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cooking, were subjected to the aroma and key odorant analysis for the first time using GC-. 3. MS-O. The extraction of the aroma compounds was carried...
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Comparative Evaluation of Key Aroma-Active Compounds in Raw and Cooked Red Mullet (Mullus barbatus) by Aroma Extract Dilution Analysis Pelin Salum, Gamze Guclu, and Serkan Selli* Department of Food Engineering, Faculty of Agriculture, Cukurova University, 01330, Adana, Turkey ABSTRACT: Raw red mullet (Mullus barbatus) and its cooked samples, obtained from steam and oven cooking, were subjected to aroma and key odorant analysis for the first time using GC-MS-O. The extraction of the aroma compounds was carried out by the direct solvent extraction−solvent assisted flavor evaporation (DSE-SAFE) method. Principal component analysis (PCA) was used to determine the relations between cooking processes and fish aroma compounds. By applying the aroma extract dilution analysis (AEDA), 8 and 13 aroma-active compounds were detected in raw and cooked fish samples, respectively. The most prominent differences between raw and cooked fish samples were as follows: 3-hydroxybutan-2-one, 2,3-octadienone, (E,E)-2,4heptadienal, linalool, γ-butyrolactone, 1-methylpyrrolidin-2-one, 2H-furan-5-one and pyrrolidin-2-one were only detected in cooked samples while hexanal and 2-phenoxyethanol in only raw fish samples. GC-MS-O results clearly indicated that cooking process results in the development of characteristics and pleasant aroma of red mullet samples due to the lipid oxidation. The most dominant aroma-active compound in the cooked fish samples was the 1-octen-3-ol which is responsible for the mushroomlike odor. KEYWORDS: Mullus barbatus, red mullet, raw, cooked, aroma-active, AEDA

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INTRODUCTION Red mullet (Mullus barbatus Linnaeus, 1758) is a member of the mullidae family. It lives in the sandy and muddy waters with a depth of 10−300 m. From the geographical distribution point of view, it inhabits the East Atlantic the European and African coasts from the British Isles to Dakar the Mediterranean sea and occasionally the Scandinavian sea. The total global production was about 14,789 tons with respect to the FAO 2014 report.1 Also, red mullet is one of the important fish species in Turkey due to its economic value.2 The quality parameters of the fish products are important to meet the consumer demands. There are many factors that affect the quality of fish and other aquatic products. The sensory properties of the fish determine the satisfaction of the consumers as well as indicate the health risks arisen from spoilage and contaminations. Therefore, one of the most important quality parameters throughout history in determining whether the fish is suitable for consumption has been the aroma of the fish.3,4 In general, the aroma of fish species is the result of the combined effect of many different volatile compounds with quantities varying between nanograms and milligrams. These volatile compounds are generally formed by the enzymatic reactions, lipid oxidation, and microbial activities.5,6 Among these, a limited number of volatile constituents namely aroma-active compounds contribute to the characteristics of the fish aroma due to their low perception thresholds. The gas chromatography-olfactometry (GC-O) method is considered to be useful for sensitively identifying these key odorant components individually.7 The specification of aromatic volatiles may display differences according to extraction techniques. Although there are a wide variety of techniques to obtain aroma compounds, within these, the solvent-assisted flavor evaporation (SAFE) is known © 2017 American Chemical Society

as a fast and careful isolation method. This technique notably stands out among the other methods with its specialty to prevent the artifact generation and aroma compound destruction primarily for complex food matrices.8 The SAFE technique was used by Mahmoud and Buettner9 for the first time for the analysis of aroma-active compounds for fresh-water fish matrices. Heat applied during the processing of fish and other seafood has a great influence on the types and quantities of aroma and aroma-active compounds.10 Heat treatments result in the formation of aroma substances with enzymatic and nonenzymatic pathways such as degradation of fatty acids, Maillard reactions and Strecker degradation in fish specimens.10,11 Several studies were conducted on the aroma and aroma-active compounds of different cooked fish and other sea product species.12−17 However, the number of studies related to the effect of heat applications on the aroma and aroma-active compounds has been limited and most of these papers were focused only on volatile compounds of fish species as affected by the heat treatment. Medina et al.18 identified volatile compounds that formed as a result of oxidation during heat ́ treatment in fish. Rodriguez et al.19 determined the volatile compounds of fresh and canned sea urchin (Paracentrotus lividus, Lamarck) and reported that the sterilization process caused important changes in the profile of the volatile compounds. Sun et al.20 investigated the effect of temperature during the heat treatment on the volatile compounds of bigeye tuna meat (Thunnus obesus) by means of electronic nose and Received: Revised: Accepted: Published: 8402

