Composition of phenolics and volatiles in strawberry cultivars and influence of preharvest hexanal treatment on their profiles

Composition of phenolics and volatiles in strawberry cultivars and influence of preharvest hexanal treatment on their profiles Azizah Misran 1,3, Priya Padmanabhan 1, J. Alan Sullivan 1, Shahrokh Khanizadeh 2, and Gopinadhan Paliyath 1,4 1 Department of Plant Agriculture, University of Guelph, Ontario, Canada N1G 2W1; and 2 Agriculture and Agri-Food Canada, 960 Carling Avenue, KW Neatby Building, Ottawa, Ontario, Canada K1A 0C6. Received 3 July 2014, accepted 29 August 2014. Published on the web 3 September 2014. Misran, A., Padmanabhan, P., Sullivan, J. A., Khanizadeh, S. and Paliyath, G. 2015. Composition of phenolics and volatiles in strawberry cultivars and influence of preharvest hexanal treatment on their profiles. Can. J. Plant Sci. 95: 115126. Biochemical changes of quality-determining components were evaluated in strawberry fruit subjected to preharvest spray treatments using a hexanal-containing formulation that is known to enhance shelf life and quality of fruits. Phenolic compounds and volatiles of fruits of four strawberry cultivars (Mira, Jewel, Kent, and St. Pierre) grown in southern Ontario were characterized by HPLC-MS and solid phase micro extraction (SPME) analysis. Qualitative and quantitative profiles of phenolic compounds varied among the cultivars. In all the cultivars, anthocyanins constituted the most prominent class of phenolic compounds. Volatile profiles of strawberry homogenate differed among the cultivars. Changes in phenolics and volatiles composition were determined in fruits of Mira and Jewel after spraying with a hexanalcontaining formulation at weekly intervals. In Jewel, preharvest hexanal spraying altered the profiles of polyphenolic components, while minimal changes were noticed in Mira. Interestingly, very few differences were identified in ester profiles of treated and untreated Mira. In general, hexanal spray application resulted in a decrease in the abundance of several volatile components including esters, ketones, and lactones in treated Jewel compared with the control. The results suggest that cultivar-specific quality changes may result from a preharvest application of hexanal formulations, which may also imply different patterns of metabolite channeling and delay of fruit ripening processes. Key words: Strawberry, phenolic compounds, volatile profiles, hexanal application Misran, A., Padmanabhan, P., Sullivan, J. A., Khanizadeh, S. et Paliyath, G. 2015. Nature des composés phe noliques et volatils chez la fraise et conséquences d un traitement a` l hexanal avant la cueillette sur leur profil. Can. J. Plant Sci. 95: 115126. Les auteurs ont e value les changements biochimiques subis par les compose s qui de terminent la qualite de la fraise traite e avant la cueillette par pulve risation de produits renfermant de l hexanal, substance qui prolonge la dure e de conservation du fruit et en rehausse la qualite. A` cette fin, ils ont caracte risé les compose s phénoliques et volatils des fruits de quatre cultivars (Mira, Jewel, Kent et St. Pierre) cultive s dans le sud de l Ontario par CHLP-SM et micro-extraction en phase solide. Le profil qualitatif et quantitatif des compose s phénoliques fluctue d une varie te a` l autre. Les anthocyanines sont les compose s les plus fréquents dans tous les cultivars. Le profil des compose s volatils de l homoge nat de fraise diffe` re selon le cultivar. Une modification de la composition des compose s phe noliques et volatils a e te observe e chez les fruits de Mira et de Jewel apre` s pulve risation hebdomadaire d une formule contenant de l hexanal. Chez Jewel, la pulve risation d hexanal avant la cueillette alte` re le profil des composés polyphe noliques, mais les changements sont minimes chez Mira. Fait intéressant, on observe très peu de diffe rences dans le profil des esters chez les fruits traite s et non traite s de Mira. En ge ne ral, la pulve risation d hexanal réduit l abondance de plusieurs compose s volatils, dont les esters, les ce tones, et les lactones chez les fruits de Jewel, comparativement au témoin non traite. Les résultats de l e tude laissent supposer que l application d une formule renfermant de l hexanal avant la cueillette peut entraıˆner des changements spe cifiques au cultivar au niveau de la qualité, ce qui pourrait signifier l acheminement des me tabolites de diverses manie` res et un retard dans le muˆrissement des fruits. Mots clés: Fraise, compose s phénoliques, profil des compose s volatils, application d hexanal Strawberry (Fragariaananassia Duch.) is one of the most popular berry fruit that belongs to the genus Fragaria in the Rosaceae family. It is one of the most 3 Current Address: Department of Crop Science, Faculty of Agriculture, University Putra, 43400, UPM Serdang, Selangor, Malaysia. 4 Corresponding author (e-mail: gpaliyat@uoguelph.ca). commonly consumed berries in the world and consequently is an important crop cultivated worldwide (Tulipani et al. 2008). It is widely appreciated for its Abbreviations: GC-MS, gas chromatographymass spectrometry; HPLC, high performance liquid chromatography; PLD, phospholipase D; SPME, solid phase micro extraction Can. J. Plant Sci. (2015) 95: 115126 doi:10.4141/cjps-2014-245 115

