Evaluation of Volatilomic Fingerprint from Apple Fruits to Ciders: A Useful Tool to Find Putative Biomarkers for Each Apple Variety

Aroma is a crucial criterion to assess the quality of apple fruits, juices, and ciders. The aim of this study was to explore similarities and differences in volatile profiles among apple fruits, juices, and ciders from different apple varieties (Festa, Branco, and Domingos) by headspace solid-phase microextraction gas chromatography–mass spectroscopy (HS–SPME/GC–MS). A total of 142 volatile organic compounds (VOCs) were identified, but only 9 were common in all analysed matrices and apple-tested varieties. Esters, alcohols, and aldehydes presented a higher concentration in apple fruits and juices, whereas esters, alcohols, and acids were dominant in ciders. Moreover, there were unique VOCs for each matrix and for each variety, highlighting the importance of the selection of apple varieties as an important factor to obtain good sensory and quality ciders, multiple benefits, and legal protection against the misuse of local products.


Introduction
Apple aroma is a crucial criterion to assess fruit quality. This organoleptic quality is due to several volatile organic compounds (VOCs) such as esters, alcohols, aldehydes, ketones, acids, and esters [1]. More specifically, 2-methyl butyl acetate, butyl acetate, and (E)-2-hexenal are reported as the most significant VOCs contributing to the typical apple aroma [2]. Most VOCs in apple juice are not genuine constituents of apples, but produced from precursors by enzymatic reactions upon squeezing [3,4]. Apple variety, environment, ripening stage, storage, and processing procedure are some factors that influence the content of VOCs in apple juices [3]. Consequently, cider, a globally popular beverage, is obtained from the partial or total alcoholic fermentation of apple juice (raw material) [5]. The level of VOCs in ciders depends on the applied technology, the microorganisms involved in the fermentation process, ageing on lees, maturation, and storage conditions [4,6,7]. Typically, ripe or overripe (senescent) apples are used in cider processing due to their softened structures, thus resulting in higher juice yields and an increase in sugar content during apple ripening [8]. In fact, in a recent study, for all studied varieties, senescent fruits provided more aromatic fermented apple beverages [9]. The VOCs formed during the fermentation process, such as 3-methyl-1-butanol, 2-phenylethanol, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl 2-methylbutanoate, 3-methylbutyl ethanoate, Foods 2020, 9,1830 3 of 18 variety) in the senescent ripening stage from 5 different trees in order to achieve as representative a sample as possible, and they were visually inspected to ensure no apparent damage or disease. The maturation index was determined (no more than 24 h after collection) using the starch-iodine test according to Blanpied et al. [15], using a reference colour-number chart, 8 being the number for senescent (over-ripe) apples.

Apple-Fruit Processing
The apples of each variety were cleaned with tap water, unpeeled, and deseeded. With the purpose of homogenizing the apple-fruit samples, all apple pieces were immediately transferred into a blender. An amount of CaCl 2 (3%, w/v) was added to avoid enzyme browning according to a previous study [16]. The mixture was stored in glass vials at 4 • C until analysis.

Apple-Juice Processing
Other servings of apple fruits (cleaned, unpeeled, deseeded, and cut into pieces) were squeezed at room temperature (22 ± 1 • C) using a hand press juicer machine for apples. An amount of CaCl 2 (3%, w/v) was added. In order to obtain a representative portion of the apple juice of a specific variety, several apple fruits of the same variety were used to make a whole juice. In this study, fresh (nonthermal processing) apple juices were used to collect data on the aroma descriptors of these juices that could be used as useful information in the traditional cider-making process. The obtained juice was divided into aliquots of 50 mL and stored in sealed glass bottles at 4 • C until further analysis.

Cider Processing
The cider (fermented apple beverage) samples were produced in 2017 for each variety (not blending) according to traditional fermentation methods in open stainless-steel tanks (100 L) at 14 ± 1 • C over the course of 3 weeks and in direct contact with lees. These ciders (from Festa, Branco, and Domingos apple varieties) were provided by specific producers (3 bottles of 750 mL for each variety) from JS, and they were obtained through fermentation by commercial Saccharomyces cerevisiae Bouquet yeast strains supplied by ENARTIS Portugal, LDA (Porto, Portugal), in an active dried form that was rehydrated and inoculated (20 g 100 L −1 ). After fermentation, sulphites (SO 2 ) with antimicrobial and antioxidant activities were added at 30 mg L −1 , and cider maturation (no more than 3 months) took place in stainless-steel tanks at a temperature of 14 ± 1 • C. Then, ciders were bottled in a dark glass after clarification that naturally occurred as the ciders stetted during maturation. The final product was transported to the laboratory in a cooler with ice and kept at 4 • C until chemical analysis for a maximum of 1 month.