June 14, 2017 August 30, 2017 September 1, 2017 September 1, 2017 DOI: 10.1021/acs.jafc.7b02756 J. Agric. Food Chem. 2017, 65, 8402−8408

Article

Journal of Agricultural and Food Chemistry

was transferred into a 500 mL Erlenmeyer flask containing 40 μg of 4nonanol as internal standard and 200 mL of high purity dichloromethane as solvent. The mixture was stirred with a magnetic stirrer for 2 h under nitrogen gas at 4−5 °C. Afterward, the aroma compounds were carefully separated from the nonvolatile compounds by SAFE technique at 40 °C.8 The SAFE extract was dried with sodium sulfate, and extract was reduced to 5 mL in a Kuderna Danish concentrator (Sigma-Aldrich, St. Louis, MO) fitted with a Snyder column (Supelco, St. Quentin, France) and then to 200 μL under a gentle stream of purified nitrogen. Extractions were also performed in triplicate. GC-MS-O Analysis of Aroma Compounds. A gas chromatography (GC) instrument with a flame ionization detector (FID) (6890 N, Agilent, DE, USA) was used to quantify the aroma compounds. Separation was carried out using a DB-WAX capillary column (60 m × 0.25 mm × 0.4 μm). The detector temperature was 250 °C. The column temperature was increased from 60 to 220 °C at a rate of 2 °C/min and then increased to 245 °C at a rate of 3 °C/min with a final hold at this temperature for 20 min. The amount of sample injected into the device was 3 μL. Helium was used as a carrier gas with a flow rate of 1.5 mL/min. Mass spectrometer (MS) (5975B VL MSD, Agilent, DE, USA) combined with GC was used to identify the aroma substances. The velocity of the helium used as the carrier gas was 1.5 mL/min. Compounds were scanned at a rate of 29−350 mass/load (m/e) at 1 s intervals with an ionization energy of 70 eV, an ion source temperature of 250 °C, and a quadrupole temperature of 120 °C. The identification of the aroma compounds was carried out by comparing the mass spectrum of the nonstandard compounds with the mass spectra of the commercial spectral database (Wiley 11.0, NIST-11, and Flavor, 2L). For the purpose of confirmation, standard aroma compounds were injected into the system under the same conditions. The linear retention index values were calculated according to the n-alkane series.23 Quantification of the aroma compounds was determined from the area of 4-nonanol, which was used as the internal standard. Representativeness Test of the Aromatic Extract. Sensory evaluations were carried out at Food Engineering Department of Cukurova University, Adana, Turkey. Sensory analyses of red mullet samples and their extracts were conducted by a group of 7 assessors (4 men and 3 women; between 24 and 46 years old). Panelists were previously trained and had knowledge and experience in sensory analysis. Sample Preparation and Presentation. After red mullet samples were homogenized with a blender, 1.5 g of the sample was taken and presented to the panelists in 25 mL specially coded and capped brown glass bottles. Extracts obtained with dichloromethane solvent were absorbed on a cardboard smelling strip (SARL H. Granger-Veyron, France) and then put aside for 1 min for solvent evaporation. Aromatized strips were presented to the panelists in 25 mL specially coded capped brown glass bottles. These preparations were completed in 15 min, and the prepared samples were presented to panelists at room temperature. Two different tests, namely, similarity and intensity tests, were performed to evaluate the closeness between the odor of fish samples and their aromatic extracts. In similarity test, panelists were asked to answer how similar the smells of the extracts and the fish samples were based on a scale. For this purpose, a scale of 100 mm with a “very different from the reference” sign on its left side and “identical to the reference” on its right side was used. Similarly, in the odor intensity test, panelists were asked to determine the odor intensity of the aromatic extract. For this purpose, similarly, a scale of 100 mm having a “no odor” sign on its left side and a “very strong odor” sign on its right side was used.24 Results were analyzed by analysis of variance with Statgraphics Plus (Manugistic, Inc., Rockville, MD, USA). Aroma Extract Dilution Analysis (AEDA). The undiluted aromatic extract (200 μL) was subjected to GC-MS-O analysis to detect the aroma-active compounds of the red mullet samples. Next, the aromatic extracts were gradually diluted 1:1 with dichloromethane solvent every time and injected for the sniffing. The analysis was completed by continuing the dilution procedure until the detection of the odor vanished.25−27 During the sniffing, to overcome the drying of