116 CANADIAN JOURNAL OF PLANT SCIENCE characteristic aroma, bright red color, juicy texture, and sweetness. Strawberries are consumed fresh or in processed forms such as jams, jellies and juices. They are in particular rich in vitamin C and are among the richest natural dietary sources of folate and manganese (Giampieri et al. 2012). Previous studies have shown that berries are ranked among the top sources of total phenolics and total antioxidant capacity with levels greater than other fruits, vegetables and cereals (Halvorsen et al. 2002). The main phenolic compounds in strawberries are anthocyanins, flavonols, flavanols, derivatives of hydroxycinnamic acid and ellagic acid (Buendı a et al. 2010; Aaby et al. 2012). Within the fruit group, strawberries have a greater antioxidant capacity (2- to 11-fold) when compared with that of apples, peaches, pears, grapes, tomatoes, oranges or kiwifruit (Wang et al. 1996). From the postharvest perspective, strawberries are extremely perishable and have a highly demanding postharvest handling requirement. Their short postharvest life is mainly due to their high susceptibility towards mechanical injury, physiological deterioration, water loss and microbial decay. In order to prevent severe postharvest losses, strawberries are often harvested at 3 4 ripe (pink) or ½ ripe stages (Pineli et al. 2011). Being a non-climacteric fruit, strawberries harvested before full ripeness are consumed without the best sensory and organoleptic characteristics. Harvest maturity and postharvest conditions are two major factors that affect the sensory and nutritional qualities of strawberries (Pineli et al. 2011). Suitable technologies and methods to enhance the postharvest shelf life of this fruit are highly desirable due to the extremely perishable nature of strawberries. Loss of fruit quality starts with the loss of membrane integrity that generally becomes the lead event during senescence and stress (Paliyath et al. 2008). Phospholipase D (PLD), a phospholipid-degrading enzyme present in strawberry is the key enzyme involved in initiating a cascade of catabolic events that leads to the eventual deterioration of the membrane, and is highly active in strawberry (Paliyath and Droillard 1992; Yuan et al. 2005, 2006). An approach of delaying the progressive membrane degradation was suggested through hexanal (a primary aldehyde) treatment through its PLD inhibition capability (Paliyath et al. 1999; Paliyath and Subramanian 2008). It was suggested that inhibition of PLD activity could eventually lead to enhanced membrane stability, and thereby increased longevity of horticultural produce. The application of hexanal, either pre- or postharvest, has shown promising results in enhancing the shelf-life of several fruits, vegetables, freshcut produce and flowers (Paliyath and Subramanian 2008; Sharma et al. 2010; Cheema et al. 2014). Currently, we are investigating the possibility of using hexanalcontaining formulations on strawberries as preharvest sprays to extend postharvest shelf life and quality. Our preliminary experiments showed some positive responses; however, more refinements and modifications in the existing methodology are required to evaluate a full functioning and effective technology with respect to strawberries. In the present investigation we used a formulation containing hexanal and applied it as a preharvest spray on strawberries at the green stage until harvest. The production of volatile compounds is one of the noticeable changes of strawberry during ripening and is an important quality factor influencing the consumer acceptability. So, as a first step, we have analyzed the composition of polyphenols and profiled the volatile components of two strawberry cultivars, Kent and Mira, in comparison with unsprayed strawberries. Another goal of this study was to characterize the polyphenolic and volatile composition of four different strawberry cultivars, Mira, Jewel, Kent and St. Pierre, through HPLC and GC/MS analyses. MATERIALS AND METHODS Plant Growth Strawberry plants (Fragariaananassa cvs. Jewel, Kent, Mira and St. Pierre) were planted in the field in matted row beds and grown according to the current agricultural practices followed in Ontario (Ontario Ministry of Agriculture, Food and Rural Affairs 2003) at the Elora Research station (University of Guelph, Elora, ON). Fertilization and irrigation were conducted at recommended rates to ensure optimum yield and quality. No pesticides or herbicides were applied. Fruits were harvested by hand-picking at commercial maturity stage. Fruit (0.5 kg fresh weight per replication) of uniform size and color were subjected to LC-MS analysis of phenolic compounds and HS-SPME/GCMS analysis of volatile compounds. Hexanal Formulation Treatment of Strawberry Cultivars Two cultivars of strawberry, Mira and Jewel, were subjected to preharvest spray treatment with a formulation containing hexanal. Plants were grown as mentioned earlier (Plant Growth section). The experiment was carried out in a completely randomized block design with four replications. Each plot was divided in half, and plants in one half of the plot were sprayed with hexanal formulation while the other half was left unsprayed (control) in a split block design. Stock hexanal formulation consisted of hexanal (1% vol/vol), geraniol (1%), a-tocopherol (1% wt/vol), ascorbic acid (1% wt/vol), cinnamic acid (0.1% wt/vol) tween 20 (10% vol/vol) and ethanol (10% vol/vol). Stock solutions were diluted with water (1 to 50 L) prior to spraying to provide a hexanal concentration of 0.02% (2 mm) in the final solution. Calcium chloride (1% wt/vol) was also added to the final spray solution. Berries were sprayed at a rate of 0.25 L per bed (1.5 m0.5 m) directed at the fruit on the plants using a pressurized nozzle sprayer. Spraying was conducted once per week for 3 consecutive weeks.

MISRAN ET AL. * COMPOSITION OF PHENOLICS AND VOLATILES OF STRAWBERRY 117 Berries harvested a week after the second spraying were used for the various experiments. One week after spraying, fruits were hand-picked at a commercial maturity stage. Intact fruits of uniform color and size without mechanical damage were selected for various experiments. Fertilization and irrigation were conducted at recommended rates to ensure optimum yield and quality. No pesticides or herbicides were applied. Total phenolic compounds, as well as the volatile profiles of the sprayed and unsprayed fruits were analyzed as described in the later sections. Extraction of Polyphenols Strawberries were homogenized in methanol using a Brinkmann Polytron @ blender (Brinkmann instruments Inc., Westbury, NY), fitted with a Polytron PTA 10 probe. The homogenate was centrifuged at 15 000g for 20 min to collect the supernatant. The extracts were dried using a stream of nitrogen to remove methanol. The dried residue