Physical˗chemical and chemical characterization of Passiflora cincinnata Mast fruits conducted in vertical shoot positioned trellis and horizontal trellises system

Passiflora cincinnata Mast. or Caatinga passion fruit is widely cultivated in the northeastern semi-arid regions of Goiás, Minas Gerais, and Bahia. The system of cultivation and field management of the Passiflora species affects the physicochemical quality of its fruits. In this study, we evaluated the chemical and physico-chemical characteristics of the fruits of 2 progenies (CPEF2220 and CBAF2334) of P. cincinnata species using the vertical and horizontal trellis system. Fruits were collected from the plants at their physiological maturation stage and tested for the fruit skin color and texture and the pH, total soluble solids, titratable acidity, pulp acidity ratio, and the contents of flavonoids, anthocyanins, and polyphenols in the seedless fruit pulp. The experimental design was completely randomized in a 2 x 2 factorial scheme (progenies x conduction system) performed in 3 replications using 4 plants in each. The analysis of variance and the means were compared using the Tukey’s test at 5% probability. The results revealed that the CBAF2334 fruits presented with greener pigments and greater flavonoids. The fruits were characterized by their green color with some yellowish nuances and a low color saturation in both the progenies. The conduction system was not found to affect the physical and physicochemical characteristics as well as the anthocyanin and polyphenol contents of the studied species. The fruits of plants cultivated on vertical trellis presented with 56.73% more flavonoid content than those cultivated on horizontal trellis.


Introduction
Passiflora cincinnata Mast. bears green-colored fruits (IMIG, 2013) and its fruit pulp is characterized to have acidic pH. This fruit is popularly known as the passion fruit of the bush (JESUS; , the passion fruit of brabo, the passion fruit of green shell, the passion fruit-shark (OLIVEIRA; RUGGIERO, 2005), and the passion fruit of the Caatinga (EMBRAPA, 2016). P. cincinnata is commonly found in the Caatinga and Cerrado biomes of Brazil, and it occurs abundantly in the northeastern semi-arid regions of Goiás, Minas Gerais, and Bahia (JESUS;. Among the several commercially important Passifloras occurring in Brazil, P. cincinnata possess significant commercial potential (FALEIRO et al., 2017). For this purpose, it is deemed important to develop genetically superior cultivars of this species and optimize the production system. Presently, there is a lack of appropriate genotypes for cultivation purpose, which needs to be resolved via genetic improvement programs (MELETTI et al., 2005;GONÇALVES, 2005). Recently, the BRS Sertão Forte (BRS SF) variety developed by Embrapa has been made available in the market; this variety is a result of the cross of 2 select plant progenies that are adapted to the semiarid region, namely, the CBAF 2334 and CPEF 2220 (EMBRAPA, 2016).
The presently available passion fruit cultivation system is based on the growth habit of this plant (BERNACCI, 2003), which needs support for conduction. Accordingly, there are 2 main types of structures: the horizontal trellis system consisting of a horizontally interwoven wire structure supported by 4 pillars (cuttings) and the vertical trellis system in which 2-3 vertically supported wires are used (LIMA et al., 2002;ZACHARIAS et al., 2016). Komuro (2008) and Monzani (2017) did not verify the differences in the physical and chemical quality of the fruits of sour passion fruits (P. edulis) in different conduction systems. Based on the time of the year of the study, Carvalho et al. (2018) found differences in some of the chemical compounds in the pulp of P. setacea species between the two conduction systems (vertical and horizontal trellis). However, similar studies are lacking for P. cincinnata.
Regarding the chemical properties of P. cincinnata, phenolic compounds are known to be present in its leaves, stems, and fruits (from peel, pulp, and seeds) (LESSA, 2011;SIEBRA, 2013;SIEBRA et al., 2014). Phenolic compounds such as flavonoids and anthocyanins are components of the flavonoid polyphenol class -a polyphenol subtype. These compounds can act as freeradical reducing and sequestering agents (CHITARRA; CHITARRA, 2005). Owing to the beneficial effects of this fruit on the human health, this plant species has gained much interest, especially with respect to its consumption and potential for the production of functional foods (GADIOLI et al., 2018;COSTA, 2017).
