)- H ´ atica, ∗ ober 2013 a 20 ıcola, Laboratorio ´ -decalactone is also ´ on Agr ´ γ omicas, Departamento de )-furanone (DMHF), to ˜ na Mackenna 4860, Macul, H The Additionally, even when However, to our knowledge, 19 ´ omica Funcional & Bioinform 16 – 2, we determined 20 major – 2013 Society of Chemical Industry 3,21 ≥ Duch. In order to identify the 12 12 c ´ atica. 8820808, La Pintana, Santiago Chile. and Herman Silva ´ olica de Chile, Casilla 306 – Correo 22, Santiago, . The main compounds identified include ) b,c ; gas chromatography–olfactometry ıcola, Laboratorio de Gen ´ ´ omicas, Departamento de Producci Fragaria x ananassa F. chiloensis ´ on Agr ´ omica Funcional & Bioinform 1000) and GC-O intensity Centro de Aromas y Sabores, DICTUC, Avda. Vicu E-mail: [email protected] Universidad de Chile, Facultad de Ciencias Agron Correspondence to:Ciencias Herman Agron Silva,de Gen Universidad de Chile, Facultad de Producci 8820808, La Pintana, Santiago, Chile Santiago, Chile Department of Chemical and Bioprocess Engineering,Pontificia College Universidad of Engineering, Cat Chile = Fragaria chiloensis Only two reports have identified the profile of volatile com- c ∗ a b pounds in esters, some alcohols and ketones. and the furanones,furanone) and 2,5-dimethyl-4-hydroxy-3(2 mesifuran (2,5-dimethyl-4-methoxy-3(2 the fruity and caramel aromas. important since it contributes to the fresh fruity aroma. the acids have small(fruity odorant impact, and the buttery 2-methylbutanoic acid aroma)aroma) contributed and to the the aroma. hexanoic acid (unpleasant 1 form chiloensis, is a strawberry that produces white fruits with Eduardo Agosin spp. ,the F. x b Mill.) and (L.) Mill.]. hiloensis The terpene F. chiloensis 11 F. chiloensis – 9 Identification of these 4 )-hex-2-enyl acetates are 3 Fragaria chiloensis E ´ (L.) Mill spp. c es Espinoza, and a good aroma derived Fragaria chiloensis Fragaria virginiana Research during the last decades ıa In ´ 2 F. virginiana Mar Duch.) is derived from a cross between : 752–759 www.soci.org a Among them, the esters methyl butanoate, 94 8 Fragaria chiloensis – 5 2014; solvent-assisted flavor evaporation (SAFE); volatile profiling; form chiloensis) is limited. Duch.

2013 Society of Chemical Industry More than 360 volatiles have been identified in Fragaria x ananassa c red color derived from from both parental species. has been dedicated topresent the in identification cultivated of red volatileof strawberry, compounds these whereas compounds the in identification the white strawberry ( chiloensis J Sci Food Agric The resulting hybrid has large fruits derived from the North American strawberry ( INTRODUCTION Strawberry, a membermost of important fruits the in the( Rosaceae world. The family, cultivated red is strawberry one of the Abstract BACKGROUND: strawberries ( Loreto Prat, unique aromas. This species, endemic to Chile, is one of the progenitors of associated with the aroma of white Identification of volatile compounds (wileyonlinelibrary.com) DOI 10.1002/jsfa.6412 Research Article Received: 11 December 2012 Revised: 12 September 2013 Accepted article published: 20 September 2013 Published online in Wiley Online Library: 21 Oct volatile compounds in white strawberry shouldconsumer reveal traits, since interesting white strawberries have a veryand characteristic pleasant aroma which leads to consumer preferencestrawberries for when white compared to red ones. considered to becontribute to important the flavor fruity active and components green that aroma. ethyl butanoate, methylmethylbutanoate, hexanoate, hexyl ethyl acetate and hexanoate, ( ethyl 2- ananassa linalool has been linked to the flowery and sweet aroma volatile compounds that might bespectrometry (GC-MS), responsible gas for chromatography–olfactometry aroma, (GC-O) these and were compared extracted, with sensory and analyses. analyzedRESULTS: Three by methods of gas extraction chromatography–mass were used:(HS-SPME) solvent-assisted and evaporation (SAFE), liquid–liquid headspace solid extraction phaseodor micro-extraction (LLE). Ninety-nine activity volatile using compounds GC-O. were Based identified by on GC-MS, the of highest which dilution 75 showed factor (FD the endemic Chilean strawberry [ Keywords: compounds in white strawberry fruit that contribute tostandards. its The aroma. identities We were chose confirmed 51 compounds by to comparisonaroma be of of tested their against white linear their strawberry commercial retention fruits indices was against reconstituted the with commercial a standards. The synthetic mixtureCONCLUSION: of The most of volatile these profile compounds. ofwoody white will be strawberry a fruit useful described reference for as future fruity, strawberry green–fresh, breeding programs. floral, caramel, sweet, nutty and Supporting information may be found in the online version of this article.