was dissolved in distilled water and loaded onto a C18 Sep-Pak cartridge (1 ml volume, Waters Corporation, MA). A total crude fraction was obtained by eluting the total loaded polyphenols directly with 100% methanol. Aliquots of eluates were stored at 208C until further use. Chemicals Gallic acid, p-coumaric acid, m-coumaric acid, o-coumaric acid, kaempferol-3-glucoside, quercetin-3- glucoside, cyanidin-3-glucoside and pelargonidin-3- glucoside used as standards for HPLC were purchased from Sigma-Aldrich, Canada. LC-MS Analysis of Phenolic Compounds Identification of fruit phenolic compounds in four strawberry cultivars was carried out using LC-MS with a negative ion detection mode program at 260 nm. Extracts of phenolic compounds filtered through a Millex HA 0.45 mm filter (Millipore Corp., Billerica, MA) were analyzed on an Agilent 1100 series HPLC-MS (Agilent Technologies, Waldbarn, Germany) equipped with an autosampler and a UV-visible detector. Electrospray ionization was performed with an API-ES mass spectrometer. The phenolic compounds were separated using XTerra MS C-18 column (5 mm, 1502.6 mm, Waters, Milford, MA) with water/formic acid (98:2, vol/ vol) (A) and methanol (B) as mobile phases. For phenolic compound and anthocyanin analysis the gradient used were as follows: B; 02 min, 7%; 230 min, 20%; 3045 min, 30%; 4550 min, 30%; 4550 min, 35%, 5060 min, 50%, 6065 min, 80%; 6567 min, 100%; 6770 min, 100%; 7073.50 min, 7%. The flow rate was 0.8 ml min 1 and chromatograms were recorded at 260 nm (phenolics) and 520 nm (anthocyanins). The injection volume was usually 20 ml (10 mg polyphenols). Each analysis was carried out in four independent replications. The phenolic compounds were identified according to their order of elution and mass spectrometric characteristics by comparing to the values in the literature. The results were expressed as mg 100 g 1 fresh-frozen wt. SPME/GCMS Analysis of Volatile Compounds Samples of strawberries (150 g) were homogenized using a Brinkman Homogenizer fitted with a Polytron PTA 10 probe and the slurry (20 g each for a total of four replicates) was then poured into a 250-mL flask (headspace volatile collection system) and tightly closed. After 30 min, a solid-phase micro-extraction (SPME) injection unit (Supelco fiber 100 nm diameter, coated with polydimethylsiloxane) was inserted into the head space to adsorb the volatiles. GC-MS analysis was conducted with a GC-MS system (Saturn 2000R Varian Inc., Palo Alto, CA). SPME fiber was inserted into GC-MS inlet maintained at 2508C and volatiles were desorbed for 5 min. The oven temperature was maintained at 408C and increased at a rate of 88C min 1 to 2208C and held for 10 min. Helium was used as the carrier gas with a flow rate of 1 ml min 1. The compounds eluted from the column were ionized by electron bombardment. Ions with a m/z ratio from 40 to 450 were recorded for analysis. Eluted volatile components were identified by library (NIST 2008) search and comparison to the stored spectra of authentic compounds. All analyses for volatiles were carried out in four independent replicates. Statistical Analysis Statistical analyses were conducted using a GraphPad Prism software (Prism 4 Statistics Guide, 2003). Means were compared using one way ANOVA followed by Tukey s comparison test to evaluate the level of significance. Significantly different means (P B0.05) were denoted by different letters. RESULTS Identification of Phenolic Compounds in the Fruit of Strawberry Cultivars Phenolic compounds identified from the selected strawberry cultivars were categorized as anthocyanins, flavonols, flavanones, ellagitannins and other simple phenolic compounds (Table 1). In general, not much significant variation in phenolic contents was noticed among the cultivars studied. A total of 20 compounds were identified in Kent, while St. Pierre, Jewel and Mira contained 17, 15 and 13 compounds, respectively. Anthocyanins were the major group of phenolic compounds identified in all cultivars. Kent had all the seven identified anthocyanin components, while Mira and St. Pierre contained four each, followed by Jewel with only three compounds. Anthocyanins identified from all cultivars included cyanidin-3-glucoside, pelargonidin-3-glucoside, pelargonidin-3-rutinoside, cyanidin-3-malonylglucoside, 5-pyranopelargonidin-3-glucoside, a cyanidin derivative, and pelargonidin-3-malonylglucoside (Table 1). Pelargonidin-3-glucoside was the predominant compound

118 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Characterization of phenolic components in strawberry fruits of Jewel, Mira, Kent, and St Pierre zy Cultivar Phenolic compounds m/z Jewel Mira Kent St. Pierre Anthocyanins Cy-3-glu 449 ND ND 3.090.6a 1.691.2a Pg-3-glu 433 57.993.4a 47.191.8b 59.690.6a 65.891.8c Pg-3-rut 579 0.590.1a 1.990.4b 2.690.1c 2.990.2c Cy-3-mal-glu 519 ND 0.490.2a 0.290.2a ND 5-pyrano-pg-3-glu 501 ND ND 0.190.2a ND Cy derivative 535 ND ND 0.790.1 ND Pg-3-mal-glu 519 17.390.8a 17.190.6a 10.790.6b 6.790.4c Flavonols Q-3-glu 477 ND ND ND 2.190.7 Q-3-mal-glu 549 0.690.1a ND 0.390.1a ND K-3-glu 447 0.790.2a ND ND 0.890.3a K-3-gluc 533 0.490.2a 1.190.6a 1.190.2a 0.490.3a K-3-mal-glu 534 1.790.4a 0.890.1ab 0.891.4ab 0.690.1b K-3-coum-glu 593 1.090.3a 0.790.8a 0.890.6a 0.490.1a Flavanols Catechin 289 1.490.4a 1.790.3a 1.990.8a 1.690.4a PA dimer 577 4.690.5b 3.290.5ab 2.091.6a 3.291.4ab PA trimer 865 2.290.4a 2.590.6a 3.291.4a 3.592.0a Ellagitannins Bis-HHDP-glu 783 2.090.4a 5.091.2b 3.690.8ab 1.291.0a Gal-HHDP-glu 633 2.890.4a 3.490.3a 2.690.2a 3.590.1a Other phenolics EA pentoside 434 ND ND 0.290.2 ND EA deoxyhexoside 447 ND ND 0.590.6a 0.790.1a p-coumarylhexose 325 1.090.3a ND 0.990.3a 2.290.2b FA hexose derivative 449 4.591.6a 6.291.2ab 6.490.9ab 8.290.9b z Data are expressed as mean9sd of four independent assays. y Data are expressed as mg 100 g 1 of fresh weight. Pg, pelargonidin; coum, coumaroyl; Cy, cyanidin; glu, glucosides; gluc, glucuronide; rut, rutinoside; mal, malonyl; Q, quercetin; K, kaempferol; PA, proanthocyanidin; HHDP, hexahydroxydiphenoyl; gal, galloyl; EA, ellagic