Volatile Compounds and Physicochemical Quality of Four Jabuticabas (Plinia sp.)

The jabuticaba is a native Brazilian fruit that has aroused worldwide interest in terms of its nutritional composition and biological activity. However, research on the profile of volatile compounds (VOCs) emitted by these fruits is rare. This study presents the first identification of VOCs from four jabuticaba species. The aim of the study was to characterize the aromatic profile of the following species: ‘Sabará’ (Plinia jaboticaba), ‘Escarlate’ (Plinia phitrantha × Plinia cauliflora), ‘Otto Andersen’ (Plinia cauliflora), and ‘Esalq’ (Plinia phitrantha). The analysis was performed by headspace solid-phase microextraction combined with gas chromatography/mass spectrometry (SPME-GC-MS). Multivariate analysis techniques applying the partial least squares-discriminant analysis (PLS-DA) and heatmap were used to compare the results. Fruit quality parameters were determined in terms of fresh mass (g), skin color, soluble solids, and titratable acidity. A total of 117 VOCs was identified including terpenoids, esters, alcohols, aldehydes, alkanes, ketones, and carboxylic acids, with 36 VOCs common to all four species. Terpenes were the majority for all jabuticabas with smaller contributions from other volatile classes, especially β-cubebene, β-elemene, and D-limonene for the ‘Otto Andersen’ jabuticaba.


Introduction
Jabuticaba (Plinia sp.) is a native Brazilian fruit belonging to the botanical family Myrtaceae, naturally occurring in the Atlantic Rainforest biome. This fruit is a globose berry about 2.0 to 3.5 cm in diameter, which develops attached to the stem. When ripe, its skin may display various colors, such as red, dark purple, and black. The pulp has a pleasant taste and is white, gelatinous, sweet exhibiting low acidity, and may contain from one to four seeds [1,2]. Currently, about nine species of jabuticaba are known, as well as some varieties, among them Plinia jaboticaba, Plinia cauliflora, Plinia trunciflora, Plinia coronate, and Plinia phitrantha [3]. The jabuticaba has aroused the interest of the food industry, due to the presence of high content of compounds exhibiting antioxidant, anti-inflammatory, and anti-hypercholesterolemic activities, mainly anthocyanins and tannins [4][5][6].
'Sabará' jabuticabas (Plinia jaboticaba) are the most cultivated in Brazil. The fruit is black, with a thin and smooth skin, and a very sweet flavor. The 'Escarlate' (Plinia phitrantha × Plinia cauliflora) jabuticaba presents side fruits [7], with a scarlet-red color, juicy, and sweet pulp. Fruits belonging to the 'Otto Andersen' (Plinia cauliflora) species are black, with abundant pulps and very sweet. The 'Esalq' (Plinia phitrantha) jabuticaba is one of the largest of their kind, reaching up to 4 cm in diameter. It is a

Results and Discussion
The four jabuticaba species were characterized in terms of basic fruit quality attributes (Supplementary material Table S1). The 'Otto Andersen' (P. cauliflora) jabuticaba was noteworthy for presenting a greater fresh mass (11.6 ± 0.3 g), as well as a larger size compared to the other species (height: 25.7 ± 0.7 mm, diameter: 26.6 ± 0.3 mm). The same specie exhibited the highest soluble solids concentration of 19.9 ± 0.2 • Brix. This value corresponds to 1.8-fold the lowest measured value (11.2 ± 0.0 • Brix), in the 'Sabará' species. This index can also be compared to Crimson Seedless table grapes (Vitis vinifera) [18], considered a fruit with a markedly sweet flavor. The dark purple color of the fruit was characterized by the low hue angle and brightness for all species. The 'Esalq' jabuticaba is noteworthy for its lowest color angle (30.3 ± 2.2) and highest brightness (30.3 ± 1.2). The chromaticity, value a* and b* of the fruit skin were up to 3-fold lower in the 'Otto Andersen' and 'Sabará' jabuticabas in relation to the 'Scarlet' and 'Esalq' fruits.
The VOC production by the four jabuticaba species totaled 117 distinct compounds ( Table 1). The partial least squares-discriminant analysis (PLS-DA) was performed to provide an overview of the aromatic profile of jabuticabas ( Figure 1B and Table S2). The main contribution of the PLS-DA was the characterization of the volatile compounds emitted by each jabuticaba, indicating a characteristic aromatic profile for each of the evaluated species, however, the PLS-DA also allowed identifying groups of volatiles shared by the four jabuticabas ( Figure 1A). Table 1. List of volatile aroma compounds found in 'Sabará' (Plinia jaboticaba), 'Escarlate' (Plinia phitrantha × Plinia cauliflora), 'Otto Andersen' (Plinia cauliflora), and 'Esalq' (Plinia phitrantha) jabuticabas by headspace solid-phase microextraction combined with gas chromatography/mass spectrometry (SPME-GC-MS).   Figure 1B,C correspond to the code of compounds listed in Table 1. The separation of clusters is shown in detail in Table S2.

