Bioactive content during the development of the acerola cv. BRS 238 (Frutacor)

The objective of this work was to determine quality, bioactive content and metabolism of vitamin C and phenolic compounds during the development of acerola BRS 238 (Frutacor). Fruits were harvested at five different stages of maturation and evaluated for physical-chemical and chemical quality characteristics, as well as for the metabolism of vitamin C and polyphenols variables. During development, there was an increase in SS/AT ratio, a decrease in chlorophyll content, increase in carotenoids content, and a decline in vitamin C and polyphenols content, alhhtough of yellow flavonoids and anthocyanins content increased. The enzyme activity of vitamin C metabolism, ascorbate oxide (AO) and ascorbate peroxidase (APX) decreased with ripening, while for phenolic metabolism, the activity of phenylalanine ammonia lyase (PAL) increased and polyphenoloxidase (PPO) decreased. It can be concluded that the fruits of aceroleira BRS 238 had a high content of bioactive compounds. For industrial extraction of bioactive compounds, fruits must be harvested at the initial stages, while for fresh consumption, they must be harvested in the final stages of development.


Introduction
Brazil is the world's largest producer, exporter and consumer of acerola as it is produced in all regions although, mainly on the Northeast region where it found the most favorable conditions for development. In this region, Pernambuco state stands out as the largest producer (Anuário da Agricultura Brasileira [AGRIANUAL] (2019); Calgaro & Braga, 2012;Instituto Brasileiro de Geografia e Estatística [IBGE] (2018); Ritzinger & Ritzinger, 2011).
The first varieties propagated vegetatively in the northeast of Brazil were Flor Branca, Okinawa and Sertaneja (Ritzinger & Ritzinger, 2011). Ever since, great advances have been made towards the development of new varieties such as Cabocla, Rubra, Tropicana, Morena, Mulata, Apodi, Cereja, Frutacor, Roxinha and Jaburu, which besides being rich in bioactive compounds and more suitable for use in natura, are more suitable for industrial pulp and juice processing and vitamin C extraction (Ritzinger, 2018).
Acerola is source of several health-promoting components as sugars and organic acids as malic, citric and tartaric acid (Riguetto, Netto, & Carraro, 2005). However, the main commercial interest of acerola comes from its high ascorbic acid or vitamin C content as varieties contain from 1,500 to 4,500 mg. 100g-¹, almost 100 times the concentrations found in citrus fruits (Moreira et al., 2009;Almeida et al., 2014). In addition to the considerable amount of vitamin C, acerola is also good source of phenolic and carotenoid compounds, associated with fruit color besides their antioxidant defense potential (Calgaro & Braga, 2012;Mariano-Nasser et al., 2017). Acerola nutritional properties depends upon several factors such as edaphoclimatic conditions, cultural practices, maturity harvesting stage, processing and post-harvest storage (Delva & Schneider, 2013). Despite the various studies carried out with acerola, there is still a lack of information regarding changes in bioactive metabolism during the development of fruits grown in the state of Ceará. Thus, this work aimed to determine the bioactive content and enzyme activity of the metabolism of vitamin C and phenolic compounds during the development of fruits from BRS 238 (Frutacor) clone produced in Ceará State.

Methodology
Aceroleira fruits (Malpighia emarginata DC) from cv. BRS 238 (Frutacor) were harvested, in October of 2018, in the experimental field of Embrapa Agroindustia Tropical, located in Pacajus, Ceará. The coordinates of the place are 4º10 'S and 38º27' W and an altitude of 60 m above sea level.
Fruits were harvested at different developmental stages ( Figure 1): 1) small and green color, 2) large and light green color, 3) large with up to 30% red color, 4) large with up to 50% red color and 5) large with up to 100% red color. After harvest, fruits were selected for uniformity of stage, and absence of injuries, sanitized, pulp was processed using a domestic centrifuge and stored at -18 oC until analysis of physical-chemical and chemical characteristics and variables of vitamin C and phenolic metabolism. Source: Authors (2020).

