Fruit Volatile Fingerprints Characterized among Four Commercial Cultivars of Thai Durian (Durio zibethinus)

Division of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangsue, Bangkok 10800, &ailand Division of Postharvest Technology, School of Bioresources and Technology, King Mongkut’s University of Technology &onburi, Bangkhuntien, Bangkok 10150, &ailand Postharvest Technology Innovation Center, Ministry of Higher Education, Science, Research, and Innovation, Bangkok 10400, &ailand


Introduction
In ailand, durian plant collection was firstly reported for 227 varieties. However, there currently are several cultivars, including "Chanee," "Kanyao," and "Monthong," in the business both in domestic and export markets [1]. "Chanee" comprises a moderate fruit size of 2.5-3 kg. e fruit shape shows swelling in the middle and is blunt at the blossom end with a big and short peduncle. When ripening, fruit is easily peeled, and the yellow flesh is a very soft fine texture, but with the thin flesh and an ample seed, it is famous for domestic markets. "Kanyao" bears a moderate fruit size of 3 kg, showing a round fruit shape and a big and long peduncle. e ripe flesh has smoothly fine texture and is yellow and sweet. "Monthong," the most famous variety, exhibits big fruit of 3-4 kg. Fruit is long, having shoulders at the stem end and protruding at the blossom end. e ripe flesh is dry and thick with a lean seed [2][3][4]. Ripe fruits of most typical durian varieties release a pungent solid smell, resulting in trouble for foreigners and under public assemblage. Recently, "Chanthaburi 1" (ICN × M 5-1-1), bred from "Chanee" and "Monthong," was officially approved and registered as a new variety by the Department of Agriculture, ailand, on 9 October 2006. e fruit is an early-season production with a harvesting time of 99-105 days after pollination. e average fruit weight is 2.5-3 kg, comprising bright yellow flesh and a sweet, delicate texture. e ripe fruit of "Chanthaburi 1" is claimed to have an extra-low smell [5]. Nevertheless, there is no analytical report yet for the volatile characterization of the fruit.
Aroma is a unique character of ripe durian fruit preferred by some but annoying for many people. Furthermore, it is seriously prohibited to take durian fruit/eat during public transportation or in assembly places such as hotels or convention halls. is matter would be a significant obstacle for the marketing of durians. Ethyl esters (fruity esters and general fruit) are the prominent esters in ripe "Monthong" flesh [6,7]. Nevertheless, this sweet smell is interrupted by sulfurous smells of sulfur-containing compounds. Ethanethiol, diethyltrisulfide, diethyldisulfide, dimethyl sulfide, 2,3butanedithiol, ethyl 1-methylethyl disulfide, 3-methyl-thiozolidine, methyl ethyl disulfide, and 1-propanethiol are such sulfur-containing compounds found in ripe durian flesh [6][7][8]. ere is no report of the relationship of durian cultivars by the aroma volatile so far. ere have been many reports of volatile components of ripe durians in "Monthong" [6][7][8][9], few in "Chanee" [6], but there is no report in "Kanyao" and "Chanthaburi 1." Furthermore, from the fruit's visual appearance, "Chanthaburi 1" fruit shape is very similar to the shape of "Kanyao," leading to confusion by visual appearance. us, fruit volatile profiles between the cultivars compared as volatile fingerprints were brought in the interest. Here, the present study was to identify odor characteristics of 4 commercial varieties. Volatiles of "Kanyao" and "Chanthaburi 1" were firstly reported, and the volatile relationship of these four varieties was then investigated.

Volatile Trapping.
e ripe aril of each cultivar was finely blended by using a high-speed homogenizer for 2 min. Homogenate at 5 g was put into a 20 mL glass vial sealed with a screw cap having a silicone laminated with polytetrafluoroethylene septum. e volatiles in the sample's headspace were trapped by SPME and analyzed by GC-MS modified from [10]. e volatiles in the headspace of the sample in a vial were trapped by solid-phase microextraction (SPME) coated with 65 µm of Polydimethylsiloxane/ Divinylbenzene (PDMS/DVB) (1 cm length), while heated at 50°C for 30 min.