From anthesis to the maturation, fruits undergo several morphological, histochemical, and biochemical differentiations (BIALE, 1964). All these processes occur as a result of the expression of genes, especially those of the MAD-box complex, such as APETALA1 (AP1), which acts mainly in relation to the petal and sepal development, APETALA3 (AP3) and PISTILLATA (PI) for the petal and stamen, AGAMOUS (AG) for stamens and carpels, SEPALLATA 1-4 (SEP 1-4) for the floral organs, and some others that act on the reproductive development of plants (CUTRI;DORNELAS, 2011). Genes such as SHATTERPROOF 1 and 2 (SHP1 and SHP2) and FRUTIFUL (PeFUL) are also attributed for their effect on fruit development (LILJEGREN et al., 2000;SCORZA et al., 2017). Thus, the gene expression interacts with the environment and gets modulated by the area of cultivation and the cultivation system; these factors can interfere in the productivity and quality of the resultant fruits due to the differences in the climate.
The different regions in Brazil share climatic differences. For instance, the Brazilian semiarid region in the northeast is characterized by the occurrence of seasonal and periodic droughts, annual average rainfall of <800 mm, and an annual average temperature of 23-27°C (MOURA et al., 2007). In Cerrado, the average annual temperature is approximately 22-23°C and the average annual rainfall is 1200-1800 mm, concentrated mostly between October and March (COUTINHO, 2018).
In this study, we evaluated the physicochemical and chemical characteristics of the fruits of 2 P. cincinnata progenies in 2 different cultivation systems at the Cerrado region of the Central Plateau.

Material and Methods
Two parental progenies (CBAF 2334 and CPEF 2220) of P. cincinnata cv. BRS Sertão Forte were sourced from the Embrapa Semiarid germplasm bank. These progenies have their genetic origin in the northeast semiarid regions and were grown at the Embrapa Cerrados Fruit Support Unit, 15°36'13.02"S; 47°43'17.34"O and at approximately 1050 m altitude, Planaltina-DF).
Holes with dimensions of 60 cm (diameter) and 60 cm (depth) were prepared using a drill bit. For planting, dolomitic limestone to raise V to 50%, 50 g/pit of P 2 O 5 (Simple Superphosphate); 20 g/pit of N (Ammonium sulphate); 60 g of K 2 O ( Potassium chloride); 100 g/well of FTE BR12; and 10 L/pit of organic matter (chicken litter) were used. The first mulching was performed after 60 days of planting, followed by others at every 45 days using 100 g/plant dosage of potassium chloride and ammonium sulphate (1:2).
The crops were implemented on April 9, 2015 and transplanted to seedlings with 3.5 months in a completely randomized design using a 2 × 2 factorial scheme (2 progenies × 2 conduction systems) with 3 replications of 4 plants per plot. The plants were distributed in 2 rows, with intervals between the plants and between rows of 2.5 m × 2.5 m in the vertical trellis system and 2.5 m × 5 m in the horizontal trellis system (Figure 1).

Figure 1. Representation of experimental area and plant distribution in vertical trellis and horizontal trellis.
The fruits were collected after they fell to the ground (physiological maturation point). Eight fruits from each plot were collected between May and June of the year 2016 based on the availability of fruit abscission, followed by storage at 10°C and 90% relative humidity for a maximum of 48 h until the physical, physicochemical, and chemical analysis of the collected fruits.
The physical features that were analyzed included the peel coloration of the fruit at 5 distinct points using a portable spectrophotometer (MiniScan EZ; HunterLab ® ) which measured the values of L (luminosity), 'a' (indicative of green or red), 'b' (indicative of yellow or blue) of which the average values of 'a' and 'b' were used to calculate chromaticity (color intensity) and hue angle (color tone) (McGuire, 1992); firmness of the fruits, making 3 holes at equal distance from the middle portion using a texturometer (CT3 4500; Brookfield Texture Analyzer ® ) configured in the normal test mode with a force of 100 g, 5-mm deformation, and the speed of 10 mm/s equipped with the TA 17-tip type 24-mm D 30°. The results were expressed in Newton (N).