Soluble solids (SS)
Soluble solids were measured as pulp was filtered using a digital refractometer (KASVI model K52-032), as results were expressed in ºBrix.

Titratable acidity (AT)
According to the methodology by Instituto Adolfo Lutz [IAL] (1985), 1.0 g of pulp was diluted in 50 mL of distilled water and titrated with a NaOH solution (0.1 M), with 1% phenolphthalein as an indicator. The basic solution was added slowly until color changed to pink and results were expressed as a percentage of malic acid.

Chlorophyll and carotenoids
Chlorophyll and carotenoids were determined according to Lichtenthaler and Wellburn (1983), with adaptations. 1.0 g of fresh pulp was vortexed for 30 s with 1 mL of 80% acetone (v/v) and 0.1 g of calcium carbonate then, centrifuged at 3000 x g for 15 min at room temperature. The supernatant was collected (300 µL) and absorbance were measured at 663, 646 and 480 nm in a microplate reader (Synergyx Mx, Biotek, USA). Results were expressed in mg.kg-1 and calculated as following:

Total soluble proteins
The content of total soluble proteins was determined according to Bradford (1976) using bovine serum albumin as a standard (BSA).

Vitamin C metabolism
Total, reduced and oxidized vitamin C Total, reduced (AsA) and oxidized (DHA) vitamin C forms were measured as proposed by Chen and Wang (2002), with modifications. 0.1 g of pulp was homogenized with 25 ml of 5% trichloroacetic acid (TCA) in an ice bath. Subsequently, the samples were centrifuged (Sigma 2-16 KL) at 15,000 x g for 12 min at 4 ° C. The supernatant was separated and used as extract for the determination of total, AsA and DHA vitamin C forms.
For total vitamin C quantification, 50 µL of the extract, 25 µL of 100 mM potassium phosphate buffer (pH 7.7) and 12.5 µL of 0.01 mM dithiothreitol (DTT), 175 µL of solution containing 10% TCA, 8.8% phosphoric acid, 0.8% 2,2-bipyridyl and 0.3% iron trichloride were maintained at 37 ° C for 60 min and the, absorbance was measured at 525 nm. DHA content was calculated as difference between total vitamin C and AsA contents. A standard curve of ascorbic acid was used to calculate the analyzes and the results were expressed in mg.kg-1.

Total extractable polyphenols (TEP)
Extract was obtained according to Larrauri, Ruperez and Saura-Calixto (1997) and total polyphenol content was determined by Folin-Ciocalteu method (Obanda, Owuor, & Taylor, 1997) adapted by Rufino et al. (2007). The content of TEP was calculated based on a standard curve of increasing doses of 98% gallic acid (0 -50 µg), used as a reference and the results expressed in mg of gallic acid equivalents (EAG) .kg-1.

Yellow flavonoids and total anthocyanins
Yellow flavonoids and total anthocyanins was determined as described by Francis (1982), calculated using molar extinction coefficient 76.6 and 98.2 mol-1.cm-1, respectively, and results were expressed in mg.kg-1.
Polyphenoloxidase activity (PPO, EC 1.14.18.1) Enzymatic extract was prepared according to Sojo, Nuñes-Delicado and García-Carmona (1998), while activity was determined acas Robinson (1987). Na activity unit (AU) was defined as a variation of 0.001 in the absorbance of the mixture at 395 nm and the results were expressed as min-1.mg-1 P.

Experimental design and statistical analysis
The experimental design adopted was the completely randomized design with five treatments and three repetitions.
The data were subjected to analysis of variance (ANOVA) and the comparison of means performed using the Tukey test at the level of 5% probability using the statistical software SISVAR, version 5.6.

