Glycosidically Bound Aroma Compounds and Impact Odorants of Four Strawberry Varieties

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ "This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS CATALYSIS, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/jf301141f. 1 Glycosidically Bound Aroma Compounds and Impact Odorants of 1 Four Strawberry Varieties 2 Ubeda, C1*; San Juan, F2; Concejero, B2; Callejón, R. M1; Troncoso, A. M1; Morales, 3 M. L1; Ferreira, V2; Hernández-Orte, P2. 4 1Área de Nutrición y Bromatología. Facultad de Farmacia. Universidad de Sevilla. 5 C/ P. García González nº 2, E- 41012. Sevilla. Spain. 6 2Laboratorio del Análisis del Aroma y Enología. Departamento de Química Analítica. 7 Facultad de Ciencias. Universidad de Zaragoza. 50009, Zaragoza, Spain. 8 *Corresponding author: [email protected]; Tel.: 34-954-556761; Fax.: 34-954-233765 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 2 Abstract 26 This paper reports the determination of glycosidically bound aroma compounds and the 27 olfactometric analysis in four strawberry varieties (Fuentepina, Camarosa, Candonga 28 and Sabrina). Different hydrolytic strategies were also studied. The results showed 29 significant differences between acid and enzymatic hydrolysis. In general terms, the 30 greater the duration of acid hydrolysis, the higher was the content of norisoprenoids, 31 volatile phenols, benzenes, lactones, Furaneol, and mesifurane. A total of 51 aglycones 32 were identified, 38 of them unreported in strawberry. Olfactometric analyses revealed 33 that the odorants with higher modified frequencies were Furaneol, γ-decalactone, ethyl 34 butanoate, ethyl hexanoate, ethyl 3-methylbutanoate, diacetyl, hexanoic acid, and (Z)- 35 1,5-octadien-3-one. This last compound, described as geranium/green/pepper/lettuce 36 (linear retention index = 1378), was identified for the first time. Differences with regard 37 to fruity, sweet, floral, and green aroma characters were observed among varieties. In 38 Candonga and Fuentepina, the green character overpowered the sweet. In the other two 39 strawberry varieties sweet attributes were stronger than the rest. 40 Keywords: Glycosides, strawberry, aroma, flavor, olfactometry. 41 42 43 44 45 46 47 48 49 50 3 INTRODUCTION 51 Strawberry is a much appreciated fruit due to its aroma, taste and health properties. It is 52 usually consumed fresh (75% of total production) but is also used in the food industry 53 as an important ingredient in jam, yogurt, syrup, tea, juice, ice cream, and other food 54 products (25% of overall production)1. Aroma is one of the most valued attributes of 55 strawberry. The aroma of this fruit includes volatile compounds, both in their free form, 56 and as non-volatile compounds, present mainly as glycoconjugates formed by a sugar 57 and an aglycone. 58 There are numerous studies concerning free volatile compounds of strawberry, with 59 more than 360 volatile flavor compounds2 identified. In order to learn more about the 60 volatile composition of strawberry, several olfactometric studies have been undertaken 61 using gas chromatography-olfactometry (GC-O) 3–5. 62 Non-volatile compounds are, moreover, potential natural sources of aroma because the 63 hydrolysis of the bonds between the sugar and the aglycone turns this molecule into an 64 aromatic compound. As ripening proceeds, the increase in these soluble sugars results in 65 an increase in the availability of precursors capable of producing aroma compounds6. 