Code
(C)  Figure 1B,C correspond to the code of compounds listed in Table 1. The separation of clusters is shown in detail in Table S2.
The principal component analysis displays each species of jabuticaba allocated in a quadrant of the factorial plane, evidencing the dissimilarity between them ( Figure 1A,B). PC1 and PC2 components were responsible for 71.4% of the total data variability, being 44.7% (PC1) and 26.7% (PC2) ( Figure 1A). Observing Figure 1C, it is possible to group the jabuticabas 'Otto Andersen' and 'Sabará' as well as 'Escarlate' and 'Esalq' in two groups by the similarity between the volatile compound's profiles. Moreover, considering that 'Escarlate' jabuticaba is the result of a breeding of the species 'Otto Andersen' and 'Esalq', the greater influence of 'Esalq' on the constitution of the aromatic profile than 'Otto Andersen' is evident.
The 'Sabará' (P. jaboticaba) jabuticaba is the most common and most commercialized species in Brazil. This species presented 12 exclusive VOCs grouped in cluster 1-C1 ( Figure 1B): Propyl acetate, cyclofenchene, ethyl isovalerate, ethyl 3-hexenoate, (Z)-3-hexen-1-ol acetate, butyl 2-butenoate, cyclopropanecarboxylic acid,3-methylbutyl ester, (Z)-3-hexenyl (E)-2-butenoate, nonanol, 2-phenylacetamide, n-(1-phenyl-2-propyl), citronellyl butyrate, ethyl cinnamate-most of which are esters, with descriptive notes such as fruity, green, and sweet (Table S2). Esters are widely produced in soft-fleshed fruits during ripening, with the dual function of attracting animals and protecting the fruits against pathogens [19]. Esters are also the most important VOC class in strawberries, for example, where they are responsible for over 90.0% of the ripe fruit's aroma [20]. Acetate esters, such as (Z)-3-hexenyl acetate, are positively evaluated by consumers regarding pear acceptance in sensory analyses, and have been described in 'Sabará' jabuticaba [21]. Additionally, acetate esters are involved in defense mechanisms against abiotic stresses in peaches" which may suggest that they also play this role in jabuticaba [22]. Three ethyl esters were identified exclusively in 'Sabará' jabuticaba. This class of compounds has been described as one of the main components of the aroma of the Passiflora genus [19]. Aliphatic esters were the major contributors to the aroma of this fruit, indicating that the lipoxygenase (LOX) pathway, which produces sequential lipid degradation, is more active than in the other assessed species. The 'Escarlate' jabuticaba (P. phitrantha × P. cauliflora) presented the smaller C2 comprising three compounds: α-muurolene, β-patchoulene, 3,6-dimethyl-4h-furo[3,2-c]pyran-4-one (Table S2), the sesquiterpene α-muurolene are the only one with aromatic notes described in the literature. The 'woody-like' aroma of this compound is widely emitted by fruits and, mainly, by flowers of different species [23][24][25][26][27]. It has also been identified in fig species, and in Ficus racemosa, characterized by being one of the volatiles emitted during the daytime, with the function of attracting pollinating wasps [28,29]. For the 'Escarlate' jabuticaba these sesquiterpenes contributed with 4.4% of the total aroma emitted, differentiating it from the other jabuticabas, where α-muurolene was not identified.
The groupings C1, C2, C3, and C4 contain the differential elements that individualize the aromatic profile of the analyzed jabuticaba species (Table S1). These differences could be attributable to the species' genetic variability, considering that all jabuticabas were grown under the same environmental conditions. On average, the compounds identified represented a small relative area when compared to the total emitted volatiles. The participation of these VOCs in the formation of jabuticaba flavor can contribute to the composition of aroma nuances, while the contribution of the aroma base may be attributed to the most abundant compounds. The compounds with a greater relative area were common to the all four species. In total, 36 VOCs belonging to various classes were identified, namely (14) sesquiterpenes, (9) sesquiterpene alcohols, (5) monoterpenes, (3) alcohols, (2) aldehydes, (1) monoterpenes alcohols, and (1) esters ( Figure 2). [36], and corresponds to 70.0% of the aroma of ylang-ylang (Cananga odorata) and frankincense (Boswellia spp.), both traditionally used for therapeutic purposes [37].
The groupings C1, C2, C3, and C4 contain the differential elements that individualize the aromatic profile of the analyzed jabuticaba species (Table S1). These differences could be attributable to the species' genetic variability, considering that all jabuticabas were grown under the same environmental conditions. On average, the compounds identified represented a small relative area when compared to the total emitted volatiles. The participation of these VOCs in the formation of jabuticaba flavor can contribute to the composition of aroma nuances, while the contribution of the aroma base may be attributed to the most abundant compounds. The compounds with a greater relative area were common to the all four species. In total, 36 VOCs belonging to various classes were identified, namely (14) sesquiterpenes, (9) sesquiterpene alcohols, (5)   Terpenoids were the major volatile organic compounds in the aroma of 'Sabará', 'Escarlate', 'Otto Andersen', and 'Esalq' jabuticabas, representing 81.5% of the total, in accordance with previously reported results [5,12,38]. This result suggests that the mevalonic acid or pyruvate and 3phosphoglycerate routes, necessary for terpene biosynthesis, are the most active pathways in mature jabuticabas in relation to the other metabolic VOC-synthesizing pathways. The differences identified for the 36 VOCs were quantitative, with some compounds being more abundant in one species compared to another. This result was similar to a study carried out with citrus species [39].
The β-cubebene sesquiterpene was the most emitted volatile compound in the analyzed jabuticabas, with the exception of 'Otto Andersen', where it appears as the second most emitted VOC. Its contribution to the total area in each species was of 16.8%, 17.8%, and 15.9% for the 'Esalq', 'Escarlate', and 'Sabará' jabuticabas, respectively. β-cubebene has a pleasant aroma translated as fruity and citrus. This compound is commonly found in the essential oils of several species [40,41] with the third most abundant member of the essential oil being obtained from cinnamon leaves (Cinnamomum osmophloeum) [42].