SPME-GC/MS. Pérez-Palacios et al.21 examined the effect of different cooking methods on the amounts of volatile compounds in breaded fish. Wang et al.11 studied the effect of heat treatment on the volatile compounds and fatty acids of Haliotis discus hannai. In addition to these research, Mall and Schieberle22 investigated the effect of two different cooking processes on the aroma-active compounds of prawns for the first time and reported that thermal processing resulted in increased quantities of volatiles generated by the protein and lipid degradation as well as the formation of new products. In the extant literature, no data are present about key odorants of the red mullet. Thus, this study has importance for being the first research about red mullet volatiles and key odorants. Therefore, the aims of the present study were (i) to evaluate the representativeness of red mullet aromatic extracts obtained by the SAFE technique, (ii) to determine the volatile compounds of the raw and heat treated (oven cooking and steam cooking) red mullets, and (iii) to characterize the key odorants of the samples by the application of AEDA method.

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MATERIALS AND METHODS

Materials. In this study, red mullet (Mullus barbatus) fish samples with 12.5 ± 4 cm length and 34.9 ± 6 g mass, obtained in October 2015 by hunting from the city of Karatas, a coastal city of Turkey in the eastern Mediterranean region, were used. Fish samples were placed into boxes with ice and transported to the Food Engineering Department of Cukurova University in 6 h and stored at −80 °C until the analysis. Dichloromethane, 4-nonanol (as internal standard), sodium chloride, and sodium sulfate were used in the study (Merck, Darmstadt, Germany). Dichloromethane was freshly distilled prior to use and water was purified by a Millipore-Q system (Millipore Corp., Saint-Quentin, France). Standard aroma compounds like hexanal, 1penten-3-ol, 3-penten-2-ol, 3-methylbutan-1-ol, limonene, 1-pentanol, 3-hydroxybutan-2-one (acetoin), 2-hexanol, 1-hexanol, nonanal, 1octen-3-ol, (E,E)-2,4-heptadienal, linalool, γ-butyrolactone, 2-phenylacetaldehyde, 1-methylpyrrolidin-2-one, 2H-furan-5-one, naphthalene, phenylmethanol, 2-phenylethanol, phenol, pyrrolidin-2-one, and 2phenoxyethanol were obtained from the Sigma-Aldrich company (Steinheim, Germany). Sample Preparation. At the beginning, the internal organs of the fish samples were removed, then the heads were separated, and the specimens were cut into fillets. The fillets were divided into three groups. Each group had 38 fillets with a total mass of about 600 g. While the first group was the control group and analyzed without any heat treatment, the second group was analyzed after oven cooking, and the third group was studied after steam cooking. Cooking time was determined with preliminary experiments, and a cooking time of 20 min was decided to be appropriate in each cooking process. The cooking process was homogeneously dispersed, and the samples were wrapped in aluminum foil. Then, the raw and cooked samples were frozen in liquid nitrogen and homogenized in small pieces by using a domestic mechanical disintegrator. For oven cooking, fillets were placed on an oven tray (Bosch Hkp 110120 Midi Oven, Munich, Germany) with a homogeneous distribution and cooked at 200 °C for 20 min. Steam cooking was carried out in a steaming basket of Thermomix (TM 31, Vorwerk Wuppertal, Germany), with a uniform distribution, and samples were steam cooked at 100 °C for 20 min. During the cooking process, oven and steaming basket temperatures were measured with a thermocouple (four multichannel K type, CK104, Verth, Taiwan). In order to obtain more accurate results, analyses were performed in three replicates. Direct Solvent Extraction−Solvent Assisted Flavor Evaporation (DSE-SAFE) of Volatile Compounds. The direct solvent extraction−solvent assisted flavor evaporation (DSE-SAFE) method was used in the isolation of aroma compounds of red mullet samples. A sample of 100 g from the frozen, crushed, and homogenized batch 8403

