Drying Treatments Change the Composition of Aromatic Compounds from Fresh to Dried Centennial Seedless Grapes

Raisin aroma is a vital sensory characteristic that determines consumers’ acceptance. Volatile organic compounds (VOCs) in fresh grapes, air-dried (AD), pre-treated air-dried (PAD), sun-dried (SD), and pre-treated sun-dried (PSD) raisins were analyzed, with 99 and 77 free- and bound-form compounds identified in centennial seedless grapes, respectively. The hexenal, (E)-2-hexenal, 1-hexanol, ethyl alcohol, and ethyl acetate in free-form while benzyl alcohol, β-damascenone, gerenic acid in bound-form were the leading compounds. Overall, the concentration of aldehydes, alcohols, esters, acids, terpenoids, ketones, benzene, and phenols were abundant in fresh grapes but pyrazine and furan were identified in raisin. Out of 99 VOCs, 30 compounds had an odour active value above 1. The intensity of green, floral, and fruity aromas were quite higher in fresh grapes followed by AD-raisins, PAD-raisins, SD-raisins, and PSD-raisins. The intense roasted aroma was found in SD-raisins due to 2,6-diethylpyrazine and 3-ethyl-2,5-dimethylpyrazine. Among raisins, the concentration of unsaturated fatty acid oxidized and Maillard reaction volatiles were higher in SD-raisins and mainly contributed green, fruity and floral, and roasted aromas, respectively.


Introduction
Raisins are considered to be significant agricultural products due to their high nutritional values. They are a rich reservoir of micronutrients such as vitamins, oligo-elements, polyphenols, and carotenoids. Along with nutritional enhancing qualities, they also boosts immunity against various diseases. These are not only used as nutritional enhancers but also consumed as snacks and are mixed in various foods while cooking, baking, and brewing. China is the third leading raisin producer, with >20 kinds of varieties cultivated for mass production [1]. The central area for raisin production is Turpan, Xinjiang, which accounts for approximately three-quarters of the entire yield of China [2]. Among raisins producing varieties of Turpan, the centennial seedless (white variety) is characterized as a

Experimental Design
A "centennial seedless" grape variety was grown in the territory of the Turpan (Xinjiang province, China), and the fully ripped "centennial seedless" grapes were harvested (total soluble solids, TSS >20 • Brix) in October 2014. The bunches of 600 kg grapes were divided into two parts, and then one cluster of grapes was soaked in a drying promoter (lipid, KOH, and Na 2 CO 3 ) for one minute. Thereafter, the unique air-drying and traditional sun-drying methods were practiced, making the raisins from the fully matured grapes (treated and/or non-treated). The drying course continued until the moisture content (<15%) remained constant for three days. The desired moisture content obtained regarding treatments such as AD (air-dried), PAD (pre-treated air-dried), SD (sun-dried), and PSD (pre-treated sun-dried) took 43,21,19, and 12 days, respectively ( Figure S1). The 1 kg samples were collected from fresh grapes >85% and dried grapes (raisin) <15%, then packed in plastic bags in triplicate and transported to the lab with dry ice covering in air freight.

Chemicals
The description of all chemicals used in this study is mentioned in File S1.

Sample Pre-Treatment
The preparation of samples was carried by minor modifications in the procedure of Wang et al. [2]. The raisins (100 g) were dipped in an equal amount of double distilled water and kept for 12 h at 4 • C. After that, they were macerated and homogenized for 240 min. Then, the pulp sample was centrifuged thrice (4 • C and 8000 rpm for 10 min) until the complete superimposition was attained [24]. The clear floating liquid was utilized for the detection of free-and bound-form compounds.

