Characterization of odour active compounds in strawberry vinegars

1 Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Ubeda, C., Callejón, R.M., Troncoso, A.M., Moreno-Rojas, J.M., Peña, F. and Morales, M.L. (2012), Characterization of odour active compounds in strawberry vinegars. Flavour Fragr. J., 27: 313-321, which has been published in final form at https://doi.org/10.1002/ffj.3103. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of SelfArchived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 2 Title: CHARACTERIZATION OF ODOUR ACTIVE COMPOUNDS IN STRAWBERRY VINEGARS Authors: C. Ubeda1, R.M. Callejón1, A.M. Troncoso1, J.M. Rojas2, F. Peña2, M.L. Morales1* Address: 1Área de Nutrición y Bromatología. Facultad de Farmacia. Universidad de Sevilla. c/ P. García González nº2, E41012. Seville. Spain. 2IFAPA Centro Alameda del Obispo. Avda. Menéndez Pidal s/n, E-14004. Córdoba. Spain. *Corresponding author: e-mail: [email protected]; Tel.: 34-954-556760; Fax.: 34-954233765 Abstract Odour-active compounds in strawberry vinegars were determined by gas chromatography equipped with an olfactometer (GC-O) using the modified frequency (MF) technique. The initial strawberry substrate was also analysed showing that ethyl 2methylbutyrate, mesifurane, β-damascenone, furaneol and γ-decalactone were preserved during the double fermentation process, presenting high MF values. The final aromatic profile of strawberry vinegars is formed both by compounds from the substrate and by those formed during alcoholic and acetous fermentation. Due to their high MF, a total of 12 odour zones, identified as acetic, butyric and isovaleric acids, methional, 3-nonen-2one, 2-phenylethanol, pantolactone+furaneol, p-vinylguaiacol, sotolon, phenylacetic acid and vanillin, were considered as possible impact odorants of strawberry vinegars. Finally, all potential impact odorants with similar sensory descriptors were grouped into 8 categories, these being: fruity, sweet, grassy, spicy, butter-lactic-cheesy, chemical, empyreumatic and miscellaneous. According to the MF percentage of these categories, 3 grassy, fruity, sweet and spicy aroma seem to have the highest influence on the overall impression of strawberry vinegars. Keywords: impact odorant, olfactometry, vinegar, strawberry, modified frequency. Introduction Because of its worldwide availability and its great array of varieties, vinegar is one of the world’s most widespread and common products. In Spain, vinegar is mainly produced by the double fermentation (alcoholic and acetous) of grapes. Present, innovations in vinegar production falls into two areas: improving production processes and employing different raw materials. Strawberries are prized for both their aroma and flavour. Spain is the world’s second largest strawberry producer. Since there are surpluses of second quality strawberry, this makes it an attractive candidate for use as a raw material in producing new kinds of vinegars. Aroma is certainly one of the most important determinants in food quality and acceptance[1]. Therefore, when introducing a new food product, the characterization of its aroma is an important aspect to be considered. Aroma is determined by a large number of compounds that are involved in different ways. Among the above, odouractive compounds play an important role in perceived aroma and especially, impact odorants, that directly influence in it. An essential tool when characterizing the olfactory impact of an odorant (its odour descriptor and intensity) is the coupling of Gas Chromatography/Olfactometry (GC/O)[2]. Among the different types of olfactometric analysis, the three main ones are: dilution methods, perceived intensity methods and frequency of detection methods. The frequency of detection has been proved to provide quantitative estimates of the sensory importance of a compound [3] and has even been proved to be accurate enough to 4 provide a real quantitative data[4], but its limited due to the fact that once the concentration becomes higher than the less sensitive member of the panel, the signal becomes saturated[5]. This