DOI: 10.1021/acs.jafc.7b02756 J. Agric. Food Chem. 2017, 65, 8402−8408

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Journal of Agricultural and Food Chemistry Table 1. Volatile Compounds of Raw and Cooked Red Mullet Samples no.

LRIa

compound

group

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

1074 1127 1177 1217 1234 1243 1277 1307 1342 1353 1359 1395 1458 1483 1493 1496 1537 1612 1650 1662 1740 1764 1861 1923 1973 2014 2130

hexanal 1-penten-3-ol 3-penten-2-ol 3-methylbutan-1-ol limonene 1-pentanol 3-hydroxybutan-2-one (acetoin) 2-hexanol 2-methyl-2-buten-1-ol 2,3-octanedione 1-hexanol nonanal 1-octen-3-ol (E,E)-2,4-heptadienal 3,5-octadien-2-one decanal linalool γ-butyrolactone 2-phenylacetaldehyde 1-methylpyrrolidin-2-one 2H-furan-5-one naphthalene phenylmethanol 2-phenylethanol phenol pyrrolidin-2-one 2-phenoxyethanol total

aldehyde alcohol alcohol alcohol terpene alcohol ketone alcohol alcohol ketone alcohol aldehyde alcohol aldehyde ketone aldehyde terpene lactone aldehyde N compound lactone naphthalene alcohol alcohol volatile phenol N compound alcohol

rawb

steam cookingb

74.6 39.3 352.4 31.4 57.3 18.5 121.5 108.3 50.6 6.2 12.6 142.8 21.6 27.2 4.1 48.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.5 b 0.3 a 9.4 a 0.1 b 2.3 a 1.9 a 2.0 a 11.3 a 2.3 a 0.4 a 0.1 a 13.1 b 0.2 a 0.5 a 0.3 a 3.3 b

9.8 5.3 22.9 13.5 10.9 3.4 7.5 12.2 30.0 171.1 1403.3

± ± ± ± ± ± ± ± ± ± ±

1.4 0.2 2.2 0.1 0.2 0.1 0.6 0.9 1.3 7.7 65

a a a a b b a b a b

55.2 73.7 416.3 18.1 65.2 35.1 162.7 106.0 74.1 11.4 24.0 57.2 45.2 42.6 7.2 16.6 6.6 16.0 10.1 33.8 29.7 7.9 3.0 7.0 3.2 46.8 72.7 1447.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.0 a 3.4 b 16.4 b 2a 2.4 b 0. 8 c 1.9 b 3.8 a 3.7 c 0. 1 c 1.4 b 0.3 a 0.9 b 0.9 b 0.1 b 1.1 a 1.1 0.2 b 0.4 c 0.6 b 1.0 b 0.3 a 0.1 a 0.3 a 0.3 a 3.5 b 0.6 a 48

oven cookingb

identificationc

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS LRI, MS, tent. LRI, MS, tent. std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS LRI, MS, tent. std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS std, LRI, MS

54.2 43.0 362.8 21.7 66.0 28.4 201.5 101.6 66.5 9.8 24.5 47.6 54.9 45.1 6.8 18.2 11.8 17.5 7.9 35.7 41.8 7.4 3.6 13.0 4.7 67.3 59.7 1422.8

5.4 a 0.5 a 17.1 a 1.8 a 0.8 b 0.1 b 1.4 c 2.3 a 0.5 b 0.2 b 1.4 b 0.9 a 0.7 c 0.4 c 0.3 b 0.7 a 0.3 0.5 b 0.4 b 0.3 b 1.4 c 0.4 a 0.2 b 0.8 b 0.1 a 2.5 c 5.9 a 45

