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Analysis of free and bound aroma compounds of pomegranate (Punica granatum L.)
LWT - Food Science and Technology 59 (2014) 461e466
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Analysis of free and bound aroma compounds of pomegranate (Punica granatum L.) Jyoti Tripathi a, Suchandra Chatterjee a, Sunita Gamre b, Subrata Chattopadhyay b, Prasad S. Variyar a, *, Arun Sharma a a b
Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 September 2012 Received in revised form 28 May 2013 Accepted 28 May 2014 Available online 11 June 2014
Volatile aroma compounds were isolated from pomegranate arils by high vacuum distillation (HVD) and solvent extraction with diethyl ether. The HVD distillate exhibited a fresh-fruity and characteristic pomegranate aroma while the total ether extract was devoid of this note in its concentrate. Gas chromatographyemass spectroscopy (GCeMS) analysis revealed the presence of 3-octen-1-yl acetate, trans3-hexen-1-ol, hexanol and 2-methyl pentanol only in the high vacuum distillate. Ether extract was dominated by 2-heptanol, 2-nonanol and 3-methyl-2-butanol. Based on olfactometric analysis of the HVD isolate, 3-octen-1-yl acetate was identified as the key odorant of pomegranate. Chemical synthesis of this compound, further confirmed its structure. Among the bound aroma compounds, 2phenylethanol (40%), alpha-terpineol (4.53%) and 2-heptanol (6.35%) were identified as the major compounds existing as glycoconjugates. Identification of the character impact compound and the occurrence of glycosidic precursors in pomegranate are being reported here for the first time. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Punica granatum Volatile aroma compounds GC-O Bound aroma compounds 3-Octen-1-yl acetate
1. Introduction Pomegranate (Punica granatum L. cv. “Ganesh”) is a fruit of the family Puniaceae, extensively cultivated in several parts of the world (Adsule & Patil, 1995). The fruit enjoys a reputation for its healthy dietetic and medicinal properties. It is reported to be rich in vitamins, polysaccharides, polyphenols and minerals (Melgarejo, Salazar, & Artes, 2000). In the traditional Indian ayurvedic system of medicine, pomegranate has been extensively used for its curative properties (Jindal & Sharma, 2004). It is consumed as a fresh fruit and is also used in the preparation of fresh juices, canned beverages, jellies and jams and for flavoring and coloring drinks (Fadavi, Barzegar, Azizi, & Bayat, 2005). Fresh fruit is characterized by its low aromatic intensity, that is further lost during processing, thus impacting consumer acceptance. Low aroma intensity of the fruit poses difficulties in the isolation and identification of its aroma compounds. Hexanal, limonene, trans-2-hexenal, cis-2-hexenal and a-terpineol have been reported to be the major aroma compounds of the fruit (Melgarejo et al., 2011). 3-methyl butanal, ethyl butyrate, isopentyl acetate, hexanol, diethyl allyl malonate, alpha-
* Corresponding author. Tel.: þ91 22 25592531. E-mail addresses: [email protected], [email protected] (P.S. Variyar). http://dx.doi.org/10.1016/j.lwt.2014.05.055 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
ionone were identified as the major aroma components in pomegranate juices from Iran by using pervaporation process (Raisi, Aroujalian, & Kaghazchi, 2008). In a study on the aroma analysis of a mixture of pomegranate and berry juices, ethyl acetate, 3methyl butanal, 3-hydroxy-2-butanone, 2-methyl-1-butanol, hexanal, furfural, 3-hexen-1-ol, ethyl hexanoate, 3-hexen-1-yl acetate were identified as the compounds contributing to the odor of pomegranate (V azquez-Araújo, Chambers, Adhikari, & CarbonellBarrachina, 2010). Recently, 23 volatile compounds were reported in pomegranate juice extracted from P. granatum L. cv. “Wonderful” using head space-solid phase micro extraction comprising mainly zquez-Araújo, Chambers, aldehydes, alcohols and terpenes (Va Adhikari, & Carbonell-Barrachina, 2011). The aroma composition of this cultivar was shown to be dominated by terpene derivatives such as limonene, a-terpineol, (Z, Z)-a-farnesene and b-caryophyllene. However, to the best of our knowledge, no studies exist on the nature of the odor active compounds of the fruit. Besides free aroma, glycosidically bound aroma compounds have been reported to occur in several foodstuffs (Sarry & Gunata, 2004). Release of these compounds during storage or processing can result in modification or enhancement in the characteristic aroma (Ananthakumar, Variyar, & Sharma, 2006; Variyar, Ahmad, Bhat, Niyas, & Sharma, 2003). Glycoconjugates thus play a crucial role in the overall food quality. To the best of our knowledge, no report
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exists till date on glycosidically bound aroma compounds of pomegranate. The present work thus aims at isolation and identification of free and bound aroma constituents of pomegranate. An attempt has been made to identify key aroma active constituents based on GC-O using olfactometric analysis. The nature of these compounds was further identified using GC/MS. The chemical synthesis of most potent aroma compound, 3-octen-1-yl acetate was also accomplished to further confirm its structure and establish its role in imparting characteristic odor to the fruit. 2. Materials and methods 2.1. Materials Commercial samples of pomegranate (cv. Ganesh) were procured from a local market. It was identified as Ganesh cv. at Agriculture Station, Akola, Maharashtra, India. All the standard compounds were obtained from Sigma/Aldrich chemical company (St. Louis, MO, USA, 98% pure), except 3-octen-1-yl acetate, which was chemically synthesized in the laboratory. Solvents (analytical reagent grade) were redistilled before use. 2.2. Methods 2.2.1. Isolation of free aroma compounds 2.2.1.1. High vacuum distillation (HVD). High vacuum distillation was carried out according to the procedure reported in literature (Sharma, Chatterjee, Kumar, Variyar, & Sharma, 2010). Pomegranate arils (250 g) were frozen in liquid nitrogen before use. The sample was placed in a glass tube (25 cm 5 cm i.d) and then connected to a distillation unit maintained under vacuum (0.133 Pa, 25 C). The distillate collected in a receiving tube (20 cm 3 cm i.d) was maintained at low temperature under liquid nitrogen. The isolate (50 mL) was extracted with diethyl ether (3 5 mL). Solvent was removed by a slow stream of nitrogen to obtain the aroma concentrate that was subjected to GC/MS analysis. 2.2.1.2. Direct solvent extraction with ether. Diethyl ether is widely used for extraction of aroma volatiles in fruits, vegetables and spices. Moreover, most of the aroma compounds reported earlier in the pomegranate or its juices are readily soluble/miscible in the diethyl ether. Therefore it was used for extraction of free aroma volatiles of pomegranate. The pomegranate arils (350 g) were frozen with liquid nitrogen and then ground in a mortar and pestle. The ground arils were soaked in diethyl ether (1 L) to extract aroma compounds. The mass was allowed to attain room temperature and the slurry was filtered through a Whatman filter paper No.1. The filtrate containing a mixture of ether-water was transferred to a separating funnel and the organic layer was collected. The residue was re-extracted twice with the organic solvent (2 500 mL) as above. The ether layers were pooled together, dried over anhydrous sodium sulfate (Merck, Darmstadt, Germany) and then reduced to a small volume (10 mL) using Kuderna-Danish apparatus. The isolate was further concentrated by a slow stream of nitrogen to obtain the aroma concentrate prior to GC/MS analysis. 2.2.2. Analysis of free aroma compounds 2.2.2.1. GCeMS and GC-O analysis. The aroma concentrates obtained after ether extraction and HVD were subjected to GCeMS and GC-O analysis on a Shimadzu GCeMS instrument (Shimadzu Corporation, Kyoto, Japan) equipped with a GC-17A gas chromatograph, provided with a DB-5 (J&W Scientific, California, USA) ((5%-Phenyl)-methylpolysiloxane, length, 30 m; id., 0.25 mm and film thickness, 0.25 mm) or a 30 m, 0.25 mm and 0.25 mm film thickness DB-Wax (J&W Scientific, California, USA) fused-silica
capillary column and an olfactory detection port (ODP-2, Gerstel, Germany). A fixed splitter (1:1) was used at the end of capillary column. One part of column flow was directed to mass detector in gas chromatograph system, while the other part was sent to an olfactory detection port (ODP). A humidified air was introduced into the sniff port up-stream at 100 mL/min near the point where the capillary column first entered the sniffing port. The air carried the capillary column effluent into a glass funnel where sensory analysis was done. The transfer line to the GC-O sniffing port was held at 280 C. Helium was introduced as make up gas in the transfer line to sniffing port at 8 mL/min. Simultaneous sniffing at the exit port of ODP and corresponding response in the mass detector allowed identification of the compound responsible for the aroma perceived based on its mass fragmentation. The operating conditions were: column temperature programmed from 60 to 200 C at the rate of 4 C/min, held at initial temperature and at 200 C for 5 min and further to 280 C at the rate of 10 C/min, held at final temperature for 20 min; injector and interface temperatures, 210 and 280 C, respectively; carrier gas helium (flow rate, 0.9 mL/min); ionization voltage, 70 eV; electron multiplier voltage, 1 kV. Peaks corresponding to various aroma notes sniffed were identified by comparing their mass fragmentation pattern with that of standard spectra available in the spectral library (Wiley/NIST Libraries) of the instrument as well as based on their retention indices on the DB-5 or DB-Wax column previously reported (Adams, 1995; Chyau, Ko, Chang, & Mau, 2003; Ferrari et al., 2004; Goodner, 2008; Sharma et al., 2010; Wang, Wang, Li, Ye, & Kubota, 2010), odor quality and mass spectral data with those of standard compounds wherever available. Though HVD resulted in extraction of true aroma of pomegranate, the quantification of the identified compounds was not possible because of very low yield of the aroma extract obtained, limiting the sensitivity of the technique. The amount of the identified aroma compounds in direct ether extract were estimated from a standard curve (R2 ¼ 0.99) of concentration versus peak area prepared using different concentrations of standard acetoin (0.01e20 mg/mL) and expressed as mg/g of pomegranate arils. 2.2.2.2. Olfactometric analysis. Analysis was carried according to the conditions reported by Guen, Prost, and Demaimay (2000). A panel of 9 judges (4 females and 5 males, aged 25e50 years) trained in odor recognition and experienced in GC-O was selected. The testing room was at 24 ± 1 C and 50 ± 5% RH; the illumination was a combination of natural and non-natural (fluorescent) light. Panelists assigned odor properties to each note detected. The number of times a note was perceived by sensory panel was defined as its detection frequency. Detection of odor by fewer than 4 panelists was considered as noise (Van Ruth & Roozen, 1994). Sniffing was divided into two parts each of 12 min. Panelists were divided in two batches consisting of four and five people. Panelists involved in first 12 min were asked to sniff another half of the sniffing in next round. The second batch sniffed the remaining 12 min of the first round. In the second round of analysis, first batch of panelists analyzed the aroma notes eluting from 12 to 24 min while the second batch analyzed the aroma compounds eluting during 0e12 min. Each person participated in the sniffing of both parts but during two distinct sessions to avoid tiredness. A final aromagram of detection frequency versus RI was obtained after summing up all the nine individual aromagrams. 2.2.3. NMR analysis The NMR spectrum was recorded on a Bruker AC-200 MHz FT NMR spectrometer (Bruker, Fallanden, Switzerland), each sample being dissolved in CDCl3 containing TMS as the internal standard.
