Interrogating Raisin Associated Unsaturated Fatty Acid Derived Volatile Compounds Using HS–SPME with GC–MS

This study proposed to investigate the generation mechanism of raisins-derived volatile compounds during unsaturated fatty acids oxidation (UFAO) using a mixture of fatty acids (FAs) and four individual FA at different time intervals (0, 4, 8, 12, 16, and 20 days; 60 °C). During the sun-drying of ‘Thompson Seedless’ grapes (Vitis vinifera L.), a total of 39 UFAO-derived volatiles were characterized by the GC-MS. Firstly a pH value of 4.2 was optimized to proceed with a raisin drying-like UFAO model reaction. Afterward, GC-MS quantification revealed 45 UFAO-derived volatiles, and the maximum numbers of compounds were identified in the interaction of all FAs (39) following linoleic acid (29), erucic acid (27), oleic acid (25), and linolenic acid (27). Pentanoic acid, (E,E)-2,4-octadienal, and n-decanoic acid were only quantified in all FAs, linoleic acid, and erucic acid, respectively. This study showed that all FAs reactions were found to be responsible for the generation of a greater number of UFAO-derived volatiles with higher concentrations.


Introduction
Conventional drying methods (sun and air) and modern drying techniques (microwave heating, freeze-drying, and oven-drying) are being used to produce raisins from fully ripened grapes [1]. According to the USDA (United State Department of Agriculture), China produces 1.90 Mt of raisins annually and contributes significantly to the global economy [2]. In the year 2016-17, China was placed third after USA and Turkey in terms of raisin production [3]. The main advantage of drying is responsible for the inhibition of microbial growth (e.g., molds, yeast, and bacteria). Moreover, the drying process also significantly affects the major physical (color, size, shape, and shrinkage) and biochemical (oxidation detrition and browning reaction) quality parameters [4]. Among the traditional methods, the sun-drying process is considered the most efficient, cost-competitive [4], and requires less time for making raisins [5,6]. Intense sun radiation and temperature differences are the key factors of the raisins' color, appearance, and aroma [7]. In regards to the chemical composition of raisins, some volatile organic compounds (VOCs) such as 2-octanol, 1-butanol, and 3-methyl-2-buten-1-ol are produced by the sun-drying method [5], whereas several other VOCs (e.g., ethyl hexanoate, 2-pentyl furan, 2,6-diethyl pyrazine, β-damascenone, β-ionone) are formed through Maillard reaction (MR), unsaturated fatty acids oxidation (UFAO), glycosidically bound (GD), and carotenoids, resulting in different kinds of flavors such as fruity, floral, fatty, and roasted (Javed et al., 2019;Wang et al., 2017). Among these chemical reactions, UFAO is the main contributor to developing an attractive raisin aroma [5].

Preparation of Free-Form Volatiles
The free-form volatiles were identified as stated in our prior research [24], with slight modifications. One hundred berries were separately chosen from fresh grapes and then the juice was collected by hand using polyethylene bags (food-grade). In the case of raisins, 100 berries were soaked overnight using an equivalent weight of distilled water. The very next day soaked samples were well mixed and macerated for 240 min, and then immediately centrifuged (8000 rpm; 4 • C for 10 min). All samples were immediately analyzed for the characterization of free-form volatiles.

Optimization of pH for Model Reaction
The buffer solution's pH was initially optimized for the UFAO model reaction. For this purpose, pyrophosphate buffer (0.2 M) was prepared by mixing tetrasodium pyrophosphate decahydrate (Na 4 O 7 P 2 .10H 2 O) and sodium phosphate monobasic dihydrate (NaH 2 PO 4 .2H 2 O) in distilled water. The pH of the pyrophosphate buffer was configured at a level of 4.2, 6.2, and 7 with the help of NaOH (2 M) and HCl (2 M). In a 20 mL reaction tube, 0.20 mM of each fatty acid (stearic acid, oleic acid, linoleic acid, linolenic acid, erucic acid) was added and blended with the help of an electric blender. The sample tubes were then kept in a controlled oven at 60 • C for 10 and 20 days. After the removal from the oven, all samples were stored at −80 • C until they were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS).