66 These non-volatile compounds have been extensively studied in grapes7–9 and in other 67 fruits such as lychee, acerola, blackberry, pineapple and mango10–14, among others. 68 Strawberry precursors have hardly been studied. After the description of the presence of 69 2,5-dimethyl-4-hydroxy-2H-furan-3-one -D-glucopyranoside in strawberry15, Wintoch 70 et al.16 analyzed the glycosidical aroma compounds from two strawberry species using 71 Amberlite XAD-2. Other research groups have focused their studies on one aglycone, 72 furaneol (2,5-dimethyl-4-hydroxy-2H-furan-3-one)17, due to its high influence on the 73 overall flavor. In addition, there have been some studies concerning the evolution of 74 these non-aromatic precursors during ripening. These studies show an increase in their 75 4 aglycones during the above mentioned stage18. Knowledge of the strawberry aromatic 76 precursors is important because it enables us to predict the final aroma of new 77 strawberry-based products. As a result, there are several different groups studying the 78 production process of strawberry fermentation products19,20. Such of analyses would 79 enable us to estimate the aromatic potential and therefore select the best raw material. 80 The aim of this study was to determine the aromatic potential of different strawberry 81 varieties with the aim of selecting the most suitable varieties for producing several 82 fermented strawberry-based food products. Therefore the aroma compounds released by 83 acid hydrolysis of glycosidic precursors isolated from 4 different varieties have been 84 determined. Free aromas were also studied by GC-O analyses to determine the most 85 important compounds, from a sensory point of view, in these varieties. 86 MATERIALS AND METHODS 87 Reagents and standards 88 Dichloromethane, ethanol and methanol were supplied by Merck (Darmstadt, 89 Germany), ethyl acetate and sodium fluoride by Fluka (Buchs, Switzerland). Sodium 90 dihydrogen-phosphate 1-hydrate, L (+) ascorbic and citric acids were purchased from 91 Panreac (Barcelona, Spain). Pure water was obtained from a Milli-Q purification system 92 (Millipore, U.S.). LiChrolut EN resins were purchased from Merck. An alkane solution 93 (C8–C28), 20 mg/L in dichloromethane, was used to calculate the linear retention index 94 (LRI) of each analyte. The chemical standards used for the identification and 95 quantification of volatile compounds were as follows: (Z)-rose oxide, linalool, α- 96 terpineol, nerol, geraniol, benzaldehyde, β-phenylethanol, 2-phenoxyethanol were 97 purchased from Fluka (Buchs, Switzerland). β-ionone was sourced from Sigma (St. 98 Louis, MO) and guaiacol, m-cresol, eugenol, 4-ethylphenol, 4-vinylguaiacol, methyl 99 vanillate, acetovanillone, zingerone, homovanillyl alcohol, phenylacetaldehyde, benzyl 100 5 alcohol, ethyl cinnamate, γ-nonalactone, γ-decalactone, (Z)-3-hexen-1-ol from Aldrich 101 (Gillingham, UK). (E)-isoeugenol, 4-vinylphenol, δ-octalactone, δ-decalactone were 102 purchased from Lancaster (Strasbourg, France). Finally, β-damascenone and vanillin 103 were supplied by Firmenich (Geneva, Switzerland) and Panreac (Barcelona, Spain) 104 respectively. 105 Samples 106 We employed freshly purchased Fragaria ananassa, var. camarosa strawberries in 107 order to optimize the extraction method and to obtain the aroma precursors extract. 