752 753 . 5 1 − − ,pH1.1)were 1 until it reached − 1 − C under vacuum (10 ◦ m DB-WAXETR capillary L under a stream of µ µ Cmin ◦ wileyonlinelibrary.com/jsfa 0.25 × C, for 20 min. After centrifugation ◦ sodium bicarbonate to separate the 1 C and column oven temperature was − ◦ 0.25 mm C, respectively. Volatiles were tentatively C, then raised 3 × ◦ ◦ C in a water bath for 15 min and extracted ◦ ) and extracted with diethyl ether (four times, 50 m DVB/Carboxen/PDMS StableFlex fiber (Supelco, 1 µ − L) from the acid and neutral–basic fractions of SAFE, . The MS quadrupole and MS source temperatures z µ C and 220 / ◦ The aqueous distillate was extracted with diethyl ether NaOH. Volatiles were isolated using the solvent-assisted solution (10 mL) plus sodium chloride (3 g) was placed in m 1 24 − L under a nitrogen stream. C) to desorb the analytes. C where it was held for 25 min. Mass spectra was obtained ◦ µ ◦ For the liquid–liquid extraction, the same method described for (four times, 50 mLwith each aqueous time). 0.5 The mol solvent L extract was treated Solvent-assisted evaporation and liquid–liquid extractions For all extraction methods,were fresh sliced white strawberry and fruitschloride blended solution. (250 Two with g) hundred and 250 fiftystandard microliters mL consisting of an in of internal 4-nonanol a (3.526 saturated mg mL calcium and separation, the organic extracts were combined and dried over anhydrous sodium sulfate. Then, the sample350 was concentrated to Headspace solid phase micro-extraction analysis A 2 cm 50/30 Inc., Bellefonte, PA,Fragaria USA) was useda 20 for mL vial tightly aroma cappednumber with extraction. a S126-0020; Teflon/silicone The septum I-CHEM, (catalog equilibrated Tennessee, at USA). 40 The sample was identified by comparing Kovats indices and the spectrometricfrom data NIST-EPA-NIH libraries (http://www.nist.gov/srd/nist1a.htm) that have more than 130,000 entries.be We tested chose against 51 their compounds commercial to standards.confirmed The by identities comparison were of their linearthe commercial retention standards indices (Supplementary against Table 1). by electron impact ionization40–450 (70 eV) scanning a mass range of mbar). flavor evaporation technique (SAFE), at 35 under stirring for 1the h at SPME the fiber same(250 was temperature. After inserted extraction, into the injection port of the GC mL each time) toThe isolate solutions containing the either acidicfraction the compounds acidic were (acid or fraction). nitrogen. concentrated the neutral–basic to 200 SAFE and LLE was used. Thediethyl obtained solution ether was (three extracted times with withatmosphere 50 with agitation mL at each 4 time) under nitrogen held for 5 min at 40 Analysis of volatile compounds by gas chromatography–mass spectrometry The analyses were carried outcoupled on to a a HP 5972A 6890 MSD Gas Hewlettequipped Packard Chromatograph, with mass a spectrometer 60 and m column (J&W Scientific, California, USA). The original concentrated samples (2 as well as fromsplitless the mode liquid–liquid using extractions a were constant injected helium in flow a of 1.9 mL min added to this solution. Then, the pH of the suspensionto was adjusted 3.6, the pH of themol natural L white strawberry mature fruit, with 2 acidic volatiles fromaqueous the neutral–basic solutions were fraction.acid adjusted The (2 mol to combined L pH 2.0 with hydrochloric The HS-SPME sample was injectedinjector in the was specific set SPME port. at The 180 240 were 150 22 Fragaria 2013 Society of Chemical Industry c Caramel, strawberry like Fruity, caramel Caramel Sweet Sweet, floral Honey . In this study, we tenta- W) at 605 m above sea level.  2.96  F. chiloensis 14 ◦ : 752–759 94 S, 73 -butyl)-4-hydroxy-4- )-furanone )-furanone t  ). Fruits from several plants were pooled. After H H -2-Hexenal Sweet 23 8.6 2014; 2) (DMHF) 3(2 diol (mesifuran) methyl-2,5- cyclohexadien-1-one dien-1,6-diol dien-1,6-diol 3(2 )-2,6-Dimethylocta-2,7- )-2,6-Dimethylocta-2,7-  -Decalactone Floral, peach E Z > Odorants detected by gas chromatography–olfactometry γ trans I 04 et al. . These results provide information that may be used in ◦ 20332133 Hydrocinnamyl alcohol21402154 Cinnamyl acetate 2,6-Di( Floral, citric Floral, sweet 16311863 Ethyl decanoate1804 Benzyl alcohol2021 2-Phenylethyl acetate 4-Hydroxy-2,5-dimethyl- Fruity, caramel Sweet, tea, floral Floral 2189 3,7-Dimethyloct-1-en-3,8- 2252 ( 2288 ( 2952 Hexadecanoic acid Chemical, lactic 12571294 Ethyl hexanoate1358 Hexyl acetate1541 2-Hexen-1-ol acetate1567 Linalool1589 Furfuryl acetate 2,5-Dimethyl-4-methoxy- Tropical fruits Fruity, strawberry like Tropical fruits, banana Tropical fruits Floral 10461224 Ethyl butanoate Sweet, sugary Table 1. (GC-O) at the highestintensity ( dilution factors (FD 500 and 1000) and GC-O RI Aroma compound Descriptor J Sci Food Agric Plants were grown under standardextraction horticultural was practices. performed Aroma on ripeFigueroa stage fruits (stage 4 according EXPERIMENTAL Strawberry fruits White strawberry fruits wereyear-old harvested plants in grown January 2011, inChile from the (38 5- fields