limitation can be partly overcome by combining the frequency of detection with a simple measurement of intensity, which also has been proved to provide reliable quantitative data[6]. As concentration increases, odour intensity may continue to increase, however the frequency of detection might not increase when all members of the panel are finally able to detect the odorant[5]. The combination of intensity and frequency of detection can be done through the so called modified frequency (MF) which is the geometric mean of the detection frequency of an aromatic zone (expressed as a percentage) and the average intensity (expressed as a percentage of the maximum intensity)[7]. This concept is useful because the discriminative capabilities of the detection frequency may be improved by taking intensity into account [8] and, moreover, because the members of a tasting panel can have widely divergent sensitivities[7]. MF, therefore, provides more reliable results. The volatile composition of strawberry has been the object of extensive study. The aroma of this fruit is generally a complex mixture of esters, furanones, and terpene alcohols with smaller amounts of lactones, aldehydes, alcohols and sulphur compounds[9-16]. Among the above, furaneol and mesifurane are considered to be the two major flavour contributors due to their low odour threshold and their high quantities[17]. These compounds have been confirmed in several studies on the odouractive compounds in different strawberry varieties. In these works, the authors have identified a considerable number of odour-active compounds and they agree on furaneol, mesifurane and γ-decalactone as being impact odorants in strawberry[18,19]. In this work we have determined odour-active compounds and the possible impact odorants by GC-O in strawberry vinegars obtained through double fermentation. 5 Moreover, an olfactometric analysis of the starting substrate has been also performed to ascertain whether the key odorants of raw material remain in the final products. Experimental Chemicals Dichloromethane and anhydrous sodium sulphate were purchased from Merck (Darmstadt, Germany), all of them were of analytical quality. Strawberry vinegar samples Vinegars analyzed in this study were produced from second quality strawberries of Camarosa variety. For that purpose, fruit was crushed and mixed with pectolytic enzymes and sulphur dioxide. Besides, 50 g/L of sucrose were added to ensure an appropriate final acidity in the resulting vinegar. This starting substrate (F8P2) was submitted to two types of alcoholic fermentations, spontaneous and inoculated with the yeast Saccharomyces cerevisiae QA23 at a concentration of 2x106 cells/mL. All resulting wines were spontaneously acetified by surface culture. Thus, we obtained two different vinegars: F8VI (from inoculated alcoholic fermentation) and F8VE (from spontaneous alcoholic fermentation). Sample extraction The liquid-liquid extraction method was performed to obtain a representative extract of the samples[9]. The procedure followed was: 5 g of anhydrous sodium sulphate was added to 50 mL of each sample and was extracted twice during 5 minutes with 5 mL of dichloromethane using a magnetic stir bar. Then, 2.5 mL of the organic phase was concentrated 5 times under a nitrogen stream. Gas Chromatography-Olfactometry conditions (GC-O) Analyses were conducted using a Varian 3800 GC (Middelburg, The Netherlands) equipped with a flame ionization detector (FID) and an OP275 olfactometer (GL 6 Science Inc., Tokyo, Japan). Two microliters of each extract were injected in splitless mode into a DB-WAX column with 60 m x 0.25 mm x 0.22 μm film thickness (J & W Scientific, Agilent Technologies Inc., Santa Clara, USA). The oven temperature program was as follows: 40° for 1 min, increasing to 220 °C at a rate of 2°C/min and held for 30 min. The column effluent was split 2:3 into a FID and a heated sniffing port. The injector and detector temperature was 220ºC. The carrier gas was H2 at a flow rate of 1 mL/min. The sensory panel was composed of three trained tasters, all of them