Odorous volatiles and methoxypyrazines responsible for the musty-peanut aroma in microwave-heated sponge gourd (Luffa cylindrica)

Abstract

Microwave heating results in the generation of odorous compounds in sliced sponge gourd (Luffa cylindrica). Herein, the odorous components of microwave-heated sponge gourd were extracted, and the volatiles were separated by a solvent-assisted flavor evaporation technique. Subsequently, gas chromatography (GC)–olfactometry analysis identified 10 compounds in the aroma extract including unsaturated aliphatic aldehydes (3-hexenal; 2-octenal), alcohols (3-hexen-1-ol; 1-octen-3-ol), and ketones (1-octen-3-one; 1,5-octadien-3-one), which might be responsible for the typical green-grassy and metallic characteristics. Microwave heating could also promote the nonenzymatic browning reaction in the softened gourd to release methional and 2-acetyl-1-pyrroline, which have boiled potato and roasted-peanut odors, respectively. Furthermore, preparative GC–mass spectrometry was used to distinguish two methoxypyrazines (3,5-dimethyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine) responsible for a highly noticeable, unpleasant musty-peanut aroma in heated sponge gourd. Moreover, 3-isopropyl-2-methoxypyrazine might also generate an unlikeable earthy odor, resulting an off-flavor, and thus could be used as one of quality markers in sponge gourd.

Introduction

Sponge gourd (Luffa cylindrica), also called luffa or hechima in Japanese, is a highly fibrous vegetable in the cucumber (Cucurbitaceae) family. The plant is widely cultivated in tropical and subtropical latitudes, and its developed gourd fruit is commonly used as a soft-cooked vegetable in the cuisines of many Asian countries and regions, including Indonesia, Malaysia, Vietnam, Thailand, India, Nepal, China, and Japan (Joshi et al., 2004; Yadav et al., 2016). In Japan, sponge gourd grows well in Okinawa Island, the country's southernmost prefecture with a humid subtropical climate, and the demand for this gourd fruit has been increasing, with an average annual commercial production of approximately 1 673 tons from 2013 to 2018 (Department of Agriculture, Forestry and Fisheries, Okinawa Prefectural Government, 2019).

The aromas of foods, including vegetables, result from natural volatile compounds produced by various biosynthesis pathways as well as compounds generated during various treatments such as processing and preservation technologies (Asikin et al., 2016; Danowska-Oziewicz et al., 2020; Raffo et al., 2018). Dinitrogenated aromatic heterocyclic compounds such as methoxypyrazines are synthesized from amino acid precursors, and the corresponding methylated structures are formed through methyl transfer reactions catalyzed by methyltransferase enzymes (Lei et al., 2018). Most methoxypyrazines are noticeably odorous and thus considered as the sources of off-flavor and taint attributes in various foods, including cheese, wine, grape juice, and cooked potato. As the unlikeable musty odors of methoxypyrazines can lower overall flavor quality and acceptance, these compounds are regarded as key unpleasant odorants in food flavor studies (Chatonnet et al., 2010; Liaw et al., 2011.; Mosneaguta et al., 2012; Pickering et al., 2008). On the other hand, the Maillard browning reaction, a nonenzymatic reaction that occurs between amino acids and reducing sugars in the presence of heat, can also generate various odorous intermediates and aroma substances in foods (Cantergiani et al., 2001; Czerny and Grosch, 2000).