of Contulmo in the south of tively identified the volatile compoundsthe that might aroma contribute of to freshextraction methods: white solvent-assisted flavor strawberry evaporation technique fruits(SAFE), using three headspace different solid-phaseliquid–liquid microextraction extraction (LLE). (HS-SPME) Then,volatile and we compounds proposed several that potent are involved in the aroma of there are no reports of gasof chromatography–olfactometry (GC-O) volatile compounds in chiloensis selectionbreedingprogramstoimprovethearomaofstrawberries. Aroma of white strawberries www.soci.org Chemicals Fifty-one aroma standardsobtained were from used the following in companies:Aldrich this Fluka and Hangzhou Analytical, study. Imaginechem Sigma- Co. They (Supplementary Table 1). were harvesting, the fruits werewhere transported they were on immediately processed. ice to the laboratory b 1 3 1 2 2 2 1 1 1 2 2 3 1 2 1 1 1 1 1 2 1 1 3 et al. 0.5 Int. a : 752–759 1000 + + + + + + + + + + + + + + + + + + + + + + 94 + + = Aroma 2014; b 1 3 1 2 2 2 1 1 1 2 2 3ND0 1 3 3 2 2ND0 1 2 1 2 2 1 1 2ND0 3 1.5 Int. a 500 FD + + + + + + + + + + + + + + + + + + + + + + + + + + + = ND 0 ND 0 J Sci Food Agric Aroma b c 1 3 1 2 2 2 2 1 1 2 1ND0ND0 2 3 1 3 3 2 2 2 1ND0ND0 1 2 1 1 2 3 0.5 ND 0 ND 0 1.5 ND 0 ND 0 0.5 ND 0 ND 0 1.5 1.5 1.5 Int. a 1FD + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + = FD GC-O Aroma § Descriptor www.soci.org L Prat 2013 Society of Chemical Industry ‡ c Extraction methods SPME, LLE Caramel, sweet, fruity SPME,LLE Fruity, woody SPME, LLE — ND 0 ND 0 ND 0 SAFE A, SAFE B, LLE Fruity SAFE A Sweet, berries SAFE A, SAFE B, SPME, LLE — ND 0 ND 0 ND 0 SPME, LLE Sweet, tea, floral SPME, LLE Sweet SPME — ND 0 ND 0 ND 0 SPMELLE — — ND 0 ND ND 0 0 ND ND 0 0 ND 0 SAFE A, SPME, LLE Floral, citric † berries -hydroxy -hydroxy p p ) Ethyl- ) Ethyl- Z Z methylbutanoate methylbutanoate acetate hydroxybutanoate hydroxybutanoate hydroxyhexanoate acetate acetate cinnamate 2-ol cinnamate alcohol o o E E Esters Alcohols Identification of compounds by gas chromatography–mass spectrometry (GC-MS) and detected by gas chromatography–olfactometry Fragaria chiloensis 993 Methyl butanoate SPME, LLE Sweet, fruity 841 Ethyl acetate SAFE A, SAFE B, SPME, LLE — ND 0 997 Isobutyl acetate LLE — ND 0 ND 0 ND 0 RI on DB WaxETR* Aroma compound Table 2. (GC-O) in 1010 Methyl 2- 1046 Ethyl butanoate SPME, LLE Nutty 1035 Ethyl 2- 109810961206 Butyl acetate1257 2-Methylbutanoate Methyl hexanoate SPME, LLE Ethyl hexanoate SPME, LLE SPME,LLE SAFE A, SPME, LLE — Tropical Sweet, fruits fruity — ND ND 0 0 ND ND 0 0 ND ND 0 0 1294 Hexyl acetate SPME, LLE Tropical fruits, banana 1276 3-Methyl-2-butenyl 1358 2-Hexen-1-ol acetate SPME Fruity, strawberry jam 1384 Methyl octanoate LLE Caramel, soy sauce 1430 Ethyl octanoate SPME, LLE Fruity, sweet 1512 Ethyl 3- 1490 Methyl 3- 1567 Furfuryl acetate SPME Tropical fruit 1631 Ethyl decanoate SPME LLE Fruity, caramel 1741 Benzyl acetate SAFE A, LLE Lactic alcohol, sweet 1668 Ethyl 3- 17821804 Methyl dodecanoate 2-Phenylethyl SAFE B, SPME, LLE — ND 0 ND 0 ND 0 18811930 Ethyl dodecanoate Hydrocinnamyl SAFE B Sweet 2010 Butyl laurate LLE Strawberry jam 2036 Ethyl tetradecanoate LLE Strawberry jam, tropical fruit 2140 Cinnamyl acetate SAFE B, SPME, LLE Floral, sweet 2302 Ethyl hexadecanoate SAFE B Caramel 2618 Benzyl benzoate LLE chemical 26462733 Ethyl octadecanoate ( LLE — ND 0 ND 0 ND 0 2962 ( 1030 2-Methyl-3-buten 1094 Isobutyl alcohol SAFE A, LLE Pungent 1136 1-Butanol SAFE A, SAFE B, LLE Fruity 1197 3-Methyl-1-butanol LLE Berries, candy 1344 1-Hexanol SPME, LLE Fresh, green 13761513 3-Hexen-1-ol 2-Ethyl-1-hexanol SPME, LLE LLE Green, can — ND 0 ND 0 ND 0 15681863 2,3-Butanediol Benzyl alcohol SAFE A, LLE SAFE A, SAFE B SPME, LLE Floral — ND 0 ND 0 ND 0 1941 2-Phenyl ethanol SPME Tea floral 2005 1-Dodecanol SAFE B, SPME Berries jam 2033 Hydrocinnamyl wileyonlinelibrary.com/jsfa

754 755 b 1 1 1 2 1 1 1 1 1 1 1 1 1 2 3 1 3 2 3 2 2 0.5 Int. a 1000 + + + + + + + + + + + + + + + + + + + + + + = Aroma b 1 2 1 2 2ND0 1 1 1 1 2ND0 1 1 1ND0 1 1 1 1ND0 2 1 3 2 3 2 1ND0 2 0.5 ND 0 1.5 ND 0 2.5 0.5 2.5 ND 0 Int. a 500 FD + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + = wileyonlinelibrary.com/jsfa Aroma b 1 2 1 2 1 1 1 1ND0ND0 2 1 1 2 2ND0ND0 1ND0ND0 1 2ND0ND0 3 3 1 3 3 2 1 2 0.5 ND 0 ND 0 1.5 0.5 1.5 1.5 2.5 ND 0 ND 0 1.5 2.5 1.5 ND 0 ND 0 1.5 1.5 2.5 2.5 Int. a 1FD + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + = FD GC-O Aroma § Descriptor ‡ 2013 Society of Chemical Industry c Extraction methods SPME Caramel SAFE A, LLE Caramel, strawberry like SAFE A, SAFE B, SPME, LLE Fruity, caramel LLE Sweet † )-furanone SPME Caramel, sweet : 752–759 H 94 -butyl)-4-hydroxy-4- )-furanone )- t H H -2-Hexenal SAFE A, SPME , LLE Sweet )-Cinnamic acid LLE — ND 0 ND 0 ND 0 )-Cinnamic acid LLE Fruity, floral Z Z 2014; methyl-2,5- cyclohexadien-1-one (DMHF) terpenes 3(2 furanone(mesifuran) diol 3(2 o o -Decalactone-Dodecalactone LLE SAFE B, LLE Berries, sweet, tea Floral, peach -Hexalactone SAFE A, SAFE B, SPME, LLE Fruity, sweet -Terpineol-Muurolene SPME, LLE SAFE B, LLE — Nuts ND 0 ND 0 ND 0 -Farnesene SAFE B, LLE Green -Hexalactone LLE — ND 0 ND 0 ND 0 E E Aldehydes and ketones trans Acids α α γ γ α δ γ Nonisoprenoids and Lactones and furanones Continued 953 2-Pentanone LLE Caramel, sweet 970 3-Methyl-3-buten-2-one LLE — ND 0 ND 0 ND 0 924 2-Ethylfuran SPME Fruity 2267 Cinnamic alcohol SAFE B, SPME, LLE Floral, sweet RI on DB WaxETR* Aroma compound Table 2. 