J. Tripathi et al. / LWT - Food Science and Technology 59 (2014) 461e466
2.2.4. Chemical synthesis 3-octen-1-yl acetate was synthesized chemically in three steps according to the procedures reported earlier (Brown & Ahuja, 1973; Vogel, 1989). The schematic of synthesis is depicted in Fig. 2 and the spectral details of products obtained at each step are given below: (i) 3-octyn-1-ol (A, 4 g, 44% yield). 1HNMR (CDCl3, 200 MHz): d 0.84-0.87 (8, 3H, t, J ¼ 6.8), 1.26-1.30 (7, 2H, m), 1.42-1.46 (6, 2H, m), 2.16-2.17 (5, 2H, m), 2.41-2.45 (2, 2H, m), 3.63-3.70 (1, 2H, t, J ¼ 6.2). EIMS: m/z (% rel. int.) 41 (57.7), 43 (18.7), 54 (100), 55 (54.4), 67 (51.1), 69 (31.2), 77 (16.6), 79 (22.4), 84 (60.5), 93 (21.5), 95 (20.5). (ii) 3-octene-1-ol (B, 0.85 g, 84% yield). 1HNMR (CDCl3, 200 MHz): d 0.84-0.87 (8, 3H, t, J ¼ 6.8), 1.26-1.30 (7, 2H, m), 1.421.46 (6, 2H, m), 2.16-2.17 (5, 2H, m), 2.41-2.45 (2, 2H, m), 3.63-3.70 (1, 2H, t, J ¼ 6.2). EIMS: m/z (% rel. int.) 41 (59.7), 43 (19.5), 54 (29.2), 55 (100), 67 (51.7), 68 (56.9), 81 (66.8), 82 (22.7), 95 (15.4), 110 (14.6). (iii) 3-octen-1-yl-acetate (C, 0.5 g, 75% yield). 1HNMR (CDCl3, 200 MHz): d 0.85-0.88 (8, 3H, t, J ¼ 6.9), 1.24-1.33 (7, 2H, m), 1.361.43 (6, 2H, m), 1.92-1.98 (5, 2H, m), 2.04 (9, 3H, s), 2.43-2.49 (2, 2H, m), 4.35-4.39 (1, 2H, t, J ¼ 7.4), 5.28-5.32 (4, 1H, dt, J ¼ 11.0, J ¼ 7.3), 5.33-5.37 (3, 1H, dt, J ¼ 11.0, J ¼ 7.3). EIMS: m/z (% rel. int.) 43 (100), 44 (12), 54 (58.8), 55 (20), 67 (30), 68 (31.2), 81 (36), 110 (22.8). 2.2.5. Reconstitution of the pomegranate aroma Diethyl ether solution (0.01 mL/mL) of standard aroma compounds as identified in high vacuum distillate (Table 1) was prepared. A synthetic pomegranate aroma was reconstituted by mixing these standard odorants in a proportion similar to that obtained by GC/MS. The total reconstituted mixture was evaluated by the trained sensory panel as described in Section 2.2.2.2 using filter paper strips (7 cm 0.5 cm) for assessing its resemblance to actual pomegranate aroma. The filter paper strips were dipped in the reconstituted aroma solution and assessors were asked to sniff and score it on a hedonic scale (1e9; 1 e not similar; 3 e weak, 5 e moderate, 7 e large, 9 e perfect matching) according to its extent of resemblance to actual pomegranate aroma as perceived in HVD isolate. It was further subjected to GC-O analysis as detailed above. 2.2.6. Isolation of aroma glycosides Pomegranate arils were separated manually from fresh pomegranate. Juice was extracted mechanically by squeezing arils (600 g)
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Fig. 2. Schematic representation of chemical reaction involved in the synthesis of 3octen-1-yl acetate. (A) 3-octyn-1-ol. (B) 3-octen-1-ol. (C) 3-octen-1-yl acetate. Reagents and conditions: (i) LiNH2/NH3/1-bromobutane/-78 C/4 h; (ii) H2/P-2 Ni/ethylenediamine/EtOH/25 C/6 h; (iii) Ac2O/pyridine/25 C/3 h.
in a blender. The aqueous solution so obtained was washed with diethyl ether to remove lipophilic substances including free aroma compounds and subsequently passed through an Amberlite XAD16 column according to the procedure reported earlier (Kumar et al., 2010). The bound aroma precursors were desorbed from the resin with methanol as eluting solvent, evaporated to dryness under vacuum and the residue was made up in methanol (0.02 g/ mL). 2.2.7. GCeMS analysis of bound aroma compounds The XAD extract was subjected to acid hydrolysis (1 N HCl, 1 h, 80 C). The hydrolysate was extracted with diethyl ether (3 20 mL) and made free of acid by repeated washing with distilled water. The organic layer was dried over sodium sulfate and then concentrated for subsequent identification of the aglycone moiety on GC/MS. Amounts of the volatile aroma compounds liberated from their glycosidic precursors were estimated from a standard curve (R2 ¼ 0.99) of concentration versus peak area prepared using different concentrations of 2-octanol (0.08e16 mg/mL) as external standard. The response factors of all the analyzed aroma compounds were assumed to be equal to one compared to that of 2octanol. The concentrations so determined on the basis of total ion current areas were expressed as ng/g of pomegranate arils. 2.2.8. Statistical analysis All data on the distribution of aroma compounds are a mean of three independent analyses, each analyzed in duplicate. Data are thus expressed as mean ± S.D. (n ¼ 6). 3. Results and discussion 3.1. Free aroma compounds
Fig. 1. Spider diagram representing the detection frequency of various aroma notes perceived by sensory panel in reconstituted aroma mixture and HVD isolate of pomegranate. ( HVD isolate; Reconstituted aroma).