108 Aromatic precursors were then determined in four different varieties of strawberry: 109 Fuentepina, Camarosa, Candonga and Sabrina. These strawberries were also employed 110 for the olfactometric studies. 111 Extraction of aroma precursors 112 In order to study the effects of different kinds of hydrolysis we prepared a precursors 113 pool from strawberries of Camarosa variety adquired in the market. The preparation 114 procedure was based on Ibarz et al.7. We used an Ultra Turrax T25 Basic mixer (Ika, 115 Labortechnik) to crush and homogenize 2 kg of strawberries with 1 L of cold Milli-Q 116 water in the presence of 0.13 M NaF, to prevent microbial growth, and 50 mg/L of 117 ascorbic acid (as an antioxidant). This mixture was then centrifuged and filtered 118 obtaining a strawberry must which was placed in PYREX flasks, adding 2 gr of 119 LiChrolut resins (previously pre-conditioned with dichloromethane, methanol and Milli- 120 Q water) per kg of strawberry. The oxygen of the flasks was evacuated using nitrogen. 121 We left the must in contact with the resins for 16h in a Heidolph PROMAX 1020 shaker 122 (Schwabach, Germany) at 90 rpm. We packaged the resin and each cartridge of 500 mg 123 was washed with 50 mL of water. It was then completely vacuum-dried and free aromas 124 were extracted with 50 mL of dichloromethane and discarded. 30 mL of an ethyl 125 6 acetate/methanol solution (9:1) were subsequently percolated through the resin. The 126 solvents were evaporated under vacuum, resuspended in a 50:50 ethanol/water solution 127 and kept at -20ºC. 128 To analyze the 4 different strawberry varieties, we followed the same technique as that 129 utilized for obtaining the precursors pool. In this case we processed 10 gr of strawberry 130 since we obtained the best results in previous studies using that quantity (data not 131 shown). The must was percolated through a 200 mg LiChrolut EN cartridge (previously 132 pre-conditioned with 10 mL dichloromethane, 10 mL methanol and 10 ml Milli-Q 133 water). After that, the column was washed with 20 mL of Milli-Q water and then was 134 completely dried. In order to eliminate all free aromatic compounds, we passed 20 mL 135 of dichloromethane through the cartridge. To recover the precursors from the resin we 136 employed 20 mL of a solution of ethyl acetate/methanol (9:1). This eluate was 137 concentrated to 1 mL under vacuum at 40 ºC and then, taken to dryness under a gentle 138 nitrogen stream. Each sample was extracted in duplicate. 139 Acid and enzymatic hydrolysis 140 Different hydrolytic conditions were performed in order to study their influence on the 141 aromatic profile of strawberry using the precursors pool previously obtained. The acid 142 hydrolyses assayed were: 15 min, 1 h and 4 h at 100 °C and one week at 45 °C. For this 143 hydrolysis we mixed 8 mL of citric buffer (0.2 M pH 2.5), 1 mL of the precursor extract 144 and 1 mL of an ethanol/water solution (50:50) (to maintain the same concentration of 145 ethanol in all the acid hydrolysis assays in a 20 mL vial. After this, the vial was sealed 146 and placed in the oven. Moreover, an enzymatic hydrolysis was performed during 16 h 147 at 38 °C. In this case we used 8.7 mL of citrate (0.1 M)/ phosphate (0.2 M) buffer 148 solution at pH=5, 1 mL of the precursor extract, which was subjected to vacuum to 149 7 remove the ethanol, and 800 L of a pectinase enzyme solution with 200 mg/mL of AR 150 2000. 