Analysis of Volatiles in Ripe Durian Flesh.
e SPME was injected into a gas chromatogram (GC 6850 series, Agilent Technologies, USA), equipped with an HP-5MS column (5% phenyl-methylsiloane capillary column, 30m × 0.248 mm I.D. with 0.25 µm thickness) and an Agilent 5913 mass selective detector with the following condition: 200°C of the injection port (splitless mode), 50°C of the column oven for 1 min and increased at a rate of 5°C·min −1 to 120°C and then to 250°C at a 10°C·min −1 rate, and 250°C of the detector. Helium was the carrier gas set to 2 mL·min −1 at 15.9 psi. iophene at 10 µL·L −1 was used as the internal standard. e spectra of the volatile profile were analyzed in the electron impact (EI) mode with an electron energy of 70 eV; a mass range of m/z 45-450; a scan rate of 0.25 s/scan; and an electron multiplier (EM) voltage of 3000 V. Spectra of the volatile profile were compared to a mass spectral database from the NIST V.14 Llibrary values (Palisade Corp., Newfield, NY, USA). ere were 3 replications for each analysis.

Calculation of Volatile Compounds.
Each volatile compound of the clear peak from the GC-MS chromatogram was analyzed for the content compared to thiophene as the internal standard. Volatile content in ng thiophene per g fresh weight was estimated by the peak area of volatiles divided by the peak area of internal standard (thiophene) and 10 µL internal standard solution (0.5 g·L −1 thiophene) to 5 g durian homogenate prior to taking SPME [11].
Volatile content ng thiophene g − 1 FW � peak area of volatile/peak area of internal standard g durian aril homogenate . (1)

Journal of Food Quality
Odor activity value (OAV) was obtained by dividing the concentration of the compound in a matrix by its odor threshold in that matrix.
us, it is generally assumed that the odorants with higher OAVs contribute more strongly to the overall aroma. OAV of each volatile compound was calculated using the following formula [12]: OAV � concentration of the volatile content odor theshold value .
2.6. Statistical Analysis. e volatile relationship of durian cultivars was analyzed using principal component analysis (PCA) by Minitab ® program ver.17 (Minitab Ltd., UK). e contents and types of ester volatiles between cultivars were analyzed using multivariations of principal components by Minitab ® .

Journal of Food Quality
Ripe flesh of most durian cultivars contains ethyl-2methylbutanoate (fruity note) and ethyl hexanoate (fruity, apple, green, and tropical fruit odor) as the active volatiles showing high OAV. Both found in all four cultivars and most commercial durians were blended with some high-OAV compounds to characterize the flavor of each durian variety. In general, ripe durian flesh exhibits the fruity sweet fragrance of both compounds. "Chanthaburi 1" contained fewer components of volatiles as well as low OAV of the volatiles. is indicates that the flesh of "Chanthaburi 1" conducted very low intensity of odors during ripening. Ethyl heptanoate (fruity, pineapple, banana-like note) was found in every cultivar except "Chanthaburi 1." Ripe flesh of "Chanee" exhibited the strongest durian aroma among 4 varieties: "Chanee" exhibited aroma of ethyl octanoate (fruity, floral odor, wine apricot note), ethyl dodecanoate (waxy, soapy, nutty, rummy), and ethyl propanoate (green fruity, apple-like) characterized as nutty, rummy, and green apple-like, "Monthong" exhibited strong apple-like fruity, aldehydic, waxy fragrance of ethyl octanoate (fruity, floral odor, wine apricot note), ethyl hexanoate (apple-like, fruity), and methyl octanoate (waxy, green, sweet, orange, aldehydic vegetable), and "Kanyao" exhibited more complex waxy, nutty, green apple-like fruity aroma of ethyl octanoate, ethyl dodecanoate (waxy, soapy, nutty, rummy note), methyl octanoate (waxy, green, sweet orange), and ethyl propanoate (green fruity, apple-like).
Diethyl disulfide and 3,5-dimethyl-1,2,4-trithiolane found in low levels in ripe durian pulp are the key compounds in durians. Although sulfur-containing compounds exhibited low OAV, compared to the esters, they exhibit an annoying pungent smell. "Chanthaburi 1" as well as "Monthong" contained sulfur-containing compounds of diethyl disulfide and 3,5-dimethyl-1,2,4-trithiolane, which exhibit a garlic-like, onion-like, pungent smell [32]. In particular, diethyl disulfide in ripe "Monthong" showing an OAV of 1.25 would release the pungent smell of "Monthong" durian as reported by Laohakunjit et al. [8] and Niponsak et al. [9]. Previous studies in Malaysia and Indonesia found that the indigenous varieties exhibited a prominent smell of sulfur-containing compounds when fully ripe showing an unpleasant odor overall [32,33].
In 4 cultivars of ai durian fruit, ripe aril sharply produced a series of ethyl esters derived from ethyl alcohol and acyls CoA of straight carbons ranging from C 4 -C 10 ( Table 1). Ethanol in the aril could be generated from anaerobic respiration under a partial hypoxic condition in aril tissue. Due to very high respiration of durian fruit during ripening, fruit husk behaving like a gas barrier makes low gas permeability to the aril. Under partial hypoxia, anaerobic respiration was induced in the aril, resulting in increased  ethanol [34][35][36][37]. Aliphatic and aromatic alcohols are typically found in Malaysian durians, whereas thiols are produced in ai durians and alcohols are not typically produced in Indonesian and Filipino durians [37]. On the other hand, with a series of straight acyl CoA reacted with the ethanol, it is supposed that β-oxidation of fatty acids would be involved in the process of ripe fruits [38] as durian pulps have high contents of fatty acids such as methyl stearate (35.93%), methyl palmitate (32.91%), methyl palmitoleate (9.50%), methyl octadecenoate (4.86%), methyl oleate (4.68%), methyl myristate (2.52%), and methyl linoleate (2.20%) [39]. Furthermore, amino acid metabolism plays a crucial role in ester production in durians. For instance, ethyl-2-methylbutanoate, a primary volatile compound, is derived from 2-methylbutanoyl-CoA through isoleucine metabolism [40]. e origination of acyls CoA in the ester production could be separated into two sources from the results. When the acyl CoA of C 4 could be derived from amino acids, acyl CoA above C 6 could be from lipid oxidations. Furthermore, alcohol acyltransferase (AAT), which modifies alcohols and acyl CoA to esters, could be essentially involved in the production of esters in most durians. Although ATT has not yet been reported in durian, it was reported to be essential for ester production during ripening in many fruits [41][42][43]. However, as a result of fewer esters in "Chanthaburi 1," the production of esters is apparently disturbed in the fruit probably by mutant functioning of the AAT or the substrate-enzyme incompatibility.