2232 1-Hexadecanol SPME, LLE Spicy 1095 Hexanal SPME, LLE Asparagus, green 2564 1-Heptadecanol SAFE B, SPME Fruity 10971155 2-Methyl-2-butenal 2-Methylpentenal SAFE A SAFE A, SAFE B, LLE Green — ND 0 ND 0 ND 0 12001179 2-Heptanone Heptanal SPME SAFE B, LLE — Humidity ND 0 ND 0 ND 0 1224 1288 Acetoin SAFE A, SAFE B, LLE Lactic 1389 Nonanal SAFE A, SAFE B, LLE Green olive 1402 2,4-Hexadienal SPME Green tea 14931526 Furfural Benzaldehyde SAFE A, SPME, LLE SPME Caramel, milk-like — ND 0 ND 0 ND 0 1443 Acetic acid SAFE A, SAFE B, SPME, LLE Vinegar 16442154 Acetophenone 2,6-Di( 2545 Benzophenone SPME, LLE15331561 Propanoic acid SAFE Isobutyric B acid — SAFE A, LLE SAFE A Berries jam Smoky, can ND Milk like, cheesy 0 ND 0 ND 0 2548 Vanilline16211674 Butanoic acid 2-Methylbutanoic acid LLE SAFE A, LLE SAFE A, SPME, LLE Lactic Vinegar, yeast Sweet 1724 Valeric acid SAFE A, SPME, LLE Yeast, mushroom 18492191 Hexanoic2276 acid Nonanoic acid 3-Methylthiopropanoic acid LLE SAFE A, LLE LLE Spicy, Rusty, pepper can, oxide — ND 0 ND 0 ND 0 27352749 Tetradecanoic acid ( LLE Lactic 1720 1712 12801541 Styrene Linalool SPME, LLE SPME, LLE — Floral ND 0 ND 0 ND 0 2133 2362 2840 ( 2022 4-Hydroxy-2,5-dimethyl- 1737 2952 Hexadecanoic acid SAFE A Chemical, lactic 1778 1690 2189 3,7-Dimethyloct-1-en-3,8- 1589 2,5-Dimethyl-4-methoxy- 1627 5-Ethyl-2(5 J Sci Food Agric Aroma of white strawberries www.soci.org b 3 2 3 1 1 3 1 1 1 2 et al. 0.5 0.5 1.5 2.5 Int. a : 752–759 1000 94 + + + + + + + + + + + + + + = 1000) and GC- Aroma = 2014; fresh weight. The b 3 2 2 3 2 2 1ND0 1 1 1 2 2 1 2.5 0.5 1.5 Int. − a 500 FD gkg µ + + + + + + + + + + + + + + + = Aroma J Sci Food Agric b 2 3 3 3 1 3 1 1 1 2 2 2.5 1.5 1.5 2.5 Int. a 1FD + + + + + + + + + + + + + + + = FD 2, were positively identified by comparing mass ≥ GC-O Aroma § strongly perceivable. = Preliminary aroma reconstitution experiment The odorants with the highest dilution factorOintensity (FD spectra, aroma andwere RIs calculated with accordingThe panelists commercial to attended standards. theirgenerated a the The Kovats specific descriptive training retention RIs strawberry terms fruit session, aroma. indices. Different required where aroma standards to they wereto presented the define panel the anddescriptors white discussed. From (apple, these discussions,cherimoya, green, nine banana, aroma pear lactic, anddescriptive caramel) pineapple, were analysis. selected citric–grapefruit, The forare aroma further listed in reference Table standards 1.to These employed reconstitute standards, when the available, modelwas were used solutions. carried The out relative bydifferent replicas quantification comparing for each the extraction method areaswith an and internal of then standard compared (4-nonanol) the in the peaks GC-MSor for of SAFE, LLE. SPME three The results are expressed in standards were added tolevels previously 1 determined in L the fresh of3.6, juice the and Milli-Q natural adjusted pH to water of pH the in whitemodel strawberry concentration fruit. solution The reconstituted wasrepeated carried twice. out The solutions asscrew were caps duo-trio and placed then tests, were in presentedsensory glass which to the vessels panel were eight with for members of orthonasal the model evaluation. solutions The were best-reconstituted comparedfruits, in to order to the determine if whitedifferences. there were strawberry any detectable fresh aromatic Descriptor ‡ 1 − www.soci.org L Prat perceivable, 3 = Lofthe Cmin ◦ µ 2013 Society of Chemical Industry c Extraction methods LLE Sweet, floral SAFE B, LLE Honey ,and4 C, respectively. The 1 ◦ − † C, then raised 3 slightly perceivable, 2 ◦ = unknown LLE Tropical fruits, floral C and 280 ◦ + -ionol m). The column effluent was split 1:1 α µ )-2,6-Dimethylocta-2,7- )-2,6-Dimethylocta-2,7- Z Z detected by sensory panel, ND, not detected by sensory panel. dien-1,6-diol dien-1,6-diol o o C and held isothermally for 25 min at this 0.25 ◦ E E Unknown 5 — Fruity, berries Unknown 6 SAFE B, LLE Floral Unknown 4 — Sweet Unknown 9 LLE Sweet, fruity Unknown 8 LLE Fruity Unknown 3Unknown 7 — LLE Chemical Sweet, tropical fruit Others Unknown 1Unknown 2 — — Green Eucalyptus not perceivable, 1 × += = Continued 0.25 mm × Intensity (Int.); 0 RI, retention index on DB WaxETR column. Aroma descriptors attributed by the sensory panel. SAFE A, acid fraction compounds from SAFE were identified by MS spectra; SAFE B, neutral/basic fraction compounds from SAFE were identified Aroma compounds. Aroma detected; Average of eight trained sensory panelists. The sensory panel was composed of eight females, 35–50 years by MS spectra; SPME,identified compounds by MS detected spectra. by SPME were identified by MS spectra; LLE, compounds detected by liquid–liquid extraction were b c ‡ § a ∗ † 2252 ( RI on DB WaxETR* Aroma compound Table 2. 