Depending on the temperature, heating changes not only the physical properties of foods but also their nutritional and biochemical compositions, which could alter the flavor quality (Asikin et al., 2016; Cantergiani et al., 2001; Mosneaguta et al., 2012). Microwave ovens, which generate electromagnetic radiation, have become commonly used in modern food processing at the household to industrial level, and heating via microwave radiation could also cause great impacts on the volatile aroma composition and sensory characteristic of foods (Danowska-Oziewicz et al., 2020; López et al., 2004). Our preliminary sensory evaluation study indicated that there was development of unpleasant odors, particularly musty aroma, in microwave-heated sponge gourd compared to that of its sliced uncooked form (data not shown). The aim of this study was thus to investigate the odorous volatiles in sliced sponge gourd after microwave heating, including the compounds responsible for a musty-peanut odor. The aroma compounds of heated sponge gourd were assessed using a gas chromatography-flame ionization detection/olfactometry (GC-FID/O) technique. The musty-peanut odor fraction was separated by preparative GC prior to structural identification by GC–mass spectrometry (MS). To the best of our knowledge, this is the first report using chromatographic analytical techniques to identify the volatile components that emit various odors including an unpleasant musty aroma in heated sponge gourd.

Materials and Methods

Sample preparation    Fresh sponge gourd fruits (Sazan variety) were obtained from a farmers' market in Yonabaru, Okinawa, Japan, in August 2018. The raw sponge gourd fruits were peeled and cut into 1 cm slices using a stainless-steel knife. Then, 150 g of cut flesh was placed in a heat-resistant glass plate, covered with plastic wrap, and then heated in a microwave at 500 W for 4 min. After cooling, the heated flesh was crushed and homogenized using an Ultra-Turrax mixer (T25, IKA Labortechnik, Staufen, Germany) for 1 min.

Aroma compound extraction    The aroma compounds were extracted from the homogenized crushed sponge gourd (150 g) by shaking with 200 mL of diethyl ether in a closed Erlenmeyer flask for 18 h at 5 °C. The mixture was transferred into a Teflon Oak Ridge centrifuge tube (Thermo Fisher Scientific, Waltham, MA, USA) and then centrifuged at 4 000 rpm for 10 min at 4 °C. Subsequently, the upper layer was filtered using Advantec No. 1 paper (Toyo Roshi Kaisha, Tokyo, Japan). The volatile aroma compounds in the extract were separated by a solvent-assisted flavor evaporation (SAFE) technique under vacuum (0.67 Pa) at 40 °C (Asikin et al., 2016; Engel et al., 1999). The volatile extract was dehydrated with 10 g of anhydrous sodium sulfate for 12 h at 5 °C and then filtered. Finally, the extract was concentrated to 100 µL using Vigreux column followed by Kuderna-Danish concentrator. The aroma extract was stored at −30 °C until analysis.

Odor assessment by GC-FID/O and compound identification by GC-MS    The compounds in the aroma extract were analyzed using a GC-FID system (Agilent 7890A, Agilent J&W, Santa Clara, CA, USA) equipped with a DB-WAX column (60 m × 0.25 mm, 0.25 µm, Agilent J&W) and an olfactory detection port (ODP) (Gerstel, Mülheim, Germany) (Asikin et al., 2016). The GC oven program was as follows: initial temperature of 40 °C for 2 min and then increased to 200 °C at a rate of 2 °C/min. The linear velocity of the helium carrier gas was maintained at 32 cm/s. The injection volume and split ratio were 1 µL and 1:5, respectively. The injector and FID temperatures were both 250 °C. The split ratio for the FID and ODP after column separation was 1:1.

The odor intensity was evaluated using direct intensity technique, and eluted odorant compounds were measured at the ODP by three trained assessors (2 males and 1 female, 21–43 years old). The assessors were selected for their sniffing ability, including odor intensity distinction, using a panel odor sensitivity screening test (Japan Association on Odor Environment). Four intensity levels of sniffed odors were recorded using an olfactory intensity device (Gerstel), as follows: very strong, 1 V; strong, 100 mV; moderate, 10 mV; and weak, 1 mV; and the intensity of assessed odors was finally perceived based on consensus among assessors.