sniffed each sample twice, assigning to each perceived odour an intensity level: 1, 2 or 3. Results were expressed as “modified frequency” (MF), calculated with the formula proposed by Dravnieks[20]. Gas Chromatography-Mass Spectrometry (GC-MS) conditions Analyses were conducted using an Agilent 6890 GC system coupled to an Agilent 5975inert quadrupole mass spectrometer and equipped with a Gerstel MP2 headspace autosampler (Müllheim an der Ruhr, Germany). The analytical column was a CP-Wax 57CB column of 50 m × 0.25 mm and 0.20 µm film thickness (Varian, Middelburg, The Netherlands). The injector port was heated to 220°C and 2 L of sample extract were injected in splitless mode with a purge flow of 70 mL/min and purge time of 2 minute. The carrier gas was He at a flow rate of 1 mL/min. The oven temperature program was the same employed in GC-O. Quadrupole, source and transfer line temperatures were 150, 230 and 250 °C, respectively. Electron ionization mass spectra data from m/z 29~350 were collected in the scan mode, with an ionization voltage of 70 eV. Identification of Aroma Compounds In the GC-MS analysis, volatile compounds were identified based on the comparison of the retention times of individual standard and computer matching with the reference mass spectra from the NIST 98 library. 7 In the GC-O analysis, compounds were identified by the comparison of their linear retention indices (LRIs) with those obtained in GC-MS analysis of extract and standards as well as with LRIs and odour qualities from Flavornet (www.flavornet.org)[21] and Pherobase (www.pherobase.com)[22] online data bases and literature[9,23-38]. Linear retention indices (LRIs) were calculated on the basis of retention times of nalkanes (C10–C32) under identical conditions for each instrument. Statistical analysis All statistical analyses were performed using Statistica software version 7.0 software package (Statsoft, Tulsa, USA). A principal component analysis (PCA) was carried out as an unsupervised method in order to ascertain the degree of differentiation between samples and which compounds were involved. Results and Discussion In this study, we only considered those odour zones which were detected in at least half of the total sniffing trials as odour-active. The odour zones and their corresponding identification are listed in Table 1, some of them were only tentatively identified (TI). Thus, GC-O analyses evidenced 79, 55 and 49 odour-active zones in substrate, F8VI and F8VE vinegars respectively. In the substrate, the odour zones that presented maxima MF (100) were those which were identified as methyl butyrate (LRI 990, TI), isoamyl alcohol (LRI 1208), cis-2nonenal (LRI 1504, TI), γ-decalactone (LRI 2150) , γ-dodecalactone (LRI 2382) and vanillin (LRI 2564, TI). Some have been previously reported as important components in strawberry aroma. Thus, methyl butyrate is one of the major esters present in this fruit[39,40]. According to Gomes da Silva and Chaves das Neves[41], prominent lactones were γ-decalactone and γ–dodecalactone, giving fruity notes. Cis-2-nonenal has been described as a frequent odorant present in different strawberry varieties[42]. On the other 8 hand, the presence of vanillin among the aroma compounds in strawberry has been scarcely observed by other authors[43,44] and until now it had been described as an odour-active compound in one variety only [45]. As expected, furaneol (LRI 2034) and its methoxy derivative[46], mesifurane (LRI 1594), the strawberry components most quoted as character impact compounds, also reached high MF; 91 and 75, respectively. Within those odour zones which reached high MF, we can highlight those that we have tentatively identified for the first time in strawberry such as 2,6-dimethylpyrazine (LRI 1305), 2-furfurylthiol (LRI 1433), guaiacol (LRI 1881), 6,7-dyhydro-7-hydroxylinalool (LRI 1971), 4-ethylphenol (LRI 2183), sotolon (LRI 2203) and ethyl hexadecanoate (LRI 2270). The first two odorants and sotolon are typical compounds produced by the Maillard reaction[47-49]. The presence of these compounds may be due to