2518 Farnesol SAFE B, LLE Sweet 2288 ( 3045 Squalene SPME, LLE Nutty 2618Not determined Vomifoliol1205 3-Oxo- Diethyl disulfide SAFE B, LLE LLE — Pungent ND 0 ND 0 ND 0 wileyonlinelibrary.com/jsfa until it reached 240 Gas chromatography–olfactometry analysis These analyses werechromatograph carried equipped with out aand flame using ionization a a detector sniffing (FID) HPThe port samples series (9000 from acid sniffer; 6890 andanalyzed Brechbuhler gas neutral–basic AG, on fractions Texas, of a SAFE(60 USA). were m DB-WAXETR capillary column (J&W Scientific) (v/v) into the FIDoutlet and splitter a (Alltech heated Associates,detector Illinois, sniffing temperatures USA). port were The with 180 injector a and fused silica of age, all of themand belonging Flavors to from the Catholic University staff of of Chile. thethan All 4 of Center them years of had experience Aromas more in quantitative descriptivewith analysis more (QDA), than 400 h of GC-O analysis.under This ISO panel 8586 has and been controlled trained by the NationalIn Standards Institute. the GC-O sessionsand the sensorially panelists described the recorded volatile the compounds.ties Aroma retention were intensi- ranked times on a four-pointthe scale value ranging 0 from represents 0 no toperceivable. 3, perception The where and concentrated basic the fraction valuewas obtained 3 diluted from is to SAFE strongly a dilutioneight factor panelists performed (FD) a of GC-O 1:500 with these and three 1:1000 samples. and the temperature. sample was injectedwas in programmed splitless mode. for The 5 oven min temperature at 40 helium column flow rate was 1.9 mL min

756 757 -butyl)- t Fragaria -butyl)-4- t -compounds are 6 C -hexalactone, ethyl , the furaneol [2,5- γ 29 -muurolene. The alcohols -farnesene. Among these, α wileyonlinelibrary.com/jsfa α 30 )-furanone and 2,6-di( H In our study, both compounds )-furanone] and mesifuran [2,5-dimethyl- 9,12,32 H Fragaria x ananassa 2 were used for a preliminary reconstitution Green–fresh notes appeared in seven odorant Some of the latter could be important to the ≥ )-furanone] are considered to be the two most 27 H The caramel odor is reported as one of the most 33,34 5,12,17 31 . Among all the compounds detected by GC-O, 75 were detected The floral zones are represented by seven odorants: benzyl hydroxy-4-methyl-2,5-cyclohexadien-1-one and 3,7-dimethyloct- 1-en-3,8-diol, respectively –The were 18 not remainingconcentrations odorants commercially (expressed were available. occur mixed as in at 4-nonanol white theand strawberry equivalent) same modifying juice, as relative using theirpanel’s commercial concentrations they suggestions. compounds according Similarities or tocomparing differences the the were sensorial aroma obtained of by reconstitutionwhite strawberry models juice. The results with of this the experiment are original as illustrated a spider diagram (Fig. 1). dodecanoate andcompounds ethyl of commercial strawberry aroma tetradecanoate. responsible for fruity impressions. The esters are key chiloensis by the sensory panelin only the 1:500 in dilution the andfor concentrated 51 each in sample compound the (1:1), is 1:1000 different dilution. 63 (Table 2). based The detection on its detection threshold Preliminary aroma reconstitution Despite the numerous compounds present in the white strawberry extract (Table 2),andanintensity only thoseexperiment with (Table a 1). dilution Fromcompounds the factor were 23 of selected notbecausethreewereunknownandtheothertwo odorant 1:1000 used zones, five in – 2,6-di( the reconstitution experiment alcohol, linalool, 2-phenylethylcinnamyl acetate, acetate, hydrocinnamyl cinnamic alcohol, cinnamic acid and compounds gamma decalactone. were The detected previously in important notesdimethyl-4-hydroxy-3(2 in 4-methoxy-3(2 important furanones in strawberriesand that contribute fruity to descriptors. caramel 4-hydroxy-4-methyl-2,5-cyclohexadien-1-one. In general,notes sweet were produceddodecanoate, ethyl by: butanoate 2-pentanone, and and acids contribute littleas to odorant the impact flavor compounds.for and They five they are, odorant however, arenotes, zones responsible irrelevant such detected as in pungent,acetic and our chemical acid, study aromas: with propanoicacid. isobutyl unpleasant alcohol, Among acid, sulfur 2-methylbutyric compounds,as well we acid as several identified others and diethyl withsulfide, unpleasant methanethiol, disulfide, valeric notes, diethyl such disulfide, as dimethyl hydrogen disulfide,thioacetate methyl andstrawberries. methyl thiobutanoate, already reported in hexanal, trans-2-hexenal and hexanol have been alreadyas reported contributors to green and fresh notes. zones, characterizedhexanal, by nonanal, 2,4 hexadienal; alcohols aldehydes (1-hexanol and1-hexanol) 2-ethyl- like and 2-methyl-2-pentenal, the sesquiterpene aroma of cultivars,concentrations. even though they are present at very low known to decrease with maturity,esters lowering green and notes, whereas furanones, responsiblerespectively, increase for with maturity. fruity and caramel notes, were detectedothers and also contributed contributed toethyl caramel hexadecanoate, to 5-ethyl-2-(5 note, (i.e. the methyl octanoate, caramel odor; but subsp. Fragaria Fragaria It is worth 2013 Society of Chemical Industry MA has also c species. It has 3,21 -norisoprenoids 27 . 