The delicate odor of pomegranate prompted us to select mild techniques for isolation of its aroma constituents. In this regard, use of HVD as a method for isolating true aroma concentrate has been reported earlier (Gholap & Bandyopadhyay, 1977). Solvent extraction is another such technique widely used for the isolation of delicate fruit aroma (Cadwallader, Tamamoto, & Sajuti, 2010). These two methods were therefore selected in the present study. Table 1 lists the major aroma compounds identified by GC/MS in the high vacuum distillate, as well as, in the ether extract of the fruit accounting for 55.44% and 36.84%, respectively of the total isolate. The remaining constituents comprised of mainly hydrocarbons that have no significant contribution to the fruit aroma. Hence they are not listed in Table 1. The high vacuum distillate possessing a strong pomegranate odor was characterized by the presence of ethyl acetate, acetoin, trans-3-hexen-1-ol, 1-hexanol, 2-methyl pentanol, a-terpineol and 1-octen-3-yl acetate and heptadecanol. The C5, C6 and C8 alcohols and carbonyl compounds that dominate the aroma profile such as trans-3-hexen-1ol, 1-hexanol, 2-methyl pentanol imparting green odor to several
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Table 1 Relative distribution of aroma isolate obtained from High Vacuum Distillation (HVD) and direct solvent extraction with ether. S.No.
Compound
Rt (min) on DB-5
RIa (RIb)
RIc (RId)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
3-Methyl butanol Hexanal Ethyl acetate Acetoin Trans-3-hexen-1-ol 1-Hexanol 2-Heptanol 2-Methyl pentanol 2-Nonanone Limonene dioxide Nonanol Alpha terpineol Farnesol isomer A 3-Octen-1-yl acetate
3.564 3.765 3.867 3.992 5.033 5.408 6.278 14.025 14.390 16.050 18.033 18.223 20.313 21.253
765 799 806 812 855 868 896 1082 1090 1132 1181 1185 1239 1264
1190 1077 882 1270 1322 1335 1338 1365 1387 1440 1651 1691 2339 1725
15.
Heptadecanol
40.189
1872
(738) (800) (807) (812) (851) (867) (1085) (1091) (1134) (1171) (1189)
2518
(1208) (1088) (898) (1271) () (1362) (1310)
HVD (Relative % area)
± ± ± ±
3.49 ± 0.84
664.96 90.52 9625.27 680.00 e e 523.34 e 60.35 565.63 547.52 407.12 625.89 e
1.23 ± 0.57
534.8018 ± 63.24
0.13 0.96 9.11 27.31
± ± ± ±
0.01 0.18 1.76 3.61
0.35 ± 0.09 (1374) (1654) (1700) (2350)
Ether extract (mg/g of arils)
12.86 ± 1.69
20.32 15.32 89.76 58.50
Odor descriptione
fruity Buttery/creamy Grassy green/earthy Green-flowery
DFf
5 5 4
± 37.90 Sweet fruity green ± ± ± ± ±
8.45 18.88 29.04 22.05 31.63
Floral/liliac
7
Strong fruity/pomegranate
8
ID method MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,
RI RI, RI, RI, RI, RI, RI RI RI RI RI RI, RI RI,
standard standard standard standard standard
standard standard
MS
Data are the mean of six replicates ± standard deviation. MS: Mass Spectroscopy. a Experimental Retention index on DB-5 column. b Literature Retention index on DB-5 column, where available. c Experimental Retention index on DB-wax column. d Literature Retention index on DB-wax column, where available. e Odor description as perceived by panelists during olfactometry global analysis. f Detection frequency (of nine panelists) for HVD aroma isolate.
fruits and vegetables, are known to be derived from linoleic and linolenic acids (De Lumen, Stone, Kazeniac, & Forsythe, 1978). The presence of C6 alcohols and aldehydes such as cis-3-hexen-1-ol, hexanol, hexanal and E-2-hexenal as the main components has been reported in Spanish cultivars of pomegranate (Melgarejo et al., 2011). The presence of (Z)-3-hexenal and hexanal was also reported in pomegranate juices by Cadwallader et al. (2010), where they have demonstrated a decrease in green aroma note of juices during storage, as a result of decline in these compounds. Alpha terpineol was another major compound in the isolate. This compound having a floral/liliac odor has been reported earlier in pomegranate by other researchers (Melgarejo et al., 2011). In a recent report, alpha terpineol has been shown to be a major aroma constituent of pomegranate (P. granatum L. cv. Wonderful) juice zquez-Araújo et al., 2011). These workers have also shown a (Va positive correlation between the presence of alpha terpineol and consumer liking for juices made by the combination of pomezquez-Araújo et al., 2010). This granate with different berries (Va compound was also detected in high amounts in the HVD isolate in the present study. Acetoin is known to have an intense creamy, fatty and buttery odor and has been reported to be the major aroma active compound of dairy products like yoghurt (Ott, Fay, & Chaintreau, 1997). The role of this compound in contributing to the characteristic odor of ash gourd has been recently demonstrated (Sharma et al., 2010). The compound has been previously reported in pomegranate juices as an aroma constituent using HSzquez-Araújo et al., 2010). Other terpenes identified SPME (Va previously such as a-pinene, b-pinene, limonene, aephellandrene, g-terpinene, menthol, b-farnesene, were also proposed to zquezcontribute to the overall aroma of pomegranate juice (Va Araújo et al., 2011). These compounds were, however, not detected in the samples presently studied. This might be due to difference in extraction procedures used. Esters are extensively reported in fruits and vegetables where they play an important role in contributing to fruity notes. Ethyl acetate known to have sweet-fruity odor is widely reported to be an aroma component of several fruits and vegetables. The presence of this compound in pomegranate juices has been earlier reported by V azquez-Araújo et al. (2010). In their studies on juices prepared by
blend of pomegranate and berries such as blueberries, blackberries, or raspberries, ethyl acetate was a major aroma constituent obtained by Head Space-SPME in all the four fruits and was suggested to contribute to the typical sweet note of fresh juices. Some esters like 3-hexen-1-yl acetate, ethyl hexanoate, identified as a constituent of aroma composition of pomegranate aroma were, however, zquez-Araújo et al., 2010). 3not detected in the present study (Va octen-1-yl acetate possessing strong fruity/pomegranate seed odor has earlier been reported to be an important aroma constituent of purple variety of passion fruit contributing to its characteristic odor (Engel & Tressl, 1983). This compound has not been reported so far as an aroma component of pomegranate. The ether extract, possessing only a mild fruity odor, was distinguished by the absence of 3-octen-1-yl acetate, trans-3hexen-1-ol, hexanol and 2-methyl pentanol (Table 1). 2-heptanol, 2-nonanol, 3-methyl butanol, hexadecanoic acid, limonene dioxide, and farnesol detected in this isolate were, however, not present in the HVD distillate. Except for limonene dioxide with a sweet citrusy herbaceous odor, the higher alcohols present in the ether extract may have little contribution to the odor of this extract. Ethyl acetate and alpha-terpeniol present in both the extracts and possessing fruity and floral notes along with limonene dioxide may thus contribute to the fruity odor of the ether concentrate. While several compounds were detected in ether extract unlike HVD, the latter possessed a characteristic odor of the fruit that was not perceived in the concentrate obtained from the ether extract. This could be attributed to the possible loss of the odor impact components during work up of the large volume of the ether extract obtained, as a result of extensive loss of volatiles during concentration. The HVD isolate alone was therefore further evaluated for identification of odor active compounds. Despite reports on the aroma composition of pomegranate and its products, no attempt has been made till date to identify key odor contributing compounds of the fruit. Presence of 3-octen-1-yl acetate, trans-3hexen-1-ol, hexanol and 2-methyl pentanol only in HVD isolates that possessed characteristic pomegranate aroma suggests the role of these compounds in contributing to the odor notes of the fruit. Attempts were therefore made to identify the role of above compounds in pomegranate aroma.