Headspace Solid-Phase Microextraction
The headspace solid-phase microextraction (HS-SPME) procedure was adopted from a previous study validated in our laboratory with apple fruits [1], with slight modifications. In short, 5 g of apple fruit or 5 mL of apple juice or cider, 5 µL of 3-octanol (as IS to the concentration of 2.94 µg L −1 ), 2 g of NaCl were added into an amber glass with constant magnetic stirring of 500 rpm. Before using, the SPME fiber was conditioned according to the manufacturer's instructions and exposed to the headspace for 45 min at 40 ± 1 • C. Then, the fiber was removed from the glass vial and immediately inserted into the GC injector port for 6 min at 250 • C for the thermal desorption of the VOCs. All analyses were performed in triplicate (n = 3).

Gas Chromatography-Quadrupole Mass Spectrometry Conditions
Chromatographic separation conditions were adopted from previous reports carried out in different apple matrices by our research team [13] using an Agilent 6890N (Palo Alto, CA, USA) Foods 2020, 9, 1830 4 of 18 gas chromatography system equipped with a BP-20 (30 m × 0.25 mm i.d. × 0.25 µm film thickness) fused silica capillary column acquired from SGE (Darmstadt, Germany) with helium (Helium N60, Air Liquid, Portugal) as carrier gas at 1 mL min −1 (column-head pressure: 13 psi). The temperature of the injector was set at 250 • C, and a splitless injector equipped with an insert of 0.75 mm i.d. was used. The temperature programme was fixed as follows: initial temperature of 40 • C, a ramp of 3 • C min −1 to 220 • C, and constant temperature was kept for 10 min at the end. The GC-qMS interface was held at 220 • C, and the manifold and quadrupole temperatures were both set at 180 • C. For MS detection, an Agilent 5975 quadrupole inert mass selective detector was used with an electron-impact (EI) energy of 70 eV and source temperature of 180 • C. The electron multiplier was set up to the autotune procedure, and acquisition mass range was set from m/z 30 to 300. The identification of VOCs was performed by comparing GC retention time and mass spectra with those of the standard, when available (Table 1); all mass spectra were also compared with the data library (NIST, 2005 software, Mass Spectral Search Program v.2.0d; Washington, DC, USA). The match factor criterion for identification was higher than 80%; Kovats index (KI) values were calculated according to the Van den Dool and Kratz equation [17]. Values were contrasted with values reported in the scientific literature for similar columns (Bianchi, 2007;Ferreira 2009), and with databases available online (The Pherobase and Flavornet). Semiquantification was performed, and VOC concentration was estimated in comparison to the added amount of 3-octanol (used as IS) according to the following equation: VOC concentration = (VOC GC peak area/IS GC peak area) × IS concentration. This approach was performed in a previous scientific study of Madeira wines [18]. Analyses were performed in triplicate, and average values of concentration (µg kg −1 (fruits) and µg L −1 (juices and ciders)) were used in further data analysis. Total ion chromatograms obtained by HS-SPME/GC-qMS analysis of apple fruits, juices, and ciders of the different varieties are shown in Figure S1.

Statistical-Data Elaboration
All experiments were carried out in triplicate, and the relative concentration is presented as mean ± standard deviation (SD). Statistical analysis was completed by use of SPSS software version 25.0 (SPSS Inc., Chicago, IL, USA) by which one-way analysis of variance (ANOVA) and the multiple-range (Tukey's) test were performed to identify significant differences among the three matrices (fruits, juices, and ciders) and among the three varieties (Festa, Branco and Domingos). Significant differences were set at p < 0.05. Before applying the chemometric approach, data from GC-qMS analyses were median-normalized and Pareto-scaled [19]. Principal-component analysis (PCA) was used for unsupervised analysis, and partial least-squares discriminant analysis (PLS-DA) for supervised analysis. All features with a variable-importance-in-projection (VIP) score higher than 1.6 and differentially expressed in univariate analysis were considered to be potential biomarkers for the discrimination of samples on the basis of apple matrices (fruits, juices, and ciders). Hierarchical-clustering analysis (HCA) was generated by Ward and Euclidean distance in order to identify clustering patterns. Statistical analysis was performed using web-based application MetaboAnalyst v. 4.0, created at the University of Alberta, Canada [20]. Table 1. Relative concentration of volatile organic compounds (VOCs) identified in apple fruits (µg kg −1 ), juices (µg L −1 ), and ciders (µg L −1 ) of different varieties (Festa, Branco, and Domingos) by headspace solid-phase microextraction gas chromatography-quadrupole mass spectroscopy (HS-SPME/GC-qMS).