13 Fragaria chiloensis Fragaria and low or undetectable Fragaria chiloensis F. virginiana : ethyl acetate, ethyl butanoate, and F. moschata and : 752–759 94 ). Alternatively, the extract methods we used might Fragaria chiloensis 2014; Three different methods for aroma extraction were although we were unable to detect it. This may be Fragaria x ananassa 25,26 27,28 Fragaria x vescana , , chiloensis Of the 99 volatile compounds identified, only 13 were identified J Sci Food Agric Identification of volatiles by gas chromatography–massspectrometry We identified 99 volatileextracted by compounds SAFE, in HS-SPME and white LLEsummarizes strawberry extraction methods; the fruits Table compounds 2 library tentatively search and identified compared with usingin the a retention the indices MS literature. available esters, alcohols, The aldehydes and volatile ketones, acids, C compounds were classified into Aroma of white strawberriesRESULTS AND DISCUSSION www.soci.org lucida butyl acetate, methyl hexanoate, ethyl hexanoate, hexenyl acetate, hexyl acetate, 2-hexenyl butanoate,acetate, benzyl 1-butanol, acetate, phenylethyl 1-hexanol and 2-heptanone. previously in used in order to maximizethe the widest variety possibilities of to volatile/odorant compounds obtain present and instrawberry. white identify Seventy-nine different compounds were extractedLLE, by 50 compoundsneutral–basic by fraction HS-SPME and and acidnumber fraction 54 of of compounds SAFE. diverse Based with upon compoundsthat the the extracted, LLE it is the maycompounds most be not complete suggested extraction detectedusing method. in the However, other the 22 methods. Therefore, LLEthat these the extracts, results demonstrated largest wereusing number the extracted LLE of method; even volatile thoughadditional the compounds compounds other than methods extracted are the LLEto extracted have method. a Therefore, more complete in volatile order profilecombination of of all the these white extractive strawberry, methods a should be performed. mentioning that methyl anthranilate (MA)a has been discriminative described key as compoundbeen found among in highvesca levels in some wildlevels species such in as spp. Sensory evaluation, gas chromatography–olfactometry GC-O results ofand the 1:1000 fold concentrated dilutions)detected led and by to diluted the 84 panelists odor-active extracts duringFromthesezones,ninecouldnotbeidentifiedbyGC-MS(unknown zones the that (1:500 gas were olfactometric analyses. compounds) (Table 2).trained The panel olfactometric analysis towhite allowed strawberry: define the fruity, atand tropical least banana fruits nine odors), (likewoody green–fresh, pineapple, sensory and guavas, floral, descriptors unpleasant.esters, caramel, The of are sweet, fruity represented by nutty, notes,acetate, 2-ethylfuran, mainly methyl methyl 2-methylbutanoate, butanoate, derived ethyl butyl from 2-methylbutanoate,hexen-1-ol acetate, 2- ethyl octanoate, methyl 3-hydroxybutanoate, ethyl 3-hydroxybutanoate, ethyl decanoate, mesifurane, furaneol, butyl laurate, gamma decalactone, isoamyland alcohol, seven 1-dodecanol unknown compounds. Theto tropical fruit six notes odorant belong hexyl zones acetate, containing only acetyl esters: acetate, ethyl furfuryl hexanoate, acetate, 1-methylethyl because we evaluated a different subspecies ( not be able to extract MA(i.e. or oxidation). may have led to modifications of MA species. and terpenes, as well as lactonesof and furanones. volatile All these families compounds have been reported in other been reported in some accessions of ,ed. et al. rugen . Holt, , :113–116 : 752–759 J Agric Food 94 175 . Recently, the J Agric Food Chem 2014; Fragaria vesca J Agric Food Chem :536–539 (1992). :120–123 (1992). Genet Resour Crop Evol :218–223 (1997). 195 ¨ oppler H, Gaschromato 195 205 species). Duch) cultivars by use of odour accessions. J Sci Food Agric Fragaria chiloensis Fragaria Z Lebensm Unters Forsch A was sequenced, opening the door to . Fragaria 38 28c:488–493 (1973). Volatile Compounds in Foods and Beverages ). Un analisis conjunto y una prueba sensorial. Fragaria ananassa Z Lebensm Unters Forsch Z Lebensm Unters Forsch :1–10 (2006). Z Lebensm Unters Forsch A 10 )-2,6-dimethylocta-2,7-dien-1,6-diol (sweet, floral The Strawberry: History, Breeding, and Physiology E :4037–4042 (2002). . Additional studies must be carried out to improve Z Naturforsch 50 Fragaria vesca )-2,6-dimethylocta-2,7-dien-1,6-diol (caramel, honey Fragaria virginiana -decalactone (floral, peach aroma), cinnamyl acetate Z γ :1185–1196 (2007). :227–232 (1997). :715–721 (2003). Fragaria chiloensis by Maarse H. Dekker, New York, pp. 333–387 (1991). in strawberries. graphisch–massenspektrometrische differenzierung von erdbeer- arten. consumidor de