UFAO Model Reaction
The pH of the reaction was optimized based on pre-experiments (4.2) for the UFAO model reaction (Figure 1). According to a prior study, when 'Thompson Seedless' grapes were dried, the pH and temperature inside the berries were similar to the optimal pH value and selected temperature. [25].

Preparation of Free-Form Volatiles
The free-form volatiles were identified as stated in our prior research [24], with slight modifications. One hundred berries were separately chosen from fresh grapes and then the juice was collected by hand using polyethylene bags (food-grade). In the case of raisins, 100 berries were soaked overnight using an equivalent weight of distilled water. The very next day soaked samples were well mixed and macerated for 240 min, and then immediately centrifuged (8000 rpm; 4 °C for 10 min). All samples were immediately analyzed for the characterization of free-form volatiles.

Optimization of pH for Model Reaction
The buffer solution's pH was initially optimized for the UFAO model reaction. For this purpose, pyrophosphate buffer (0.2 M) was prepared by mixing tetrasodium pyrophosphate decahydrate (Na4O7P2.10H2O) and sodium phosphate monobasic dihydrate (NaH2PO4.2H2O) in distilled water. The pH of the pyrophosphate buffer was configured at a level of 4.2, 6.2, and 7 with the help of NaOH (2 M) and HCl (2 M). In a 20 mL reaction tube, 0.20 mM of each fatty acid (stearic acid, oleic acid, linoleic acid, linolenic acid, erucic acid) was added and blended with the help of an electric blender. The sample tubes were then kept in a controlled oven at 60 °C for 10 and 20 days. After the removal from the oven, all samples were stored at −80 °C until they were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS).

UFAO Model Reaction
The pH of the reaction was optimized based on pre-experiments (4.2) for the UFAO model reaction (Figure 1). According to a prior study, when 'Thompson Seedless' grapes were dried, the pH and temperature inside the berries were similar to the optimal pH value and selected temperature. [25]. Fatty acid oxidation reactions were carried out in pyrophosphate buffer at pH 4.2, with each reaction mixture containing 0.3 mL of fatty acids being blended in a 20 mL reaction tube using an electric blender. The following are the seven reaction mixtures: (1) control; (2) stearic acid; oleic acid (3); linoleic acid (4); linolenic acid (5); erucic acid (6) and (7) all fatty acids with the duplicate. After that, all the sample tubes were kept in a controlled oven at 60 • C for 20 days. The reaction samples were removed from the oven at an interval of 4 days, cooled at room temperature, and then kept in a refrigerator at −80 • C. Before analysis in GC-MS, samples were kept at room temperature for 2 h.

GC-MS Analysis
A GC (7890; Agilent Technologies, USA) paired with an MS (5975; Agilent) and fitted including an HP-INNOWAX (60 m × 0.25 mm id) capillary column with such a 0.25 µm film thickness was used for the detection of VOCs in raisins and UFAO model reaction, using a previous procedure (Wang et al., 2015). Helium was applied as a carrier gas (1 mL/min), and the SPME (solid-phase microextraction) was injected in splitless mode. The oven temperature was first set at 50 • C for 1 min, then suddenly raised to 220 • C (3 • C/min) and maintained for 5 min at 220 • C. After that, it was progressively increased to 250 • C (5 • C/min) and kept constant for 5 min.
The mass spectra were obtained in the electron impact mode at 230 • C and 70 eV for the source temperature and ionization energy (EI) mode, respectively. After acclimating to a mass range of 20-450 m/z, full-scan mode and selected ion mode (autotune) were both used for the acquisition. The retention times of n-alkane (C6-C24) were used to determine retention indices in a similar chromatographic pattern. VOCs were identified based on retention indices of known standards, and the mass spectra were matched to the NIST-08 collection. In the absence of a reference standard, a preliminary identification was made using the mass spectrum of the NIST-08 library and retention indices from earlier studies.