J. Tripathi et al. / LWT - Food Science and Technology 59 (2014) 461e466
3.2. Identification of odor active compounds GC-O method based on detection frequency (Olfactometric analysis) was employed to characterize the aroma active compounds of pomegranate. This technique has been widely used for studying the most potent odorants in food and food products (Guen et al., 2000; Sharma et al., 2010). It is a rapid method and does not require trained panelists. HVD isolate was injected on to a DB-5 column connected to an olfactory detection port (ODP). In the present study, five odor active compounds were detected by at least four assessors. Table 1 presents odor descriptors perceived by the panel in HVD isolate in order of elution of the components through the column. Different aroma notes such as flowery, pomegranate peel, fruity, pomegranate seed and lemon were perceived at the sniffing port by a sensory panel made up of nine judges who were familiar with pomegranate aroma. The nine judges constituting the sensory panel agreed that the aroma of the isolate was typical pomegranate like. The odor detection frequency of HVD volatiles analyzed by GC-O is shown in Table 1. The highest detection frequency was obtained for peaks 12 (fruity-floral) and 14 (fruity/ pomegranate like) while other peaks 3 (fruity), 5 (grassy green/ earthy), 6 (green flowery) and 8 (sweet fruity/green) showed relatively lower frequencies. The peak corresponding to 3-octen-1yl acetate, eluting at Rt 21.77 min closely resembled the aroma perceived during chewing of pomegranate arils. The detection frequency of this note was quite high (Table 1). To further ascertain the identity of this peak, synthesized 3-octen-1-yl acetate was subjected to GC/MS and GC-O analysis under the same conditions as above. The synthetic 3-octen-1-yl acetate eluted at same Rt as in HVD isolate. Also the mass fragmentation pattern of synthetic 3octenyl acetate was similar to that of peak eluting at Rt 21.77 min. Thus 3-octen-1-yl acetate was identified as the major compound contributing to the characteristic odor of the fruit. In order to further confirm the role of the above compounds in contributing to the odor of pomegranate, a comparative sensory analysis of reconstituted pomegranate aroma mixture with HVD isolate was performed. Response of sensory panel clearly indicated a close resemblance of total reconstituted aroma to that of the isolate obtained by HVD (score 8 on hedonic scale). The detection frequencies of various notes in the HVD isolate and reconstituted mixture by sensory panel obtained from olfactometric analysis are represented as a spider graph in Fig. 1. It is clear from Fig. 1 that the detection frequencies of characteristic pomegranate aroma note in HVD isolate and reconstituted mixture are similar (8). This characteristic pomegranate aroma, perceived in a region corresponding to 3-octen-1-yl acetate, confirmed this compound as the key odorant of the fruit. The other aroma notes also had nearly same detection frequencies in both the samples studied, indicating the similarity between reconstituted mixture and HVD isolate. 3.3. Glycosidically bound aroma compounds Table 2 provides the distribution of aglycones liberated from the total bound fraction of pomegranate after hydrolysis. The compounds were identified by their kovats indices, comparison of their mass fragmentation pattern with the standard spectral library available in the GC/MS instrument and literature data which was further confirmed by available authentic standards. 2-phenyl ethyl alcohol accounted for 40% of the total bound aroma compounds. It possesses floral, rose like aroma and is an important volatile component of a variety of fruits and beverages, including muscat of bornova wines (Selli, Canbas, Cabaroglu, Erten, & Gunata, 2006), red wine vinegars (Charles et al., 2000) and lychee (Chyau et al., 2003). Other major aglycones existing as glycosides include alpha-terpineol (0.541 ng/g) and 2-heptanol (0.764 ng/g). Alpha
465
Table 2 Quantitative distribution of free volatile components obtained from bound aroma precursors. S.No.
Compound
RIa (RIb)
RIc (RId)
Concentration (ng/g of arils)
ID Method
1. 2.
2-Heptanol 2-Octanol
896 997 (997)
1338 (1310) 1352 (1332)
0.764 ± 0.381 0.023 ± 0.009
3.
2-Methyl benzaldehyde 2-Nonanol 2-Phenyl ethyl alcohol Alpha terpineol 2-Methoxy-4vinyl phenol
1125
1498
0.193 ± 0.021
MS, RI MS, RI, standard MS
1170 (1171) 1182
1651 (1654) 1902 (1901)
0.003 ± 0.001 3.792 ± 1.056
MS, RI MS, RI
1185 (1189)
1691 (1700)
0.541 ± 0.107
1313
2185 (2175)
0.008 ± 0.002
MS, RI, standard MS, RI
4. 5. 6. 7.
Data are the mean of six replicates ± standard deviation. a Experimental Retention index on DB-5 column. b Literature Retention index on DB-5 column, where available. c Experimental Retention index on DB-WAX column. d Literature Retention index on DB-WAX column where available.
terpineol, a monoterpene alcohol, with fruity odor is widely reported to be present in fruits and vegetables in free and bound form (Sharma et al., 2010). It has also been previously reported to be present in the volatile fraction of pomegranate juices (Melgarejo et al., 2011). 2-heptanol, possessing mushroom/earthy odor is reported to be present in grape musts and virgin olive oil (Gomez, Martinez, & Laencina, 1994; Morales, Luna, & Aparicio, 2005). It is reported to contribute to the aroma of some varieties of grape juices such as Monastrell and Cabernet Sauvignon (Morales et al., 2005). Other constituents existing as glycosides such as 2-methyl benzaldehyde (odor reminiscent of bitter almond), 2-octanol (fresh green, woody, earthy), 2-nonanol (fruity green, fatty odor), 2-methoxy-4-vinyl phenol (spicy/smoky) were also identified in the present study (Table 2). Many of them have also been reported to be present as their glycoconjugates in several fruits (Winterhalter & Skouroumounis, 1997). No compound possessing a typical odor of the pomegranate fruit was detected as its glycoside precursor. 4. Conclusion The present study establishes the role of 3-octen-1-yl acetate as the key aroma compound of pomegranate. The characteristic odor of the fruit was found to arise from a combination of 3-octen-1-yl acetate along with alpha terpineol, hexanol, ethyl acetate, trans3-hexen-1-ol and acetoin. Presence of glycosidically bound aroma precursors is reported here for the first time. The role of these compounds in the overall aroma of the fruit, if any, needs further investigation. References Adams, R. P. (1995). Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Illinois, USA: Allured Publishing Corporation. Adsule, R. N., & Patil, N. B. (1995). Handbook of fruit science and technology. In D. K. Salunkhe, & S. S. Kadam (Eds.), Pomegranate (pp. 455e464). New York: Marcel Dekker Publishers. Ananthakumar, A., Variyar, P. S., & Sharma, A. (2006). Estimation of aroma glycosides of nutmeg and their changes during radiation processing. Journal of Chromatography A, 1108, 252e257. Brown, C. A., & Ahuja, V. K. (1973). “P-2 nickel” catalyst with ethylenediamine, a novel system for highly stereospecific reduction of alkynes to cis-olefins. Journal of the Chemical Society, Chemical Communications, 553e554. http://dx.doi.org/ 10.1039/C39730000553. Cadwallader, K. R., Tamamoto, L. C., & Sajuti, S. C. (2010). Aroma components of fresh and stored pomegranate (Punica granatum) juice. Flavors in non carbonated beverages. In ACS symposium series (pp. 93e101).
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Charles, M., Martin, B., Ginies, C., Etievant, P., Coste, G., & Guichard, E. (2000). Potent aroma compounds of two red wine vinegars. Journal of Agricultural and Food Chemistry, 48, 70e77. Chyau, C. C., Ko, P. T., Chang, C. H., & Mau, J. L. (2003). Free and glycosidically bound aroma compounds in lychee (Litchi chinensis Sonn.). Food Chemistry, 80, 387e392. De Lumen, B. O., Stone, E. J., Kazeniac, S. J., & Forsythe, R. H. (1978). Formation of volatile compounds in green beans from linoleic and linolenic acids. Journal of Food Science, 43, 698e708. Engel, K. H., & Tressl, R. (1983). Formation of aroma components from nonvolatile precursors in passion fruit. Journal of Agricultural and Food Chemistry, 31, 998e1002. Fadavi, A., Barzegar, M., Azizi, M. H., & Bayat, M. (2005). Physicochemical composition of 10 pomegranate cultivars (Punica granatum) grown in Iran. Food Science and Technology International, 11, 113e119. Ferrari, G., Lablanquie, O., Cantagrel, R., Ledauphin, J., Payot, T., Fournier, N., et al. (2004). Determination of key odorant compounds in freshly distilled cognac using GC-O, GC-MS, and sensory evaluation. Journal of Agricultural and Food Chemistry, 52, 5670e5676. Gholap, A. S., & Bandyopadhyay, C. (1977). Characterisation of green aroma of raw mango (Magnifera indica L.). Journal of Science of Food and Agriculture, 28, 885e888. Gomez, E., Martinez, A., & Laencina, J. (1994). Localization of free and bound aromatic compounds among skin, juice and pulp fractions of some grape varieties. Vitis, 33, 1e4. Goodner, K. L. (2008). Practical retention index models of OV-101, DB-1, DB-5 and DB-wax for flavor and fragrance compounds. LWT e Food Science and Technology, 41, 951e958. Guen, S. L., Prost, C., & Demaimay, M. (2000). Critical comparison of three olfactometric methods for the identification of the most potent odorants in cooked mussels (Mytilus edulis). Journal of Agricultural and Food Chemistry, 48, 1307e1314. Jindal, K. K., & Sharma, R. C. (2004). Recent trends in horticulture in the Himalayas. Indus Publishing, ISBN 8173871620. Kumar, K. K., AnanthaKumar, A., Ahmad, R., Adhikari, S., Variyar, P. S., & Sharma, A. (2010). Effect of gamma-radiation on major aroma compounds and vanillin glucoside of cured vanilla beans (Vanilla planifolia). Food Chemistry, 122, 841e845. Melgarejo, P., Salazar, D., & Artes, F. (2000). Organic acids and sugars composition of harvested pomegranate fruits. European Food Research and Technology, 211, 185e190. zquez-Araújo, L., Hernandez, F., Martinez, J. J., Melgarejo, P., Sanchez, A. C., Va Legua, P., et al. (2011). Volatile composition of pomegranates from 9 Spanish
