Pattern Recognition of Varieties of Peach Fruit and Pulp from Their Volatile Components and Metabolic Profile Using HS-SPME-GC/MS Combined with Multivariable Statistical Analysis

A fruit’s aroma profile, composed of a complex mixture of volatile organic compounds, is among the core attributes related to the overall taste and consumer preference. Prunus persica L. is a preferred summer fruit with a distinct, favorable olfactory characteristic. The volatile compositions of both peach fruits and fruit pulps from eight peach cultivars (four native and four introduced) was investigated to compare their composition and assess flavor-contributing compounds. In total, 65 compounds were profiled after a HS-SPME-GC-MS analysis: 16 esters, 14 aldehydes, 5 alcohols, 7 hydrocarbons, 7 ketones, 8 acids, and 8 terpenes. The most common compounds were esters, acids, and aldehydes. Although the same compounds were identified in both fruit and pulp, their %TIC (total ion current) differed in favor of the whole fruit. Following the metabolic profiling of the whole fruit and fruit pulp, a total of 44 compounds were identified from the studied varieties. Among them, amino acids, organic acids, sugar alcohols, saccharides, fatty acids, and phenolic acids were identified as existing groups. According to the provided principal component analysis (PCA) and hierarchical cluster analysis (HCA), the relative %TIC of the identified volatile compounds fluctuated depending on the studied cultivar. No differences were visible in the PCA biplots, which suggested that the polar and lipid metabolites do not provide significant variations when considering different parts of the fruit, contrary to the volatile compounds. The obtained results could successfully be applied in the metabolic chemotaxonomy of peaches and the differentiation of the metabolites present in different parts of the peach.


Introduction
Fruit consumption is very important for general health and wellbeing, with a number of national strategies focusing on its increased intake. Although components like sugar content, element composition, fibers, phenolic acids, and organic acids will contribute positively to the health of the average individual, volatile compounds are the ones that will provoke the desire to eat. Volatile compounds are extremely important when the consumer makes the final decision about foods that will be eaten. The peach fruit is one of the most preferred fruits worldwide, consumed both fresh and processed. The volatile profile of peaches has been studied with various aims throughout the years [1 -3].
Different methods are applied in volatolomics. Currently, the HS-SPME-GC-MS has been recognized as a useful method that drastically minimizes interference during identification [4]. Prior to analysis, the extraction process has to be chosen carefully in order to result in a proper determination [5]. The taste/aroma qualities of fruits, and peaches Organic acids are generally important to the specific tastes of fruits due to the sourness and acidity they provide [27]. The eight studied varieties ranged in terms of shikimic acid, malic acid, citric acid, quinic acid, and succinic acid, with the WF having higher levels compared to the FP. The primal organic acid in all samples (FP and WF) was shikimic acid, while the least present was L-ascorbic acid. Shikimic acid has been previously reported as most present in peach varieties [28]. Similarly, malic acid, citric acid, quinic acid, and succinic acid are regularly reported as the dominant ones [29][30][31].
Phenolic compounds are considered to be widely distributed in plants [32]. The current results (Table 1) reveal the presence of six phenolic acids, while only one of the varieties ("July Lady") lacked p-coumaric acid. Studies acknowledge the importance of phenolic acids due to their biological functions [33]. Here, once more the whole fruit sample is found to be richer in phenolic acids compared to the fruit pulp. This proves that the fruit skin should be an object of research and consumed where appropriate due to its ability to accumulate health-contributing compounds. Protocatechuic acid, chlorogenic acid, and caffeic acid are most present in the WF and FP of the "Gergana" variety, p-coumaric acid and sinapic acid in the "Filina", and ferulic acid in the "Flat Queen". By default, chlorogenic acid is reported as the most abundant in plant samples [34], which is further supported by the current results. Chlorogenic acid is reported as a possible preventive of cardiovascular disease [35].
The amount of amino acids established in the current study reveals that peaches cannot be considered protein contributors in the daily diet. A total of 18 amino acids have been identified in the peach varieties object of analysis. From all studied samples, the two nectarine varieties ("Gergana" and "Morsiani 90") are the only ones that contain all essential amino acids in even limited quantities. Aspartic acid is the least detected in all samples. The same trend as for other compounds, where the WF is richer than the FP, is observed for the amino acids.
In accordance with earlier studies [36], linoleic acid is reported as one of the most abundant fatty acids. Other fatty acids (FA) found in greater quantities are the palmitic, oleic, and stearic acids. The WF samples are documented as richer in FAs compared to the FP ones. The "Laskava", "July Lady", and "Morsiani 90" varieties hold the most FAs compared to the other studied varieties. There is a predominance of long-chain saturated fatty acids and the presence of more PUFA compared to MUFA. Based on existing reports [37,38], these findings hint at the possibility of more profound studies on the topic of the possible health effects of the fatty acid content in peaches.