Physico-chemical and chemical analysis during the development of acerola BRS 238
Soluble solids (SS) content of acerola BRS 238 increased signficantly during ripening (Figure 2). At stage 2, the SS content was at 9.5 °Brix and it increased significantly to 11 °Brix at stadium 5. Ribeiro and Freitas (2020) found values that SS increased from 7.6 to 8.6 ºBrix during maturation of cv. Junko. Figueiredo Neto et al. (2014) studying the cv. Flor-Branca, Okinawa and Sertaneja found the highest SS contents for ripe acerola cv. Okinawa, 12.70 °Brix. The increase in SS content can be explained, mainly by the accumulation of sugars through gluconeogenesis or the hydrolysis of polysaccharides such as starch (Oliveira, 2012).    Pigments chlorophyll a and b and carotenoids changed during acerola devopment ( Figure 6). Chlorophyll was gradually degradated, while carotenoids increased. Chlorophyll a content in stage 1 was 357.88 and declined significantly to 27.77 mg.kg-¹ in stage 5, while chlorophyll b decline was slower from stage 1 to 5, from 200.30 to 82.32 mg.kg-1, respectively. Carotenoids increased significantly from 8.23 in stage 1 to 127.85 mg.kg-¹ in stage 5, however the main increase was observed after stage 4. The results of this work indicate that the synthesis of carotenoids in acerola BRS 238 is concomitant with the degradation of chlorophyll, explaining the color change of fruits from green to red. Lima et al. (2005) evaluated acerolas at different maturity stages and reported that carotenoid content varied between 3.2 to 406 mg.kg-¹ depending on the genotype, maturity stage harvest time. Carotenoids are important for human nutrition as they are precursors of vitamin A, moreoveor, they are also potent antioxidants, which characterizes it as a bioactive compound (Delgado-Vargas,

Vitamin C metabolism during the development of acerola BRS 238
Total vitamin C significantly reduced its content from 32,425.29 mg.kg-¹, in stage 1 to 20,106.10 mg.kg-¹ in stage 5 (Figure 7), as well as the reduced (AsA) and oxidized (DHA) forms from 30,294.40 and 2,130.89 to 19,215.33 and 890.77 mg.kg-¹, respectively. Moreover and despite the decline observed, acerola BRS 238 still presents considerably high vitamin C levels. Vitamin C is a bioactive compound that presents human health benefits in both reduced AsA and oxidized DHA forms and its contents vary according to variety, cultivation and harvest conditions, in addition to maturity (Gomez & Lajolo, 2008;Rabelo, 2016). Research, Society andDevelopment, v. 10, n. 2, e42410212640, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12640 9 The content and forms of vitamin C may be influenced by synthesis as well as by degradtion due to enzymatic oxidation or auto-oxidation. Enzymatic oxidation can be mediated by oxidoreductase enzymes such as ascorbate oxidase that catalyzes the oxidation of AsA to monodehydroascorbate (MDHA) by reducing molecular oxygen (O2) to water; and ascorbate peroxidase, which uses two AsA molecules to reduce oxygen peroxide (H2O2) to water, forming MDHA (Foyer & Noctor, 2011). MDHA may be further oxidized to DHA form.
AO activity peaked at stage 4 reaching 6.62 µmol AsA.min-1.mg-1 P ( Figure 8A), while APX activity declined significantly from 0.20 to 0.15 µmol H2O2. min-1.mg-1 P from stage 3 to 5 ( Figure 8B). The increase in AO activity at stage 4 influences significantly the total vitamin C, AsA and DHA contents (Figure 7), while APX exerted a greater influence on stage 3 ( Figure 8B). APX is the most important enzyme involved with H2O2 neutralization, in chloroplasts, and promotes its detoxification through the donation of electrons from the ascorbate that reduce H2O2 to water, while forming MDHA (Zimmermann & Zentgraf, 2005). Source: Authors (2020).