Linear retention index calculated on DB-WAX capillary column. bConcentration: results are the means of three repetitions as μg/kg. cMethods of identification: LRI, linear retention index; MS tent., tentatively identified by MS; std, chemical standard; when only MS or LRI is available for the identification of a compound, it must be considered as an attempt of identification. a

mm by Hallier et al.;15 for muscle of turbot (Psetta maxima), the scores were determined between 58 and 61 mm by Serot et al.;40 and for smoked salmon muscles, 72 ± 14 and 72 ± 17 mm on a 100 mm scale was reported by Varlet et al.28 Also, similarity and intensity scores of French rainbow trout were found between 51.1 and 58.7 mm by Selli et al.;16 and for gray mullet extracts, the values were from 62.7 to 70.8 mm by Cayhan and Selli.24 Volatile Compounds of Raw and Cooked Red Mullet. The volatile compounds of the red mullet samples are given in Table 1 with the linear retention index (LRI) values on a DBwax column. Analyses were made in triplicate, and the means and the standard deviations were calculated. A total of 26 aroma compounds were identified in the raw fish samples (R) including 11 alcohols, five aldehydes, three ketones, two lactones, one terpene, two nitrogen compounds, one volatile phenol, and one naphthalene. In the heat treated fish samples (steam cooked (S) and oven cooked (O)), a total of 27 aroma substances were identified including 11 alcohols, five aldehydes, three ketones, two lactones, two terpenes, two nitrogen compounds, one naphthalene, and one volatile phenol. Significant proportions of the volatiles were alcohol compounds in the red mullet samples. Alcohols constituted 58.20%, 60.46%, and 54.80% of the total aroma substances in raw, steamed, and oven cooked fish samples, respectively. Aldehydes were the second largest volatile group in the raw (18.51%) and steam cooked (12.55%) samples while ketones were the second in the oven cooked samples (15.33%).

the nose, a special device was used to deliver moist air to the olfactometry port and therefore the sensitivity of the panelists to the odor was increased. The dilution grade was expressed based on “flavor dilution (FD)” factor. The aroma substance with high FD values correlated to the high degree of aroma activity. AEDA was performed by three trained panelists. Statistical Analyses. Experimental data were subjected to the analysis of a Duncan multiple range test using the SPSS 17 software package with 95% confidence. Moreover, principal component analysis (PCA) was used to group samples with all aroma properties (p < 0.05). The XLSTAT (2013) statistical program (Addinsoft, New York, NY, USA) was used for PCA analysis.

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RESULTS AND DISCUSSION Representativeness of the Extract. According to the results of the representativeness tests, the DS-SAFE extracts showed 80.7 mm of similarity and 63.5 mm of odor intensity on a 100 mm scale. Consequently, the extraction from the DSSAFE method was found reliable for olfactometric analysis. In the literature, no study was conducted to investigate the possibility of using the DS-SAFE method for assessing the key odorants of fish samples except the one by Mahmoud and Buettner.9 The authors studied the aroma-active and off-odor compounds in German rainbow trout (Oncorhynchus mykiss) using the SAFE technique. However, there was no information on the representativeness of the aromatic extracts obtained by this method. In comparing the findings of the present study with previous studies, similarity scores of cooked silurus (Silurus glanis) flesh extracts were found between 22 and 55 8404

DOI: 10.1021/acs.jafc.7b02756 J. Agric. Food Chem. 2017, 65, 8402−8408

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Journal of Agricultural and Food Chemistry

Figure 1. PCA biplot of the volatile compounds of raw and cooked red mullet samples.