Preparation of Free-and Glycosidically Bound-Form Volatiles
The clear floating fluid was directly used for the detection of free-form compounds. In contrast, the method of Wen et al. [25] was used for the detection of glycosidically bound volatilities with minor amendments. All samples were run in triplicate.
The Cleanert (PED-SPE) column was employed to detach the glycosidically bound VOCs. Firstly, the column was activated with water (10 mL) and methanol (10 mL) separately, and then, 1 mL of the sample was added. The majority of the free-form VOCs were removed by 5 mL of dichloromethane, whereas the sugar and acids were rinsed by water (5 mL). After that, glycosidically bound compounds were eluted in a 50 mL flask using 20 mL of methanol. Finally, the bound-form VOCs were obtained by removing the solvent with a rotary evaporator (30 • C). Subsequently, the bound-form compounds were enzymatically hydrolyzed by pouring 5 mL of citrate/phosphate buffer solution (2 M; pH 5) into a vial. The process was carried out for 16 h (40 • C) in an incubator with the action of AR2000 [25,26].

Solid-Phase Microextraction Condition (SPME)
The both (free-and bound-form) VOCs of raisins were extracted by following solidphase microextraction condition (SPME) situations: 5 mL of clear supernatant and 10 µL of internal standard (4-methyl-2-pentanol; 1.0018 mg/L) were mixed in a flask holding a magnetic stirrer. Then, NaCl (1.3 g) was poured, and the flask was tightly plugged with a silicon base polytetrafluoroethylene (PTFE) stopper and then equilibrated on a hotplate (60 • C; 40 min) with constant agitation. After that, the VOCs were extracted by extraction coating fiber Carboxen/Polydimethylsiloxane/ Divinylbenzene (CAR/PDMS/DVB) in the headspace of the flask for 40 min. Finally, the fiber was desorbed into the gas chromatography (GC) injection port for 8 min.

GC/MS Analysis
The isolation and detection of the aromatic compounds of the raisins were executed in a GC-7890 attached with a MS-5975 and fitted out with a 60 m × 0.25 mm (HP-INNOWax) capillary column with a film thickness of 0.25 µm (J&W Scientific, Folsom, CA, USA). The condition of the GC/MS temperature was similar to our previous study [2,5]. The carrier gas "helium" was adjusted at a flow rate (1 mL per min) with the splitless GC inlet mode for SPME extract injection. The oven temperature was adjusted, as mentioned in our previous studies [2,5,13].
Mass spectra were achieved in an electron impact manner with an ionization energy and source temperature of 70 eV and 230 • C, respectively. The mass range (20-450 m/z) was used as full-scan mode, and then the process proceeded with an auto-tuning condition for the selective ion mode. The retention indices (RI) were obtained by the retention time (RT) of C6-C24 n-alkane in similar chromatographic circumstances. Identification was based on the RI of available standards and matching with mass spectra in the NIST-08 library. Wherein the unavailability of reference standards, a tentative determination was performed by comparing mass-spectra with NIST-08 library and retention indices present in the aforementioned literature.

Quantification of VOCs
The method of quantification was followed as reported by Wu et al. [27], with little modification. The standard amount of the acids and sugars in the raisin supernatant was used to prepare an artificial raisin suspension. The simulated solution was prepared by adding 5 g/L of tartaric acid and 400 g/L of sugars (glucose) in distilled water, and then a 4.2 pH was maintained with 1 M of sodium hydroxide solution. For the spike, the standard compounds (known concentration) were mixed in HPLC-grade ethanol and were further diluted into two-fold dilution with 15 levels by using an artificial raisin suspension. The 15 levels of standard VOC solutions were run in a similar way to the raisin supernatant. The regression coefficients of calibration curves quantified the aroma compounds at above 0.99. Without standard compounds, the contents of volatiles were measured by comparison with the standard curves holding a similar number of C-atoms or identical functional group. The quantitative data of the identified VOCs were checked by particular ion peak areas regarding the internal standard.

OAVs Calculation
Odor activity value (OAV) is the proportion of the VOCs content to its odor threshold in a water-based solution, and compounds with >1 OAVs are considered as flavor contributors (aroma compounds) [16,28]. The identified aroma compounds were classified into six standard classes based on flavor such as floral, green, fruity, fatty, roasted, and chemical aromas. The OAVs were calculated by following previously reported criteria [2,19,29].

Statistical Procedures
The data analysis was executed using SPSS software. A one-way ANOVA test was used to analyze the significant impact of different drying treatments on VOCs, employing least significant difference LSD tests at p < 0.05. The principal component analysis was executed under the concentration of UFAO and MR volatiles using R-software.

