Ripening of bananas using Bowdichia virgilioides Kunth leaves 2

2 Rivaildo da Costa Nascimento1+; Oliveiros de Oliveira Freire1; Ana Rosa dos Santos Domingos1; Mirelly 3 Souza dos Santos Falcão1; Lylian Souto Ribeiro2; José Cola Zanuncio3; Wellington Souto Ribeiro3+ 4 1Departamento de Agroecologia e Agropecuária, Sítio Imbaúba s∕no, Campus II, Universidade Estadual 5 da Paraíba, 58117-000 Lagoa Seca, Paraíba, Brasil. E-mail: rivaagro10@gmail.com, 6 oliveirossenar@gmaill.com, anarosasantos09@gmail.com, mirellyfalcao.mf@gmail.com. 7 2Departamento de Fitotecnia de Ciências Ambientais, Campus II, Universidade Federal da Paraíba, 8 58397-000 Areia, Paraíba, Brasil. E-mail: lilyan0490@yahoo.com.br 9 3Departamento de Entomologia/BIOAGRO, Universidade Federal de Viçosa, 36570-900 Viçosa, Minas 10 Gerais, Brasil. E-mail: zanuncio@ufv.br, wellington.souto@ufv.br. 11 12 +These authors contributed equally to this work 13 14 Corresponding Author: 15 Wellington Souto Ribeiro and José Cola Zanuncio 16 Ph Rolfs, s∕n, Viçosa, Minas Gerais, 36570-900, Brasil. 17 Email address: wellington.souto@ufv.br 18 19 20 21 22 23 24 25 26

then covered with plastic sheeting without air exchange between the external and internal environments. The tarp is left for 24 hours or longer depending on the fruit quantity. Masonry tanks of 1 m 2 are also used with this method (personal farmer communication, 2017).
The sustainable management of B. virgilioides plants can facilitate the use of its leaves to induce banana ripening more economically. The objective was to test the empirical method of banana maturation using B. virgilioides leaves compared to the conventional method with CaC 2 .

Results
Ethylene concentration was higher in atmosphere of the treatments with 4, 6, 8, and 10 g of leaves B. virguloides, followed by treatment with CaC 2 in the laboratory ripening trial. In the field ripening trial, the ethylene concentration was higher in atmosphere of the treatment with leaves B. virguloides, followed by treatment with CaC 2 . Acetylene (C 2 H 2 ) was only detected on treatment with CaC 2 in the laboratory and field ripening trial. Respiratory rate, ascorbic acid content, malic acid, pH, and chlorophyll in 'Pacovan' bananas matured with B. virgilioides leaves and CaC 2 do not differed (p < 0.05) in the laboratory (6, 8, and 10 g) and field ripening trial. The respiratory rate and ascorbic acid content of matured 'Pacovan' bananas was higher with B. virgilioides leaves and CaC 2 than control in the laboratory (6, 8, and 10 g) and field ripening trial. The malic acid, pH, and total chlorophyll concentration of ripened 'Pacovan' bananas of B. virgilioides leaves and CaC 2 was lower than control in the laboratory (6, 8, and 10 g) and field ripening trial (Table 1).