butyrolactone were volatile compounds of the lactone group detected in the present study. Cooking treatments increased the amounts of both lactones (Table 1). These compounds can be formed by enzymatic, thermal, and oxidative pathways in food samples.30 3,5-Octadien-2-one, 2,3-octadienone, and 3hydroxybutan-2-one were ketone group compounds identified in the fish samples. Ketone compounds were reported to be found in fish and other seafood as a result of lipid oxidation, amino acid disruption, and Maillard reactions.31 3-Hydroxybutan-2-one was the most abundant compound among the ketones (Table 1), and its amount increased significantly after heat treatment (p < 0.05). Similarly, Min et al.32 reported that the amount of 3-hydroxybutan-2-one increased in heat-treated foods as a result of oxidative degradation of saturated fats or nonenzymatic amino-carbonyl reactions. Also, 3,5-octadien-2one was formed as a result of α-linolenic acid degradation.33 This compound was previously identified as a volatile compound in cooked salmon (Salmo salar), barramundi (Lates calcarifer), and Senegalese sole (Solea senegalensis).34−36 In addition, it was reported that the amounts of two nitrogen compounds (1-methylpyrrolidin-2-one and pyrrolidin-2-one) detected in fish samples increased with the rise of the temperature.37 Some alcohols and aldehydes were positioned on the negative F1 axis in Figure 1. Among the alcohol compounds, 1-octen-3-ol was very important because of the low threshold value of perception (1.2 μg/kg).35 This compound is generally formed from arachidonic acid as a result of 12-lipoxygenase enzyme activity and was detected in several kinds of fish species.15,38,39 2-Phenylacetaldehyde and (E,E)-heptadienal were aldehydes which were located on the negative F1 axis position (Figure 1). It was previously reported that 2phenylacetaldehyde was formed by the Strecker degradation of the Maillard reactions from the phenylalanine while 2,4-

Principal component analysis (PCA) was applied in order to determine the relations between cooking processes and volatile compounds in the fish samples. Based on the results of the PCA, two different principal components were determined, and these two components described 100% of the total variability of the experimental data (F1 was 80.97%, and F2 was 19.03%). The PCA biplot of the volatile compounds detected in the raw and cooked fish samples is shown in Figure 1, and the aromaactive compounds are underlined on this graph. According to the PCA biplot, fish samples were well categorized as raw and cooked samples. As seen in Figure 1, the raw samples were positioned on the positive F1 axis while the heat-treated samples were on the negative F1 axis. It was also found that the n-aldehydes decanal, hexanal, and nonanal in the raw red mullet samples were located very close to each other in the PCA biplot. PUFAs were reported to be the precursor components of these compounds in fish species.28 As a result of cooking, aldehydes in fish and seafood could disappear with carbonyl-amino reactions and thermal processes could lead to various flavor compounds.29 Accordingly, Sun et al.20 determined that, when fresh bigeye tuna fish (Thunnus obesus) meat is exposed to 100 °C temperature, the abundance of octanal and nonanal compounds increased. However, if the intensity of the heat treatment was increased up to 150 °C, the amount of these compounds decreased to nearly undetectable levels.20 But, our study results show that the amount of these compounds decreased after both steam (mean temperature: 96 °C) and oven cooking (mean temperature: 208 °C) (Table 1). Furthermore, naphthalene, phenol, 3methylbutan-1-ol, and 2-phenoxyethanol were positively correlated with the raw red mullet samples according to the PCA analysis results (Figure 1). Compounds such as lactones, ketones, pyrroles and terpenes in both oven and steam cooked red mullet samples were positioned on the negative F1 axis. 2H-Furan-5-one and γ8405

DOI: 10.1021/acs.jafc.7b02756 J. Agric. Food Chem. 2017, 65, 8402−8408

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Journal of Agricultural and Food Chemistry