Results and Discussion
A total of 100 VOCs were successfully identified, including 14 aldehydes, 19 alcohols, 16 esters, nine acids, 22 terpenes, seven ketones, five furans and benzenes, four pyrazines, and two phenols, of which three VOCs (α-Terpinene, geranial, and 2,3-diethyl, pyrazine) had not been previously reported in raisins (Table S1). The remaining 97 compounds were mentioned in different studies regarding raisin varieties, drying treatments, and storage [2,3,5,13,[20][21][22]. The concentration (average ± standard deviation) of both (freeand bound-form) VOCs of the different treats (AD, PAD, SD, and PSD) are presented in Table S1. There were 99 and 77 compounds that existed in free-and bound-form, respectively. The additional 22 VOCs subsisted only in the free-form because they can not be linked to the sugar through the "glycosidic bond" deprived of similar groups [2].
Overall, the concentration of bound-form VOCs in aldehydes, alcohols, esters, acids, terpenes, ketones, furans, benzenes, and pyrazines was significantly lower than free-form volatiles ( Figure 1). The hydrolyzation of glycosidically bound compounds is the reason for less volatile content, which changes into an intense flavor [18]. Only the phenolic group had more content in bound-form as a result of the higher content of 4-vinylguaiacol. Some compounds such as 3-methyl-2-buten-1-ol, ethyl decanoate, and 2,3-diethyl, pyrazine only existed in bound-form, which might be due to the strong glycosidic bounding of the sugar with hydroxide group. Among the bound-form volatiles, pentanal, hexanal, 3-methyl-1butanal, nonanoic acid, acetic acid, and geranic acid had a concentration above 500 µg/g ( Table S1). The concentration of all bound-form compounds was more plentiful in fresh grapes than dried, except styrene, 1-hexanol, and nonanal. The compounds such as octanal, methyl hexadecanoate, and 2-acetylfuran were only found in fresh grapes. At the same time, 1-butanol, ethyl octanoate, ethyl decanoate, (E)-2-nonenal, and (E,E)-2,4-heptadienal were not identified in fresh grapes (Table S1).  [2,3,5,13,[20][21][22]. The concentration (average ± standard deviation) of both (free-and bound-form) VOCs of the different treats (AD, PAD, SD, and PSD) are presented in Table  S1. There were 99 and 77 compounds that existed in free-and bound-form, respectively. The additional 22 VOCs subsisted only in the free-form because they can not be linked to the sugar through the ''glycosidic bond'' deprived of similar groups [2]. Overall, the concentration of bound-form VOCs in aldehydes, alcohols, esters, acids, terpenes, ketones, furans, benzenes, and pyrazines was significantly lower than free-form volatiles ( Figure 1). The hydrolyzation of glycosidically bound compounds is the reason for less volatile content, which changes into an intense flavor [18]. Only the phenolic group had more content in bound-form as a result of the higher content of 4-vinylguaiacol. Some compounds such as 3-methyl-2-buten-1-ol, ethyl decanoate, and 2,3-diethyl, pyrazine only existed in bound-form, which might be due to the strong glycosidic bounding of the sugar with hydroxide group. Among the bound-form volatiles, pentanal, hexanal, 3-methyl-1-butanal, nonanoic acid, acetic acid, and geranic acid had a concentration above 500 μg/g (Table S1). The concentration of all bound-form compounds was more plentiful in fresh grapes than dried, except styrene, 1-hexanol, and nonanal. The compounds such as octanal, methyl hexadecanoate, and 2-acetylfuran were only found in fresh grapes. At the same time, 1-butanol, ethyl octanoate, ethyl decanoate, (E)-2-nonenal, and (E,E)-2,4heptadienal were not identified in fresh grapes (Table S1).