the same formation pathway, since strawberry has reducing sugars as well as amino acids. Moreover, sniffers also perceived some off-odour attributes tentatively identified as volatile phenols with high MF: guaiacol and 4-ethylphenol. Several authors point to these compounds as off-flavours in different food matrices[50,51]. Moreover, they are related to fruit contamination caused by different microorganisms[52,53]. In addition to the above-mentioned compounds, we found other important odour zones with MF higher than 70, providing fruity notes, corresponding to typical strawberry esters (ethyl butyrate (LRI 1029) and ethyl hexanoate (LRI 1234, TI)). Furthermore, we identified linalyl valerate/citronellol (LRI 1766, TI) and nerolidol (LRI 2003) around this MF value, giving grassy and citric aroma characters. Among the 38 odour zones with a significant contribution to the aroma (MF>70), we were, even tentatively, unable to identify four of them. 9 The comparison of olfactometric analysis results of substrate and vinegars showed that a total of 29 odorants present in the strawberry puree were detected both in vinegars produced from wines whose substrate had been inoculated and those which had been obtained from spontaneous fermentation. These odorants were, therefore, preserved throughout the double fermentation process. Most of them, however, presented lower MF values in vinegar than in the substrate due to the losses and transformations undergone during the alcoholic and acetous fermentations[54,55]. The aromatic profile of wine vinegars is the result of the permanence of those volatile compounds from the raw material (grape varietals aroma), synthesized by yeast during alcoholic fermentation and those formed during the acetous fermentation. The results showed that after the double fermentation, some characteristic compounds of strawberry[27,56] such as: γ-decalactone, furaneol, mesifurane, β-damascenone (LRI 1821) and ethyl 2-methylbutyrate (LRI 1040) were preserved from the raw material to both vinegars. Surprisingly, moreover, they reached high MF values. On the other hand, the impact odorants of strawberry, ethyl butyrate and γdodecalactone, were only perceived in vinegar produced from wine obtained by inoculation of the substrate while methyl butyrate was perceived only in vinegar from wine obtained by spontaneous fermentation. These results indicate that the overall odour impression of vinegar would evoke aromatic notes of the raw strawberry material used. Moreover, we observed the appearance of odour zones, or an increase in the MF scores of other odour zones corresponding to compounds usually formed in acetous fermentation[9,57]. Therefore, aromatic notes such as butter (diacetyl, LRI 970) and pungent (acetic acid, LRI 1405), appeared in the vinegars. The cheese odour, identified as isovaleric acid (LRI 1669), increased its MF (53 in the substrate), reaching the 16 [31] Y. Wang, C. Finn, M. C. Qian, J. Agric. Food Chem. 2005, 53, 3563. [32] S. Deterre, B. Rega, J. Delarue, M. Decloux, M. Lebrun, P. Giampaoli, Flavour Fragr. 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Data scores (MF) (a) and variable loadings (b) plots on the planes made up of the first two principal components (PC1 against PC2). The compound names corresponding to the number of variables are located in Table 1. Figure 2. Contribution of each aroma category as a percentage of the total MF of odour zones that might be potential impact odorants in at least one sample. 