The fresh, herbaceous, and waxy aroma of β-elemene ranks second among the most important emissions for jabuticabas, except for 'Otto Andersen', where it appears in the fourth place. β -elemene is a sesquiterpene extensively studied due to its biological functions. However, the anticancer activity is by far its most relevant function. β-elemene is capable of causing apoptosis and inhibiting the proliferation of several cancer cells until the most aggressive brain tumor [43]. Currently, Curcuma wenyujin, belonging to the Zingiberaceae family, is the plant most applied to the extraction of βelemene [44]. Our results suggest that jabuticabas, mainly the 'Esalq' and 'Escarlate' species, are a Terpenoids were the major volatile organic compounds in the aroma of 'Sabará', 'Escarlate', 'Otto Andersen', and 'Esalq' jabuticabas, representing 81.5% of the total, in accordance with previously reported results [5,12,38]. This result suggests that the mevalonic acid or pyruvate and 3-phosphoglycerate routes, necessary for terpene biosynthesis, are the most active pathways in mature jabuticabas in relation to the other metabolic VOC-synthesizing pathways. The differences identified for the 36 VOCs were quantitative, with some compounds being more abundant in one species compared to another. This result was similar to a study carried out with citrus species [39].
The β-cubebene sesquiterpene was the most emitted volatile compound in the analyzed jabuticabas, with the exception of 'Otto Andersen', where it appears as the second most emitted VOC. Its contribution to the total area in each species was of 16.8%, 17.8%, and 15.9% for the 'Esalq', 'Escarlate', and 'Sabará' jabuticabas, respectively. β-cubebene has a pleasant aroma translated as fruity and citrus. This compound is commonly found in the essential oils of several species [40,41] with the third most abundant member of the essential oil being obtained from cinnamon leaves (Cinnamomum osmophloeum) [42].
The fresh, herbaceous, and waxy aroma of β-elemene ranks second among the most important emissions for jabuticabas, except for 'Otto Andersen', where it appears in the fourth place. β -elemene is a sesquiterpene extensively studied due to its biological functions. However, the anticancer activity is by far its most relevant function. β-elemene is capable of causing apoptosis and inhibiting the proliferation of several cancer cells until the most aggressive brain tumor [43]. Currently, Curcuma wenyujin, belonging to the Zingiberaceae family, is the plant most applied to the extraction of β-elemene [44]. Our results suggest that jabuticabas, mainly the 'Esalq' and 'Escarlate' species, are a natural source of this compound, which displays the potential to be used as a raw material for its extraction, as reported for Nigella damascena [45]. D-limonene is the most abundant compound in the 'Otto Andersen' jabuticaba, representing 12.1% of the total emitted volatile compounds. This VOC is an important component of fragrances, used in cosmetic, pharmaceutical, and food products due to its 'citric-like' aroma [46] typical of oranges. This monoterpene has aroused growing interest with regard to the human health, where over sixty scientific articles have been published regarding its biological activity [47]. It exhibits an anti-inflammatory action [48], and is considered promising for the development of drugs to prevent gastric damage [49]. Other studies have reported its action, and its potential use in chemopreventive procedures against different types of cancers [50,51].
Linalool was abundant in 'Otto Andersen' jabuticabas, representing 9.0% of the total emitted VOC, 4-fold higher than the average of the other three species. The amount of linalool has been related to the regulatory action of methyl jasmonate acid in other species [55][56][57][58][59]. In addition, differences in the emitted amounts may change its specific functions and interactions with linalool itself or its derivatives, due to its multifunctional nature [60]. Linalool is a sesquiterpenoid alcohol with a floral aroma emitted by flowers, fruits [61], and even fungi [62], widely used in the composition of various fragrances. It is also the key compound of the aroma of conventional fruits such as tomatoes (Solanum licopersum) [63], citrus [64], and strawberries (Fragaria × ananassa) [65].
The compounds ethyl acetate, β-ocimene, o-cymene, and ethanol were abundant in the 'Otto Andersen' and 'Sabará' jabuticabas. For ethanol, the average of these two species was 17.3-fold higher than the average of the 'Escarlate' and 'Esalq' jabuticabas.
Some odoriferous compounds have low odor detection thresholds. Among those reported for the four jabuticaba species, benzaldehyde was observed in relatively low amounts ( Figure 2) but may have a significant impact on the aroma due to the low detection threshold (0.093 mg Kg −1 ) (Table S3). Other compounds with a low threshold of detection were identified with larger relative areas, such as D-limonene (0.04 mg Kg −1 ) and linalool (0.001 mg Kg −1 ) (Table S3).
Considering the physicochemical quality and aromatic profile of each of the jabuticabas analyzed, there is great potential and diversity to meet distinct consumer demands, and also support genetic improvement programs. The 'Otto Andersen' (P. cauliflora) jabuticaba stands out for its fresh mass, size, and concentration of soluble solids compared to other species. The typical dark purple color of the skin of the jabuticaba fruit was slightly more intense in the 'Esalq' species (P. phitrantha) and can be a positive factor in consumer choice. Regarding the composition of the aromatic profile, jabuticaba 'Sabará' is interesting for gathering the greatest number of exclusive esters of sweet and fruity notes. The 'Escarlate' jabuticaba is distinguished by the intense presence of terpenes, mainly sesquiterpenes. Jabuticabas are recognized for the presence of bioactive compounds such as ellagitannins and anthocyanins [67], now adding the existence of significant differences in their aromatic profile. The groupings of the volatile compounds of each jabuticaba suggest that changes in the regulatory aspects of the metabolic routes of each variety occur, mainly related to the concentration and availability of substrates, the type of substrate, and enzymatic activity [68]. The volatile profile of jabuticabas is constituted mainly by terpenes, both in the number of detected compounds and in relative area abundance. Among them, β-cubebene, β-elemene, d-limonene, γ-elemene are potentially characteristic candidates of the aroma of members from the Plinia genus. Additionally, it is important to note that volatiles abundant in jabuticaba, such as monoterpene d-limonene and sesquiterpene β-elemene, exhibit therapeutic efficiency proven in several studies. Therefore, jabuticabas can be a reference for studies on new possibilities of the uses of different species, prioritizing the extraction of compounds that can benefit human health.