Compounds were identified using a GC-MS system (Agilent 7890A-5975C, Agilent J&W) with the above-described column and GC parameters. For MS detection, the ionization energy was 70 eV, the ionization source and interface temperatures were both 230 °C, and the MS scan range (m/z) was 33–450 amu. Peak identification was performed by comparing the linear retention indices (RIs) with a homologous series of n-alkanes (C7–C20) and the MS fragmentation patterns with the National Institute of Standards and Technology (NIST) MS Spectral Library, Version 2008.

Musty-peanut odor fraction collection by preparative GC-MS/O and structural confirmation by GC-MS    The musty-peanut odor fraction was collected from the aroma extract using a preparative GC-MS/O system (Agilent 7890B-5977B) equipped with DB-WAX column (30 m × 0.32 mm, 0.50 µm, Agilent J&W), an ODP, and a Tenax-TA™ and thermal desorption unit (Tenax-TDU) separation line (Gerstel). The oven temperature was initially maintained at 60 °C for 2 min and then increased to 220 °C at a rate of 5 °C/min. The flow rate of the helium carrier gas was maintained at 1.2 mL/min. The injection volume was 1 µL in splitless mode, and the injector temperature was 250 °C. The odor fraction was collected in a Tenax-TDU tube over multiple elutions (10–20 times). The fraction containing the musty-peanut odor was then injected into another Agilent 7890B-5977B GC-MS system equipped with an HP-5MS column (30 m × 0.25 mm, 0.25 µm, Agilent J&W) for compound identification. Desorption from the TDU was carried out by increasing the temperature from 30 to 250 °C and then holding for 3 min, and the temperature of the cooled injection system was increased from −50 to 250 °C. The GC parameters were the same as those described above. For MS detection, the ionization energy was 70 eV, the ionization source and interface temperatures were both 230 °C, and the MS scan range (m/z) was 35–350 amu. Peak identification was performed by comparing the RIs and the MS fragmentation patterns with the MS spectral library and the spectra of coinjected standards. Authentic 3-isopropyl-2-methoxypyrazine and 3,5-dimethyl-2-methoxypyrazine standards were purchased from Sigma–Aldrich (St Louis, MO, USA) and Merck KGaA (Darmstadt, Germany), respectively.

Results and Discussion

The aroma extract of microwave-heated sponge gourd emitted a mixture of odorants that comprised green-grassy, metallic, burnt, roast, musty, boiled potato, and beany odors at different intensity levels. GC-FID/O analysis via high-polarity capillary DB-WAX column (Fig. 1a; Table 1) revealed a number of chromatographic peaks, three of which had high odor intensities corresponding to musty-peanut (Peak No. 7), boiled potato (Peak No. 8), and metallic beany (Peak No. 9). Peak Nos. 8 and 9 were identified as methional and 1-octen-3-ol, respectively, as confirmed by GC-MS. Although the chemical identity of Peak No. 7 was somewhat difficult to determine owing to a low MS fragment intensity and a weak FID signal, this compound was suspected to be a methoxypyrazine. Such compounds typically produce a strong unpleasant musty odor that could be responsible for an undesirable flavor in heated sponge gourd. Furthermore, the GC-FID/O analysis showed metallic-mushroom (1-octen-3-one; Peak No. 2) and burnt-roasted-peanut (2-acetyl-1-pyrroline; Peak No. 3) odors at moderate levels in heated sponge gourd. Additionally, weak green-grassy odors were derived from 3-hexenal and 3-hexen-1-ol (Peak Nos. 1 and 5, respectively) as well as a moderate metallic green-grassy odor from 1,5-octadien-3-one (Peak No. 4) and a simple grassy scent from 2-octenal (Peak No. 6).

Fig. 1.

(a) Typical GC-FID/O chromatogram (DB-WAX column) of microwave-heated sponge gourd aroma extract. Assessed aroma descriptions of peaks are given in Table 1. (b) Typical GC-MS/O chromatogram (HP-5MS column) of the musty-peanut odor fraction. Detailed aroma descriptions of the methoxypyrazines (Peak Nos. 7-1 and 7-2) are given in Table 2. The rectangles overlapping the chromatographic peaks indicate the panelist responses to sniffed odors in the ODP (base width = duration; height = intensity, as shown on the secondary y-axis).