The pulp was separated from the fruit using a blunt blade food mixer, followed by sieving. The physicochemical analyses of the pulp were performed according to the methodologies described by the ADOLFO LUTZ INSTITUTE (2008). For instance, soluble solids (SS) content was determined using a digital refractometer (HI 96801; Hanna ® ); pH with a pH meter equipped with an electrode and potentiometer (HI 221; Hanna ® ); titratable acidity using potentiometric volumetry by titrating the standard 0.1 N sodium hydroxide solution in 2-mL of the pulp diluted in 48 mL of distilled water using a manual dispenser (776 Dosimat ΩMetrohm ® ) and a pH meter (HI 2211; Hanna ® ); the results are expressed in per gram of citric acid and the ratio in SS/titratable acidity.
Chemical analyses of the anthocyanin and flavonoid contents (LEES; FRANCIS, 1972) were performed using 5 g of the sample (seedless pulp) homogenized in ethanol and 1.5 N (85:15 v/v) HCl solution in 50-mL volumetric flask. The samples were stored under light shelter in a refrigerated environment for 16 h. Next, the sample was filtered and subjected to absorbance measurement on a spectrophotometer (Biomate 3; ThermoFisher ® ) at a wavelength of 374 ηm for flavonoids and 535 ηm for anthocyanins.
The polyphenols content was determined by using the spectrophotometric method of total extractable polyphenols dosage using the Folin Ciocalteau reactive, following the procedures of obtaining the extracts by Larrauri et al. (1997) and that of reading by Obanda and Owuor (1997). The moisture in the pulp was removed by drying 5 g of the sample from each frozen experimental unit and freeze drying for 24 h; the results obtained were used to calculate the chemical compounds on dry matter basis.
Statistical verification of the significance of the treatments was performed by applying analysis of variance (ANOVA). Residual normality assumptions were verified by the Shapiro-Wilk test (MIOT, 2017) and the homogeneity of variance by the Levene test (LEVENE, 1960). Tukey's test was used to compare the means at a 5% probability level.
Associations among the physical, physicochemical, and chemical variables of the fruit were determined by the Pearson's Linear Correlation analysis. All analyzes were performed using the R statistical software (version 3.5.0) (R, 2018).

Results and discussions
The positive effects of the selected progenies CPEF 2220 and CBAF 2334 in the 2 conduction systems were evaluated, but no effect of the interaction was noted between the progenies and the adopted conduction systems. The values concerning the color and texture characteristics (firmness) of the fruits are presented in Table 1.
The fruits showed slight difference in their coloration, as the only color component that showed differences was in the values of "a", which represented color variation from red (+) to green (-). Negative 'a' values (Table 1) indicated the predominance of the green fruit color (MCGUIRE, 1992), and the differences showed that the fruits of progeny CBAF 2334 had a color that tended more toward green. The values of "b" indicate values within the yellow (+) scale of the Cielab system (MCGUIRE, 1992).  The fruits of these two progenies shared the same color shade, because there were no significant differences in the hue angle values (Table 1). The values presented indicated the yellowish-green color of the fruits, because the yellow color was indicated by 90º and the green one by 180º, which together with the saturation of these colors (MCGUIRE, 1992;MENDONÇA et al., 2003), provided the practical visualization of the fruit color tending toward green ( Figure 2). Several authors have classified these fruits as green (OLIVEIRA; RUGGIEIRO, 2005;WONDRACEK, 2009;MAGALHÃES, 2010;IMIG, 2013). P. cincinnata fruits have also been characterized as being green in color, but with yellowish nuances of a low intensity and/ or color saturation (according to the chroma values) and intermediate brightness of black and white.
The data shown in Table 2 indicate that the fruits of the 2 progenies tested, irrespective of the conduction system adopted, did not present any significant differences for the evaluated physicochemical characteristics. However, in comparison with the results reported by Lima et al. (2017), who worked with P. cincinnata pulp, there are values close to pH (2.91 and 2.83, respectively), but higher by 24% in the values of SST (11.84 and 9.53, respectively) and by 9.8% in pulp acidity (4.59 and 4.18, respectively), thus determining a higher value for the acidity ratio (2.56 and 2.36, respectively). These values were similar for those observed in some other progenies and cultivars of P. edulis Sims. For instance, according to Medeiros et al. (2009) and Abreu et al. (2009), the progeny Marília Selection of Cerrado and BRS Gigante Amarelo, cultivated in the Federal District, presented with the pH of 2.74 and 3.08, titratable acidity of 4.81 and 6.85, and brix of 13.93 and 12.68, respectively. The proximity of the values indicates the possibility to study the application of pulp in the composition of blends (NEVES et al., 2011) of P. edulis and P. cincinnata, because there was not much change in the physicochemical quality of the blend.
