima et al., 1991) confirmed that no squid flavour wasgenerated in a squid extract when ribose or sulphur-containingamino acids were not present. Therefore, both studies suggest astrong involvement of 2-furfuylthiol and 2-methyl-3-furanthiol inthe characteristic aroma of squid.

The two acids shown in Table 1, butyric and 3-methylbutyricacid, were revealed to have completely different degrees of rele-vance when their OAVs were calculated. The 3-methylbutyric acidcontribution may be considered negligible because its OAV wasbelow 1. However, the butyric acid concentration exceeded 7 timesits threshold and can thus be considered a net contributor to theboiled squid odour. According to Table 2, the last potential con-tributors to the squid aroma were two enolones: 4,5-dimethyl-3-hydroxy-2(5H)-furanone with an OAV of 3.1 and a “spicy”descriptor, and 2,5-dimethyl-4-hydroxy-3(2H)-furanone with anOAV below 1 and a “burnt sugar” descriptor. The latter wasdescribed in cooked squid aroma by Kubota et al. (1996), having ahigh dilution value in their GCeO experiment. Both compounds arecommon in other roasted food and broths (Bueno et al., 2011; Leeet al., 2001; Tonsbeek, Plancken, & Vonderwe, 1968).

The contribution to the aromaof the three last compounds shownin Table 2 may be considered of little relevance because all of themappeared in concentrations far below their corresponding odourthresholds. 2-acetylpyrazine and 4,5-dimethylthiazole have beendescribed in canned squid (Yakush et al., 1987) and as potent odor-ants of cooked squid (Kubota et al.,1996), but it isworthnoticing thatthe concentration of 4,5-dimethylthiazole found in our study was1000 times lower than that found by Kubota et al. and therefore itsimportance in the aroma of our sample should be negligible.

4. Conclusions

The aroma generated by cooking squid is due to at least thirteenvolatile molecules of which ten have been identified and quantified.The work has determined that the flavour of cooked squid is notcaused by a single compound with the characteristic squid odour,but by a group of compounds mainly produced in Maillard re-actions. 2-acetyl-1-pyrroline, identified for the first time in squid, isthe most important odorant in the aroma of squid broth, based onits high OAV. Other net contributors to squid flavour are threesulphur compounds (3-(methylthio)propanal, 2-furfurylthiol, 2-methyl-3-furanthiol), two enolones (sotolon and furaneol) andbutyric acid. 4,5-dimethylthiazole and 2-acetylpyrazine, proposedas potent odorants in a previous study, are not relevant in thearoma of Illex argentinus squid.

Acknowledgements

This work has been funded by the Anfaco-Cecopesca company.V.C. has received a grant from Gobierno de Aragon.

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