Table 1. Assessed odorous volatiles of microwave-heated sponge gourd aroma extract

Peak No.a	Compoundb	RI	Aroma	Odor intensity
1	3-Hexenal	1141	Green-grassy	Weak
2	1-Octen-3-one	1302	Metallic mushroom	Moderate
3	2-Acetyl-1-pyrroline	1337	Burnt-roasted-peanut	Moderate
4	1,5-Octadien-3-one	1374	Metallic green-grassy	Moderate
5	3-Hexen-1-ol	1390	Green-grassy	Weak
6	2-Octenal	1426	Grassy	Moderate
7	Suspected methoxypyrazine	–	Musty-peanut	Strong
8	Methional	1453	Boiled potato	Strong
9	1-Octen-3-ol	1458	Metallic beany	Strong

^a  Typical GC-FID/O chromatogram is presented in Figure 1a.

^b  Compound was identified based on comparison of MS fragmentation pattern from GC-MS analysis with NIST MS Spectral Library and RI on DB-WAX column.

The compounds responsible for the musty odor of microwave-heated sponge gourd were definitively identified by collecting the relevant fraction through preparative GC-MS/O followed by GC-MS analysis via non-polar HP-5MS column and RI confirmation. In contrast to the use of DB-WAX column, GC-MS analysis through non-polar columns such HP-5MS which are loaded with (5%-phenyl)-methylpolysiloxane phase could separate the odorous compounds. The fraction comprised two methoxypyrazine compounds, namely, 3,5-dimethyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine (Peak Nos. 7-1 and 7-2, respectively), with unpleasant musty-peanut odors (Fig. 1b). The similarity indices of the MS fragmentation patterns of these two compounds in the MS library database were 93% and 97%, respectively. For 3,5-dimethyl-2-methoxypyrazine, a molecular ion base peak at m/z 138 [M] and characteristic ions at m/z 82 [M−56, loss of C₄H₈], m/z 109 [M−29, loss of CHO], and m/z 120 [M−18, loss of H₂O] were detected (Table 2). In contrast, 3-isopropyl-2-methoxypyrazine showed a fragment ion at m/z 152 [M] with characteristic ions at m/z 124 [M−28, loss of C₂H₄] and m/z 137 [M−15, loss of CH₃]. In addition to produce a musty-peanut odor, 3,5-dimethyl-2-methoxypyrazine also emitted chocolate, green-leafy, and beany aromas, whereas 3-isopropyl-2-methoxypyrazine might also be responsible for an unpleasant earthy odor in heated sponge gourd. The separation and structural identification results of the current study could be certainly used as important basis for further food technology studies such reducing the amounts of the unwanted odorous compounds, particularly methoxypyrazines, in processed sponge gourd.

Table 2. Aromas and characteristic ions of methoxypyrazines responsible for the musty-peanut odor of microwave-heated sponge gourd

Peak No.a	Compoundb	RI		CAS No.	Aroma	Characteristic ions (m/z)
		DB-WAX	HP-5MS
7-1	3,5-Dimethyl-2-methoxypyrazine	1430	1053	92508-08-2	Musty-peanut, chocolate, green-leafy, beany	82, 109, 120, 138
7-2	3-Isopropyl-2-methoxypyrazine	1437	1096	25773-40-4	Musty-peanut, earthy	124, 137, 152

^a  Typical GC-MS/O chromatogram is presented in Figure 1b.

^b  Compound was identified based on comparison of MS fragmentation pattern from GC-MS analysis with NIST MS Spectral Library and the spectra of coinjected standards, and RI on DB-WAX and HP-5MS columns.