Aldehydes
The concentration of all aldehyde compounds was higher in fresh grapes, except (E,E)-2,4-nonadienal, (E)-2-heptenal, and benzaldehyde. The compounds (E)-2-nonenal and (E,E)-2,4-heptadienal came from the oxidation of linoleic acid [30], and decanal produced by the oxidation of oleic acid [31] were not identified in fresh grapes ( Table 1). The content of VOCs was comparatively higher in fresh grapes as compared to all raisins,

Aldehydes
The concentration of all aldehyde compounds was higher in fresh grapes, except (E,E)-2,4-nonadienal, (E)-2-heptenal, and benzaldehyde. The compounds (E)-2-nonenal and (E,E)-2,4-heptadienal came from the oxidation of linoleic acid [30], and decanal produced by the oxidation of oleic acid [31] were not identified in fresh grapes ( Table 1). The content of VOCs was comparatively higher in fresh grapes as compared to all raisins, which were prepared in different ways. Among raisins, the SD and PSD treatments were produced in higher concentrations than AD and PAD, excluding (E)-2-heptanal and phenylacetaldehyde ( Table 1). The higher content in SD and PSD is might be due to the intense light with high temperature [3,5].

Ester
The ethyl hexanoate, γ-nonalactone, and ethyl acetate provided the fruity note to the raisins. Their concentration was significantly higher in PAD-raisins than fresh grapes, AD-raisins, SD-raisins, and PSD-raisins. The volatiles, such as ethyl nonanoate and methyl salicylate, were only identified in PAD-raisins (Table 1). Among raisins, methyl hexadecanoate and β-Ionone existed in air-dried raisins while diethyl succinate was specified to pre-chemical treated raisins.

Acids
The pentanoic acid, hexanoic acid, and octanoic acids were produced by the oxidation of methyl linoleic acid [32] and with a higher concentration in SD-raisins, fresh grapes, and AD-raisins, respectively. Dodecanoic acid and 2-ethyl hexanoic acid were only identified in PAD-raisins with a concentration of more than ten µg/g (Table 1).

Terpenoids
Terpenoid is one of the leading classes in this study, markedly providing fruity and floral flavor to fruits [33]. It is also responsible for the fragrance in both grapes and raisins [2]. All terpenes compounds were significantly higher in fresh grapes as compared to dried raisins, except nerol oxide, and nerolidol 2, lilac alcohol, and cosmene didn't quantify in grapes. Among raisins, the amount of VOCs was comparatively higher in AD-raisins, and least were recorded in SD-raisins ( Table 1). The high temperature and light intensity in the sun-drying method might cause less concentration in terpenes [3,5]. Geraniol, geranial, linalool, and β-damascenone were significantly assisted in the fruity and floral aroma of raisins [34].
There were three pyrazine VOCs, 2-ethyl-6-methyl pyrazine, 5-ethyl-2,3-dimethyl pyrazine, and 2,6-diethylpyrazine which were already reported in raisins [2,21]. During the drying process, the pyrazine compounds are produced through the Maillard reaction and are responsible for a strong roasted aroma to dried grapes. 2-Ethyl-6-methylpyrazine was significantly higher in fresh grapes and could be originated by the condensation process of aspartic acid with galactose, fructose, and glucose [35]. Moreover, 2,6-diethyl pyrazine and 5-ethyl-2,3-dimethylpyrazine were generated from the reaction of aspartic acid and [36], and their amount was significantly higher in SD-raisins but was not found in fresh grapes (Table 1).

Ketones, Benzenes and Phenol
All the ketone compounds found in this study were also previously observed in dried grapes [2,21,22]. They did not majorly contribute to aroma [2] due to the high threshold odor value. 3-Octen-2-one, 2,3-butanedione, and acetoin did not exist in fresh grapes, and their concentration was significantly higher in SD-raisins following PSD-raisins, AD-raisins, and PAD-raisins (Table 1).
A high amount of benzene compounds were noted in fresh grapes. Among raisins, styrene, o-cymene, and toluene were statistically at par while naphthalene and 2-methylnaphthalene had higher amounts in AD-raisins. The identified benzene compounds in grapes and raisins produced a chemical note and it cannot contribute to aroma due to the low concentration and high threshold, as mentioned in the previous study [3]. Only two phenolic compounds are identified in fresh and dried grapes, whichhave already been reported in three raisin varieties dried by air [2]. The 4-ethenyl-2-methoxyphenol was produced from the breakdown of ferulic acid [37] and was only identified in fresh centennial grapes, but it cannot be found in raisins as mentioned in earlier studies [38].