Quantification
The quantification method was improved following our prior findings by making necessary modifications [26]. The raisin imitation solution was constructed using the acid and sugar concentrations found in the real raisin solution. In distilled water, a mock solution was prepared with glucose (400 g/L) and tartaric acid (5 g/L), and the pH (4.2) was maintained using a NaOH solution (1 M). For the spike, ethanol (HPLC-standard) was mixed with the known concentration of standard VOCs, which were then diluted into a 15-level using a simulated raisin solution. The raisin supernatant was evaluated in the same way as the standard VOC solutions (15 levels). All aroma compounds had regression coefficients of calibration curves that were over 0.99 in terms of quantification. In the absence of standard compounds, the quantification of compounds was estimated by comparison with those standard curves that hold an identical number of C-atoms or the same functional groups. The amount of detected aroma volatiles was checked by distinguished ion peak areas for 4-methyl-2-pentanol as "internal standard" (1.0018 mg/L).

Statistical Analysis
The volatiles identified from 'Thompson Seedless' were statistically analyzed, and the statistical significance of the drying period was evaluated by one-way ANOVA at a significance level of p < 0.05 with SPSS software, version 20.0 (IBM corp., Chicago, IL, USA). The heatmaps were created using the Metabo-Analyst 5.0 software (McGill University, Montreal, QC, Canada; http://www.metaboanalyst.ca/) using one-factor analysis for VOC concentrations. To standardize the data, autoscaling was employed (mean-centered and divided by the standard deviation of each variable).

Free-Form Volatile Compounds Formed during Raisins Drying
Among UFAO-derived compounds, pentanal, hexanal, octanal, (E)-2-hexenal, (E,E)-2,4-heptadienal, and hexanoic acid were the major bioactive compounds formed in the drying process. In addition, several other compounds such as hexanal, (E)-2-hexenal, 1pentanol, 1-hexanol, ethyl hexanoate, and ethyl octanoate originated from linoleic acids [18,19,27], which were shown to be more concentrated in fresh grapes and showed a decline with the prolonged drying process (Table 1). These findings suggest that these volatile compounds were easily volatilized under the sun-drying process due to the higher potency of light and temperature [5,6,17]. Regardless of these, the content of other compounds from aldehydes such as (E,E)-2,4-nonadienal and (E,E)-2-octenal produced by linoleic acids [18,19] and alcohols (1-heptanol, 1-octanol and (E)-2-octen-1-ol) generated from oleic acids [28,29] were significantly higher on the eighth day of drying. Whereas the acidic compounds such as pentanoic acid, hexanoic acid, and heptanoic acid came from methyl linoleic acid [30], increased with the drying time of 'Thompson Seedless' grapes and attained the peak values on the 12th day and then gradually declined in concentration (Table 1). These findings demonstrated that the volatility of 'Thompson Seedless' raisins dried in the sun changed significantly as a consequence of moisture changes and sun drying. Some UFAO-derived compounds such as ethyl nonanoate, butyrolactone, and dodecanoic acid were not characterized in fresh grapes; however, they were observed on 4, 4, and 12 days, respectively (Table 1). 2-octanol and 2-nonanol were discovered exclusively in fresh grapes, which may be attributed to their low concentration and ease of volatilization during the drying process.