la( Region MetropolitanaEcon hacia Agraria la frutilla Blanca some strawberry ( threshold values. and Reinehart and Winston, New York, p. 447 (1966). in wild and cultivated 54 odorants in fresh strawberryand juice by sensory quantitative45 measurements studies on model mixtures. cultivars, ‘Annelie’ and ‘Alaska Pioneer’, obtained by backcrossingcultivated of strawberries with wild strawberries, (1982). aroma compounds through aminoChem acid metabolism. assessment of strawberries ( 51 flavour – discrimination of aroma typescompounds. by quantification of volatile F. chiloensis 6 Larsen M and Poll L, Odour thresholds of some important compounds 4 Adasme C, Spiller A and Diaz J, Determinacion de preferencias del 5 Latrasse A, Fruits III, in 7 Larsen M, Poll L and Olsen C, Evaluation of the aroma composition of 2 Ulrich D, Komes D, Olbricht K and Hoberg E, Diversity of aroma patterns 3 Drawert F, Tressl R, Staudt G and K 1 Darrow GM, 8 Schieberle P and Hofmann T, Evaluation of the character9 impact Hirvi T and Honkanen E, The volatiles of two new strawberry 10 Perez A, Olias R, Luances P and Sanz11 C, Biosynthesis Azodanlou R, of Darbellay strawberry C, Luisier J, Villettaz J and Amado12 R, Quality Ulrich D, Hoberg E, Rapp D and Kecke S, Analysis of strawberry odor) and hexadecanoic acid (chemical, lactic odor) were identified in the reconstitution of the aroma of genome of (floral, sweet), ( aroma), ( fruits odor), hexylacetate acetate (fruity, strawberry (tropicalfruits jam odor), fruits linalool odor), (sweet odor), odor), furfuryldecanoate mesifuran (fruity (caramel 2-hexen-1-ol acetate and odor), caramel ethyl (tropical odors),odor), benzyl alcohol 2-phenylethyl (spice, acetate floral (sweet, tea(caramel, strawberry and like odor), floral hydrocinnamyl alcohol odors), (floral, citric DMHF aroma), SUPPORTING INFORMATION Supporting information may be found inarticle. the online version of this REFERENCES ACKNOWLEDGEMENTS Loreto Prat isat a Universidad member AndresMillennium of Bello. Nucleus the in This Biotechnology Plant065-F. research We Cell would PhD like was Biotechnology to thank Program Dr (PCB) supported Lee Meiselde ICM from Chile, INTA, by Universidad for P06- her critical reading of the manuscript. new genes involved inany metabolic case, the pathways results obtained related in this to workfor will aroma. be future a In strawberry useful breeding reference programs. , fruits fruit. www.soci.org L Prat under Fragaria , a diploid 2013 Society of Chemical Industry Fragaria lactic c green fruits. The dashed Fragaria chiloensis F. chiloensis Fragaria ananassa Fragaria chiloensis 2: ethyl butanoate pineapple ≥ Fragaria vesca Fragaria vesca apple and However, there are reports 2 Fragaria chiloensis . 3 2 1 0 F. chiloensis model citric-grapefruit The volatile profile of Fragaria virginiana caramel Furthermore, some octoploids would have 2 . , Fragaria vesca 37 – , reported here, revealed 99 volatile compounds, pear 35 cherimoya and Aroma reconstitution of banana chiloensis It has been reported previously that The sensory profiles of white strawberry juice and the Fragaria chiloensis the same conditions, as well asthe a comparative genes genomic that analysis regulate of the production of these compounds. reconstitution modelexception showed of a the greenjuice good note, but not which agreement, in isconcentration the perceived were with model. estimated in and This the the not differencewe absolutes natural did may and/or not because be include because all compounds.the the The white overall similarity strawberry between juicescale and from the 0 model to wasbetween 3. the marked fruit Although 2.4 and the the on reconstitutedconsidered panel a formula, qualitatively their was to aroma able was be tofruits. similar discriminate to the juice from the fresh strawberry, has moreFragaria volatile ananassa compounds than the octoploid that indicate thatstrawberries. environment is important in the aroma of compounds that are not present in diploids anddescribed in vice this versa. work, Also, the methods as used for themake determination can differences. Further insightrevealed into by these comparing possibilities the volatile may profiling be of wileyonlinelibrary.com/jsfa We identified 99 different volatile compoundsOnly in 75 ofthe these training compounds sensorial wereas panel. described fruity, These as green, odorantsodors odorants floral, that have by play caramel, notes2). important such sweet, roles Twenty and in weredilution some the factor potent complete unpleasant of aroma aroma 1:1000 (Table compounds and an detected intensity with a CONCLUSION (nutty odor), 2-hexenal (fresh odor), ethyl hexanoate (tropical Figure 1. red line (thin) shows the modelblue based on line white (thick) strawberry juice.fruits shows The using solid the the reconstitution compounds3 suggested model represent by for the the gas sensoryon chromatography–olfactometry panel. the 0, intensity. The 1, figure names 2 represent and descriptive terms to define the according to the sensory panel. spp. ananassa significantly more than what has been reported for