Thus, 10 aroma compounds were detected in heated sponge gourd by GC-FID/O analysis. The volatiles could be intrinsically produced in the gourd through biogenesis pathways prior to heating but could also be generated during the heating process. Volatile aldehydes, alcohols, and ketones are mostly generated through natural biosynthesis during fruit development. The formation of hydrocarbon odorous substances in green gourd vegetables, including sponge gourd, could also be affected by genetic variations, pre- and postharvest environmental conditions, and postharvest treatments (López et al., 2004.; Raffo et al., 2018). The combination of six unsaturated aliphatic components, namely, 3-hexenal, 2-octenal, 3-hexen-1-ol, 1-octen-3-ol, 1-octen-3-one, and 1,5-octadien-3-one, which are typical flavor attributes in green vegetables (Seo and Baek, 2009), could provide the primary green-grassy and metallic characteristics of sponge gourd. Additionally, 1-octen-3-ol and 1-octen-3-one could provide particular beany and mushroom notes, respectively, in sponge gourd, similar to the aroma profiles of vegetables such as perilla and rocket leaves (Raffo et al., 2018; Seo and Baek, 2009). These unsaturated aliphatic volatiles could be generated at post-slicing process prior to heating that cause tissue injuries and cell damages, which initiate the activity of endogenous enzymes such lipoxygenase, and this enzymatic activity could also be further raised during initial stage of cooking (Raffo et al., 2018).

Microwave heating is a modern thermal processing method that is widely used for cooking vegetables by softening tissues in a relatively short time. However, this technique could also initiate various reactions in sponge gourd that produce not only compounds with likable flavors but also various putrid odors, resulting in an off-flavor. Methional, which is a primary product of the Strecker degradation reaction, might contribute to the distinct boiled potato notes of this gourd vegetable. Methional has also been reported to emit onion and sulfurous aromas that could cause unpleasant odors in microwave-heated sponge gourd (An et al., 2019; Villière et al., 2015). However, the perceived odor threshold of this methionine-derived compound is much higher than those of 3,5-dimethyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine (0.2 vs. 0.0004 and 0.0002 ppb, respectively) (Czerny and Grosch, 2000; Landaud et al., 2008; Young et al., 1996). Accordingly, these methoxypyrazines, which were characterized by high odor intensity levels and smaller odor thresholds, could be the major volatiles responsible for the unpleasant musty-peanut odor in microwave-heated sponge gourd (Fig. 1a).

The two odorous methoxypyrazines detected in heated sponge gourd could be derived from amino acid precursors such as leucine, phenylalanine, alanine, and valine, via methyltransferase-catalyzed methylation of the corresponding hydroxy pyrazine intermediates (Chatonnet et al., 2010; Lei et al., 2018). The concentrations of these amino acids in raw sponge gourd were 2.22, 2.69, 4.99, and 6.16 mg/100 g, respectively (Supplementary data, Table S1). Dinitrogenated aromatic heterocycles such as 3-isopropyl-2-methoxypyrazine are known to cause off-flavors in various food products, such as earthy tones in roasted coffee beans and ladybug taint in grape juice (Cantergiani et al., 2001; Pickering et al., 2008). This pyrazine derivative is also considered to produce undesirable musty, earthy, and green odors in bell pepper and Okinawan spinach (Shimizu et al., 2011; Wampler and Barringer, 2012). In contrast, 3,5-dimethyl-2-methoxypyrazine is a wine contaminant that emits fungal and corky aromas (Chatonnet et al., 2010). Nevertheless, the pleasant chocolate aroma of 3,5-dimethyl-2-methoxypyrazine as well as its typical green-leafy and beany odors could complement the application of microwave heating treatment to the softened gourd. Additionally, 2-acetyl-1-pyrroline, which was detected at a moderate level and is a product of the nonenzymatic Maillard browning reaction, could also generate burnt- and roasted-peanut aromas that might compensate to some extent for any unpleasant odors produced during the microwave processing of heated sponge gourd.