Optimization of pH for Model Reaction
pH is a key factor for any chemical reaction that plays a vital role in the generation of volatile compounds and also influences their concentration. For optimization of the raisin drying model reaction, the pre-experiment was carried out at three different pHs (4.2, 6.2, and 7). Amongst 27 UFAO-derived compounds, 25, 21, and 21 compounds were found at pH 4.2, 6.2, and 7, respectively (Table 2). Retention Indices (RI): Kovats retention indices were calculated based on a n-alkane series (C6-C24) on the poly (ethylene glycol) Retention Indices (RI): Kovats retention indices were calculated based on a n-alkane series (C6-C24) on the poly (ethylene glycol) (PEG) column under the same chromatographic conditions. Identification method (ID-M): 1, identified, mass spectrum and RI were in accordance with standards; 2, tentatively identified, mass spectrum matched in the standard NIST 2008 library and RI matched with NIST Standard Reference Database (NIST Chemistry WebBook). NF = Not found; Mean ± standard deviation (n = 3) of the same compounds followed by different letters are significantly different (p < 0.05), the free volatiles were compared separately.  Overall, the concentration of volatiles from the aldehydes, acids, and alcohols group was highest at pH 4.2, whereas the concentration of furan compounds (2-pentyl furan) was utmost at pH 7 ( Figure 1) because it is more favorable for the generation of furan compounds [27]. Different raisins varieties were found to contain 1-octen-3-one [10]; however, in a 10-day reaction, it was only produced at 6.2 pH ( Table 2). All of the ester compounds (methyl hexadecanoate, ethyl hexanoate, ethyl octanoate, methyl octanoate, ethyl nonanoate, γ-nonalactone, and butyrolactone), and some compounds from acids (1-nonanol, 2-nonanol, 2-ethyl-1-hexanol and (Z)-3-Hexen-1-ol), ketones (3-Octen-2-one and 2,6-dimethyl-4-heptanone) and terpene (3,4-dimethyl-2,4,6-octatriene) were found in 'Thompson Seedless' during drying. On the other hand, nonanoic acid and n-decanoic acid were not identified in 'Thompson Seedless' raisins but were reported in 'Centennial Seedless' raisins [9,10] and also quantified in an optimization study (Table 2).

Control and Stearic Acid
Only 2,6-dimethyl-4-heptanone was found in control and stearic acid on all reaction days (0, 4, 8, 12, 16, and 20). The 2,6-dimethyl-4-heptanone has been detected in raisins dried by air [10] or sun [6], as well as during storage [5]. The source of 2,6-dimethyl-4heptanone was not cited in the literature and our outcome indicated that it could not be generated from fatty acids; however, it might be produced due to the heat of the reaction.

Oleic Acid (C18:1)
Twenty-five compounds originating from the oleic acid, including 11 aldehydes, 5 esters, 5 alcohols, 3 acids, and 1 furan were enlisted in Table 3. Most of the compounds were generated on the eighth day of the model reaction; however, octanal and nonanal were detected on the day of the reaction, which might be they were easily oxidized (Supplementary Table S2). Heptonic acid was only identified on sixteen-day and its concentration was very low (Figure 2A). As compared to linoleic acid, it was seen in all reaction days with higher content, except 0-day (Supplementary Table S2). These findings indicate that linoleic acids are the main source of heptonic acid.

Linolenic Acid (C18:3)
Among other fatty acids, the least number of compounds (14) were characterized in linolenic acid which might be due to the triple bond that needs more energy to produce volatile compounds. Only, (E,E)-2,4-heptadienal and (E,E)-3,5-Octadien-2-one were produced at the start of reactions, and also were found on other days (4, 8, 12, 16, and 20). Whitfield and Mottram (1992) reported that heptanal was produced by oleic acid and linolenic acid and it was one of the concentrated compounds which was found on the eighth day ( Figure 3).  Overall, the concentration of compounds generated by oleic acid was increased as the reaction time proceeded and highly concentrated at 20 days, except 2-pentyl furan, (E,E)-2,4-decadienal, heptonic acid and 1-octanol (Figure 2A). The higher amount of 2pentyl furan, and (E,E)-2,4-decadienal, heptonic acid and 1-octanol were recorded at 12 and 16 days, respectively (Figure 2A). Among 25 VOCs from oleic acids, some compounds such as octanal, decanal, 1-heptanol, and methyl octanoate were already reported [18,19,28].
The compounds produced by linoleic acid were more concentrated at 4 days and then their content showed a decreasing trend with the time of model reaction. On the other hand, some compounds viz. nonanoic acid, ethyl octanoate, 1-octen-3-one, 2-heptanol, (E)-2-hexenal, (E)-2-octen-1-ol, 2-pentyl furan, heptanoic acid, and octanoic acids were increased gradually with reaction time and found the highest content on the last day of the reaction (20 days; Figure 2B).

Linolenic Acid (C18:3)
Among other fatty acids, the least number of compounds (14) were characterized in linolenic acid which might be due to the triple bond that needs more energy to produce volatile compounds. Only, (E,E)-2,4-heptadienal and (E,E)-3,5-Octadien-2-one were produced at the start of reactions, and also were found on other days (4, 8, 12, 16, and 20). Whitfield and Mottram (1992) reported that heptanal was produced by oleic acid and linolenic acid and it was one of the concentrated compounds which was found on the eighth day (Figure 3).