acid; FA, ferulic acid; ND, not detected. a, b Means followed by same letter in the same row are not significantly different at PB0.05 using Tukey s test. in all cultivars, accounting for 87% of the total anthocyanins in St. Pierre and 71% in Mira. Among the cultivars, St. Pierre had the highest pelargonidin-3- glucoside content of 65.8 mg 100 g 1 FW followed by Kent (59.6 mg), Jewel (57.9) and Mira (47.1 mg 100 g 1 FW). Pelargonidin-3-rutinoside was highest in St. Pierre (2.9 mg 100 g 1 FW) followed by Kent (2.6 mg 100 g 1 FW), Mira (1.9 mg 100 g 1 FW) and Jewel (0.5 mg 100 g 1 FW). Cyanidin-3-glucoside was undetectable in Jewel and Mira, while Kent had 3.0 mg and St. Pierre had 1.6 mg 100 g 1 FW. Two other pigments, 5-pyranopelargonidin-3-glucoside and a cyanidin derivative were detected only in Kent, but in very low amounts of 0.1 mg and 0.7 mg 100 g 1 FW, respectively. Cyanidin-3-malonyl-glucoside was identified only in Mira and Kent. Flavonols detected in strawberry cultivars were two quercetin and four kaempferol derivatives (kaempferol- 3-glucoside, kaempferol-3-glucuronide, kaempferol-3- malonylglucoside and kaemferol-3-coumaroylglucoside, Table 1). Jewel and St. Pierre contained the highest number of flavanols (five compounds) followed by Kent with four and Mira with only three compounds. Quercetin- 3-glucuronide was detected only in St. Pierre at a concentration of 2.1 mg 100 g 1 FW whereas quercetin- 3-malonylglucoside was identified in both Jewel (0.6 mg) and Kent with 0.3 mg 100 g 1 FW. Kaempferol- 3-glucoside was present only in Jewel (0.7 mg 100 g 1 FW) and St. Pierre (0.8 mg 100 g 1 FW). Other kaempferol derivatives were found in all cultivars in the range of 0.41.7 mg 100 g 1 FW. Flavan-3-ols group identified in strawberry included catechin, proanthocyanidin dimer and proanthocyanidin trimer. Catechin and proanthocyanidin trimer were detected in all cultivars, but there were no significant differences in their quantity among cultivars. The lowest proanthocyanidin dimer content was found in Kent with 2.0 mg 100 g 1 FW. Two different ellagitannins, namely bis-hexahydroxydiphenoyl-glucose (bis-hhdp-glucose) and galloyl-hhdpglucose, were detected in all cultivars (Table 1). Mira contained the highest bis-hhdp-glucose content (5.0 mg 100 g 1 FW) compared with the other three cultivars (1.23.6 mg 100 g 1 FW). No significant difference was observed in galloyl-hhdp-glucose level among the cultivars (2.63.5 mg 100 g 1 FW). Other phenolics identified in strawberry cultivars included ellagic acid derivatives, p-coumaroyl hexose and a ferulic acid hexose derivative. A pentoside of ellagic acid was identified

MISRAN ET AL. * COMPOSITION OF PHENOLICS AND VOLATILES OF STRAWBERRY 119 only in Kent, while a deoxyhexoside of ellagic acid was detected in both Kent and St Pierre. Identification of Volatile Compounds in the Fruit of Strawberry Cultivars The analysis of volatile compounds from fruits of four different cultivars using Head Space (HS)-SPME/GC- MS was performed. The compounds identified belong to different classes as listed in Table 2. The highest number of volatiles was identified in Mira with 27 compounds followed by St. Pierre and Jewel with 23 and 21 compounds, respectively. Kent had the simplest volatile mixture with only 14 identified compounds. In this study, caryophyllene oxide was tentatively identified as the major volatile compound present in all cultivars based on mass spectral comparison to authentic compounds in NIST library. Caryophyllene oxide constituted approximately 75% of total volatiles in the fruits of St. Pierre and Kent, while in Mira and Jewel, the relative proportions of caryophyllene oxide decreased to 50 and 35%, respectively (Table 2). Esters comprised the second most abundant class within the volatiles, with 25 compounds detected across the four cultivars. In all cultivars, the detected Table 2. Volatile compounds of the fruits of different strawberry cultivars (Jewel, Mira, Kent and St Pierre) z Cultivars Volatile compound RT Jewel Mira Kent St. Pierre Esters Methyl butanoate 3.19 ND y 0.690.1a 0.390.1b 0.590.1a Methyl-3-methyl butanoate 3.96 ND 0.0290.01a 0.190.1a 0.190.04a Ethyl butanoate 4.34 0.490.04a 0.490.4a ND 0.290.1a Butyl acetate 4.57 0.590.1a 0.190.02b ND ND Propyl butanoate 5.04 ND ND ND 0.190.02a Ethyl-3-methyl butanoate 5.27 0.290.1a 0.190.02a 0.390.1a ND Isoamyl acetate 5.71 0.590.2a 0.190.02b 0.190.1b ND Methyl hexanoate 6.63 0.490.2a 1.9490.5b 0.290.1a 190.1bc Butyl butanoate 8.06 0.790.3a 0.290.2b 0.190.1b ND Ethyl hexanoate 8.11 1.790.4a 1.391.4a 2.291.5a 0.390.1a (z)-hexenyl acetate 8.25 0.290.1a ND 0.190.1a 0.190.2a 2-Hexyl acetate 8.37 0.890.4a 0.5 90.1ab 0.290.1b 0.190.02b Isopropyl hexanoate 8.83 ND 0.190.04a ND 0.290.1a Butyl-3-methyl-butanoate 9.06 ND 0.190.1a ND ND Isopentyl-2methyl-proponoate 9.25 1.190.6a 0.190.04b ND 0.290.04b Hexyl butanoate 11.83 0.790.6a 0.590.2a 0.290.1a 0.190.04a Hexyl butyrate 11.90 0.490.2a 0.190.1a 0.290.2a 0.190.04a Methyl salicylate 12.02 0.490.1a 0.490.2a ND 0.0490.02b Hexyl-3-methyl- butanoate 12.73 ND 0.0290.02 ND ND Isopentyl hexanoate 12.88 Traces 0.190.2a 0.190.02a 0.190.02a Octyl butanoate 15.26 ND 0.990.8 ND ND Octyl-2-methyl-butanoate 16.06 ND 0.490.1 ND ND Octyl hexanoate 18.26 ND 0.290.2 ND ND Heptyl butanoate 12.16 ND ND ND 0.190.02 (4E)-4-hexenyl butyrate 11.72 0.790.2 ND ND ND Terpenoids Linalool 10.10 0.590.1a 0.290.1b 0.190.02b 0.290.02b Nerolidol 18.0 0.290.2a 0.590.4a ND 0.190.01a Caryophyllene oxide 17.20 7.094.8a 10.192.8a 15.691.6b 15.690.2b Aldehydes (E)-2-hexenal 5.33 ND ND ND 0.190.1 (E)-2-nonenal 11.30 ND 0.190.02a ND 0.290.04a Ketone Gamma decalactone 16.64 3.390.2a 0.890.4b ND 0.590.1 Lactone 2-Heptanone 5.98 0.190.1 ND ND ND Furanones Mesifurane 9.32 0.390.2a 0.390.1a ND 0.190.02a Acid Hexanoic acid 7.80 0.290.2a ND ND 0.390.2 z Values (peak area of volatiles from 20 g of slurry) represents the percent relative area and were obtained from four independent analyses. y ND, not detected. ac Means9SD followed by same letter in the row are not significantly different at PB0.05 using Tukey s test.