These findings are important for revealing the primary causes of the musty-peanut odor in microwave-heated sponge gourd. The identification of methoxypyrazines as responsible for the off-flavor of heated gourd will lead to further research on comparison of comprehensive volatile flavor profiles of fresh and cooked sponge gourd of different cooking practices, e.g. slicing, heating, and so on, as well as to practical efforts in developing cooking strategies when using sponge gourd as an ingredient with the intention of reducing or masking potent undesirable flavors. These unpleasant musty-peanut compounds could also be used as odorous markers in further research on improving microwave and other heating technology applications for the thermal processing of sponge gourd.

In conclusion, sponge gourd was found to contain several key odorous compounds that could be derived from both pre- and postharvest treatments, including combined effects of post-slicing enzymatic reaction and microwave heating. Unsaturated aliphatic aldehydes, alcohols, and ketones might contribute the typical green-grassy and metallic characteristics of sponge gourd. Additionally, methional might contribute to the boiled potato notes in heated sponge gourd. Moreover, musty-peanut odors were strongly detected from two methoxypyrazine compounds, namely, 3,5-dimethyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine. In particular, 3-isopropyl-2-methoxypyrazine might generate an unlikeable earthy odor, thus causing an off-flavor in microwave-heated sponge gourd.

Acknowledgements    We are grateful to Masahiro Horiuchi and Kumi Kasamatsu (Takata Koryo Co. Ltd.) for technical advice on preparative GC and MS confirmation. We thank Yasutaka Shimabukuro and Eriko Arakaki (University of the Ryukyus) for technical assistance. This work was supported by a grant for a commissioned project study (JPJ005336) from the Ministry of Agriculture, Fishery and Forestry of Japan.

Conflict of interest    There are no conflicts of interest to declare.