e Relationship of Durian Cultivar Relied on Aroma
Volatiles. All 4 varieties showed that ethyl esters were the major components in the ripe flesh. Ethyl acetate and ethyl nonanoate were found in "Chanthaburi 1" but not in the parent, "Chanee" and "Monthong," whereas, on the other hand, ethyl butanoate and ethyl heptanoate found in the parent were not found in "Chanthaburi 1". Methyl-2-methylbutanoate was detected only in ripe "Monthong" flesh.
Ester compounds as the major volatiles were taken to calculate the relationship between cultivars. e differences in essential substances between durian species may be due to genetics and the environment. Genetic factors influence the formation of precursors, enzymes, and odor generation [44]. e durian of "Chanthaburi 1," a hybrid variety, has overall odor characteristics related to the parent variety, "Chanee," and the father species is "Monthong." Nevertheless, by considering the odor, "Chanthaburi 1" has a mild odor while still unripe, similar to the odor of "Kanyao." Although identifying the essential substances in "Chanthaburi 1" durian exhibited a more minor odor type than the strong aroma varieties, the essential substances (OAV) in the "Chanthaburi 1" exhibited characteristics related to both "Chanee" and "Kanyao." e relative content of the ester was obtained according to the dendrogram (Figure 2) of each essential substance. e volatile contents in "Chanthaburi 1" were related to "Monthong" when considering the ester composition. e ester compounds in "Chanthaburi 1" were correlated well with the "Monthong" variety, consistent with the species characteristics that 'Monthong' was the father. However, the relationship of ester compounds in "Chanee" was close to that in "Kanyao." Principal component analysis (PCA) using the ester compounds from Table 1 was operated to correlate and classify the essential components of the four durian varieties. Ester compounds were classified in the same component, with an eigenvalue greater than 1, and the component was equal to 2 (data not shown) with Minitab© 17 displayed in the score plot and biplot (Figure 3). e main component and the secondary components were associated with the ester compounds of the four durian varieties. When looking at the main components, "Chanthaburi 1" durian was related to "Chanee," and from the secondary, "Chanthaburi 1" was, on the other hand, related to the "Monthong" variety which corresponds to the ester characteristics of the parents. But, "Kanyao" has characteristics that are clearly different from those of "Chanthaburi 1" by both components. In addition, the ester characteristics of "Chanthaburi 1" as shown in Figure 3(c) were ethyl acetate, ethyl nonanoate, and methyl-2-methyl-2-butenoate, which exhibit a rum-like, grape, and cognac, as well as caramel note. For "Chanee," it can be seen from Figure 3(c) that the distinctive esters were propyl-2methylbutanoate, ethyl-2-methyl pentanoate, and methyl heptanoate showing winey, apple, pineapple, green, melon and waxy flavors, cognac rum wine, intensely fruity, and orris-like. In "Monthong," the ester characteristics were methyl-2-methylbutanoate, ethyl-2-hexenoate, and methyl-2-hexenoate. e scent characteristics are sweet fruity, apple-like odor, green, pineapple, apple, green, banana, honey. On the other side, "Kanyao" exhibited a distinctive scent of methyl hexanoate, propyl propanoate, and methyl dodecanoate, showing fruity, pineapple, complex fruity odor, apple and banana, waxy soapy, nutty, and coconut mushroom. When considering the OAV value of each durian species, if the OAV is greater than 1, it can be expected to exhibit a unique aroma. e OAV value of "Chanthaburi 1" was clearly similar to that of "Chanee," the mother variety, and close to that of "Kanyao" (Table 2 and Figure 4). e OAV values showed that "Chanthaburi 1" had the dominant esters, ethyl acetate (10.0) and ethyl nonanoate (225.7), which exhibited fruity, sweet, grape and rum-like, slightly fatty, oily, fruity, scent characteristics of nutty, reminiscent of cognac with a rosy fruity note. Nevertheless, "Kanyao" has outstanding OAV values of ethyl octanoate (4,241.9) and ethyl dodecanoate (4,138.9) at high, which is likely to be another distinctive scent, characterized by long stems showing fruity, fatty, floral odor (wine apricot note), waxy, sweet, musty, pineapple, dairy, sweet, waxy soapy rummy, and nutty floral. e distinctive OAV value is of methyl heptanoate (6.7) because it is found only in "Chanee," showing sweet, fruity, and green, with a waxy apple-like note. e higher levels of OAV were found in "Monthong" and "Kanyao" durians, but less common in "Chanthaburi 1" was ethyl octanoate (204.9), which showed a pleasantly fruity, floral odor (wine apricot note). e OAV values were different from the ester relative content, which was the relative content of the volatile compounds present in each durian species, indicating that "Chanthaburi 1" was Journal of Food Quality 7    consistent with "Monthong" and "Chanee," the father and mother, respectively. Nevertheless, if the OAV value was considered, "Chanthaburi 1" was close to "Kanyao" more than "Monthong" (Figure 4). According to the observation from the odor characteristics, the odor of "Chanthaburi 1" is mild, similar to that of "Kanyao," which the OAV value can explain to some extent. Based on information on the composition of these essential substances, it could greatly benefit the status of ai durians in terms of the choice of eating fresh fruit and the choices to use ripe durian pulp as an ingredient of food or dessert which requires the durian odor.
e study could increase the opportunities of ai durian transport channels to the world.