cultivars using head space solid phase microextraction. Journal of Food Science, 76, S114eS120. Morales, M. T., Luna, G., & Aparicio, R. (2005). Comparative study of virgin olive oil sensory defects. Food Chemistry, 91, 293e301. Ott, A., Fay, L. B., & Chaintreau, A. (1997). Determination and origin of the aroma impact compounds of yogurt flavor. Journal of Agricultural and Food Chemistry, 45, 850e858. Raisi, A., Aroujalian, A., & Kaghazchi, T. (2008). Multicomponent pervaporation process for volatile aroma compounds recovery from pomegranate juice. Journal of Membrane Science, 322, 339e348. Sarry, J. E., & Gunata, Z. (2004). Plant and microbial glycoside hydrolases: volatile release from glycosidic aroma precursors. Food Chemistry, 87, 509e521. Selli, S., Canbas, A., Cabaroglu, T., Erten, H., & Gunata, Z. (2006). Aroma components of cv. Muscat of Bornova wines and influence of skin contact treatment. Food Chemistry, 94, 319e326. Sharma, J., Chatterjee, S., Kumar, V., Variyar, P. S., & Sharma, A. (2010). Analysis of free and glycosidically bound compounds of ash gourd (Benincasa hispida): identification of key odorants. Food Chemistry, 122, 1327e1332. Van Ruth, S. M., & Roozen, J. P. (1994). Gas chromatography/sniffing port analysis and sensory evaluation of commercially dried bell peppers (Capsicum annuum) after rehydration. Food Chemistry, 51, 165e170. Variyar, P. S., Ahmad, R., Bhat, R., Niyas, Z., & Sharma, A. (2003). Flavoring components of raw Monsooned Arabica Coffee and their changes during radiation processing. Journal of Agricultural and Food Chemistry, 51, 7945e7950. zquez-Araújo, L., Chambers, E., IV, Adhikari, K., & Carbonell-Barrachina, A.A. Va (2010). Sensory and physicochemical characterization of juices made with pomegranate and blueberries, blackberries, or raspberries. Journal of Food Science, 75, S398eS404. zquez-Araújo, L., Chambers, E., IV, Adhikari, K., & Carbonell-Barrachina, A.A. Va (2011). Physico-chemical and sensory properties of pomegranate juices with pomegranate albedo and carpellar membranes homogenate. LWT e Food Science and Technology, 44, 2119e2125. Vogel, A. I. (1989). Aliphatic compounds. In B. S. Furniss, A. J. Hannaford, P. W. G. Smith, & A. R. Tatcheli (Eds.), Vogel’s textbook of practical organic chemistry (p. 513). England: Longman Scientific & Technical Publishers. Wang, X., Wang, D., Li, J., Ye, C., & Kubota, K. (2010). Aroma characteristics of cocoa tea (Camellia ptilophylla Chang). Bioscience Biotechnology and Biochemistry, 74, 946e953. Winterhalter, P., & Skouroumounis, G. (1997). Biotechnology of Aroma Compounds (pp. 73e105). Berlin, Germany: Springer.
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Analysis of free and bound aroma compounds of pomegranate (Punica granatum L.) Jyoti Tripathi a, Suchandra Chatterjee a, Sunita Gamre b, Subrata Chattopadhyay b, Prasad S. Variyar a, *, Arun Sharma a a b
Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 September 2012 Received in revised form 28 May 2013 Accepted 28 May 2014 Available online 11 June 2014
Volatile aroma compounds were isolated from pomegranate arils by high vacuum distillation (HVD) and solvent extraction with diethyl ether. The HVD distillate exhibited a fresh-fruity and characteristic pomegranate aroma while the total ether extract was devoid of this note in its concentrate. Gas chromatographyemass spectroscopy (GCeMS) analysis revealed the presence of 3-octen-1-yl acetate, trans3-hexen-1-ol, hexanol and 2-methyl pentanol only in the high vacuum distillate. Ether extract was dominated by 2-heptanol, 2-nonanol and 3-methyl-2-butanol. Based on olfactometric analysis of the HVD isolate, 3-octen-1-yl acetate was identified as the key odorant of pomegranate. Chemical synthesis of this compound, further confirmed its structure. Among the bound aroma compounds, 2phenylethanol (40%), alpha-terpineol (4.53%) and 2-heptanol (6.35%) were identified as the major compounds existing as glycoconjugates. Identification of the character impact compound and the occurrence of glycosidic precursors in pomegranate are being reported here for the first time. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Punica granatum Volatile aroma compounds GC-O Bound aroma compounds 3-Octen-1-yl acetate
1. Introduction Pomegranate (Punica granatum L. cv. “Ganesh”) is a fruit of the family Puniaceae, extensively cultivated in several parts of the world (Adsule & Patil, 1995). The fruit enjoys a reputation for its healthy dietetic and medicinal properties. It is reported to be rich in vitamins, polysaccharides, polyphenols and minerals (Melgarejo, Salazar, & Artes, 2000). In the traditional Indian ayurvedic system of medicine, pomegranate has been extensively used for its curative properties (Jindal & Sharma, 2004). It is consumed as a fresh fruit and is also used in the preparation of fresh juices, canned beverages, jellies and jams and for flavoring and coloring drinks (Fadavi, Barzegar, Azizi, & Bayat, 2005). Fresh fruit is characterized by its low aromatic intensity, that is further lost during processing, thus impacting consumer acceptance. Low aroma intensity of the fruit poses difficulties in the isolation and identification of its aroma compounds. Hexanal, limonene, trans-2-hexenal, cis-2-hexenal and a-terpineol have been reported to be the major aroma compounds of the fruit (Melgarejo et al., 2011). 3-methyl butanal, ethyl butyrate, isopentyl acetate, hexanol, diethyl allyl malonate, alpha-
* Corresponding author. Tel.: þ91 22 25592531. E-mail addresses: [email protected], [email protected] (P.S. Variyar). http://dx.doi.org/10.1016/j.lwt.2014.05.055 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
ionone were identified as the major aroma components in pomegranate juices from Iran by using pervaporation process (Raisi, Aroujalian, & Kaghazchi, 2008). In a study on the aroma analysis of a mixture of pomegranate and berry juices, ethyl acetate, 3methyl butanal, 3-hydroxy-2-butanone, 2-methyl-1-butanol, hexanal, furfural, 3-hexen-1-ol, ethyl hexanoate, 3-hexen-1-yl acetate were identified as the compounds contributing to the odor of pomegranate (V azquez-Araújo, Chambers, Adhikari, & CarbonellBarrachina, 2010). Recently, 23 volatile compounds were reported in pomegranate juice extracted from P. granatum L. cv. “Wonderful” using head space-solid phase micro extraction comprising mainly zquez-Araújo, Chambers, aldehydes, alcohols and terpenes (Va Adhikari, & Carbonell-Barrachina, 2011). The aroma composition of this cultivar was shown to be dominated by terpene derivatives such as limonene, a-terpineol, (Z, Z)-a-farnesene and b-caryophyllene. However, to the best of our knowledge, no studies exist on the nature of the odor active compounds of the fruit. Besides free aroma, glycosidically bound aroma compounds have been reported to occur in several foodstuffs (Sarry & Gunata, 2004). Release of these compounds during storage or processing can result in modification or enhancement in the characteristic aroma (Ananthakumar, Variyar, & Sharma, 2006; Variyar, Ahmad, Bhat, Niyas, & Sharma, 2003). Glycoconjugates thus play a crucial role in the overall food quality. To the best of our knowledge, no report