The investigated volatile compositions of both peach fruit and fruit pulp from eight peach cultivars are presented in Table 2. In total, 65 compounds were profiled after an HS-SPME-GC-MS analysis: 16 esters, 14 aldehydes, 5 alcohols, 7 hydrocarbons, 7 ketones, 8 acids, and 8 terpenes. The highest content of volatile compounds was attributed to the "Morsiani 90" variety ( Table 2) (both pulp and whole fruit), while the lowest belonged to the "Ufo 4" (whole fruit) and "July Lady" (fruit pulp). More volatiles were generally present in the whole fruit of peaches, contrary to flat peaches and nectarines, where more volatiles were identified in the fruit pulp. None of the metabolites was identified in only either pulp or whole fruit. However, the whole fruit was richer in terms of %TIC of the identified compounds, which undoubtedly points to the importance of the peel for the flavor experience and is a favorable argument for consuming the fruit without peeling it. The chemical families that were more abundant in the fruit pulp were hydrocarbons, alcohols, and ketones.   The compositions and concentrations of volatile compounds and metabolites in the peach fruits and pulps are shown in Figure 1. The profile shows that the samples consist of the same chemical classes. However, total emission is not only variety-dependent but also fluctuates depending on the presence or absence of the fruit skin.
Some of the identified volatiles in the studied peach varieties belong to the so-called microbial volatiles [39], which are natural substances that enhance plant growth, productivity, and disease resistance. A few of them are butanoic acid (high % of TIC in the "Laskava" and "July Lady" varieties), benzaldehyde (relatively low in all studied samples), hexanol (relatively low in all studied samples), and myrcene.
Saccharides and fatty acids are the most frequent compounds in the WF and FP of the studied peaches, with different percentage distributions (Figure 1). The least found compounds are the sugar alcohols. Aldehydes and esters were the most present compounds in the whole fruits of the studied varieties (Figure 1). Alcohols, on the other hand, were the least found in all samples. Previous research on the WFs of peach varieties has established a similar trend in the results [2]. Analogous compound groups have also been identified in a recent study of famous Chinese peach cultivars [40]. The compositions and concentrations of volatile compounds and metabolites in the peach fruits and pulps are shown in Figure 1. The profile shows that the samples consist of the same chemical classes. However, total emission is not only variety-dependent but also fluctuates depending on the presence or absence of the fruit skin. Some of the identified volatiles in the studied peach varieties belong to the so-called microbial volatiles [39], which are natural substances that enhance plant growth, productivity, and disease resistance. A few of them are butanoic acid (high % of TIC in the "Laskava" and "July Lady" varieties), benzaldehyde (relatively low in all studied samples), hexanol (relatively low in all studied samples), and myrcene.
Saccharides and fatty acids are the most frequent compounds in the WF and FP of the studied peaches, with different percentage distributions (Figure 1). The least found compounds are the sugar alcohols. Aldehydes and esters were the most present compounds in the whole fruits of the studied varieties (Figure 1). Alcohols, on the other hand, were the least found in all samples. Previous research on the WFs of peach varieties has Considering their abundance, the most important aldehydes for the WF and FP are (E)-2-hexenal, hexanal, heptanal, and nonanal. Contrary to the current results, benzaldehyde was identified as a major aldehyde contributor in a study of American peach cultivars [3]. Heptanal has its highest quantity in the early-ripening variety "Filina". Hexanal and (E)-2-hexenal are compounds which are associated with the maturation process, and their quantities are lower in the FPs of all studied varieties. Hexanal was found to be naturally occurring within the fruit and is considered important for the volatile profile of nectarines, in particular [41]. Its %TIC in the two studied nectarine varieties is rather similar, even though the ripening period is different. Both hexanal and E-2-hexenal are also regularly identified in apple cultivars and are associated with a specific leafy-sweet odor [42].
According to other research, the acid content in peaches is rather low [43]. The most dominant acids in the investigated peach whole fruits and pulps are dodecanoic acid and nonanoic acid. Ketones come in relatively low amounts, with γ-octalactone and γdodecalactone being the most abundant. Lactones are usually isolated in peach volatiles, and they are flavor-contributing [26]. Lactones are also linked to a specific peach-like odor and flavor [44]. According to the established results, the "Ufo 4" and "July Lady" should exhibit the most distinct peach-associated aroma. It should also be mentioned that the fruit pulp contains more lactones compared to the whole fruit. Γ-Dodecalactone is also a major lactone isolated in other research [3]. Lactones, particularly delta-lactones, are as well implicated in the peach aroma. Recently, benzaldehyde, linalool, and C 10 lactones were found to increase in the final period of peach ripening, while C 6 aldehydes decreased [2,45].