Contribution of Apple Matrices (Fruit, Juice, and Cider) on Volatile Profile
As stated above, this study allowed for identifying common VOCs among 3 matrices studied from 3 different varieties (Figure 2), and other specifics for each commodity (Table 1). In order to differentiate apple-fruit, -juice, and -cider samples by volatilomic profile, principal-component analysis (PCA) was performed (Figure 3).

Contribution of Apple Matrices (Fruit, Juice, and Cider) on Volatile Profile
As stated above, this study allowed for identifying common VOCs among 3 matrices studied from 3 different varieties (Figure 2), and other specifics for each commodity (Table 1). In order to differentiate apple-fruit, -juice, and -cider samples by volatilomic profile, principal-component analysis (PCA) was performed (Figure 3).  Table 1.
Thus, effective separation according to apple matrices was achieved. The closeness of the samples on the PCA score plot indicated a similar volatile profile, and the PCA biplot showed the relationship between loadings (VOCs) and variables (fruits, juices, and ciders). The variance of PC1 and PC2 was 41.6% and 19.1%, respectively, representing 60.7% of the total variability of data, allowing for good differentiation among apple fruits, juices, and ciders. Combining the variable-importance-in-projection (VIP) values from PLS-DA higher than 1.6 (data not shown), 15 VOCs were selected as putative markers for discrimination among apple fruits, juices, and ciders.  Table 1.
The main chemical families of VOCs found in ciders that formed the fermentation bouquet were esters and alcohols, and aldehydes and ketones to a lesser extent (Figure 1). In this way, among esters, methyl octanoate (71), ethyl nonanoate (96), ethyl decanoate (110), diethyl butanedioate (117), . Heat-maps of putative markers identified in apple fruits, juices, and ciders generated by Euclidean distance through Ward agglomerative method (peak number attribution shown in Table 1).
The main chemical families of VOCs found in ciders that formed the fermentation bouquet were esters and alcohols, and aldehydes and ketones to a lesser extent (Figure 1). In this way, among esters, methyl octanoate (71), ethyl nonanoate (96), ethyl decanoate (110), diethyl butanedioate (117), ethyl 9-decenoate (119), and 2-phenylethyl acetate (125) were identified in all cider samples from the 3 investigated varieties (Table 1). Acids could also be important odour compounds in the ciders, such as octanoic acid (139), with a sweaty cheese aroma that was only found in ciders, but not in fruits or juices. However, this VOC was semiquantified below its odour threshold (OT~3000 µg L −1 ). Among alcohols detected in ciders, 2 (3-methyl-1-pentanol (62) and 3-(methylthio)-1-propanol (120)) were identified in the ciders from the 3 varieties. Regarding the terpenoids detected only in ciders, β-damascenone (126), characterized by a woody, sweet, fruity, green, and floral aroma, was found in 2 of the 3 studied varieties, ranging between 13.2 and 17.8 µg L −1 . Additionally, in the current study, styrene (47) was only identified in ciders and was quantified for the first time. It is a terpenoid with sweet, balsamic, floral, and plastic odour attributes, and it has only been detected in ciders from all studied varieties, the Festa variety being the samples with the highest relative concentration (67.5 µg L −1 ).
On the other hand, there were several VOCs that were not identified in cider samples from the three varieties (Table 1). For example, α-farnesene (122) was identified in apple fruits and juices, but not detected in ciders. Moreover, toluene (18) with the sweet aroma is another VOC found in apple fruits and juices, but not detected in cider samples. Additionally, some VOCs, such as hexanal (22) and 2-hexenal (41), found in apple fruits and juices from the three different varieties, were not identified in ciders (Table 1). Likewise, the ketone family were decreased in ciders in comparison with in the fruits and juices (Figure 1). Four ketones (2-propanone (2), 3-octanone (46), 6-methyl-5-hepten-2-one (64) and 1,3-dihydroxy-2-propanone (140)) were not found in ciders, but they were identified in fruits and juices.

Impact of Apple Variety on Volatile Profile
Food-authenticity issues may be solved by the detection and eventual quantification of specific metabolites that are able to discriminate among specific varieties, as shown in Table 2. In this way, for example, in apple-fruit samples, α-farnesene (122) was detected in all varieties, but (Z,E)-α-farnesene (121) was only identified in 2 varieties (Festa and Branco). The same applied in the case of linalool (95), which in the current study was only identified in Branco fruit samples. Furthermore, another terpenoid (estragole (113) with sweet, phenolic, anise, spicy, green, herbal, and minty aroma descriptors) was only detected in apple fruits from the Branco variety, as can be seen in Tables 1 and 2. Thus, these VOCs could serve as authenticity indicators to verify apple-fruit variety. Another VOC only detected in the Branco variety (fruit and juice) was 2-nonenal (94). The Domingos apple sample was the variety with more unique VOCs in comparison with those in the other varieties (Festa and Branco) ( Table 2). In fact, benzothiazole (134) was a unique VOC to the Domingos apple juice and regarding the VOCs that were only present in Domingos ciders, such as pentyl acetate (33), decanal (89), citronellol (123), geranylcetone (129), or nerolidol (138) ( Table 2). This find provides us with a clear overview of the importance of the selection of apple varieties as a crucial factor for the cider-making process to obtain a cider of good sensory and quality properties.