Metabolism of phenolic compounds during the development of acerola BRS 238
Results show that total extractable polyphenols (TEP) decline significantly with fruit development from 50,829.40 mg EAG.kg-¹ FP, at stage 3, to 27,900.96 mg EAG.kg-¹ FP, at stage 5 ( Figure 9A). Despite the decline observed in phenolics, yellow flavonoid content increased significantly from stage 4 to 5, from 53.00 to 74.48 mg.kg-¹ ( Figure 9B), as did the anthocyanin content increasing from 44.28 to 136.08 mg.kg-¹, from stage 4 to 5 ( Figure 9C). These results coincide with those stages when fruits color changed from 75 to 100% red skin, indicating the greater synthesis of these compounds. Polyphenols, among several other functions, act as antioxidant compounds by suppressing singlet oxygen or free radicals preventing cell damage (Rice-Evans, Miller, Bolwell, Bramley, & Pridham, 1995;Luo et al., 2011). In general, the antioxidant activity is due to their ability to donate hydrogens from the hydroxyl groups positioned along the benzene ring, in order to prevent the oxidation of lipids and other biomolecules (Foti, Piattelli, Amico, & Ruberto, 1994;Alamed, Chaiyasit, McClements, & Decker, 2009). Flavonoids as yellow flavonoids and anthocyanins are phenolic compounds mainly associated with pigmentation of fruits. In addition to physiological functions in plants, they offer health benefits such as anti-allergic, antiviral, anti-tumor, anti-inflammatory and antioxidant functions. (Vendramini & Trugo, 2004;Huber, Rodriguez-Amaya, 2008;Rodriguez-Amaya, Kimura, & Amaya-Farfan, 2008).
The content of phenolic compounds depends on the balance between their synthesis and their degradation, and phenylalanine ammonia lyase (PAL) is the main enzyme of its biosynthesis while they can be degradated enzymatically by polyphenoloxidase (PPO) or by self-oxidation. PAL activity increased significantly from stage 1, with 86.07 to 134.34 µmol acid. transcin. h -¹.mg -¹ P at stage 5 ( Figure 10A). At stage 3, PAL activity increases significantly concomitantly to TEP content ( Figure 9A), indiactive of greater synthesis. PPO phenolic-degradative activity declined from 7,559.52 AU min-¹.mg-¹ P, at stage 1 to 5,438.65 AU min-¹.mg-¹ P, at stage 5 ( Figure 10B). PPO is located in plastids, while its substrates, phenolic compounds are in vacuoles and from the moment there is some type of damage to the plant tissue, decompartmentalization occurs and this enzyme comes into contact with its substrate (Murata, Noda , & Homma, 1995;Parkin, 2010;Liu et al., 2011). When the enzyme meets its substrate, oxidation reactions that form quinones generally occur, leading to non-enzymatic reactions that polymerize and give rise to dark colored pigments (melanins) (Zeraik, Souza, Fatibello -Son, & Leite, 2008). The action of this enzyme causes loss of quality and changes the flavor, in addition to cause enormous economic losses (Araujo, 2004).

Conclusions
It can be concluded that acerola BRS 238 (Frutacor) has a high SS/AT ratio that gives a sweeter flavor to this fruit compared to other clones, and ripening led to decrease in vitamin C and polyphenols content due the activity of enzymes of their respective metabolism. Although these compounds decrease their content in the final stages of development, there are still found in considerably high content. Thus, for vitamin C industrial extraction, fruit should be harvested at stage 1, while fresh fruit marketing of the fruit, stages 4 and 5 are the most recommended.
In future studies that address the subject, it is suggested that a deeper study be done on the enzymes that are part of vitamin C metabolism, specifically the activity of the enzymes L-galactone-1,4-lactone dehydrogenase, Monodehydroascorbate reductase, Dehydroascorbate reductase and Glutathione reductase since they are part of the recycling of this vitamin in the plant and directly interfere in its metabolism. With these it would be possible to better discuss the results seen in this work.