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exists till date on glycosidically bound aroma compounds of pomegranate. The present work thus aims at isolation and identification of free and bound aroma constituents of pomegranate. An attempt has been made to identify key aroma active constituents based on GC-O using olfactometric analysis. The nature of these compounds was further identified using GC/MS. The chemical synthesis of most potent aroma compound, 3-octen-1-yl acetate was also accomplished to further confirm its structure and establish its role in imparting characteristic odor to the fruit. 2. Materials and methods 2.1. Materials Commercial samples of pomegranate (cv. Ganesh) were procured from a local market. It was identified as Ganesh cv. at Agriculture Station, Akola, Maharashtra, India. All the standard compounds were obtained from Sigma/Aldrich chemical company (St. Louis, MO, USA, 98% pure), except 3-octen-1-yl acetate, which was chemically synthesized in the laboratory. Solvents (analytical reagent grade) were redistilled before use. 2.2. Methods 2.2.1. Isolation of free aroma compounds 2.2.1.1. High vacuum distillation (HVD). High vacuum distillation was carried out according to the procedure reported in literature (Sharma, Chatterjee, Kumar, Variyar, & Sharma, 2010). Pomegranate arils (250 g) were frozen in liquid nitrogen before use. The sample was placed in a glass tube (25 cm 5 cm i.d) and then connected to a distillation unit maintained under vacuum (0.133 Pa, 25 C). The distillate collected in a receiving tube (20 cm 3 cm i.d) was maintained at low temperature under liquid nitrogen. The isolate (50 mL) was extracted with diethyl ether (3 5 mL). Solvent was removed by a slow stream of nitrogen to obtain the aroma concentrate that was subjected to GC/MS analysis. 2.2.1.2. Direct solvent extraction with ether. Diethyl ether is widely used for extraction of aroma volatiles in fruits, vegetables and spices. Moreover, most of the aroma compounds reported earlier in the pomegranate or its juices are readily soluble/miscible in the diethyl ether. Therefore it was used for extraction of free aroma volatiles of pomegranate. The pomegranate arils (350 g) were frozen with liquid nitrogen and then ground in a mortar and pestle. The ground arils were soaked in diethyl ether (1 L) to extract aroma compounds. The mass was allowed to attain room temperature and the slurry was filtered through a Whatman filter paper No.1. The filtrate containing a mixture of ether-water was transferred to a separating funnel and the organic layer was collected. The residue was re-extracted twice with the organic solvent (2 500 mL) as above. The ether layers were pooled together, dried over anhydrous sodium sulfate (Merck, Darmstadt, Germany) and then reduced to a small volume (10 mL) using Kuderna-Danish apparatus. The isolate was further concentrated by a slow stream of nitrogen to obtain the aroma concentrate prior to GC/MS analysis. 2.2.2. Analysis of free aroma compounds 2.2.2.1. GCeMS and GC-O analysis. The aroma concentrates obtained after ether extraction and HVD were subjected to GCeMS and GC-O analysis on a Shimadzu GCeMS instrument (Shimadzu Corporation, Kyoto, Japan) equipped with a GC-17A gas chromatograph, provided with a DB-5 (J&W Scientific, California, USA) ((5%-Phenyl)-methylpolysiloxane, length, 30 m; id., 0.25 mm and film thickness, 0.25 mm) or a 30 m, 0.25 mm and 0.25 mm film thickness DB-Wax (J&W Scientific, California, USA) fused-silica
capillary column and an olfactory detection port (ODP-2, Gerstel, Germany). A fixed splitter (1:1) was used at the end of capillary column. One part of column flow was directed to mass detector in gas chromatograph system, while the other part was sent to an olfactory detection port (ODP). A humidified air was introduced into the sniff port up-stream at 100 mL/min near the point where the capillary column first entered the sniffing port. The air carried the capillary column effluent into a glass funnel where sensory analysis was done. The transfer line to the GC-O sniffing port was held at 280 C. Helium was introduced as make up gas in the transfer line to sniffing port at 8 mL/min. Simultaneous sniffing at the exit port of ODP and corresponding response in the mass detector allowed identification of the compound responsible for the aroma perceived based on its mass fragmentation. The operating conditions were: column temperature programmed from 60 to 200 C at the rate of 4 C/min, held at initial temperature and at 200 C for 5 min and further to 280 C at the rate of 10 C/min, held at final temperature for 20 min; injector and interface temperatures, 210 and 280 C, respectively; carrier gas helium (flow rate, 0.9 mL/min); ionization voltage, 70 eV; electron multiplier voltage, 1 kV. Peaks corresponding to various aroma notes sniffed were identified by comparing their mass fragmentation pattern with that of standard spectra available in the spectral library (Wiley/NIST Libraries) of the instrument as well as based on their retention indices on the DB-5 or DB-Wax column previously reported (Adams, 1995; Chyau, Ko, Chang, & Mau, 2003; Ferrari et al., 2004; Goodner, 2008; Sharma et al., 2010; Wang, Wang, Li, Ye, & Kubota, 2010), odor quality and mass spectral data with those of standard compounds wherever available. Though HVD resulted in extraction of true aroma of pomegranate, the quantification of the identified compounds was not possible because of very low yield of the aroma extract obtained, limiting the sensitivity of the technique. The amount of the identified aroma compounds in direct ether extract were estimated from a standard curve (R2 ¼ 0.99) of concentration versus peak area prepared using different concentrations of standard acetoin (0.01e20 mg/mL) and expressed as mg/g of pomegranate arils. 2.2.2.2. Olfactometric analysis. Analysis was carried according to the conditions reported by Guen, Prost, and Demaimay (2000). A panel of 9 judges (4 females and 5 males, aged 25e50 years) trained in odor recognition and experienced in GC-O was selected. The testing room was at 24 ± 1 C and 50 ± 5% RH; the illumination was a combination of natural and non-natural (fluorescent) light. Panelists assigned odor properties to each note detected. The number of times a note was perceived by sensory panel was defined as its detection frequency. Detection of odor by fewer than 4 panelists was considered as noise (Van Ruth & Roozen, 1994). Sniffing was divided into two parts each of 12 min. Panelists were divided in two batches consisting of four and five people. Panelists involved in first 12 min were asked to sniff another half of the sniffing in next round. The second batch sniffed the remaining 12 min of the first round. In the second round of analysis, first batch of panelists analyzed the aroma notes eluting from 12 to 24 min while the second batch analyzed the aroma compounds eluting during 0e12 min. Each person participated in the sniffing of both parts but during two distinct sessions to avoid tiredness. A final aromagram of detection frequency versus RI was obtained after summing up all the nine individual aromagrams. 2.2.3. NMR analysis The NMR spectrum was recorded on a Bruker AC-200 MHz FT NMR spectrometer (Bruker, Fallanden, Switzerland), each sample being dissolved in CDCl3 containing TMS as the internal standard.
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2.2.4. Chemical synthesis 3-octen-1-yl acetate was synthesized chemically in three steps according to the procedures reported earlier (Brown & Ahuja, 1973; Vogel, 1989). The schematic of synthesis is depicted in Fig. 2 and the spectral details of products obtained at each step are given below: (i) 3-octyn-1-ol (A, 4 g, 44% yield). 1HNMR (CDCl3, 200 MHz): d 0.84-0.87 (8, 3H, t, J ¼ 6.8), 1.26-1.30 (7, 2H, m), 1.42-1.46 (6, 2H, m), 2.16-2.17 (5, 2H, m), 2.41-2.45 (2, 2H, m), 3.63-3.70 (1, 2H, t, J ¼ 6.2). EIMS: m/z (% rel. int.) 41 (57.7), 43 (18.7), 54 (100), 55 (54.4), 67 (51.1), 69 (31.2), 77 (16.6), 79 (22.4), 84 (60.5), 93 (21.5), 95 (20.5). (ii) 3-octene-1-ol (B, 0.85 g, 84% yield). 1HNMR (CDCl3, 200 MHz): d 0.84-0.87 (8, 3H, t, J ¼ 6.8), 1.26-1.30 (7, 2H, m), 1.421.46 (6, 2H, m), 2.16-2.17 (5, 2H, m), 2.41-2.45 (2, 2H, m), 3.63-3.70 (1, 2H, t, J ¼ 6.2). EIMS: m/z (% rel. int.) 41 (59.7), 43 (19.5), 54 (29.2), 55 (100), 67 (51.7), 68 (56.9), 81 (66.8), 82 (22.7), 95 (15.4), 110 (14.6). (iii) 3-octen-1-yl-acetate (C, 0.5 g, 75% yield). 1HNMR (CDCl3, 200 MHz): d 0.85-0.88 (8, 3H, t, J ¼ 6.9), 1.24-1.33 (7, 2H, m), 1.361.43 (6, 2H, m), 1.92-1.98 (5, 2H, m), 2.04 (9, 3H, s), 2.43-2.49 (2, 2H, m), 4.35-4.39 (1, 2H, t, J ¼ 7.4), 5.28-5.32 (4, 1H, dt, J ¼ 11.0, J ¼ 7.3), 5.33-5.37 (3, 1H, dt, J ¼ 11.0, J ¼ 7.3). EIMS: m/z (% rel. int.) 43 (100), 44 (12), 54 (58.8), 55 (20), 67 (30), 68 (31.2), 81 (36), 110 (22.8). 2.2.5. Reconstitution of the pomegranate aroma Diethyl ether solution (0.01 mL/mL) of standard aroma compounds as identified in high vacuum distillate (Table 1) was prepared. A synthetic pomegranate aroma was reconstituted by mixing these standard odorants in a proportion similar to that obtained by GC/MS. The total reconstituted mixture was evaluated by the trained sensory panel as described in Section 2.2.2.2 using filter paper strips (7 cm 0.5 cm) for assessing its resemblance to actual pomegranate aroma. The filter paper strips were dipped in the reconstituted aroma solution and assessors were asked to sniff and score it on a hedonic scale (1e9; 1 e not similar; 3 e weak, 5 e moderate, 7 e large, 9 e perfect matching) according to its extent of resemblance to actual pomegranate aroma as perceived in HVD isolate. It was further subjected to GC-O analysis as detailed above. 2.2.6. Isolation of aroma glycosides Pomegranate arils were separated manually from fresh pomegranate. Juice was extracted mechanically by squeezing arils (600 g)
463
Fig. 2. Schematic representation of chemical reaction involved in the synthesis of 3octen-1-yl acetate. (A) 3-octyn-1-ol. (B) 3-octen-1-ol. (C) 3-octen-1-yl acetate. Reagents and conditions: (i) LiNH2/NH3/1-bromobutane/-78 C/4 h; (ii) H2/P-2 Ni/ethylenediamine/EtOH/25 C/6 h; (iii) Ac2O/pyridine/25 C/3 h.
