Identification, Comparison and Classification of Volatile Compounds in Peels of 40 Apple Cultivars by HS–SPME with GC–MS

Aroma is an important quality indicator for apples and has a great influence on the overall flavour and consumer acceptance. However, the information of the aroma volatile compounds in apple peels is largely unknown. In this study, evaluation of volatile compounds in peels of 40 apple cultivars was carried out using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). A total of 78 volatile compounds were identified, including 47 esters, 12 aldehydes, 5 alcohols, 3 ketones, 1 acid and 10 others. Eight volatile compounds were common in all apple cultivars. Cultivar Changfu No. 2 contained the highest number of volatile compounds (47), while Qinyue contained the least (20). Honey Crisps had the highest volatile content, at 27,813.56 ± 2310.07 μg/kg FW, while Huashuo had the lowest volatile content, at 2041.27 ± 120.36 μg/kg FW. Principal component analysis (PCA) clustered the 40 apple cultivars into five groups. Aroma is cultivar-specific, volatile compounds of hexyl butyrate, hexyl 2-methylbutyrate and hexyl hexanoate, together with hexanal, (E)-2-hexenal, 1-hexanol, estragole and α-farnesene could be proposed for apple cultivar classification in the future.


Introduction
Aroma, which is one of the most important quality indicators for fruits, has a great influence on the overall flavour and consumer acceptance [1]. It is generally a complex mixture of volatile compounds whose composition and concentrations are specific to the species, and often the variety, of fruit [2,3]. Volatile compounds, which determine the aroma profile of fruits, directly contribute to perceived odour and flavour attributes. Knowledge of these volatile compounds forms the basis of breeding programs aiming to improving fruit aroma. As an important trait of fruit quality, more attention has been paid to the study of aroma volatiles in recent years.
Apples (Malus×domestica Borkh.) are one of the most widely cultivated and frequently consumed fruits in the world [4]. Aroma is an important standard for evaluating the quality and characteristics of apples, and the aroma volatile compounds in apples have been studied for more than 50 years. Although more than 300 volatile compounds have been identified in apples, including alcohols, aldehydes, acids, ketones, terpenoids, sesquiterpenes, and esters, only a subset of 20-30 compounds significantly contribute to the typical apple aroma [5,6]. Among these, esters are the most abundant compounds. The esters, especially those with even-numbered carbon chains including combinations of acetic, butanoic, and hexanoic acids with ethyl, butyl, and hexyl alcohols, are the major contributors to apple volatiles. Butyl acetate, hexyl acetate, 2-methylbutyl acetate, and ethyl 2-methyl-butanoate are the crucial esters due to their high content and impact on the aroma of several apple varieties [7]. Alcohols are another important group of compounds, after esters, which affect the aroma of ripe apples, with the most abundant being 2-methyl-1-butanol, 1-butanol, 1-hexanol and 1-propanol [8,9]. Aldehydes are abundant in pre-climacteric apples, but after ripening, some aldehydes become almost imperceptible [10]. More than 25 aldehydes, mostly hexanal, trans-2-hexenal, and butanal, have been identified in apples [8]. During apple ripening, the volatile compounds are converted from aldehydes to esters to such an extent that esters can account for more than 80% of all aromatic compounds in some cultivars, such as Golden Delicious and Golden Reinders [9,11]. Aroma is cultivar-specific; therefore, study of the volatile profile at the variety level is necessary. Volatile compounds have been investigated at the germplasm level for peach (Prunus persica), pear (Pyrus ussuriensis), and melon (Cucumis melo) [12][13][14]. However, there are few studies on the comparative analysis of volatile compounds in a number of apple cultivars.
There are some microextraction techniques for the determination of volatile compounds, such as continuous sample drop flow microextraction [15], dispersive liquid-liquid microextraction [16] and solid-phase microextraction (SPME) [17]. The determination of volatile compounds in apples requires a suitable selective, sensitive analytical method. Although the lifetime of the microfiber is short, SPME, a simple, solvent-free method for the extraction of volatile compounds, combined with gas chromatography-mass spectrometry (GC-MS), has been widely used for the qualitative and quantitative analysis of volatile compounds in apple fruit [18,19].
In this study, HS-SPME combined with GC-MS was used to determine the composition and concentration of the volatile compounds in 40 apple cultivars. This work evaluated the aroma profiles of apple peels at cultivar levels, and these results could be valuable for future breeding programs, aiming to produce apple cultivars with enhanced aroma quality.

Plant Materials
The 40 apple cultivars used in this study are listed in Figure 1 and Table 1, along with some basic compositional parameters. The apples were harvested in 2019 from the experimental station of Northwest A and F University, Baishui County, Shaanxi Province, China (35 • 21 N, 109 • 55 E). Orchard management procedures such as irrigation, pruning, disease control and fertilisation, were similar for all cultivars. Fruits were sampled at full ripening and maturity was determined by taste, ground colour, starch index and days after pollination. Three biological replicates from three trees of each cultivar were prepared, with 4-6 fruits per replicate. Fruit peels (<1 mm in thickness) were collected from each apple with an apple peeler, immediately frozen in liquid nitrogen, and stored at −80 • C until analysis.  Table 1. Bars = 2 cm.