151 Otherwise, for the analysis of the four varieties of strawberry, the dry extract was 152 reconstituted in 10 mL of citric buffer (0.2 M pH 2.5, 10% EtOH) and was subjected to 153 hydrolysis at 100 ºC for one hour. Before any hydrolysis was undertaken, the remaining 154 oxygen was displaced from the vial with nitrogen in order to prevent oxidation of the 155 compounds during the process. Each hydrolysis was done in duplicate. 156 Extraction of volatiles released in the hydrolysis 157 After the hydrolysis, the solution was percolated through a 50 mg LiChrolut EN 158 cartridge (previously pre-conditioned with 6 mL dichloromethane, 2 mL methanol and 2 159 mL of citric buffer solution). Then was washed with 1 mL Milli–Q water and dried. To 160 elute the aromatic compounds, 700 µL of dichloromethane were passed through the 161 column and collected in a Kuderna Danish Supelco (Bellefonte, PA, USA), adding 14 162 µL of the internal standard 4-methyl-2-pentanol (402.6 µg/g). Finally was concentrated 163 to 100 µL with a gentle nitrogen stream. 164 Preparation of the olfactometry extract 165 To obtain a representative extract of each strawberry variety for the olfactometry 166 analyses we followed the method used by Ferreira et al.21. 80 g of the fruit were crushed 167 and placed in a purge and trap system22. A Lichrolut EN cartridge was placed on the top 168 of the bubbler flask. A nitrogen stream of 500 mL/min was applied to the sample for 169 100 min, releasing the free volatile compounds of strawberry in the headspace being 170 trapped by the cartridge. Finally these compounds were eluted with 3.2 mL of 171 dichloromethane containing 5% methanol. The extract was concentrated to a final 172 volume of 200 μL. 173 174 8 GC-MS and GC-O analytical conditions 175 Gas chromatographic analysis of the volatiles released in the hydrolysis was performed 176 with a CP-3800 chromatograph coupled to a Saturn 2200 ion trap mass spectrometric 177 detection system from Varian (Sunnyvale, CA, USA). A DB-WAXetr capillary column 178 (J&W Scientific, Folsom, CA, USA) (60 m × 0.25 mm I.D., film thickness 0.5 μm) 179 preceded by a 3 m × 0.25 mm uncoated (deactivated, intermediate polarity) precolumn 180 from Supelco (Bellefonte, PA, USA) was used. Helium was the carrier gas at a flow rate 181 of 1 mL/min. The oven temperature program was 3 min at 40 °C, 10 °C/min up to 90 182 °C, 2 °C/min up to 230 °C and finally held at this temperature for 37 min. Initially the 183 injector was kept at 35 °C for 0.3 min and a pressure pulse of 25 psi for 2.60 min was 184 applied. The injector was then heated to 250 °C at rate of 200 °C/min. The splitless time 185 was 2.60 min. The injection volume was 4 μL. The global run time was recorded in full 186 scan mode (40–220 m/z mass range). The chromatographic data were analyzed by 187 Varian Saturn GC–MS Version 6.3 software23. 188 To carry out the olfactometric analyses we followed the protocol described in Ferreira et 189 al., (2009). The sensory panel was composed of six expert sniffers. Each strawberry 190 extract was smelled once a day by each panelist. Sniffing time was approximately 30 191 min. The experiments were carried out in a Thermo 8000 series GC equipped with a 192 flame ionization detector (FID) and a sniffing port (ODO-1 from SGE) connected by a 193 flow splitter to the column exit. The chromatographic conditions were the same as 194 described in Campo et al., (2005). Tasters were asked to score the intensity of each 195 aromatic stimulus using a 4-point scale (0=not detected, 1=weak, 2=clear but not 196 intense note, 3=intense note). 