758 759 Nat ). Duch. J Agric J Agric . Fragaria Hortscience J Food Sci :19–22 (1979). Acta Hortic ISHS chiloensis 27 Fragaria vesca :487–496 (2007). :529–538 (2001). spp. :383–390 (2005). 72 11 29 :1248–1254 (2004). Fragaria x ananassa :139–151 (1999). 52 65 wileyonlinelibrary.com/jsfa J Food Sci J Agric Food Chem :78–97 (2005). Turk J Agric For HortTechnology Fragaria chiloensis 28 Food Chem J Agric Food Chem )-Cinnamic acid derivatives and phenolics from E :87–93 (2008). L., compared to those of cultivated berries, :316–321 (1981). :8512–8518 (2005). J Food Qual -furan-3-one and its derivatives: analysis, synthesis and 127 29 53 cv. Senga Sengana. H :109–118 (2011). , The genome of woodland strawberry ( 43 , Comparison of methodologies for the identification of aroma :579–584 (2006). et al :268–272 (2004). :1022–1026 (2000). et al compounds in strawberry. hydroxy-2 biosynthesis – A review. Plant Breed compounds in a model population of x ananassa Food Chem compounds in69 strawberry puree during heating. aroma is influenced by cultivar,35 maturity, and storage. date effects onstrawberries. flavour and other quality attributes of California breeding by sensory and708 instrumental methods. cultivated strawberries: The role of the sulfur compounds. composition of small fruit. Genet O, selected aroma-active compoundssolid-phase in micro strawberries extraction by gas chromatographywith headspace sensory and descriptive correlation analysis. characteristics and volatile constituents of strawberryduring (cv. maturation. Cigaline) Hirschmann G, ( Food Chem Fragaria vesca Chilean strawberry fruits, 25 Kaskas E, Kaskas S, Koch-Dean M,26 Schwab W, Zabetakis Larkov I, O, Gramshaw Lavid N, JW and Robinson D, S. 2,5-Dimethyl-4- 27 Olbricht K, Grafe C, Weiss K and Ulrich D, Inheritance of aroma 34 Schulbach KF, Rouseff RL and Sims CA,35 Changes Forney in CF, volatile Kalt sulfur W and Jordan MA,36 The composition Pelayo-Zaldivar of C, strawberry Ebeler SE and Kader, AA, Cultivar and harvest 28 Ulrich D, Hoberg E and Olbricht K, Flavour control in strawberry 33 Dirinck P, De Pooter H, Willaert G and Schamp N, Flavor quality of 37 Forney CF, Horticultural and other factors affecting aroma volatile 38 Shulaev V, Sargent DJ, Crowhurst RS, Mockler T, Veilleux RE, Folkerts 29 Jetti RR, Yang E, Kurnianta A, Finn C and Qian MC, Quantification of 30 Menager I, Jost M and31 Aubert Cheel C, J, Theoduloz Changes C, Rodriguez in J, Saud physicochemical G, Caligari P and Schmeda- 32 Pyysalo T, Honkanen E and Hirvi T, Volatiles of wild strawberries, , ) Food et al Fragaria Fragaria Fragaria J Jpn Soc 2013 Society of Chemical Industry c J Agric Food Chem Fragaria chiloensis :9123–9132 (2009). -adenosylmethionine:furaneol S :421–434 (1997). 57 :210–220 (2008). :4568–4573 (1999). 74 Chem Mikrobiol Technol Lebensm 49 47 :517–531 (1990). :237–241 (1999). 50 :1133– 1142 (2002). 209 50 : 752–759 J Agric Food Chem , Aroma development during ripening of a new and versatile technique for the careful and 94 J Agric Food Chem ± J Agric Food Chem et al fruit and participation of an alcohol acyltransferase :300–305 (2005). :377–385 (2009). ): Characterization of some cultivars and influence of ). , Softening rate of Chilean strawberry ( J Sci Food Agric 2014; 74 Postharvest Biol Technol 113 et al :4025–4030 (2002). :3129–3136 (2002). :141–148 (1992). -methyltransferase activity in ripening fruits. evaporation direct isolation of aroma compoundsEur from Food complex Res food Technol matrices. fruits reflects the expression of polygalacturonase and pectategenes. lyase Herrera R, odorants contributing to ‘Toyonoka’using strawberry an aroma adsorptive in column extracts andHort Sci aroma dilution analysis. ananassa freezing. chiloensis (FcAAT1) gene. Comparison of phenolic composition and antioxidanttwo properties of native Chilean and oneChem domestic strawberry genotypes. Aroma biosynthesis in strawberry: O 50 of strawberryClassification aroma and discrimination through50 of SPME/GC varieties. and J Agric ANN Foodstrawberry varieties analyzed methods. Chem by purge-and-trap headspace GC-MS. J Agric Food Chem biosynthesis. of fresh strawberry flavour. ananassa 14 hyphenated purge-and-trap gas chromatographysensory techniques and analysis in the aroma profiling of strawberries ( PDS, 24 Engel W, Bahr W and Schieberle P, Solvent assisted flavour 21 Gonzalez M, Gaete-Eastman C, Valdenegro M, Figueroa C, Fuentes L, 20 Douillard C and Guichard E, The aroma of strawberry ( 19 Fukuhara K, Li X, Okamura M, Nakahara K and Hayata Y, Evaluation of 22 Simirgiotis MJ, Theoduloz C, Caligari P and Schmeda-Hirschmann G, 14 Urruty L, Giraudel J, Lek S, Roudeillac P and Montury M, Assessment 15 Hakala M, Lapvetelainen T and Kallio H, Volatile compounds of selected 16 Zabetakis I and17 Fischer Holden N M, and Hammerschmidt Strawberry FJ, A flavour: contribution Analysis to the and analysis 18 Gomes da Silva M and Chaves das Neves H, Complementary use of 23 Figueroa CR, Pimentel P, Gaete-Eastman C, Moya M, Herrera R, Caligari 13 Lavid N, Schwab W, Kafkas E, Koch-Dean M, Bar E, Larkov O, J Sci Food Agric Aroma of white strawberries www.soci.org