Physiological Characteristics Measurement
Single fruit weight was measured by an electronic balance (Mettler-Toledo Inc., Greifensee, Switzerland). The apple fruits' total soluble solid (TSS) and titratable acidity (TA) was determined by a hand refractometer (Atago, Tokyo, Japan) and a digital fruit acidity meter (GMK-835F Perfect, Berlin, Germany), respectively.

HS-SPME Procedure
HS-SPME was applied for the extraction and concentration of volatile compounds in apple peels. All the extractions were performed using a divinylbenzene/carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) fibre with a thickness of 50/30 µm (Supelco, Bellefonte, PA, USA). For the extraction of volatile compounds, 5 g of apple peel was placed into a 50 mL screw-cap headspace vial containing a magnetic stirring rotor and 1 g NaCl spiked with 10 µL (0.4 mg/mL) 3-nonanone (internal standard). Subsequently, the headspace bottle was equilibrated at 50 • C for 10 min on a metal heating platform with agitation. Prior to use, the new SPME fibre was conditioned in the GC injector port for 0.5 h at 240 • C. Then, the fibre was inserted into the headspace with continuous heating and agitation (200 rpm) for 30 min to adsorb volatile substances. After extraction, it was introduced into the heated injector port of the chromatograph for desorption at 250 • C for 2.5 min.

GC-MS Analysis
A Thermo Trace GC Ultra gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with an HP-INNOWax capillary column (60 m × 0.25 mm × 0.25 µm) was used for analysis. The oven temperature was programmed as follows: 40 • C held for 3 min, raised to 150 • C at 5 • C/min, then increased at 10 • C/min to 220 • C and held for 5 min. Helium, the carrier gas, was circulated at 1.0 mL/min at a constant flow rate in splitless mode. The temperature of the ion source and transfer line were both maintained at 240 • C. MS fragmentation was performed under an electron ionisation of 70 eV with the scan range of 35-450 m/z.

Qualitative and Semi-Quantitative Analysis
Xcalibur 3.2 software was used to process the data collected from the GC-MS. Volatile compounds were identified by comparing retention indices (RI) and retention times (RT) to those of compounds in the NIST/EPA/NIH Mass Spectral Library database (NIST, 2014). Linear retention indices were calculated under the same chromatographic conditions after injection of a C7-C30 n-alkane series (Supelco, Bellefonte, PA, USA). Based on the total ion chromatogram, the content of each volatile compound was quantified as 3-nonanone equivalent (internal standard) by the peak area.

Statistical Analysis
All the data were the mean of three replicates. Excel 2010 software was conducted for statistical analysis and charting of data. Principal component analysis (PCA) was executed using Origin 2017 software (OriginLab Corporation, Northampton, MA, USA).

Identification and Determination of Volatile Compounds in Forty Apple Cultivars
The identification of volatile compounds and studies of diversity among cultivars were performed based on the retention indices obtained from GC-MS. A total of 78 volatile compounds were identified and quantified in 40 apple cultivars, including 47 esters, 12 aldehydes, 5 alcohols, 3 ketones, 1 acid and 10 other compounds (Table 2) (Table 3). Eight volatile compounds (E17 hexyl acetate, E26 butyl caproate, E27 hexyl butyrate, E28 hexyl 2-methylbutyrate, E40 hexyl hexanoate, A1 hexanal, A4 2-hexenal and O8 α-farnesene) were present in peels of all apple cultivars (Tables S1 and S2). As shown in Table 2, hexyl butyrate (E27), hexyl 2-methylbutyrate (E28), hexyl hexanoate (E40), and 2-hexenal (A4) and α-farnesene (O8) were the most abundant compounds (average content > 700 µg/kg FW) in the apple cultivars, which is in agreement with the results of previous studies [20][21][22].  Aroma is a complex mixture of many volatile compounds, and the amount and content of aroma substances showed different patterns among various apple cultivars [18,23]. In this study, differences were also observed in the total content of volatile compounds among the 40 apple cultivars, ranging from 2041.27 ± 120.36 µg/kg FW to 27,813.56 ± 2310.07 µg/kg FW ( . The above analysis indicates that the volatiles were dependent, to a great extent, on the cultivars, which is consistent with a previous study [18]. Golden Delicious (GD) has been reported to have the high volatile compound content [24]. However, the total content of volatiles in cultivar GD in this study (4436.74 ± 425.36 µg/kg FW) was not high. This result might be attributed to geographical variations, such as territory, climate, water and other environmental factors.

Composition and Concentration of Volatile Compounds
Esters, aldehydes, alcohols, ketones, acids and other volatiles constitute the aroma of different apple cultivars [2,5,18]. The composition and concentrations of volatile compounds in the peels of 40 apple cultivars are shown in Table S3. The percentage of each type of volatile in peels of 40 apple cultivars are presented in Figure 2 and Table S4. The total content of each type of volatile in apple cultivars are presented in Table 4.