120 CANADIAN JOURNAL OF PLANT SCIENCE esters included methyl hexanoate, ethyl hexanoate, hexyl butanoate, 2-hexyl acetate, hexyl butyrate and isopentyl hexanoate, of which ethyl hexanoate was the most prominent (Table 2). Aldehydes, 2-hexenal and 2-nonenal, were found in varying amounts among the cultivars. Interestingly, 2-hexenal was detected only in St. Pierre, but that was only about 0.3% of the total volatiles. Only Mira and St. Pierre had 2-nonenal as part of their volatiles. Only one lactone, gamma decalactone, an important strawberry aroma component, was identified in Jewel at a significantly higher amount, and it was also present in smaller amount in Mira and St. Pierre, but not in Kent. Another volatile component, 2-heptanone was detected only in Jewel. Hexanoic acid was identified in Jewel and St. Pierre. Mesifurane, which belongs to the furanones, and generally imparts fruity and caramel like characteristics to the fruit, was detected in Jewel, Mira and St. Pierre. The terpenoid, linalool was significantly higher in Jewel compared with the other cultivars. Effect of Preharvest Spray Treatment with Hexanal Containing Formulation on Phenolic Compounds of Strawberry Fruits Two strawberry cultivars (Jewel and Mira) were subjected to preharvest spray treatment with a hexanal-containing formulation. Phenolic compounds in control and hexanaltreated strawberry fruit (Table 3) were identified by high performance liquid chromatography electro-spray ionization mass spectrometry (HPLC-ESI-MS). Significantly higher pelargonidin-3-glucoside content of 26.5 mg 100 g 1 FW was detected in the spray treated fruit of Jewel than the control (21.5 mg 100 g 1 FW). But in Mira, similar levels of pelargonidin-3-glucoside were recorded both in the control as well as in the treated fruit. Increased pelargonidin-3-malonylglucoside content was detected in Jewel fruit sprayed with hexanal formulation than in the control. Flavonols were unaffected by hexanal formulation treatment in both cultivars. A significant decline in flavanol content in sprayed fruit compared with the control was detected in Jewel (Table 3). Ellagitannins also showed elevated levels in control fruit of Jewel, compared with the formulation-sprayed fruit. In Mira, Bis-HHDP glucose showed an increase in treated fruit compared with the control. Other phenolic compounds also increased with respect to spray treatment in Jewel. A decline in the level of other phenolic compounds in response to spray treatment was noticed in Mira. Effect of Preharvest Spray Treatment with Hexanal-containing Formulation on Volatile Compounds in Strawberry Fruits Table 4 depicts the changes in profiles of volatile components of strawberry cultivars in response to preharvest hexanal-containing formulation treatment. Decreased levels of several volatile components, including Table 3. Comparison of phenolic compounds of unsprayed (control) strawberries and those subjected to pre harvest spraying with hexanal containing formulation zy Jewel Phenolic compounds Control Treatment Control Treatment Anthocyanins Pg-3-glucoside 21.591.0a 26.591.8b 16.992.4c 16.891.2c Pg-3-malonylglucoside 19.791.0a 15.196.0a 15.591.6a 16.293.0a Pg-3-rutinoside Trace Trace 1.690.1a 1.790.3a Cy-3-glucoside Trace Trace 0.690.1a 0.690.6a Flavonols Q-3-malonylglucoside 0.890.4a 0.590.2a ND ND K-3-glucoside 0.590.2a 0.490.04a ND ND K3-glucuronide 0.490.3a 0.590.1a ND ND K3-malonylglucoside 1.390.1a 1.590.1a 0.890.1b 0.790.2b K3-coumaroylglucoside 0.590.6a 0.690.2a 0.790.3a 0.390.1a Flavanols PA dimer 6.390.3a 3.290.9b 5.690.3a 5.091.0a PA trimer 3.690.3a 1.890.8b 3.990.2a 3.590.4a Catechin 2.691.8a 1.490.8b 3.090.5b 2.590.3b Ellagitannins Bis-HHDP-glucose 6.190.2a 5.990.2a 4.191.3b 4.491.0b G-HHDP-glucose 4.691.2a 3.991.0a 7.091.8a 6.192.0a Other Compounds FA hexose derivatives 3.490.6a 4.690.6b 9.292.5c 8.891.5c Mira z Data are expressed as mean9sd of four replicate assays. y Data are expressed as mg 100 g 1 of fresh weight. Pg, pelargonidin; Cy, cyanidin; Q, quercetin; K, kaempferol; PA, proanthocyanidin; G, galloyl; HHDP, hexahydroxydiphenoyl; FA, ferulic acid; ND, not detected. ac Means followed by same letter in the same row are not significantly different at PB0.05 using Tukey s test.

MISRAN ET AL. * COMPOSITION OF PHENOLICS AND VOLATILES OF STRAWBERRY 121 esters, ketone, lactone, and acid group, were detected in Jewel fruit subjected to preharvest spray application compare with the control. Among the esters, compounds such as ethyl butanoate, butyl acetate, ethyl-3-methyl butanoate, isoamyl acetate, methyl hexanoate, ethyl hexanoate, hexyl acetate and hexyl butanoate were significantly affected by the treatment. Enhanced levels of butyl acetate, isoamyl acetate, ethyl hexanoate, hexyl acetate and hexyl butanoate were detected in control fruit of Jewel compared with the treated fruit. On the other hand, compounds such as ethyl butanoate, ethyl-3- methyl butanoate, methyl hexanoate, isopentyl-2-methyl propanoate were decreased by the hexanal formulation treatment and detected only in the control (Table 4). Twenty-two different esters were identified in Mira in comparison to only 13 in Jewel. Interestingly, ester profiles of treated fruit of Mira did not show much variation from the control. But, levels of ethyl butanoate and ethyl-3-methyl butanoate were significantly higher in fruit sprayed with hexanal formulation compared with the control. While ester compounds such as isopentyl hexanoate and octyl butanoate were higher in the control Table 4. Comparison of profiles of volatile compounds in unsprayed (control) strawberries and those subjected to pre harvest spraying with hexanal containing formulation z Jewel Volatile Compounds Control Treatment Control Treatment Esters Methyl butanoate ND y ND 0.490.1a 0.590.1a Methyl-3-methyl butanoate ND ND 0.190.01a 0.190.1a Ethyl butanoate 0.190.1a ND 0.390.1a 0.690.2b Butyl acetate 0.390.1a 0.190.1b 0.190.02a 0.190.02a Ethyl-3-methyl butanoate 0.190.1a ND 0.0290.02b 0.190.02a Isoamyl acetate 0.490.1a 0.290.2b 0.190.004b 0.0190.02c Methyl hexanoate 0.190.02a ND 1.390.3b 1.490.3b Butyl butanoate 0.190.1a 0.190.2a 0.190.1a 0.290.4a Ethyl hexanoate 0.390.1a 0.190.04b 1.490.4c 2.090.4c (z)-hexenyl acetate 0.390.1a 0.290.2a ND ND 2-Hexyl acetate 1.290.7a 0.590.7b 0.390.1b 0.390.04b Isopropyl hexanoate ND ND 0.290.1a 0.190.1a Butyl-3-methyl butanoate ND ND 0.190.02a 0.190.04a Isopentyl-2methylproponoate 0.490.1a ND 0.190.1a 0.190.02a Hexyl butanoate 0.590.2a 0.290.1b 0.490.1a 0.390.2a Hexyl butyrate 0.190.04a 0.390.3a 