in a blender. The aqueous solution so obtained was washed with diethyl ether to remove lipophilic substances including free aroma compounds and subsequently passed through an Amberlite XAD16 column according to the procedure reported earlier (Kumar et al., 2010). The bound aroma precursors were desorbed from the resin with methanol as eluting solvent, evaporated to dryness under vacuum and the residue was made up in methanol (0.02 g/ mL). 2.2.7. GCeMS analysis of bound aroma compounds The XAD extract was subjected to acid hydrolysis (1 N HCl, 1 h, 80 C). The hydrolysate was extracted with diethyl ether (3 20 mL) and made free of acid by repeated washing with distilled water. The organic layer was dried over sodium sulfate and then concentrated for subsequent identification of the aglycone moiety on GC/MS. Amounts of the volatile aroma compounds liberated from their glycosidic precursors were estimated from a standard curve (R2 ¼ 0.99) of concentration versus peak area prepared using different concentrations of 2-octanol (0.08e16 mg/mL) as external standard. The response factors of all the analyzed aroma compounds were assumed to be equal to one compared to that of 2octanol. The concentrations so determined on the basis of total ion current areas were expressed as ng/g of pomegranate arils. 2.2.8. Statistical analysis All data on the distribution of aroma compounds are a mean of three independent analyses, each analyzed in duplicate. Data are thus expressed as mean ± S.D. (n ¼ 6). 3. Results and discussion 3.1. Free aroma compounds
Fig. 1. Spider diagram representing the detection frequency of various aroma notes perceived by sensory panel in reconstituted aroma mixture and HVD isolate of pomegranate. ( HVD isolate; Reconstituted aroma).
The delicate odor of pomegranate prompted us to select mild techniques for isolation of its aroma constituents. In this regard, use of HVD as a method for isolating true aroma concentrate has been reported earlier (Gholap & Bandyopadhyay, 1977). Solvent extraction is another such technique widely used for the isolation of delicate fruit aroma (Cadwallader, Tamamoto, & Sajuti, 2010). These two methods were therefore selected in the present study. Table 1 lists the major aroma compounds identified by GC/MS in the high vacuum distillate, as well as, in the ether extract of the fruit accounting for 55.44% and 36.84%, respectively of the total isolate. The remaining constituents comprised of mainly hydrocarbons that have no significant contribution to the fruit aroma. Hence they are not listed in Table 1. The high vacuum distillate possessing a strong pomegranate odor was characterized by the presence of ethyl acetate, acetoin, trans-3-hexen-1-ol, 1-hexanol, 2-methyl pentanol, a-terpineol and 1-octen-3-yl acetate and heptadecanol. The C5, C6 and C8 alcohols and carbonyl compounds that dominate the aroma profile such as trans-3-hexen-1ol, 1-hexanol, 2-methyl pentanol imparting green odor to several
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Table 1 Relative distribution of aroma isolate obtained from High Vacuum Distillation (HVD) and direct solvent extraction with ether. S.No.
Compound
Rt (min) on DB-5
RIa (RIb)
RIc (RId)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
3-Methyl butanol Hexanal Ethyl acetate Acetoin Trans-3-hexen-1-ol 1-Hexanol 2-Heptanol 2-Methyl pentanol 2-Nonanone Limonene dioxide Nonanol Alpha terpineol Farnesol isomer A 3-Octen-1-yl acetate
3.564 3.765 3.867 3.992 5.033 5.408 6.278 14.025 14.390 16.050 18.033 18.223 20.313 21.253
765 799 806 812 855 868 896 1082 1090 1132 1181 1185 1239 1264
1190 1077 882 1270 1322 1335 1338 1365 1387 1440 1651 1691 2339 1725
15.
Heptadecanol
40.189
1872
(738) (800) (807) (812) (851) (867) (1085) (1091) (1134) (1171) (1189)
2518
(1208) (1088) (898) (1271) () (1362) (1310)
HVD (Relative % area)
± ± ± ±
3.49 ± 0.84
664.96 90.52 9625.27 680.00 e e 523.34 e 60.35 565.63 547.52 407.12 625.89 e
1.23 ± 0.57
534.8018 ± 63.24
0.13 0.96 9.11 27.31
± ± ± ±
0.01 0.18 1.76 3.61
0.35 ± 0.09 (1374) (1654) (1700) (2350)
Ether extract (mg/g of arils)
12.86 ± 1.69
20.32 15.32 89.76 58.50
Odor descriptione
fruity Buttery/creamy Grassy green/earthy Green-flowery
DFf
5 5 4
± 37.90 Sweet fruity green ± ± ± ± ±
8.45 18.88 29.04 22.05 31.63
Floral/liliac
7
Strong fruity/pomegranate
8
ID method MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,
RI RI, RI, RI, RI, RI, RI RI RI RI RI RI, RI RI,
standard standard standard standard standard
standard standard
MS
Data are the mean of six replicates ± standard deviation. MS: Mass Spectroscopy. a Experimental Retention index on DB-5 column. b Literature Retention index on DB-5 column, where available. c Experimental Retention index on DB-wax column. d Literature Retention index on DB-wax column, where available. e Odor description as perceived by panelists during olfactometry global analysis. f Detection frequency (of nine panelists) for HVD aroma isolate.
fruits and vegetables, are known to be derived from linoleic and linolenic acids (De Lumen, Stone, Kazeniac, & Forsythe, 1978). The presence of C6 alcohols and aldehydes such as cis-3-hexen-1-ol, hexanol, hexanal and E-2-hexenal as the main components has been reported in Spanish cultivars of pomegranate (Melgarejo et al., 2011). The presence of (Z)-3-hexenal and hexanal was also reported in pomegranate juices by Cadwallader et al. (2010), where they have demonstrated a decrease in green aroma note of juices during storage, as a result of decline in these compounds. Alpha terpineol was another major compound in the isolate. This compound having a floral/liliac odor has been reported earlier in pomegranate by other researchers (Melgarejo et al., 2011). In a recent report, alpha terpineol has been shown to be a major aroma constituent of pomegranate (P. granatum L. cv. Wonderful) juice zquez-Araújo et al., 2011). These workers have also shown a (Va positive correlation between the presence of alpha terpineol and consumer liking for juices made by the combination of pomezquez-Araújo et al., 2010). This granate with different berries (Va compound was also detected in high amounts in the HVD isolate in the present study. Acetoin is known to have an intense creamy, fatty and buttery odor and has been reported to be the major aroma active compound of dairy products like yoghurt (Ott, Fay, & Chaintreau, 1997). The role of this compound in contributing to the characteristic odor of ash gourd has been recently demonstrated (Sharma et al., 2010). The compound has been previously reported in pomegranate juices as an aroma constituent using HSzquez-Araújo et al., 2010). Other terpenes identified SPME (Va previously such as a-pinene, b-pinene, limonene, aephellandrene, g-terpinene, menthol, b-farnesene, were also proposed to zquezcontribute to the overall aroma of pomegranate juice (Va Araújo et al., 2011). These compounds were, however, not detected in the samples presently studied. This might be due to difference in extraction procedures used. Esters are extensively reported in fruits and vegetables where they play an important role in contributing to fruity notes. Ethyl acetate known to have sweet-fruity odor is widely reported to be an aroma component of several fruits and vegetables. The presence of this compound in pomegranate juices has been earlier reported by V azquez-Araújo et al. (2010). In their studies on juices prepared by
blend of pomegranate and berries such as blueberries, blackberries, or raspberries, ethyl acetate was a major aroma constituent obtained by Head Space-SPME in all the four fruits and was suggested to contribute to the typical sweet note of fresh juices. Some esters like 3-hexen-1-yl acetate, ethyl hexanoate, identified as a constituent of aroma composition of pomegranate aroma were, however, zquez-Araújo et al., 2010). 3not detected in the present study (Va octen-1-yl acetate possessing strong fruity/pomegranate seed odor has earlier been reported to be an important aroma constituent of purple variety of passion fruit contributing to its characteristic odor (Engel & Tressl, 1983). This compound has not been reported so far as an aroma component of pomegranate. The ether extract, possessing only a mild fruity odor, was distinguished by the absence of 3-octen-1-yl acetate, trans-3hexen-1-ol, hexanol and 2-methyl pentanol (Table 1). 2-heptanol, 2-nonanol, 3-methyl butanol, hexadecanoic acid, limonene dioxide, and farnesol detected in this isolate were, however, not present in the HVD distillate. Except for limonene dioxide with a sweet citrusy herbaceous odor, the higher alcohols present in the ether extract may have little contribution to the odor of this extract. Ethyl acetate and alpha-terpeniol present in both the extracts and possessing fruity and floral notes along with limonene dioxide may thus contribute to the fruity odor of the ether concentrate. While several compounds were detected in ether extract unlike HVD, the latter possessed a characteristic odor of the fruit that was not perceived in the concentrate obtained from the ether extract. This could be attributed to the possible loss of the odor impact components during work up of the large volume of the ether extract obtained, as a result of extensive loss of volatiles during concentration. The HVD isolate alone was therefore further evaluated for identification of odor active compounds. Despite reports on the aroma composition of pomegranate and its products, no attempt has been made till date to identify key odor contributing compounds of the fruit. Presence of 3-octen-1-yl acetate, trans-3hexen-1-ol, hexanol and 2-methyl pentanol only in HVD isolates that possessed characteristic pomegranate aroma suggests the role of these compounds in contributing to the odor notes of the fruit. Attempts were therefore made to identify the role of above compounds in pomegranate aroma.