In previous test periods, independent of the dry or rainy season, these same authors did not find any significant differences in the flavonoid content of the fruits collected from plants in a vertical trellis versus horizontal trellis system. Thus, if the results presented in Table 3 were evaluated in the rainy season, they could present different results due to the effect of the weather. Cohen et al. (2008) also verified the influence of the cropping system and the harvest season on yellow passion fruit cv. BRS Cerrado Sun. The authors observed higher levels of flavonoids and polyphenols in a single cultivation conducted in the conventional fertilizer system and the vertical trellis systems than when cultivated with cassava.
Thus, it is extremely important to study the effect of the conduction system for every species, because the flavonoid levels differ among species, considering that quantities of these compounds as reported for P. tenuifila cultivated in the DF region (SOZO et al., 2013) were higher and those for P. setacea (CAMPOS, 2010;CARVALHO et al., 2018) were lower than those presented by P. cincinnata (Table 3) on the wet weight basis. Table 3 presents the results of the chemical analyses, wherein a significant difference was noted only for the flavonoid content.
The flavonoid contents were significantly higher in fruits sourced from plants grown on a vertical trellis and the CBAF 2334 progeny (Table 3). Carvalho et al. (2018) verified the opposite effect of the conduction system in P. setacea. They reported that, in the second year of the cultivation during the rainy season (February/March), lower values of flavonoid contents were recorded for fruit pulp produced in the vertical trellis system when compared to that in the horizontal trellis system, possibly due to the higher humidity and the lower sun exposure in that particular season. Lower anthocyanins values were reported by Lessa (2011) in the pulps of P. cincinnata, P. setacea, and P. edulis, which did not differ from each other, with variations from 0.06 × 10 -3 to 0.098 × 10 -3 mg/100 pulp.
The values presented in Table 3 for polyphenols ranged from 43.61 to 56.11 mg/100 g, corresponding on a wet basis, and they were higher than that presented by Cohen et al. (2008) for the fruit pulp of P. edulis cv. BRS Sol do Cerrado that presented in the conventional cultivation 36.27 mg of polyphenols/100 g of pulp. Lessa (2011) used the Badiale-Furlong methodology and verified higher average values of phenolic compounds, ranging from 190.25 to 210.85 mg of gallic acid/100 g of fresh sample in P. cincinnata, P. setacea, and P. edulis. According to Lessa (2011), P. cincinnata showed the highest content of phenolic compounds. Carvalho et al. (2018) used the same methodology for P. setacea and found that the average polyphenol content was 78.5 mg/100 g fruit pulp (wet basis). Sozo et al. (2013) reported 80.88 mg of polyphenols/100 g of pulp on wet basis in P. tenuifila, which was higher than that for P. cincinnata in this study (as presented in Table 3). Table 4 shows the correlations between the evaluated characteristics.
Significant correlations also indicated that lower the pH, higher are the soluble solids (SS) and total titratable acidity (TTA) values, which may have occurred due to the lower concentration of free hydrogen ions in the fruit pulp, as indicated by pH (INSTITUTO ADOLFO LUTZ, 2008). Therefore, the formation of organic acids and total sugars may have interfered with the amount of free hydrogen ions in the pulp.
In addition, increases in TTA may occur with decreasing pH, depending on the concentration of organic acids in the pulp. Regarding the amount of soluble solids dissolved in the pulp, they are mainly composed of sugars and some other dissolved substances; hence, as the exact quantity is not known (CHITARRA; CHITARRA, 2005), a negative correlation was observed with polyphenols. Table 4 indicates that higher content of these compounds correlates with a lower amount of soluble solids in the fruit pulp.

Conclusions
The fruits of P. cincinnata were characterized by their green color with yellowish nuances and low intensity and/or color saturation and intermediate brightness to black and white. Of all, the fruits of progeny CBAF 23334 have greater intensity of the green color and greater flavonoid content in the pulp when compared to that in the fruits of progeny CPEF2220.
The type of conduction system did not affect the physical and physicochemical characteristics and the anthocyanins and polyphenols content of the fruits, although the system in the vertical trellis provided the production of fruits of P. cincinnata with higher flavonoid content.