0.190.02a 0.190.04a Methyl salicylate 0.290.7a 0.290.1a 0.190.1a ND Hexyl-3-methyl butanoate ND ND 0.0290.04 0.0290.02 Isopentyl hexanoate ND ND 0.590.3 0.290.1 Octyl butanoate ND ND 0.990.2 0.690.3 Octyl-2-methyl butanoate ND ND 0.390.1 0.390.02 Octyl hexanoate ND ND 0.190.04 0.190.04 Total 4.1 1.9 5.94 7.23 Terpenoids Linalool 3.490.6a 1.791.0b 0.790.1c 0.790.1c Nerolidol 3.390.4a 1.790.1b 0.390.4c 1.592.4b Caryophyllene oxide 5.192.0a 14.792.6b 11.690.1b 10.093.2b Total 11.8 18.1 12.6 12.2 Aldehydes (E-Z)-2,6-nonadienal ND ND 0.190.04a 0.190.02a (E)-2-nonenal ND ND ND 0.190.04 Total 0.1 0.2 Ketone Gamma decalactone 3.992.6a 0.390.4b 0.490.1b 0.390.1b Lactone 2-Heptanone 0.190.1 ND ND ND Furanones Mesifurane 0.390.1a 0.390.2a 0.290.04a 0.390.1a Acid Hexanoic acid 0.490.3a 0.190.1b ND ND Mira z Values (peak area of volatiles from 20 g of slurry) represents the percent relative area and were obtained from four independent analyses. y ND, not detected. ac Means9SD followed by same letter in the row are not significantly different at PB0.05 using Tukey s test.

122 CANADIAN JOURNAL OF PLANT SCIENCE than the treated fruit, terpenoids, namely linalool and nerolidol, were lower in treated Jewel. However, the hexanal formulation treatment increased the levels of carophyllene oxide to nearly threefold. In Mira, the trend was reversed; the level of nerolidol was higher in treated fruit than in the control. Mesifurane was not significantly affected by the treatment in both cultivars. But in Jewel, gamma decalactone, 2-heptanone and hexanoic acid levels were significantly decreased in the hexanal formulation treatment compared with the control. However, these compounds in Mira were less affected by the hexanal treatment compared with Jewel. DISCUSSION The demand for strawberries and consequently strawberry production has increased steadily, primarily because of their nutritional value and high antioxidant potential (Henning et al. 2010). Characterization of polyphenolic profiles of four cultivars demonstrates cultivar variation in the composition and distribution of various polyphenolic compounds. This finding indicates that composition of phenolic compounds in strawberries can vary with genetic background. Several researchers have reported a wide variation in the composition and distribution of phenolic compounds among the different strawberry genotypes (Wang et al. 2002; Rekika et al. 2005; Wang et al. 2014). In the present study, anthocyanins were the most abundant phenolic compounds in all the cultivars, and pelargonidin-3-glucoside was identified as the prominent anthocyanin component in all cultivars tested. In line with these findings, several previous studies conducted with strawberry cultivars also reported that pelargonidin-3-glucoside was detected as the major anthocyanin component (Lo pez-da-silva et al. 2007; Buendı a et al. 2010; Aaby et al. 2012; Xie et al. 2014). Higher pelargonidin-3-glucoside content (4767 mg 100 g 1 FW) was observed in the present study compared with values of 1636 mg 100 g 1 FW in a previous study conducted in several strawberry cultivars (Buendı a et al. 2010). The content of pelargonidin- 3-malonylglucoside ranged from 7 mg 100 g 1 FW in St Pierre to 17 mg 100 g 1 FW in Jewel and Mira. According to Tulipani et al. (2008), the contribution of pelargonidin-3-malonylglucoside can vary between 0 and 15% of total anthocyanins. Cyanidin-3-glucoside content (1.63.0 mg 100 g 1 FW) was lower than those reported in other studies (0.54.2 mg 100 g 1 FW) (Lo pez-da-silva et al. 2007; Buendı a et al. 2010; Aaby et al. 2012). The proportion of pelargonidin-3-rutinoside observed in this study was lower than previously reported levels in other cultivars (Lo pez-da-silva et al. 2007). Another anthocyanin compound, 5-pyranopelargonidin-3-glucoside, was detected only in Kent. It was first identified in Camarosa (Andersen et al. 2004) and has been further characterized in Carisma, Oso Grande and Tudnew strawberry cultivars (Lo pez-da-silva et al. 2007). In strawberries, anthocyanins have been found to significantly contribute to the total antioxidative activity (Wang et al. 1996) and are primarily glycosides of pelargonidin and cyanidin (Lo pez-da-silva et al. 2002). Quercetin-3-glucuronide was the predominant flavonol; however, it was detected only in St. Pierre. Flavonol content and composition were significantly influenced by the cultivar, as noticed previously (Buendı a et al. 2010). In accordance with previous reports (Ma a tta-riihinen et al. 2004), quercetin and kaempferol dervatives were the flavonols identified in four cultivars. In general, the level of ellagitannins (4.78.4 mg) detected in this study was relatively lower than that in some previous reports. Mira showed the highest ellagitannin content and St. Pierre had the lowest. In a previous study, the range of galloyl-hhdp-glucose was 1.64.0 mg 100 g 1 FW (Buendı a et al. 2010), which is in agreement with our finding (2.83.5 mg 100 g 1 FW). Both compounds were also detected by Aaby et al. (2007), but not in the study of Seeram et al. (2006). Buendı a et al. (2010) reported ellagitannin values ranging from 9.7 to 23 mg 100 g 1 FW in cultivars grown in southern Spain. But the relative amount of ellagitannins constituted B9% of the total phenolic content in this study. Except Mira, p-coumaroyl hexose was identified in all cultivars (0.2 mg 100 g 1 FW). Concentration of p-coumaroyl hexose varied from 1.7 to 13.6 mg 100 g 1 FW in 27 strawberry cultivars studied (Aaby et al. 2012). A ferulic acid hexose derivative was also identified and it was relatively abundant in St. Pierre compared with Jewel. In a previous study, 0.62.1 mg 100 g 1 FW of ferulic acid hexose derivative was detected in 15 strawberry cultivars (Buendı a et al. 2010). It has also been shown that phenolic compounds play a crucial role in extending shelf life and enhancing the quality of fresh fruits by delaying senescence induced by oxidative-degradation (Khanizadeh et al. 2009). Therefore, preservation of antioxidant levels during the postharvest storage is an important option for supporting increased antioxidant intake. Volatile compounds are responsible for the unique aroma and flavor of fresh strawberries. Production of volatile compounds is one of the noticeable changes that occur during ripening of strawberry and is an important quality factor influencing the consumer acceptability. The unique flavor of strawberry fruits is mainly determined by a complex blend of esters, ketones, acids, aldehydes, alcohols, lactones and sulfur compounds (Perez et al. 1992; Jetti et al. 2007). Even though these volatiles comprise about 0.01 to 0.001% of the fresh fruit weight, they have a significant impact on the overall flavor of strawberry (Buttery 1981). Strawberry aroma is dependent on many factors, and studies report that genotypes or cultivar exhibit considerable variations in the profiles of the volatiles they produce (Forney et al. 2000). In this study, cultivars exhibited distinct differences in the chemical composition of volatiles. In the present study, the tentatively identified compound, caryophyllene oxide, was the most abundant volatile component in all cultivars. Caryophyllene oxide has been isolated from different plants (Jun et al. 2011;