three aroma-active alcohols were detected in the raw red mullet samples. These were hexanol (green), 1-octen-3-ol (mushroom), and 2-phenoxyethanol (chemical). 2-Phenoxyethanol was previously identified as an aroma-active compound in German rainbow trout (Oncorhynchus mykiss).9 While 2phenoxyethanol had an FD factor value of 8, the FD factor values of the other two alcohols were found to be 2. Three unknowns may have contributed to the overall aroma of the raw red mullet: LRI, 1109 (FD = 2); LRI, 1620 (FD = 8); and LRI 1637 (FD = 2); providing green, cucumber, and burnt notes, respectively (Table 2). It was also found in the current study, that after cooking, the number of aroma-active compounds and their FD factors changed sharply. It was also determined that these changes were mostly caused by the lipid oxidation. 1-Octen-3-ol (FDS = 216; FDO = 512) was identified as the most dominant aromaactive compound in the cooked red mullets providing mushroom odor (Table 2). It was also reported as the key aroma compound in many fish species.41−43,45 Although the FD factor of 1-octen-3-ol was low in the raw red mullet samples, it significantly increased as a result of cooking processes. This finding is in agreement with previous findings on Senegalese sole muscle by Moreira et al.11 Similarly, it was found that the FD value of 1-hexanol (FDS,O = 16), which was an another alcohol compound, increased after cooking. This compound was found to have contributed green odor to the cooked samples (Table 2). It was previously known that 1-hexanol arose from lipoxygenase and hydroperoxide lyase activities.46 Two lactone compounds, γ-butyrolactone (FDS = 32; FDO = 64) and 2H-furan-5-one (FDS = 8; FDO = 16), detected with high FD factors in the cooked samples were described as having fatty and cooked-buttery odors, respectively (Table 2). However, both of these compounds were not detected as aroma-active compounds in the raw red mullet samples. Similarly, Mall and Schieberle22 reported that the FD values of some of the lactones resulting from the breakdown of carbohydrate or proteins by heat treatment in shrimps were significantly higher than those of the untreated fresh shrimp. In addition, in the heat-treated fish samples, two of the compounds of the aldehyde group were aroma-active. (E,E)2,4-Heptadienal (cooked vegetable odor) was another important aroma-active compound which was produced by lipid oxidation (Table 2). The FD value of this compound was determined to be 16 in both cooked fish samples. But this aldehyde was not detected in the raw fish samples. Similarly, Mall and Schieberle22 found that the “-dienal” group of aldehydes increased the aroma-activity status of shrimps due to heat treatment. This compound was previously identified in sardines (Sardina pilchardus), salmon (Salmo salar), and rainbow trout (Oncorhynchus mykiss).41,42,45 Regarding the ketone compounds, 3-hydroxy-2-butanone and 2,3-octanedione compounds were found to be the key odorants in the heated samples. 3-Hydroxybutan-2-one caused the unpleasant oily odor note while 2,3-octanedione gave the pleasant cooked odor in the heated samples. 3-Hydroxybutan-2-one was also reported to be an aroma-active compound in pine mushrooms (Tricholoma matsutake Sing.), and the FD value of this compound increased after cooking.47 Two nitrogenous compounds (pyrrolidin-2-one and 1-methylpyrrolidin-2-one) were identified as aroma-active compounds after cooking. This is the first time that these key odorants were reported in fish species. Of these compounds, pyrrolidin-2-one was found to have an FD value of 8 with popcorn odor note (Table 2).

heptadienal was produced from linolenic acid by 12 hydroperoxides.28 Aroma-Active Compounds in the Raw and Cooked Red Mullet Samples. The aroma-active compounds of the raw (R), steamed (S), and oven (O) cooked red mullet samples are shown in Table 2 with the odor notes and flavor dilution Table 2. Aroma-Active Compounds of the Raw (R), Steamed (S), and Oven (O) Cooked Red Mullet Samples with the Odor Notes and Flavor Dilution (FD) Factor Values FDc no.

LRIa

compound

1 2 3 4

1074 1109 1173 1277

5 6 7 8

1353 1359 1458 1483

hexanal unknown unknown 3-hydroxybutan-2-one (acetoin) 2,3-octanedione 1-hexanol 1-octen-3-ol (E,E)-2,4-heptadienal

9 10

1495 1537

decanal linalool

11 12 13 14

1612 1620 1637 1662

15

1764

γ-butyrolactone unknown unknown 1-methylpyrrolidin-2one 2H-furan-5-one

16 17

2014 2130

pyrrolidin-2-one 2-phenoxyethanol

odor descriptionb green green cooked fish oily cooked green mushroom cooked vegetable green flowery/ green oily cucumber burnt oily/fish oil cooked/ buttery popcorn chemical

(R)

(S)

(O)