19 Table 1. Odour active compounds in strawberry substrate and obtained vinegars. LRI DB-Wax LRI CP-Wax or/and literature Odour Descriptor Odorantd Fa FMb Fa FMb Fa FMb Identificationc Substrate Inoculated Spontaneous 934 - strawberry acid unknown - - - - 3 41 - 963 961e plastic, synthetic ethyl propionate - - 3 41 3 41 MS, OD 970 969[9];970[23,24] butter diacetyl - - 4 67 4 67 LRI, OD 990 990[21] strawberry methyl butyrate1 6 100 - - 6 100 LRI, OD 1012 1012e; 1014[25] medicinal isobutyl acetate 4 47 - - - - LRI, MS, OD 1020 1022[21] fruit, strawberry, pineapple methyl isovalerate2 6 71 - - - - LRI, OD 1029 1029e;1032[24] banana, strawberry, soap ethyl butyrate3 5 75 5 53 - - LRI, MS, OD 1040 1040e,1041[26] banana, fruit ethyl 2-methylbutyrate4 6 82 6 58 6 82 LRI, MS, OD 1055 1055e,1056[26] fruit, strawberry, banana ethyl isovalerate 4 67 3 41 3 41 LRI, MS, OD 1066 1067[22];1069[27] metallic, rubber, sweat hexanal5 5 75 - - - - LRI 1069 - toasted bread, coffee unknown6 5 75 - - - - - 1083 1083e,1084[9,28] rubber, sweat, latex isobutanol 4 67 - - - - LRI, MS, OD 1105 1105e banana isoamyl acetate 3 41 - - - - MS, OD 1144 1146e,1145[21] /1145[21] boiled potato, vegetable, grass 1-butanol/myrcene7 6 82 3 58 3 41 LRI, MS, OD/ LRI, OD 1166 1169[29] green, plastic, rubber, limonene 3 41 - - - - LRI, OD 1183 1181[30]/1185[25] sweet, medicinal, aspirin methyl hexanoate/ethyl 3methylpentanoate 4 47 - - - - LRI, OD 1208 1205,1208[21]; 1212e rancid, rubber, chemical isoamyl alcohols(2-methyl-1butanol+3-methyl-1-butanol)8 6 100 4 47 6 58 LRI, MS, OD 1220 1220[21] plastic 2-hexenal 4 67 - - - - LRI, OD 1224 - chicken, cooked, synthetic, vegetable unknown - - 3 41 - - - 20 1234 1230e,1240[30] strawberry, blackberry, citrus, violet ethyl hexanoate9 5 75 - - - - LRI, MS, OD 1247 - vegetable, plastic, flower unknown 3 41 - - - - - 1264 1269[31] boiled potato, boiled vegetable α-terpinolene 4 58 - - - - LRI,OD 1289 - metallic, paint unknown - - - - 3 41 - 1302 1303[25];1306[24] plastic, cooked vegetable, metallic 1-octen-3-one 5 53 - - - - LRI, OD 1305 1308[21] toasted corn, boiled potato 1-octen-3-one+2,6dimethylpyrazine10 5 75 4 47 5 53 LRI, OD/ LRI, OD 1317 1315[25] barbecue 2-methyl-3-furanthiol - - 3 58 5 53 LRI, OD 1327 1331[33] toasted, chicken soup, dairy product, plastic 2-acetyl-1-pyrrolidone - - - - 6 58 LRI, OD 1347 1345[21]/1345[21] vegetable, grass 2-octenal/3-nonenal 3 41 - - - - LRI, OD 1350 1350[28]; 1355[34]; 1357e plastic 1-hexanol 3 58 - - - - LRI, MS, OD 1373 1368[24];1377[34]/ 1377[21] green leaf , fish dimethyl trisulfide/trans-2-hexenol11 6 71 - - - - LRI, OD/LRI, OD 1391 - baked potato, vegetable unknown 3 58 - - - - - 1396 1394[25, 30]; 1396e baked potato, mushroom cis-3-hexenol12 5 75 3 41 3 41 LRI, MS, OD 1405 1405e pungent acetic acid13 - - 6 82 6 82 MS, OD 1419 1424[25] anise, sweet, plastic doll ethyl cyclohexanoate 5 53 - - - - LRI, OD 1433 1436[25] toast, coffee 2-furfurylthiol14 6 82 - - - - LRI, OD 1435 1432e;1436[21] fruit, strawberry, lemon ethyl octanoate 3 58 3 41 - - LRI, MS, OD 1450 1449[35]/1452[25] baked potato methional15 - - 6 100 4 82 LRI, OD 1463 1568 strawberry, banana, vanilla, sweet benzaldehyde - - 3 41 - - MS, OD 1500 - toast, burned, hot iron unknown 3 41 - - - - - 1502 - vegetable, baked potato, orange unknown 3 58 - - - - - 1504 1502[35] toasted, vegetable, river water cis-2-nonenal16 6 100 4 47 3 41 LRI, OD 1508 1511[36] river water, vapour 3-nonen-2-one17 4 67 5 91 5 91 LRI, OD 1535 1534e plastic, rancid 2-(methylthio)ethanol 3 58 - - - - MS, OD 1560 1560[30] grass, boiled green beans, flower linalool 4 67 - - - - - 1563 1563[21]; 1565e cheese, vomit isobutyric acid - - - - 4 58 LRI, MS,OD 21 1581 1575[21] violet, flower, vegetable, soap E,Z-2,6-nonadienal18 6 82 4 75 3 71 LRI, OD 1594 1584[22]; 1592e caramel, sweet, cotton candy mesifurane19 5 75 3 41 6 71 LRI, MS, OD 1611 - river water, rancid unknown 3 58 - - - - - 1616 - cut grass, river water unknown 3 50 - - - - - 1619 1621[25] toasted, peanut, rancid 2-acetylpyrazine 3 58 - - - - LRI, OD 1623 1623e; 1632[35] cheese, vomit butyric acid 6 82 6 82 6 82 LRI, MS, OD 1648 1645[21];1647e river water, fruit compote, sweet, plastic acetophenone 3 41 - - 3 41 LRI, MS, OD 1669 1665[21] cheese isovaleric acid20 5 53 6 100 6 100 LRI, OD 1692 1692e fruit, flower, plastic, vapour γ-hexalactone 3 41 - - - - MS, OD 1700 1702e; 1711[22] cut grass, soap, plastic, flower α-terpineol 4 67 4 47 - - LRI, MS, OD 1715 1718e;1706[37] plastic doll, anise benzyl acetate21 5 75 - - - - LRI, MS, OD 1721 1720[21] mint, lemon, vegetable carvone 5 65 5 53 3 58 LRI, OD 1731 1731e;1738[38] plastic, river water methionol 3 58 4 67 - - LRI, MS, OD 1740 - plastic, bitumen, pneumatic