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Principal component plot showing the scores for the samples of acid and 425 enzymatic hydrolysis. 426 Figure 2. GC-MS Chromatographic profile of the strawberry variety Fuentepina. 427 Figure 3. Sensory profile plot of Fuentepina, Camarosa, Candonga and Sabrina 428 varieties considering fruity, sweet, green and floral characteres. 429 430 19 Table 1. Concentration (g/kg of Strawberries Except Where Indicated x) of Volatile Compounds Released After Harsh Acid and Enzymatic Hydrolysis of the Strawberry Precursors Pool. 0 min 15 min 1 h 4 h 1 week 45 °C Enzymatic Terpenes α-terpinolene nd 0.62±0.10 a 2.94±0.36 b 4.51±0.14 c 0.27±0.01 d 1.17±0.09 f (Z)-rose oxide nd nd 0.02±0.00 a 0.25±0.01 b nd nd (R/S)-linalool nd 75±2 a 3.50±0.30 b nd 5.07±0.13 c 105±2 d α-terpineol nd 27±1 a 111±13 b 50±1 c 77±2 d 1.28±0.14 e nerol nd 6.20±0.79 a 12.72±0.39 b nd nd 2.18±0.22 c geraniol 4.46±0.36 a 29±1 b 4.91±0.63 a nd 3.74±0.35 a 5.95±0.56 a farnesol nd 12±1 nd nd nd nd linalool acetate nd 0.23±0.04 nd nd nd nd terpinen-4-ol x nd nd 3.01±0.27 a 2.84±0.09 a 0.68±0.04 b nd δ-terpineol x nd nd 6.74±0.41 a 6.05±0.38 a 3.93±0.05 b nd neric acid nd nd 0.20±0.02 a 0.50±0.04 b nd 1.01±0.03 c Norisoprenoids β-damascenone nd 0.46±0.02 a 1.30±0.11 b 2.28±0.08 c 0.59±0.02 d nd β -ionone 0.15±0.01 a nd nd nd nd 0.25±0.01 b 1,1,6-trimethyl-1,2- dihydronaphthalene(TDN) x nd 0.28±0.01 a 1.68±0.01 b 2.77±0.09 c 0.59±0.02 d 1.11±0.01 e tert-1-(2,3,6-trimethylphenyl) buta-1,3-diene (TPB) x nd 0.34±0.01 a 6.35±0.09 b 7.51±0.44 b 2.13±0.04 c 0.50±0.00 d 3-oxo-β-ionone x nd 1.33±0.03 a 4.51±0.42 b 4.65±0.21 b 2.77±0.14 c nd actinidols x nd 0.24±0.02 a 5.81±0.52 b 6.62±0.23 b 4.09±0.15 c 0.25±0.02 a norisoprenoid 1 x nd nd 2.81±0.22 a 4.15±0.10 b 0.27±0.03 c nd 3-oxo-α-ionol nd nd 0.63±0.07 a nd nd 75±2 b Volatile phenols guaiacol nd nd nd 0.70±0.09 a nd 0.91±0.04 a m-cresol nd nd nd nd nd 0.22±0.01 eugenol 0.62±0.01 a 0.70±0.08 a 1.35±0.17 b 6.06±0.86 c 1.07±0.02 b 18±1 d 4-ethylphenol nd 0.08±0.00 a nd nd nd 1.11±0.14 b 4-vinylguaiacol 4.38±0.08 a 5.73±0.79 a 116±11 b 151±14 b 35±2 c 352±8 d (E)-isoeugenol 1.79 ±0.09 a 1.38±0.07 a 0.91±0.14 b,c 1.33±0.05 a,b 0.68±0.01 c 3.70±0.57 d 4-vinylphenol 121± 2 a 247±16 b 12606±1440 c 20904±3263 c,e 6231±120 d 27863±2764 e Vanillin derivatives vanillin 0.50±0.01 a 1.08±0.01 b 2.22±0.23 c 3.81±0.10 2.36±0.06 8.21±0.68 methyl vanillate 0.10±0.00 a nd nd nd nd 1.16±0.09 b acetovanillone nd nd nd 0.56±0.09 a nd 2.19±0.00 b homovanillyl alcohol nd nd nd nd 1.18±0.00 nd homovanillinic acid x 5.21±0.10 a 4.10±0.24 b nd nd nd 83±4 c Benzenes benzaldehyde 0.74±0.06 a 1.86±0.01 b 3.69±0.35 c 8.13±0.68 d 3.07±0.12 c 11±1 e phenylacetaldehyde 0.67±0.01 a 0.87±0.10 a 3.24±0.19 b 4.35±0.27 c nd 4.46±0.43 c benzyl alcohol 1.69±0.21 a 3.14±0.37 b 21±1 c 59±1 d 10±1 e 1361±40 f β-phenylethanol nd 1.90±0.08 a 4.54±0.05 b 9.61±0.27 c 3.21±0.19 d 97±4 e ethyl cinamate nd nd 7.09±0.09 a 23±1 b 18±1 c 3.21±0.19 d 2-phenoxyethanol 1.03±0.04 a 1.38±0.24 a,c 0.64±0.11 b 0.96±0.01 a 0.54±0.06 b 1.85±0.14 c benzoic acid 