Discussion
There are serious economic and quality reasons to certify the authenticity of varieties used in different food commodities. Moreover, as food processing progresses, for example, from apple fruits to ciders, it becomes extremely difficult to distinguish between varieties [13]. In this respect, a volatilomic pattern may be a useful tool to discriminate between food commodities and varieties. The main precursors of VOCs in apple fruits are fatty acids that are catabolized through β-oxidation and the lipoxygenase (LOX) pathway, which produce aldehydes, alcohols, and esters. Among these, aldehydes are predominant in immature apples, whereas alcohols and esters prevail in ripe/over-ripe fruits [22]. Regarding the different investigated variables (such as apple variety, ripening stage, and yeast strain), apple variety proved to be the primary attribute influencing the quality and aroma properties of apple ciders [23]. Three sources of VOCs in ciders, namely, apple juices, yeast, and yeast metabolism, were reported [4]. In the current study, the main chemical families of VOCs found in ciders that conferred the fermentation bouquet were esters and alcohols, and aldehydes and ketones to a lesser extent, as previously reported [24]. Regarding the different chemical families of VOCs, esters positively contribute to the aroma profile of ciders, bringing fruity and floral sensory properties [25]. More specifically, ethyl hexanoate (sweet, fruity, pineapple, waxy, fatty, estery, green, and banana odour descriptions) was reported as a VOC that increases in ciders in comparison with apple juices [26]. This VOC was associated with the fermentative process and the involved yeast strains [27], and, together with ethyl decanoate and ethyl octanoate, determines fruity and floral aromas in fermented fruit beverages [9]. However, there are other VOCs, such as 2-hexenal and 1-hexanol, which were described as the main contributors to the green odour of apple fruits and juices [4,28].
Regarding the fermentation process of apple juices, Antón et al. [25] found 3-methyl-1-pentanol to be a VOC that increases its concentration in cider samples from spontaneous fermentation in comparison with ciders from commercial Saccharomyces cerevisiae. This might be justified by yeast species associated with the spontaneous fermentation of both Saccharomyces and non-Saccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) that could affect concentrations of VOCs in ciders [29]. Styrene is another VOC reported in apple brandy and cider, with odour threshold values ranging between 3.6 to 80 µg L −1 [30], and in apple fruits [31]. The formation of this VOC may be because high cinnamic acid content and yeast pitching rate, in combination with open fermentation management, cause quick and increased styrene formation during fermentation, as was previously reported for wheat beer [32]. Thus, styrene may be used as an important indicator to monitor the cider-making process (as well as in beers) and management with food authentication purposes. In contrast, other VOCs were not detected in cider samples, such as toluene, which may be due to the toluene degradation pathway of S. cerevisiae (M00418 KEGG pathway) producing benzyl alcohol and benzaldehyde. Both VOCs were found in the current study in ciders. Recently, toluene was reported in apples for the first time [31] and was also identified in apple-juice samples from Madeira as a putative biomarker for the discrimination of the geographical origin of apple juices [13]. Furthermore, the conversion mechanism of benzyl alcohol to toluene in fruit juices was also recently reported [33].
On the other hand, there are VOCs that allow for distinguishing apple varieties. In this sense, (Z,E)-α-farnesene was only identified in 2 varieties (Festa and Branco) about 100 times less than another isomer ((E,E)-α-farnesene) [34]. In this context, in a previous study, (Z,E)-α-farnesene was able to differentiate banana plant cultivars since this VOC was detected in the Pacific plantain cultivar, but not identified in Cavendish cultivar, whereas (E,E)-α-farnesene was detected in both banana cultivars [35]. Hence, (Z,E)-α-farnesene might be used as a putative marker to discriminate apple-fruit varieties for food authenticity purposes. The same applies in the case of linalool, which, in the banana study mentioned above, was only detected in Pacific plantain and, in the current study, was only identified in Branco fruit samples; this VOC showed insect-and disease-control properties [35], with benefits for the quality of apple fruits. Both linalool and estragole could differentiate basil varieties [36]. In addition, 2-nonenal, with waxy and fatty aroma descriptors, was previously used to distinguish among 10 different fresh jujube varieties by HS-SPME/GC-MS coupled with E-nose [37]. Benzothiazole was also identified as a putative marker for distinguishing apple varieties from Madeira in a previous study on apple juices recently carried out by our research group [13].