unknown 4 67 - - 4 58 - 1766 1765[21]/1762[21]; 1769[34] rancid, flower, citric, fresh linalyl valerate/citronellol22 5 75 - - 3 41 LRI, OD/ LRI, OD 1785 1784[21] mint, plastic ethyl salicylate 3 58 - - - - LRI, OD 1810 - plastic, peanut, barbecue unknown23 3 58 5 75 4 67 - 1821 1818[25] fruit preserve, quince compote, roast apple -damascenone24 6 82 5 75 3 58 LRI, OD 1824 1823e;1829[21] mint, flower, jasmine 2-phenylethanol acetate - - 4 58 - - LRI, MS, OD 1836 licorice, curry, spicy unknown25 - - 6 82 - - - 1844 1838e;1842[21] plastic, sweat, dung, rancid ethyl dodecanoate26 5 75 - - - - LRI, MS, OD 1861 1864[25] river water, olive, clove, barbecue guaiacol27 6 82 4 67 4 67 LRI, OD 1877 1880e boiled potato, metallic, mint, violet benzyl alcohol 4 58 - - 4 58 MS, OD 1915 1916[25] rose, hyacinth 2-phenylethanol28 5 53 6 71 5 75 LRI, MS, OD 1919 1920e coconut, sweet hydroxycinnamyl acetate - - 3 41 - - MS, OD 1926 - cut grass, lima beans, gasoline unknown 29 5 75 - - - - - 1941 - chamomile, urine unknown - - 4 67 - - - 1957 - lemon, baked potato unknown 3 41 - - - - - 22 1967 1967[34] ripe fruit, quince compote, roast apple δ-octalactone - - 4 47 - - LRI, OD 1971 1972e lemon, washing powder, green beans, grass 6,7-dihydro-7-hydroxylinalool30 6 82 - - - - MS 1988 - caramel, metallic, sweet, honey unknown 3 41 - - 4 58 - 2003 2009[21]/2006[27]; 2010[22] soap, vegetable trans-nerolidol/cis-nerolidol31 5 75 3 41 - - LRI, MS, OD 2034 2034e;2033[21]/ 2033[33] cotton candy, caramel, quince pantolactone+furaneol32 6 91 6 100 5 75 LRI, MS, OD 2054 - sweat, grass, stagnant water unknown 33 5 75 - - - - - 2064 - green beans, baked potato, rancid oil unknown34 5 75 - - - - - 2085 2084[35]/2091[25] tempera, burned plastic p/m-cresol 35 - - 5 75 - - LRI, OD 2150 2157e;2165[38] fruit, blackberry, peach γ-decalactone36 6 100 6 100 4 67 LRI, MS, OD 2155 - mentholated unknown - - 3 58 2 47 - 2167 2170e; 2159[28]; 2176[25] clove, sponge cake eugenol37 6 82 3 41 - - LRI, MS, OD 2183 2182[35]; 2185[25] tempera, cucumber 4-ethylphenol38 4 75 5 75 3 58 LRI, OD 2196 2198[21] coconut, clove, toasted p-vinylguaiacol39 4 75 6 82 5 91 LRI, OD 2203 2203[22]; 2204[25] licorice, curry sotolon40 3 71 6 82 4 82 LRI, OD 2219 2216[22] liquor δ-decalactone41 - - - - 3 71 LRI, MS, OD 2230 2223[21]/2234[38] grass, banana, fruit, honey o-aminoacetophenone - - 3 41 3 58 LRI, OD 2241 2247[21] spice, coconut, flower, roast vegetable abhexone42 4 67 5 75 3 58 LRI, OD 2270 2269e tempera, barbecue, burned plastic ethyl hexadecanoate43 4 75 5 75 - - MS, OD 2319 - mint, lemon, toothpaste, fruit unknown 4 47 3 58 3 58 - 2382 2384[21]; 2385e apple, apricot, strawberry, sweet, milk γ –dodecalactone 44 6 100 4 58 - - LRI,MS, OD 2396 - banana, flower, clove unknown 4 47 - - 3 58 - 2437 2426[21] river water, clove, spicy, barbecue δ –dodecalactone45 4 82 6 58 - - LRI, OD 2455 2452e sweet, cinnamon coumaran - - 4 58 - - MS, OD 2490 - plastic, bleach, rancid unknown 4 67 - - - - - 2545 - sweet, coconut unknown - - 4 67 - - - 2556 2251[21];2553e[37] rose, honey phenylacetic acid46 4 75 6 100 5 91 LRI, OD 2564 2569[22] vanilla, caramel vanillin47 6 100 5 75 4 75 LRI, OD 23 2600 2598[21] honey, vanilla methyl vanillate - - 3 58 - - LRI, OD 2636 - Compote, banana, strawberry unknown 3 41 - - - - - 2644 2640[21] honey acetovanillone - - 3 71 - - LRI, OD aF: Frequency of occurrence bMF: Modified frequency. cLRI: Indentified by Lineal Retention Index; MS: Identified by matching mass spectra of GC-MS analysis of extract with those from the NIST 98 library; OD: Odour descriptor. dCorresponding number of this compound in Figure 1. eLRI in column CP-Wax 57CB of compound identified by GC-MS. 24 Figure 1a Substrate Inoculated Spontaneous -6 -4 -2 0 2 4 6 8 Factor 1: 73,42% -5 -4 -3 -2 -1 0 1 2 3 4 5 Factor 2: 26,58% 25 Figure 1b 1, 4 3 8,10 15 18 19 13,17,20,40 22 23 28 31 32 35 37 39 41 42 43 46 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 Factor 1 : 73,42% -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 Factor 2 : 26,58% 36 27,29,30,33,34,47 2,5,6,9,11,12,14,21,26 16 45 24 45 7 38 25