7.10±0.93 a 10±1 b 113±17 c 210±19 d 44±3 e 240±7 d dihydromethyl-eugenol x nd nd 0.20±0.03 a 0.41±0.01 b 0.18±0.01 a 3.20±0.17 c Lactones 20 δ-octalactone y nd 0.47±0.01 a 1.09±0.01 b,c 1.47±0.01 b 1.08±0.00 c nd γ-nonalactone y nd 0.68±0.01 a nd nd nd 0.86±0.00 b γ-decalactone y nd 0.10±0.01 a 7.54±0.00 b 17±1 c nd 1.08±0.03 d pantolactone 2.49±0.03 a 1.18±0.11 b 6.49±0.49 c 8.47±0.98 c nd nd Miscellaneous (Z)-3-hexen-1-ol nd 1.16±0.01 a 2.03±0.01 b 2.19±0.09 b 2.17±0.12 b 13±1 c (E)-2-hexen-1-ol 4.37±0.52 a 4.95±0.52 a 2.87±0.15 b 2.90±0.10 b 3.00±0.17 b 5.80±0.17 c ethyl decanoate 4.34±0.05 a 4.38±0.02 a 4.36±0.02 a nd 4.28±0.00 a nd 2-ethylhexanoic acid 1.30±0.22 a 1.20±0.06 a 1.13±0.01 a 1.07±0.01 a 1.87±0.22 a,b 2.19±0.09 b 4-methoxy-2,5-dimethyl- 3(2H)furanone (mesifurane) x 50±5 a 338±4 b 339±19 b 315±23 c 251±6 d 307±9 c 4-hydroxy-2,5-dimethyl- 3(2H)furanone (furaneol) x 23±1 a 58±2 b 74±1 c 102±3 d 60±1 b 74±3 c cinnamic acid x 338±18 a 586±61 b 2917±226 c 7657±555 d 1828±80 e 6209±119 d nd: non detected. x Chemical standard not available. Tentatively identified. Data are relative areas (to 4-Methyl-2-pentanol x 1000). y Data are the relative areas (to 4-Methyl-2-pentanol x 1000). Chemical standard available,but the degradation of the products did not allow quantification. Concentrations of the same compound with different letter show significant differences (p < 0.05). 21 Table 2. Concentration (g/kg of Strawberries Except Where Indicated x) of Volatile Compounds Released After Harsh Acid Hydrolysis of the Precursor Extract from Each Strawberry Variety. Fuentepina Camarosa Candonga Sabrina Terpenes α-terpinolene (1) 0.58±0.05a 0.39±0.01b 0.24±0.01c 0.19±0.01d (Z)-rose oxide (2) 0.02±0.00 nd nd nd (Z)-linalool oxide x (3) 1.16±0.13 a nd nd 7.68±0.34 b (E)-linalool oxide x (4) 1.02±0.03 a nd nd 4.81±0.46 b (R/S)-linalool (5) 9.21±0.23 a 13±1 a 32±3 b 48±2 c α-terpineol (6) 100±4 a 63±6 b 89±10 b 78±5 b nerol (7) 0.82±0.09 a 0.93±0.13 a 3.83±0.43 b 6.03±0.42 c Geraniol (8) 18±2 a 22±1 a,b 28±2 b 45±5 c Farnesol nd nd 9±1 a 18±2 b δ-terpineol x (9) 1.19±0.09 a 0.48±0.03 b 0.59±0.01 b 0.34±0.01 c Norisoprenoids β-damascenone (10) 2.00±0.18 a 1.75±0.00 a 1.14±0.14 b 0.65±0.01 c β –ionone nd 0.92±0.01 a nd 0.67±0.04 b 1,1,6-trimethyl- 1,2-dihydronaphthalene (TDN) x (11) 1.09±0.08 a 0.46±0.03 b 0.42±0.01 b 0.08±0.01 c tert-1-(2,3,6-trimethylphenyl) buta-1,3-diene (TPB) x (12) 3.84±0.07 a 4.34±0.45 a 0.96±0.09 b 0.58±0.02 c 3-Oxo-β-ionone x (13) 2.01±0.18 a 1.48±0.03 a 0.74±0.04 b 0.44±0.01 c Actinidols x (14) 2.84±0.32 a 2.24±0.04 a 0.91±0.01 b 0.75±0.01 c norisoprenoid 1 x (15) 0.69±0.04 a 0.73±0.01 a 0.27±0.01 b 0.05±0.01 c Volatile phenols m-cresol nd 0.65±0.02 a 0.47±0.03 b nd Eugenol nd 0.91±0.01 a 0.17±0.01 b 0.27±0.03 c 4-vinylguaiacol (16) 76±1 a 31±2 b 31±2 b 26±1 b 4-vinylphenol (17) 8565±92 a 994±73 b 9602±90 c 2426±242 d Vanillin derivatives Vanillin (18) 2.96±0.08 a 4.12±0.03 b 1.46±0.10 c 1.75±0.19 c Zingerone (19) 0.76±0.01 a nd nd 1.07±0.07 b Benzenes Benzaldehyde (20) 6.82±0.11 a 4.94±0.17 b 3.80±0.35 b 4.74±0.30 b Phenylacetaldehyde (21) 3.60±0.28 a 2.66±0.08 b 2.16±0.03 c 2.16±0.03 c benzyl alcohol (22) 37±1 a 20±1 b 14±1 c 8.45±0.49 d β-phenylethanol (23) 9.39±0.62 a 7.55±0.18 a 6.09±0.29 b 6.17±0.37 b Ethyl cinamate (24) 8.71±0.69 nd nd nd 2-phenoxyethanol (25) 5.20±0.42 a 7.95±0.67 b 3.29±0.42 c 5.42±0.42 a benzoic acid (26) 