Effect of Drying Method on UFAO-and MR-VOCs
The 41 and 10 VOCs of UFAO and MR, respectively, were analyzed using principal components (PCs) analysis to determine the effect of different drying treatments (AD, PAD, SD, and PSD). Regarding drying treatments, the first two PCs of UFAO and MR corresponded to 83.85% and 88.42%, respectively. The UFAO compounds were well separated regarding drying treatments. The majority of the UFAO compounds belonged to SD and PSD raisins following AD and PAD raisins (Figure 2A). Of the total variance of PC1, 62.02% was characterized by MR compounds, and they were closely linked to SD treatments ( Figure 2B). The phenylacetaldehydes and benzaldehyde were considered to belong to AD, while 5-hydroxymethyl-2-furaldehyde belonged to PSD methods.
The aromatic series of centennial seedless as fresh and dried (AD, PAD, SD, and PSD) grapes are presented in Figure 3. Based on aroma character, the 30 aroma-producing compounds were classified into six categories, including green, fatty, roasted, chemical, fruity, and floral aromas.
The flavor characteristics (green, fruity, floral, and chemical) were relatively higher in fresh grapes than dried grapes. Among raisins, the strength of fruity and floral fragrances was abundant in AD-raisins as compared to others. Correspondingly, floral and fruity (tropical tree fruit) is the main character of AD-raisins in storage [5], as well as in sweet wine prepared by Garnacha Tintorera grapes [40]. The β-damascenone (threshold value 0.09 µg/L) was the major contributor of floral and fruity notes [2,41]. Besides, β-ionone is responsible for the floral aroma and their intensity was higher in fresh grapes, AD-raisins and PAD-raisins, was not generated in SD-and PSD-raisins. The terpene compounds, especially geraniol and linalool, β-damascenone, and β-ionone, were reported as a major contributor to the fruity and floral aroma in litchis [27], blackberries [42], and raisins [2,3]. The green aroma had the most intense aroma as compared to fruity, floral, roasted, and chemical and they were mainly strengthened by hexenal, (E,E)-2,4-heptadienal and octanal, particularly higher in fresh grapes. Whereas, the roasted aroma came from MR during the drying of the grapes, mainly produced through 2,6-diethylpyrazine and 3-ethyl-2,5dimethylpyrazine, and were more intense in SD-raisins but did not contribute to fresh grapes. These two volatile compounds were found as outstanding aromatic compounds in roasted coffee [43] and dried raisins [2].  [3]; e Wu et al., [19]). OTV (odor threshold value); OAV (odor active value); AD (air-dried); PAD 81 (pre-treated air-dried); SD (sun-dried); PSD (pre-treated sun-dried). 82 The aromatic series of centennial seedless as fresh and dried (AD, PAD, SD, and PSD) 83 grapes are presented in Figure 3. Based on aroma character, the 30 aroma-producing com-84 pounds were classified into six categories, including green, fatty, roasted, chemical, fruity, 85 and floral aromas.

Conclusions
Volatile compounds in fresh grapes and dried raisins (AD-raisins, PSD-raisins, SDraisins, and PSD-raisins) were detected and evaluated. The terpenoids and aldehydes were the main class of VOCs that were responsible for the aroma in both grapes and raisins. The aroma profile in fresh grapes and dried raisins was quite different. The main characteristic aroma was green, fruity, and floral, which were mainly produced in fresh grapes and AD-raisins, while the intensity of roasted and fatty aromas was more compelling in SDraisins. Further studies will focus on the model reaction of UFAO and MR during the drying of grapes.
Supplementary Materials: The following are available online at https://www.mdpi.com/2304-815 8/10/3/559/s1, Figure S1: The effect of drying treatments on the changing of TSS during the drying of centennial Seedless raisins, Table S1: The concentration of free-and glycosidically bound VOCs in fresh and dried centennial Seedless grapes, File S1: List of chemicals.