Linolenic Acid (C18:3)
Among other fatty acids, the least number of compounds (14) were characterized in linolenic acid which might be due to the triple bond that needs more energy to produce volatile compounds. Only, (E,E)-2,4-heptadienal and (E,E)-3,5-Octadien-2-one were produced at the start of reactions, and also were found on other days (4, 8, 12, 16, and 20). Whitfield and Mottram (1992) reported that heptanal was produced by oleic acid and linolenic acid and it was one of the concentrated compounds which was found on the eighth day ( Figure 3). In the erucic acid reaction, 27 compounds were generated comprising 11, 6, 5, and 5 coming from aldehydes, esters, alcohols, and acids, respectively. The compounds such as heptanal, octanal, nonanal, 1-heptanol, and decanal originated from oleic acid and linoleic acids [18,19,30] and were identified in all days of the model reaction (Table 3) and 'Thompson Seedless' grapes (Table 1), as well. Apart from these, some compounds (ethyl hexanoate, ethyl hexadecnoate, and n-decanoic acid) were only found within 20 days of the reaction (Supplementary Table S2).
All compounds that were produced by erucic acids were more concentrated at 20 days of reaction ( Figure 4A), except 1-octanol and (E,E)-2,4-decadienal because they were not only found at 20 days (Supplementary Table S2). The concentration of most generated In the erucic acid reaction, 27 compounds were generated comprising 11, 6, 5, and 5 coming from aldehydes, esters, alcohols, and acids, respectively. The compounds such as heptanal, octanal, nonanal, 1-heptanol, and decanal originated from oleic acid and linoleic acids [18,19,30] and were identified in all days of the model reaction (Table 3) and 'Thompson Seedless' grapes (Table 1), as well. Apart from these, some compounds (ethyl hexanoate, ethyl hexadecnoate, and n-decanoic acid) were only found within 20 days of the reaction (Supplementary Table S2).
All compounds that were produced by erucic acids were more concentrated at 20 days of reaction ( Figure 4A), except 1-octanol and (E,E)-2,4-decadienal because they were not only found at 20 days (Supplementary Table S2). The concentration of most generated compounds was increased with the time of reaction. The identified ester compounds were higher in concentrations as compared to other identified compounds in erucic acid reactions.

All Fatty Acids
The reaction which contains all fatty acids has produced more volatile compounds (39) than the reaction carried out by single fatty acids. In this reaction, mostly volatiles were produced at 4 days of reaction while pentanoic acid, ethyl hexanoate, and 1-octen-3-one were found at 20 days of reaction. Pentanoic acid was the only compound identified in all fatty acids on the last day of the reaction (Supplementary Table S2). Among 39 volatiles, heptanal, octanal, nonanal, (E)-2-heptenal, (E)-2-octanal, and 1-octen-3-ol were those compounds identified in all the reaction days of the experiment. compounds was increased with the time of reaction. The identified ester compounds were higher in concentrations as compared to other identified compounds in erucic acid reactions.

All fatty Acids
The reaction which contains all fatty acids has produced more volatile compounds (39) than the reaction carried out by single fatty acids. In this reaction, mostly volatiles were produced at 4 days of reaction while pentanoic acid, ethyl hexanoate, and 1-octen-3-one were found at 20 days of reaction. Pentanoic acid was the only compound identified in all fatty acids on the last day of the reaction (Supplementary Table S2). Among 39 volatiles, heptanal, octanal, nonanal, (E)-2-heptenal, (E)-2-octanal, and 1-octen-3-ol were those compounds identified in all the reaction days of the experiment.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12030428/s1. Figure S1: Effect of sun-drying method on the moisture loss of 'Thompson Seedless' grape. Table S1: Descriptions of identified UFAO-derived compounds during drying in 'Thompson Seedless' grape. Table S2: Formation of UFAO compounds by the reaction of different fatty acids in a raisin-like Model Reaction.