MISRAN ET AL. * COMPOSITION OF PHENOLICS AND VOLATILES OF STRAWBERRY 123 Raja Rajeswari et al. 2011). It has been used as a preservative and has been tested in vitro for antibacterial and antifungal activity (Yang et al. 1999). Recently, caryophyllene oxide has shown cytoxicity against several cancer cell lines (Jun et al. 2011). Previous studies reported that esters comprised 2590% of the total number of volatiles in ripe strawberry fruit (Perez et al. 1992; Forney et al. 2000). Among the esters, butanoates comprised the predominant proportion of esters. Esters such as ethyl 2-methylbutanoate, hexyl acetate, methyl hexanoate and E-2-hexenyl acetates are considered as principal flavor components that contribute to the fruity note of strawberry aroma (Ulrich et al. 1997). While terpenes and sulfur compounds normally comprise less than 10 and 2%, respectively, both may contribute to aroma (Scherier 1980; Dirinck et al. 1981). Linalool, a common terpene alcohol was detected in all cultivars analyzed and it imparts a fruity, floral and berry -like aroma notes to strawberries (Du et al. 2011). The presence of nerolidol was found in Jewel, Mira and St. Pierre. Nerolidol was also found in five cultivars including Elsanta, Elvira, Dania, Pandora and Senga Sengana, but its presence was inconsistent (Larsen et al. 1992). Only one lactone, gamma decalactone known for its spicy, floral and fruity characteristics was identified in the present study. This is one of the key strawberry volatiles and has been found in high concentrations in some cultivars (Douillard et al. 1989). E-2-hexenal, which is thought to be responsible for green or fresh fruity aroma, was detected only in St Pierre at low concentration. A low concentration of 2-hexenal was also found in cultivated strawberries of Senga and Korona (Ulrich et al. 1995). Since SPME fiber has poor extraction efficiency for short-chain carboxylic acid, small molecules of volatile compounds in strawberry were difficult to detect (Jetti et al. 2007). All the cultivars except Kent showed the presence of mesifurane with no variation among them. Mesifurane is a furanone and it generally imparts the caramel-like, aroma to strawberry fruit. Several studies have identified the presence of mesifurane, although it is also not detected in many cultivars (Jetti et al. 2007). According to our findings, volatile profile of each cultivar was unique with respect to the composition and distribution of flavor components. Mira showed a remarkable volatile profile characterized by the presence of isopropyl hexanoate, octyl-2-methyl butanoate, octyl butanoate, hexyl-3-methyl butanoate, and butyl-3-methyl butanoate. St. Pierre was unique because it was the only cultivar to produce 2-hexenal, propyl butanoate and heptyl butanoate and only Jewel had 2-heptanone and (4E)- 4-hexenyl butyrate. The volatile profiles of strawberry are complex and vary depending on cultivars. There has been considerable debate as to which volatiles are the most important in producing the characteristic aroma of strawberries, but this is highly variable and their content depends mainly on stage of ripeness, varietal differences, climate and location (Larsen et al. 1992; Forney et al. 2000). Earlier research demonstrated the potential of formulations containing hexanal to enhance and preserve the shelf life and quality of perishable produce such as fruits, flowers and vegetables (Paliyath and Murr 2007; Paliyath and Subramanian 2008). Hexanal is a component of GRAS (generally regarded as safe) status and several lines of evidence suggest that hexanal treatment has been effective in maintaining the quality and shelf life of the fruits. Recently we have confirmed that in addition to phospholipase D inhibition, hexanal causes changes in gene expression, many of which are specific to delaying the production and action of ethylene, and inhibition of cell wall degradation (Tiwari and Paliyath 2011; Cheema et al. 2014; Padmanabhan et al., unpublished data). It appears that hexanal spray treatments can lead to metabolite channeling that leads to enhanced levels of quality enhancing ingredients. Hexanal strongly inhibited mesophilic bacteria at 4 o C under modified atmosphere packaging (Lanciotti et al. 1999) and also reduced the decay of apple fruit inoculated with conidia of Penicillium expansum (Fan et al. 2006). According to Utto et al. (2008), application of hexanal vapor reduced the infection of tomatoes by Botrytis cinerea whilst maintaining tomato quality after harvest. Thus hexanal treatments result in broad-spectrum benefits to fruits. While other ingredients in the present composition may not provide as direct an effect in enhancing shelf life as hexanal, they may provide additional protection (e.g., as free radical scavengers, or by physical protection of cell wall and membrane as induced by calcium salts), thus enhancing the effect of the formulation (Sharma et al. 2010; Cheema et al. 2014). As evidenced in the results of the present study, little variation in the profiles of phenolic compounds was detected between control and spray-treated fruits of Mira. On the other hand, some alteration in the profiles of polyphenolic components was noticed between control and spray-treated fruit of Jewel. In Jewel, pelargonidin-3- glucoside, the most prominent anthocyanin component was increased in response to formulation spraying, while pelargonidin-3-malonylglucoside level declined in treated fruit. Hexanal formulation used for spraying also contained cinnamic acid and it might have induced the synthesis of anthocyanins, especially pelargonidin-3-glucoside through UDP-glucose:flavonoid-3-O-glucosyltransferase (3-GT) activity in sprayed fruit. Sharma et al. (2010) observed that sweet cherries sprayed with hexanal containing EFF formulation showed bright red color in contrast to a purple red color of unsprayed fruits. The level of anthocyanins and some of phenolic compounds in sweet cherries were also maintained after spraying with hexanal formulation, up to 30 d in cold storage (Sharma et al. 2010). Strawberries treated with methyl jasmonate in conjunction with ethanol showed higher total phenolic and anthocyanin contents than ethanol-treated or untreated strawberries (Ayala-Zavala