16 4

32 8

32 16 256 16

32 16 512 16

2 4

2 8

32

64

64 2

64 2

8

16

8

8

2 8

2 2

16

8 2

8

a

Linear retention index calculated on DB-WAX capillary column. Odor description as perceived by panelists during olfactometry. cFD factor is the highest dilution of the extract at which an odorant was determined by aroma extract dilution analysis.

b

factor (FD) values of these compounds. A total of 8 and 13 different aroma-active compounds were identified in the raw samples and the cooked samples, respectively. These 13 key odorants were reported for the first time in red mullet, among the 27 volatile compounds. It was previously reported that fresh saltwater fishes are almost odorless when compared to freshwater and euryhaline fishes.5 These fishes generally have a little odor immediately after they are caught. In the current study, in the raw red mullet fish samples, decanal (FD = 16) was found as the potent aroma-active compound and it gave green odor note to the red mullet fish (Table 2). It was formerly reported that decanal was mainly the result of the oxidation of oleic acid.28 This compound was previously identified as an aroma active compound in turbot (Psetta maxima), rainbow trout (Oncorhynchus mykiss), salmon (Salmo salar), sea bream (Sparus aurata), and cooked gray mullet (Mugil cephalus).24,40−43 Another aroma-active aldehyde compound identified in the raw fish samples was hexanal. It was reported to be produced by the oxidation of n-6 PUFAs.44 This compound gave a fresh green odor to the red mullet samples, but the FD value was very low. Hexanal was previously identified as an aroma active compound in brown trout (Salmo trutta), sardines (Sardina pilchardus), salmon (Salmo salar), and rainbow trout (Oncorhynchus mykiss).16,42,44,45 In addition, 8406

DOI: 10.1021/acs.jafc.7b02756 J. Agric. Food Chem. 2017, 65, 8402−8408

Article

Journal of Agricultural and Food Chemistry Krings et al.48 found that this compound also gave a popcorn odor note in cocoa powders. It was known that the amount of pyrrolidinone compounds increased with heat treatment. Wu et al.37 studied the effects of sugar, fat, and monosodium glutamate on model systems and found that pyrrolidin-2-one was formed by the heat treatment and this compound was formed as a result of a series of reactions from glutamic acid. The other aroma-active compound in cooked red mullet was linalool (flowery/green odor). The FD factor of this compound was 4 for the S samples and 8 for the O samples. In a previous study, it was reported that it was available in cooked pine mushrooms (Tricholoma matsutake Sing.) as key odorant and its FD value increased by the cooking process.47 In addition, in the current study, two unknown compounds were detected in the cooked samples. Unknown LRI 1637 (FDS = 64; FDO = 64) was the most intense, with high FD (64) values in both cooked samples. The odor of this compound was burnt note. Another unknown compound was LRI 1173 (FDS = 16; FDO = 32), which resulted in a cooked odor note. In conclusion, the aroma-active compounds of raw and cooked red mullets were successfully characterized by the application of aroma extract dilution analysis. Decanal, exhibiting a green note, was one of the most potent odorants detected in the raw red mullet samples. Between the two cooking methods (oven cooking and steam cooking) employed in the study, differences in the formation of individual key odorants were not significant, with the exception of 1-octen-3ol, γ-butyrolactone, and 2H-furan-5-one, which had higher FD factors in the oven cooked fish. The highest FD factor of 1octen-3-ol detected in cooked fish samples suggested that it might be the most dominant aroma-active compound responsible for the mushroom-like odor. It is also worth noting that the aroma of oven cooked samples was more intense and their FD values of key odorants were higher than those of steam cooked fishes.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: + (90) 322 338 6173. Fax: + (90) 322 338 6614. ORCID

Serkan Selli: 0000-0003-0450-2668 Funding

We thank the Cukurova University Research Fund (No.: FYL2016-5484) for its financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to thank Dr. Muharrem Keskin from Mustafa Kemal University, Hatay, Turkey, for his outstanding editing and proofreading. The authors would also like to thank the reviewers for their valuable and constructive comments, which were utilized to improve the quality of the paper.

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DOI: 10.1021/acs.jafc.7b02756 J. Agric. Food Chem. 2017, 65, 8402−8408