Aldehydes
Aldehydes were the second most abundant volatiles in this study, accounting for between 8.25% (Jonagold, JNG) and 69.23% (COP) of the total volatile content in the apple cultivars ( Figure 2; Table S4). More than 25 aldehydes have been identified in apples [8,36]. In this study, 12 types of aldehyde compound were identified ( Table 2). Aldehyde content varied greatly among the apple cultivars and ranged from 662.21 ± 80.12 µg/kg FW (18.59% of total volatiles) in FJ to 4905.73 ± 520.41 µg/kg FW (22.60% of total volatiles) in Jiguan (JG) ( Table 4; Table S4). Hexanal (A1) and (E)-2-hexenal (A4) were the most predominant constituent aldehydes (average content > 200 µg/kg FW) in all apple cultivars in this study, which was in agreement with a previous report [28].

Principal Component Analysis of Volatile Compounds
Principal component analysis (PCA), an unsupervised clustering method, is often used to provide a partial visualisation of data in a reduced-dimension plot [43,44]. PCA was used extract important information from the 78 volatile compounds detected in the 40 apple cultivars. As shown in Figure 3, the first two principal components accounted for 63.92% of the variation in the data, with PC1 and PC2 explaining 38.24% and 25.68% of the total variance, respectively. Scatter plots of the 40 apple cultivars are shown in Figure  3A, and the corresponding loadings establishing the relative importance of the variables are shown in Figure 3B. The 40 cultivars were divided into five groups based on the relationships between cultivars (scores) and their volatile compounds (loadings). The first group included five cultivars (RXH, JNG, CM, HY, HC), which contained high relative contents of butyl acetate (E7), hexyl acetate (E17), butyl caproate (E26), butyl heptanoate (E35), and estragole (O10). The second group contained eight cultivars (MI, FJ, RL, RD, MYK, JZ, RGL, PNM) characterised by high relative contents of 2-methylbutyl acetate (E8), amyl propionate (E13), 2-methylbutyl 2-methylbutyrate (E19), hexyl 2-methylbutyrate (E28), 2-methylbutyl hexanoate (E30), and 2-methylbutanoic acid (O4). The third group was composed of three cultivars (HF, HG, JNT), which contained high levels of propyl butyrate (E9), 2-methyl-1-butanol (B3), and 1-hexanol (B5). The fourth group included five cultivars (GS, HS, RX, COP, and ID) with low relative contents of esters and high relative contents of aldehydes such as 2-hexenal (A4), (E,E)-2,4-heptadienal (A9). The fifth group contained the other 19 cultivars, and showed no consistency in the composition of volatile compounds. Among these cultivars, JNG in group 1 was characterised by high levels of butyl acetate (E7), hexyl acetate (E17), and butyl caproate (E26), in agreement with previous studies [25]. However, hexyl acetate (E17) was the major ester compound and was present in high levels in cultivar GD, which did not cluster into Group 1, possibly due to the influence of the content of other esters, such hexyl butyrate (E27) and hexyl hexanoate (E40). Cultivar FJ, one of the most widely cultivated apples in China, clustered into group 2 based on high relative content of 2-methylbutyl acetate (E8), amyl propionate (E13), 2-methylbutyl 2-methylbutyrate (E19), and hexyl 2-methylbutyrate (E28). 2-methylbutyl acetate is the main compound in the aroma profile of Fuji apples [45]. Granny Smith apples have low volatile emission compared with other apple varieties [46]. In this study, GS had low total content of volatile compounds, but the high relative content of 2-hexenal (A4) clustered it into group 4. As expected, group 5 contained the highest number of apple cultivars, and these cultivars had different types and contents of volatile compounds. These differences in the volatiles in cultivars contributed to diversity among apple varieties. According to PCA analysis results in this study, the most abundant esters in apple peels (hexyl butyrate, hexyl 2-methylbutyrate and hexyl hexanoate), together with hexanal, (E)-2-hexenal, 1-hexanol, estragole and α-farnesene could been proposed for apple cultivar classification in the future.

Conclusions
In this study, the identification, comparison and classification of volatile compounds in peels of 40 apple cultivars was carried out using HS-SPME combined with GC-MS. A total of 78 volatile compounds were detected in 40 apple cultivars. Eight volatile compounds were common in all the apple cultivars. Aroma profiles showed large differences among the cultivars. Cultivar Changfu No. 2 contained the highest number of volatile compounds, while Qinyue contained the least number of compounds. Honey Crisps had the highest volatile content, while Huashuo had the lowest volatile content. PCA clustered the 40 apple cultivars into five groups.
Overall, this study offered useful information for evaluating the profiles of volatile compounds in the peels of different apple cultivars and provided a reference for future breeding and improvement in apple flavour.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/foods10051051/s1, Table S1: Number of apple cultivars for each identified volatile compound.  Table S2. The content (µg/kg FW) of eight common volatile compounds in peels of 40 apple cultivars. Table S3: The contents (µg/kg FW) of identified volatiles in the peels of 40 apple cultivars. Table S4: Percentage (%) of each type of volatiles in apple cultivars.