Samples
The ripe jabuticabas from four varieties Sabará, Escarlate, Esalq, and Otto Andersen were harvested in November 2019 in Casa Branca, São Paulo, Brazil, latitude 21 • 46 26 S, longitude 47 • 05 11 W. After collection, the fruits were frozen at −20 • C and transported to the Laboratory of Postharvest of Horticultural Crops in Piracicaba, São Paulo, Brazil (LPV-ESALQ/USP). The fruits (approximately 1 kg of each jabuticaba) were selected according to the appearance, maturation stage, and absence of defects, to ensure homogeneity and uniformity and were frozen at −80 • C until analysis.

Quality Parameters
Fresh mass was determined on an analytical balance and the results expressed in grams (g). Skin color was obtained with a Minolta ® CR-300 colorimeter, with two readings taken on opposite sides of each fruit. The results were expressed as color angle ( • Hue), luminosity (L *), chromaticity (C *), a * coordinates and b * coordinates.
The height, diameter, and shape of the fruit were measured with a digital caliper with two readings taken at equidistant points for each attribute. The results were expressed in millimeters (mm).
The soluble solid content in the pulp was quantified using an Atago Palette PR-101 digital refractometer [69]. The results were expressed as • Brix. The pulp's titratable acidity was determined by the neutralization titration, using 2.0 g of filtered pulp homogenized in 18 mL of distilled water. The samples were titrated with a standard 1 N sodium hydroxide solution (NaOH) to pH 8.1 [70]. The results were expressed as % of citric acid equivalents.