A total of five alcohols were Identified in all studied samples. The relative cumulative alcohol content ranged from 2.69 %TIC ("Filina", WF) to 6.73 %TIC ("Evmolpiya", FP). The highest alcohol content is found in the "Evmolpiya" variety, in both the WF and FP. The content of benzyl alcohol, which is a natural component in peaches [46], gradually increases for the varieties that ripen in the months of July and August and then decreases in the late-ripening "Flat Queen" and "Evmolpiya" varieties. The "Morsiani 90" does not fall into this trend, as it is the latest ripening (mid-September) variety used in this study, and a steady increase in its benzyl alcohol content is registered. This may suggest that not only the ripening period, but the cultivar as well contributes to the volatile peculiarities of each variety.
Esters contributed largely to the overall volatile components identified in this study. Moreover, the most abundant esters (average content >3.00%TIC) as determined by GC-MS were ethyl tiglate, ethyl hexanoate, ethyl benzoate, and 2-phenyl ethyl butanoate. Methyl decanoate and benzyl butanoate were present in relatively low amounts (average content <1.5% TIC) in the FPs of each variety. Consistent with previous reports [47,48], ethyl tiglate was the most abundant in most of the WFs of the cultivars analyzed in this study.
The estimated amount of terpenes identified in the studied peach varieties is less than that estimated in the peach cultivar "Cresthaven" grafted onto five different rootstocks [49]. However, a resemblance in the compounds found in the highest quantities (limonene, linalool, and ocimene) is visible. This indicates that the cultivar, seasonal dynamics, and growing conditions influence the volatiles emitted by the same fruit, but there are some specific compounds that can be found in all cultivars.
Among the seven identified hydrocarbons, the amount of tetradecane stood out from the others. It was also observed that the nectarine samples were richer in tridecane compared to flat peaches and peaches. These results comply with and extend the ones published about peach volatile emissions from Chinese samples [50].
Aroma is very valuable for the consumer, and specific attention should be paid to odor-important compounds. Thus, if key odor-active compounds for peaches have to be identified, lactones will be among them [51]. Although past research [52] on the relative VOC concentrations and sensory attributes of peaches found higher concentrations of monoterpenes and esters compared to lactones in peaches, the latter are major contributors to the specific fruity, peach-like aroma [53]. Terpenoids and aldehydes are seen as insignificant contributors [53]. The sensory quality of peaches and fruit in general is also dependent on the odor threshold [54]. This may be the reason for odor-active substances' inability to produce flavor intensity among the overall volatile compounds.
With reference to the abovementioned knowledge, fruit skin is identified as an extremely important contributor to the overall sensory experience in terms of lactone content. Consumption of peaches with their skin will not only contribute to the zero-waste approach promoted nowadays but will also maximize the sensory experience of the consumer.

Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) of HS-SPME-GC-MS Data
PCA is a commonly used dimensionality-reduction method that provides visualizations of data. As is evident from Figure 2, the studied cultivars were divided into different groups when the WF or only the FP was being evaluated. This undoubtedly proves the importance of the fruit skin to the overall volatile contribution.
The applied multivariable analysis aided in the identification of the volatiles that contribute to the differentiation of the peach varieties' WFs and FPs. High positive load scores in PC1, which distinguish the "Flat Queen" from the other studied peaches, are shown by dodecane, nonanal, and ethyl octanoate. The high negative scores in PC1 clearly differentiate the "July Lady" and "Laskava" whole fruits from the others. When the fruit pulp is assessed, high positive load scores in PC1 distinguish again the "Flat Queen" from the other studied varieties in terms of 1-octen-3-yl-butanoate. The "Ufo 4" pulp appeared different from the rest due to the negative scores of several compounds in PC2.
identified, lactones will be among them [51]. Although past research [52] on the relative VOC concentrations and sensory attributes of peaches found higher concentrations of monoterpenes and esters compared to lactones in peaches, the latter are major contributors to the specific fruity, peach-like aroma [53]. Terpenoids and aldehydes are seen as insignificant contributors [53]. The sensory quality of peaches and fruit in general is also dependent on the odor threshold [54]. This may be the reason for odor-active substances' inability to produce flavor intensity among the overall volatile compounds.
With reference to the abovementioned knowledge, fruit skin is identified as an extremely important contributor to the overall sensory experience in terms of lactone content. Consumption of peaches with their skin will not only contribute to the zero-waste approach promoted nowadays but will also maximize the sensory experience of the consumer.

Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) of HS-SPME-GC-MS Data
PCA is a commonly used dimensionality-reduction method that provides visualizations of data. As is evident from Figure 2, the studied cultivars were divided into different groups when the WF or only the FP was being evaluated. This undoubtedly proves the importance of the fruit skin to the overall volatile contribution. The applied multivariable analysis aided in the identification of the volatiles that contribute to the differentiation of the peach varieties' WFs and FPs. High positive load scores in PC1, which distinguish the "Flat Queen" from the other studied peaches, are shown by dodecane, nonanal, and ethyl octanoate. The high negative scores in PC1 clearly differentiate the "July Lady" and "Laskava" whole fruits from the others. When the fruit pulp is assessed, high positive load scores in PC1 distinguish again the "Flat Queen" from the other studied varieties in terms of 1-octen-3-yl-butanoate. The "Ufo 4" pulp appeared different from the rest due to the negative scores of several compounds in PC2.
PC1 and PC2 explain 44.4% of the total variation in the peaches' whole fruits, while the differences in the pulps when considering PC1 and PC2 account for 58.1% of the variation. The clustering results also confirm the differences when taking into account the WF or only the FP. The conducted analysis of the data revealed that peaches, flat peaches, and nectarines can fall into the same cluster when the contribution of their volatiles is being assessed.
Considering the clade arrangement in Figure 2A,B, it can be concluded that when the fruit pulp is being evaluated, more differences are visible between cultivars compared to evaluation of the whole fruit. In both dendrograms, there are seven clades consisting of PC1 and PC2 explain 44.4% of the total variation in the peaches' whole fruits, while the differences in the pulps when considering PC1 and PC2 account for 58.1% of the variation. The clustering results also confirm the differences when taking into account the WF or only the FP. The conducted analysis of the data revealed that peaches, flat peaches, and nectarines can fall into the same cluster when the contribution of their volatiles is being assessed.
Considering the clade arrangement in Figure 2A,B, it can be concluded that when the fruit pulp is being evaluated, more differences are visible between cultivars compared to evaluation of the whole fruit. In both dendrograms, there are seven clades consisting of different arrangements when evaluating the WF and FP. For example, the fruit pulp leaves the "Morsiani 90" and "July Lady" as clustered together, being the most different from the others. On the other hand, the whole fruit reveals the "Filina" and "Flat Queen" as the most different from the others, but similar to each other. Here, once more, the importance of the skin is observed in the differences of the data visualization.
The same approach was used in terms of the polar and lipid metabolites, and the results are presented in Figure 3. PC1 and PC2 explain 63.4% of the total variation in the peaches' whole fruits and FPs. No differences are visible in the PCA biplots, which suggests that the polar and lipid metabolites do not provide significant variations when considering different parts of the fruit, contrary to the volatile compounds. Saccharides, being the most frequent compounds in the WFs and FPs of the studied peaches, exhibit high positive load scores.
Considering the clade arrangement, it can be assumed that when the fruit pulp is being evaluated, more differences are visible between cultivars compared to evaluation of the whole fruit. In both dendrograms ( Figure 3A,B) there are six clades consisting of the same arrangements when evaluating WFs and FPs. The "July Lady" is simplicifolious, which is significantly different from the arrangement when volatiles are being evaluated. The "Flat Queen" and "Morsiani 90" are revealed as the most different from the others, but they are similar to each other. different arrangements when evaluating the WF and FP. For example, the fruit pulp leaves the "Morsiani 90" and "July Lady" as clustered together, being the most different from the others. On the other hand, the whole fruit reveals the "Filina" and "Flat Queen" as the most different from the others, but similar to each other. Here, once more, the importance of the skin is observed in the differences of the data visualization. The same approach was used in terms of the polar and lipid metabolites, and the results are presented in Figure 3. PC1 and PC2 explain 63.4% of the total variation in the peaches' whole fruits and FPs. No differences are visible in the PCA biplots, which suggests that the polar and lipid metabolites do not provide significant variations when considering different parts of the fruit, contrary to the volatile compounds. Saccharides, being the most frequent compounds in the WFs and FPs of the studied peaches, exhibit high positive load scores.
(A)  Considering the clade arrangement, it can be assumed that when the fruit pulp is being evaluated, more differences are visible between cultivars compared to evaluation of the whole fruit. In both dendrograms ( Figure 3A,B) there are six clades consisting of the same arrangements when evaluating WFs and FPs. The "July Lady" is simplicifolious, which is significantly different from the arrangement when volatiles are being evaluated. The "Flat Queen" and "Morsiani 90" are revealed as the most different from the others, but they are similar to each other.