131±12 a 80±7 b 129 ±3 a 116±5 a Lactones δ-octalactone y (27) 2.89±0.15 a 2.10±0.18 b 14±0 c 7.65±0.93 d γ-nonalactone y (28) 1.89±0.13 a 1.94±0.16 a 1.42±0.07 b 1.45±0.16 b γ-decalactone y (29) 12±1 a 5.55±0.45 b 23±1 c 26±2 c Pantolactone (30) 1.66±0.01 a 1.28±0.01 a 0.93±0.01 b 0.84±0.01 c Miscellaneous (Z)-3-Hexen-1-ol (31) 5.26±0.41 a 5.21±0.08 a 4.85±0.04 b 4.10±0.00 c (E)-2-Hexen-1-ol (32) 19±2 a,b 24±1 a 17±1 b 18±1 b ethyl decanoate (33) 16±1 a 17±0 a 16±0 a 16±0 a 2-ethylhexanoic acid (34) 13±1 a 14±1 a,b 13±1 a 15±1 b 4-methoxy-2,5-dimethyl-3(2H)furanone (mesifurane) x (35) 5.07±0.02 a 22±1 b 34±1 c 42±1 d 22 4-hydroxy-2,5-dimethyl-3(2H)furanone (furaneol) x (36) 8.15±0.01 a 39±1 b 16±1 c 19±1 c cinnamic acid x (37) 1678±36 a 178±24 b 850±49 c 877±61 c nd: non detected. (num): peak number in Figure 2. x Chemical standard not available. Tentatively identified. Data are relative areas (to 4-Methyl-2-pentanol x 1000). y Data are the relative areas (to 4-Methyl-2-pentanol x 1000). Chemical standard available,but the degradation of the products did not allow quantification. Concentrations of the same compound with different letter show significant differences (p < 0.05). 23 Table 3. Odour Active Compounds of the Four Strawberry Varieties Analysed. LRI VF5- MSDBWax Odour descriptor Identity % Modified frequency Fue Cam Cdo Sab 918 solvent, gas, glue n.i 0 0 31 0 972 dairy product, sweet, buttery diacetyl 55 61 55 78 1007 fruity, strawberry, sweet isobutyl acetate 0 48 33 24 1033 fruity, strawberry, sweet ethyl butanoate 69 59 75 73 1052 fruity, sweet, anis, cream ethyl 2-methylbutanoate 50 46 29 0 1066 fruity, apple, anis, green, metallic ethyl-3- methylbutanoate 61 33 69 73 1180 rubber, moisture, gas n.i 0 0 0 34 1191 fruity, anis methylhexanoate 17 0 0 33 1236 fruity, raspberry, strawberry, anis ethyl hexanoate 43 33 62 55 1303 mushroom, metallic, chlorine, cucumber 1-octen-3-one 51 33 45 53 1312 spicy, green, barbecue, yeast 2-methyl-3-furanthiol 55 0 50 36 1346 floral, sweet, strawberry (Z)-rose oxide 38 0 0 0 1378 geranium, green, pepper, lettuce (Z)-1,5-octadien-3-one 82 51 80 29 1380 tropical, pineapple, citrus, green methyloctanoate * 0 0 61 0 1458 vinegar acetic acid 67 38 48 38 1548 green, grass, sweet, cucumber (E)-2-nonenal 41 0 0 0 1552 garbage, sulfur, peanuts, barbecue n.i 0 0 49 0 1563 floral, lemon (R/S)-linalool 33 0 0 0 1570 unpleasant, fatty acid, vomit, vinegar n.i 0 0 0 40 1597 tropical, sweet, caramel, cotton candy mesifurane * 43 33 35 45 1609 strawberry n.i 31 0 0 0 1626 burnt hair 2-acetyl pyrazine 73 61 75 53 1631 cheese, vomit, feet butyric acid 27 17 35 43 1676 cheese, feet, sweat, milk isovaleric acid 59 67 61 61 1730 fruity, honey, berry, tropical, sweet, floral phenyl acetate * 0 31 22 38 1826 sweet, floral, rose -damascenone 0 26 0 41 1850 soil, green, spicy, pepper, peanuts, dry grass hexanoic acid * 65 54 66 58 1865 camphor, barbecue, spicy guaiacol 45 76 35 59 2052 caramel, strawberry, sweet furaneol 82 82 80 85 2100 leather, animal, stable p-m-cresol 31 47 33 36 2170 peach, sweet, strawberry γ-decalactone 80 26 85 83 2221 animal, spicy, licorice sotolon 45 76 0 31 2294 latex, spicy, burnt n.i 0 0 0 53 2420 coconut, vanillin γ/δ-dodecalactone 0 29 25 33 n.i: not identified * Tentatively identified by Lineal Retention Index and odour descriptor. Fue: Fuentepina; Cam: Camarosa; Cdo: Candonga; Sab: Sabrina. 24 Figure 1.