References

•  An,  K.,  Liu,  H.,  Fu,  M.,  Qian,  M.C.,  Yu,  Y.,  Wu,  J.,  Xiao,  G., and  Xu,  Y. (2019). Identification of the cooked off-flavor in heat-sterilized lychee (Litchi chinensis Sonn.) juice by means of molecular sensory science. Food Chem., 301, 125282.
•  Asikin,  Y.,  Hirose,  N.,  Tamaki,  H.,  Ito,  S.,  Oku,  H., and  Wada,  K. (2016). Effects of different drying–solidification processes on physical properties, volatile fraction, and antioxidant activity of non-centrifugal cane brown sugar. LWT-Food Sci. Technol., 66, 340-347.
•  Cantergiani,  E.,  Brevard,  H.,  Krebs,  Y.,  Feria-Morales,  A.,  Amadò,  R., and  Yeretzian,  C. (2001). Characterisation of the aroma of green Mexican coffee and identification of mouldy/earthy defect. Eur. Food Res. Technol., 212, 648-657.
•  Chatonnet,  P.,  Fleury,  A., and  Boutou,  S. (2010). Origin and incidence of 2-methoxy-3,5-dimethylpyrazine, a compound with a “fungal” and “corky” aroma found in cork stoppers and oak chips in contact with wines. J. Agric. Food Chem., 58, 12481-12490.
•  Czerny,  M. and  Grosch,  W. (2000). Potent odorants of raw Arabica coffee. Their changes during roasting. J. Agric. Food Chem., 48, 868-872.
•  Danowska-Oziewicz,  M.,  Narwojsz,  A.,  Draszanowska,  A., and  Marat,  N. (2020). The effects of cooking method on selected quality traits of broccoli and green asparagus. Int. J. Food Sci. Technol., 55, 127-135.
• Department of Agriculture, Forestry and Fisheries, Okinawa Prefectural Government. (2019). “Annual Report of Distribution of Horticultural Products in Okinawa Prefecture.” Department of Agriculture, Forestry and Fisheries, Okinawa Prefectural Government, Okinawa, pp. 1-41 (in Japanese).
•  Engel,  W.,  Bahr,  W., and  Schieberle,  P. (1999). Solvent assisted flavour evaporation – A new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur. Food Res. Technol., 209, 237-241.
•  Joshi,  B.K.,  Hari,  B.K.C.,  Tiwari,  R.K.,  Ghale,  M.,  Sthapit,  B.R., and  Upadhyay,  M.P. (2004). “Descriptors for sponge gourd [Luffa cylindrica (L.) Roem.]”. NARC, LIBIRD and IPGRI, Kathmandu.
•  Landaud,  S.,  Helinck,  S., and  Bonnarme,  P. (2008). Formation of volatile sulfur compounds and metabolism of methionine and other sulfur compounds in fermented food. Appl. Microbiol. Biotechnol., 77, 1191-1205.
•  Lei,  Y.,  Xie,  S.,  Guan,  X.,  Song,  C.,  Zhang,  Z., and  Meng,  J. (2018). Methoxypyrazines biosynthesis and metabolism in grape: A review. Food Chem., 245, 1141-1147.
•  Liaw,  I.W.,  Miracle,  R.E.,  Jervis,  S.M.,  Listiyani,  M.A., and  Drake,  M.A. (2011). Comparison of the flavor chemistry and flavor stability of mozzarella and cheddar wheys. J. Food Sci., 76, C1188-C1194.
•  López,  M.G.,  Guzmán,  G.R., and  Dorantes,  A.L. (2004). Solid-phase microextraction and gas chromatography–mass spectrometry of volatile compounds from avocado puree after microwave processing. J. Chromatogr. A, 1036, 87-90.
•  Mosneaguta,  R.,  Alvarez,  V., and  Barringer,  S.A. (2012). The effect of antibrowning agents on inhibition of potato browning, volatile organic compound profile, and microbial inhibition. J. Food Sci., 77, C1234-C1240.
•  Pickering,  G.J.,  Karthik,  A.,  Inglis,  D.,  Sears,  M., and  Ker,  K. (2008). Detection thresholds for 2-isopropyl-3-methoxypyrazine in Concord and Niagara grape juice. J. Food Sci., 73, S262-S266.
•  Raffo,  A.,  Masci,  M.,  Moneta,  E.,  Nicoli,  S.,  del Pulgar,  J.S., and  Paoletti,  F. (2018). Characterization of volatiles and identification of odor-active compounds of rocket leaves. Food Chem., 240, 1161-1170.
•  Seo,  W.H. and  Baek,  H.H. (2009). Characteristic aroma-active compounds of Korean perilla (Perilla frutescens Britton) leaf. J. Agric. Food Chem., 57, 11537-11542.
•  Shimizu,  Y.,  Imayoshi,  Y.,  Kato,  M.,  Maeda,  K.,  Iwabuchi,  H., and  Shimomura,  K. (2011). New eudesmane-type sesquiterpenoids and other volatile constituents from the roots of Gynura bicolor DC. Flavour Fragrance J., 26, 55-64.
•  Villière,  A.,  Le Roy,  S.,  Fillonneau,  C.,  Guillet,  F.,  Falquerho,  H.,  Boussely,  S., and  Prost,  C. (2015). Evaluation of aroma profile differences between sué, sautéed, and pan-fried onions using an innovative olfactometric approach. Flavour, 4, 24.
•  Wampler,  B. and  Barringer,  S.A. (2012). Volatile generation in bell peppers during frozen storage and thawing using selected ion flow tube mass spectrometry (SIFT-MS). J. Food Sci., 77, C677-C683.
•  Yadav,  B.S.,  Yadav,  R.,  Yadav,  R.B., and  Garg,  M. (2016). Antioxidant activity of various extracts of selected gourd vegetables. J. Food Sci. Technol., 53, 1823-1833.
•  Young,  W.F.,  Horth,  H.,  Crane,  R.,  Ogden,  T., and  Arnott,  M. (1996). Taste and odour threshold concentrations of potential potable water contaminants. Water Res., 30, 331-340.