124 CANADIAN JOURNAL OF PLANT SCIENCE et al. 2005). Another interesting finding is that a significant reduction in the levels of flavanols such as PA dimer, PA trimer, and catechin were detected in sprayed fruits compared with unsprayed fruit of Jewel. In strawberries, accumulation of flavanoids is a noticeable feature during the early stages of fruit development (Halbwirth et al. 2006). Preharvest spraying also increased the amount of ferulic acid hexose in the treated Jewel. Since strawberry aroma or flavor is a significant factor that affects consumer acceptability and organoleptic quality, preservation of aroma is of utmost importance during postharvest handling and shipping. Also, loss of desirable flavor can reduce the quality and marketability of fresh strawberries. Preharvest spray application of formulation containing hexanal resulted in a decrease in the abundance of several volatile components including esters, ketone, lactone, and acid group in treated Jewel than the control. Interestingly, esters such as ethyl butanoate, ethyl-3-methyl butanoate, methyl hexanoate, isopentyl-2-methyl propanoate were identified only in unsprayed fruits. Significant reduction in the levels of esters such as ethyl butanoate, butyl acetate, ethyl-3- methyl butanoate, isoamyl acetate, methyl hexanoate, ethyl hexanoate, hexyl acetate and hexyl butanoate was detected in sprayed fruits with respect to the control. In agreement with this observation, Ayala-Zavala et al. (2005) reported higher levels of methyl hexanoate and hexyl acetate in control than in strawberries treated with ethanol, methyl jasmonate, and methyl jasmonate ethanol vapors during storage. Similarly, Blanch and Ruiz del Castillo (2011) also noticed that some of the most important aroma volatiles; ethyl butanoate, butyl acetate, hexyl acetate, and 2,5-dimethyl-4-hydroxy-2-H-furan- 3-one were not affected by an ethanol/methyl jasmonate spray treatment. In contrast, levels of ethyl hexanoate and methyl hexanoate are enhanced with respect to treatments with ethanol/methyl jasmonate. High levels of gamma decalactone, 2-heptanone and hexanoic acid levels were present in the control compared with the sprayed berries. In Jewel, hexanal spraying resulted in enhancement in carophyllene oxide to nearly threefold compared with the control. Monoterpenes such as linalool, and sesquiterpenes such as nerolidol and carophyllene oxide are the key compounds in the aroma profile in strawberry (Zabetakis and Holden 1997). The increase in some of the terpenoid component such as caryophyllene oxide after hexanal treatment may contribute to a lowered degree of pathogen infections in strawberries (Paliyath, unpublished data). In Mira, only minor modifications to the volatile profile of spray treated fruit were detected in comparison with Jewel, which showed some significant changes. Enhanced levels of ethyl butanoate and ethyl-3-methyl butanoate were detected in response to spray treatment compared with the control. While ester compounds such as isopentyl hexanoate and octyl butanoate were higher in the control than the treatment. In fruits, increased volatile production is associated with ripeness. So a decrease in the level of volatile components with respect to spraying is indicative of a delay in the ripening process. Both phenolics and volatiles have nutritional as well as physiological functions within the fruit. An alteration in the ratio of sugars to acids (phenolic, TCA intermediates) can alter the disease susceptibility (more sugar) of fruits, as well as sensory perception. Similarly, some volatiles, such as caryophyllene derivatives (terpene derivatives), have antifungal properties, but also provide a strong spicy aroma to the fruits. While some of the ester volatiles may be reduced in some treated fruits (indicative of lower phospholipid degradation, fatty acid biosynthesis, or a reduction in respiration), not all fruits may respond this way. Thus, the responsiveness of fruits to hexanal formulation, and interactions of specific pathways that lead to the biosynthesis of phenolics and volatiles, can affect the organoleptic quality characteristics of treated fruits. To conclude, our study shows that variations existed in total phenolic and volatile components of fruits of selected strawberry cultivars. Preharvest spraying of fruits of Mira and Jewel with hexanal formulation altered the profiles of phenolics and volatiles. Experiments are underway to determine the effectiveness of preharvest spraying of hexanal formulation in prolonging the shelf life of strawberries of various cultivars. It would also be interesting to find out whether any relation exists between various polyphenolic fractions and the postharvest shelf life of strawberry. ACKNOWLEDGEMENTS This research was supported by the Ontario Ministry of Agriculture, Food and Rural Affairs and a visiting fellowship to Dr. Azizah Misran from the Government of Malaysia. The authors also thank Valsala Kallidumbil and Ramany Paliyath for their excellent technical assistance. Aaby, K., Mazur, S., Nes, A. and Skrede, G. 2007. Characterization of phenolic compounds in strawberry (Fragaria ananassa Duch.) fruits by HPLC detectors and contribution of individual compounds to total antioxidant capacity. J. Agric. Food Chem. 55: 43954406. Aaby, K., Mazur, S., Nes, A. and Skrede, G. 2012. Phenolic compounds in strawberry (Fragariaananassa Duch.) fruits: Composition in 27 cultivars and changes during ripening. Food Chem. 132: 8697. Andersen, O. M., Fossen, T., Torskangerpoll, K., Fossen, A. and Hauge, U. 2004. Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin. Phytochemistry 65: 405410. Ayala-Zavala, J. F., Wang, S. Y., Wang, C. Y. and Gonzalez-Aguilar, G. A. 2005. Methyl jasmonate in conjunction with ethanol treatment increases antioxidant capacity, volatile compounds and postharvest life of strawberry fruit. Eur. Food Res. Technol. 221: 731738. Blanch, G. P. and Ruiz del Castillo, M. L. 2011. Changes in strawberry volatile constituents after pre-harvest treatment with natural hormonal compounds. Flavour Fragr. J. 27: 180187.