Sample Preparation
A total of 3 g of pulp and skin from 150 fruits of each species of jabuticaba were crushed in an analytical mill (IKA A11) with liquid N 2 and placed in 20 mL glass flasks containing 7 mL of a 30% (w/v) NaCl solution. The flasks were then sealed with stainless steel lids with silicone septa and stored at −20 • C for GC-MS analysis.

GC-MS Analysis
The volatile compounds were analyzed applying the solid phase microextraction method (SPME), consisting of five replicates each [71]. The vials were thawed in a water bath at 40 • C, under agitation, for 10 min, to accumulate volatile compounds in the headspace. After the equilibrium time, the septum was punctured with a needle, allowing for exposure of the SPME fiber (divinylbenzene, carboxene, and polydimethylsiloxane 50/30 µm, Supelco, Inc., (Sigma-Aldrich, Belfonte, PA, USA)) for 50 min.
Once captured, the volatile compounds were desorbed from the fiber by exposure to the heat of the chromatography injector (200 • C) for 5 min.

Statistical Analyses
An analysis of variance (one-way ANOVA) and the Tukey test (p < 0.05) were applied to the physical-chemical characterization data for comparison between species-sample size (N = 5). A partial least squares-discriminant analysis (PLS-DA) and hierarchical clustering and heatmap, prepared using the Metaboanalyst 4.0 program [72] were used to verify the difference between the VOC profiles.