Conclusions
irty-three esters and three sulfur-containing compounds were the main volatiles found and affected the flavor character of the ripe pulp of four varieties of ai durians, "Chanee," "Monthong," "Kanyao," and "Chanthaburi 1." Ethyl esters were the major esters as ethyl-2methylbutanoate and ethyl hexanoate were the crucial essential substances found in all four varieties. e overall aroma character of the durian was a mixture combined of fruity-like apple/pineapple with rum, butter, oily, and waxy odors. Although ripe durians produced few sulfurcontaining volatiles, the compounds exhibit a sulfurous pungent smell. Using the volatile ester profiles, "Chanthaburi 1" correlated with "Chanee," the mother breed, and "Monthong," the father breed. "Kanyao" was different from "Chanthaburi 1." However, with high OAV values concerned, "Chanthaburi 1" was obviously associated with "Chanee," but the odor character was more similar to "Kanyao" than "Monthong."

Data Availability
e data used to support the findings of this study are included within the supplementary information file.

Conflicts of Interest
ere are no conflicts of interest in this study. ailand (NRCT) (Grant no. 04/2563). e authors appreciate the Postharvest Technology Innovation Center, Ministry of Higher Education, Science, Research, and Innovation, Bangkok, for providing them some scientific facilities.

Supplementary Materials
Supplementary Figure 1