J. Tripathi et al. / LWT - Food Science and Technology 59 (2014) 461e466
3.2. Identification of odor active compounds GC-O method based on detection frequency (Olfactometric analysis) was employed to characterize the aroma active compounds of pomegranate. This technique has been widely used for studying the most potent odorants in food and food products (Guen et al., 2000; Sharma et al., 2010). It is a rapid method and does not require trained panelists. HVD isolate was injected on to a DB-5 column connected to an olfactory detection port (ODP). In the present study, five odor active compounds were detected by at least four assessors. Table 1 presents odor descriptors perceived by the panel in HVD isolate in order of elution of the components through the column. Different aroma notes such as flowery, pomegranate peel, fruity, pomegranate seed and lemon were perceived at the sniffing port by a sensory panel made up of nine judges who were familiar with pomegranate aroma. The nine judges constituting the sensory panel agreed that the aroma of the isolate was typical pomegranate like. The odor detection frequency of HVD volatiles analyzed by GC-O is shown in Table 1. The highest detection frequency was obtained for peaks 12 (fruity-floral) and 14 (fruity/ pomegranate like) while other peaks 3 (fruity), 5 (grassy green/ earthy), 6 (green flowery) and 8 (sweet fruity/green) showed relatively lower frequencies. The peak corresponding to 3-octen-1yl acetate, eluting at Rt 21.77 min closely resembled the aroma perceived during chewing of pomegranate arils. The detection frequency of this note was quite high (Table 1). To further ascertain the identity of this peak, synthesized 3-octen-1-yl acetate was subjected to GC/MS and GC-O analysis under the same conditions as above. The synthetic 3-octen-1-yl acetate eluted at same Rt as in HVD isolate. Also the mass fragmentation pattern of synthetic 3octenyl acetate was similar to that of peak eluting at Rt 21.77 min. Thus 3-octen-1-yl acetate was identified as the major compound contributing to the characteristic odor of the fruit. In order to further confirm the role of the above compounds in contributing to the odor of pomegranate, a comparative sensory analysis of reconstituted pomegranate aroma mixture with HVD isolate was performed. Response of sensory panel clearly indicated a close resemblance of total reconstituted aroma to that of the isolate obtained by HVD (score 8 on hedonic scale). The detection frequencies of various notes in the HVD isolate and reconstituted mixture by sensory panel obtained from olfactometric analysis are represented as a spider graph in Fig. 1. It is clear from Fig. 1 that the detection frequencies of characteristic pomegranate aroma note in HVD isolate and reconstituted mixture are similar (8). This characteristic pomegranate aroma, perceived in a region corresponding to 3-octen-1-yl acetate, confirmed this compound as the key odorant of the fruit. The other aroma notes also had nearly same detection frequencies in both the samples studied, indicating the similarity between reconstituted mixture and HVD isolate. 3.3. Glycosidically bound aroma compounds Table 2 provides the distribution of aglycones liberated from the total bound fraction of pomegranate after hydrolysis. The compounds were identified by their kovats indices, comparison of their mass fragmentation pattern with the standard spectral library available in the GC/MS instrument and literature data which was further confirmed by available authentic standards. 2-phenyl ethyl alcohol accounted for 40% of the total bound aroma compounds. It possesses floral, rose like aroma and is an important volatile component of a variety of fruits and beverages, including muscat of bornova wines (Selli, Canbas, Cabaroglu, Erten, & Gunata, 2006), red wine vinegars (Charles et al., 2000) and lychee (Chyau et al., 2003). Other major aglycones existing as glycosides include alpha-terpineol (0.541 ng/g) and 2-heptanol (0.764 ng/g). Alpha
465
Table 2 Quantitative distribution of free volatile components obtained from bound aroma precursors. S.No.
Compound
RIa (RIb)
RIc (RId)
Concentration (ng/g of arils)
ID Method
1. 2.
2-Heptanol 2-Octanol
896 997 (997)
1338 (1310) 1352 (1332)
0.764 ± 0.381 0.023 ± 0.009
3.
2-Methyl benzaldehyde 2-Nonanol 2-Phenyl ethyl alcohol Alpha terpineol 2-Methoxy-4vinyl phenol
1125
1498
0.193 ± 0.021
MS, RI MS, RI, standard MS
1170 (1171) 1182
1651 (1654) 1902 (1901)
0.003 ± 0.001 3.792 ± 1.056
MS, RI MS, RI
1185 (1189)
1691 (1700)
0.541 ± 0.107
1313
2185 (2175)
0.008 ± 0.002
MS, RI, standard MS, RI
4. 5. 6. 7.
Data are the mean of six replicates ± standard deviation. a Experimental Retention index on DB-5 column. b Literature Retention index on DB-5 column, where available. c Experimental Retention index on DB-WAX column. d Literature Retention index on DB-WAX column where available.
terpineol, a monoterpene alcohol, with fruity odor is widely reported to be present in fruits and vegetables in free and bound form (Sharma et al., 2010). It has also been previously reported to be present in the volatile fraction of pomegranate juices (Melgarejo et al., 2011). 2-heptanol, possessing mushroom/earthy odor is reported to be present in grape musts and virgin olive oil (Gomez, Martinez, & Laencina, 1994; Morales, Luna, & Aparicio, 2005). It is reported to contribute to the aroma of some varieties of grape juices such as Monastrell and Cabernet Sauvignon (Morales et al., 2005). Other constituents existing as glycosides such as 2-methyl benzaldehyde (odor reminiscent of bitter almond), 2-octanol (fresh green, woody, earthy), 2-nonanol (fruity green, fatty odor), 2-methoxy-4-vinyl phenol (spicy/smoky) were also identified in the present study (Table 2). Many of them have also been reported to be present as their glycoconjugates in several fruits (Winterhalter & Skouroumounis, 1997). No compound possessing a typical odor of the pomegranate fruit was detected as its glycoside precursor. 4. Conclusion The present study establishes the role of 3-octen-1-yl acetate as the key aroma compound of pomegranate. The characteristic odor of the fruit was found to arise from a combination of 3-octen-1-yl acetate along with alpha terpineol, hexanol, ethyl acetate, trans3-hexen-1-ol and acetoin. Presence of glycosidically bound aroma precursors is reported here for the first time. The role of these compounds in the overall aroma of the fruit, if any, needs further investigation. References Adams, R. P. (1995). Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Illinois, USA: Allured Publishing Corporation. Adsule, R. N., & Patil, N. B. (1995). Handbook of fruit science and technology. In D. K. Salunkhe, & S. S. Kadam (Eds.), Pomegranate (pp. 455e464). New York: Marcel Dekker Publishers. Ananthakumar, A., Variyar, P. S., & Sharma, A. (2006). Estimation of aroma glycosides of nutmeg and their changes during radiation processing. Journal of Chromatography A, 1108, 252e257. Brown, C. A., & Ahuja, V. K. (1973). “P-2 nickel” catalyst with ethylenediamine, a novel system for highly stereospecific reduction of alkynes to cis-olefins. Journal of the Chemical Society, Chemical Communications, 553e554. http://dx.doi.org/ 10.1039/C39730000553. Cadwallader, K. R., Tamamoto, L. C., & Sajuti, S. C. (2010). Aroma components of fresh and stored pomegranate (Punica granatum) juice. Flavors in non carbonated beverages. In ACS symposium series (pp. 93e101).
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