Discussion
The highest ethylene concentrations in the treatments with 4, 6, 8, and 10 g of B. virgilioides leaves are due to a cumulative effect of the gas produced by fruits and leaves. Ethylene concentration increases under atmospheric conditions modified by gas exchange limitation and autocatalysis 17 . The ethylene detected in the treatment with CaC 2 was the autocatalytic produced by the banana fruits and induced by the calcium carbide. CaC 2 may increase respiration rate, ethylene autocatalytic, chlorophyll degradation, carotenoid synthesis, starch conversion to sugar, increased activity of cell wall enzymes degradation, color change, texture, fruit aroma, and taste 18 . Acetylene is an ethylene analog used to initiate fruit ripening 19 . However, acetylene has lower biological activity than ethylene and higher concentrations for the same exposure period and for the same responses are needed 20 . In bananas, 0.01 ml L −1 of ethylene at 18 °C for 24 h began to ripen, while 1.0 ml L −1 of acetylene was required for a similar effect in several Florida hose cultivars 19 . The C 2 H 2 was only detected on treatment with CaC 2 due to the presence of this compound which is industrially produced and only releases C 2 H 2 when reacted with water 21 .
The increase in the respiratory rate of 'Pacovan' bananas ripened with 6, 8, and 10 g of B. virgilioides leaves and CaC 2 is due to the climacteric induction of respiration by the ethylene and acetylene emanated by B. virgilioides leaves and CaC 2 , respectively. Phosphofructokinase activity, which regulates this pathway 13 , produces energy (ATP) from starch degradation and hexose oxidation resulting in climacteric respiration 22 . In addition, the fruit exposure to ethylene and acetylene produced by B. virgilioides leaves and CaC 2 , respectively, may have increased the activity of the enzymes synthase and oxidase of ACC 23 inducing climacteric respiration and accelerating maturation. www.nature.com/scientificreports www.nature.com/scientificreports/ The highest ascorbic acid content (AsA) in bananas ripened with 6, 8, and 10 g of B. virgilioides leaves and CaC 2 is due to the higher demand for AsA 24,25 in these fruits, presumably by the most oxidized redox cell state 26,27 . The early fruit ripen, induced by ethylene and acetylene, produces reactive oxygen species 28 increasing the demand for AsA reacting with superoxide, hydroxyl and peroxyl radicals, hydrogen peroxide, hypochlorite and singlet oxygen 29 . However, the biosynthesis of AsA is an antioxidant response by the D-glucosone, D-galacturonate, myo-inositol and D-mannose/L-galactose [30][31][32] . The AsA accumulation in these fruits may also be associated with the low oxidation of this pH-dependent molecule, with maximum at pH 5 and 11.5, being faster in alkaline media 33 pathways but not necessarily related to its accumulation 34 .
The reduction of ripen 'Pacovan' banana acidity (malic acid) with 6, 8, and 10 g of B. virgilioides leaves and CaC 2 is due to the oxidation of organic acids during fruit ripening by the increase in tricarboxylic acid cycle activity 35 . These acids were more rapidly and extensively degraded during the climacteric respiration 36 induced by ethylene and acetylene, emanating from the B. virgilioides leaves and CaC 2 , respectively.
The pH reduction in 'Pacovan' bananas can be explained by the increase in the ascorbic acid content exceeding the titratable acidity reduction in the fruits matured with 6, 8, and 10 g of B. virgilioides leaves and CaC 2 . The AsA accumulation reduced the pH of these fruits due to the acidic character of this molecule attributed to the enodiol group (-HOC=COH-). The hydrogens of the enodiol group can dissociate, resulting in the strong ascorbic acid acidity and therefore are potential reducing agents 27 .
The lowest concentration of total chlorophyll in the 'Pacovan' banana peel ripe fruit induced with 6, 8 and 10 g of B. virgilioides leaves is due to the structural decomposition of chlorophyll by chlorophyllases, stimulated by ethylene 37 and acetylene emanated from leaves and CaC 2 , respectively. The increase in the activity of these enzymes in these treatments coincides with the climacteric increase in fruit respiration 38 , which was also induced by ethylene and acetylene. Ethylene and acetylene its analogues accelerate the chlorophyll losses 39 and regulates the yellowing of banana peels 40 . The drop of total chlorophyll concentration in banana fruits induced by CaC 2 was similar due lower biological activity than ethylene and higher concentrations for the same exposure period and for the same responses are needed 20,41 .

Conclusion
The method used by Borborema producers in the Paraíba state, Brazil to ripen 'Pacovan' bananas with Bowdichia virgilioides leaves is safer and has the same results than those obtained with carbide. www.nature.com/scientificreports www.nature.com/scientificreports/

Material and Methods
Location and raw material. Banana bunches of the 'Pacovan' variety, from agroecological production, were harvested in the early hours in the morning. Banana fruits were selected and standardized according to size, absence of physiological defects and infections, at the maturation stage 3 with yellowish green color 42 . Part of the harvested fruits were transported to the laboratory and part remained in the field. Banana ripening was evaluated with B. virgilioides leaves (harvested according to the producers orientation) and calcium carbide (CaC 2 ) in the field and laboratory.
Laboratory ripening trial. 'Pacovan' banana bites were scrapped with a stainless-steel knife and the fruits were, individualized in trays of expanded polystyrene for 30 min, to reduce the ethylene effect produced in their wound. The treatments were 2.0 g of B. virgilioides leaves + plastic film coating (T1); 4.0 g of B. virgilioides leaves + plastic film coating (T2); 6.0 g of B. virgilioides leaves + plastic film coating (T3); 8.0 g of B. virgilioides leaves + plastic film coating (T4); 10.0 g of B. virgilioides leaves + plastic film coating (T5); CaC 2 (g 50 kg −1 ) + coating with plastic film (T6); internal control (only coated with plastic film) (T7) and external control (without coating with plastic film) (T8) per banana. Treatments were stored at 27 ± 2 °C and relative humidity of 87 ± 5%. The fruits remained under these conditions for 48 hours and were then evaluated. The experiment was developed in triplicate.
Field ripening trial (empirical method). Two and a half kilograms of Bowdichia virgilioides leaves were placed covering the entire soil and 100 kg of banana fruits are placed over them. The bananas and the leaves were then covered with tarp without air exchange between the external and internal environments. The tarp was left for 24 hours (personal farmer communication, 2017). The same procedure was performed by replacing the leaves with CaC 2 (0,5 g kg −1 of fruit). The control consisted only of the fruits covered by the tarp.
Ethylene and acetylene quantification. Ten air samples were withdrawn with syringes from the atmosphere beneath the tarp. The syringes needle tip were sealed with rubber and immediately taken to the laboratory where were injected into a GC-14B (ShimadzuCrop Kyoto Japan), with Porapak-Q packaged column and flame ionization detector for ethylene and acetylene analysis.
Respiratory rate. Banana fruits were placed in hermetically sealed containers with 10 mL of 0.5 N NaOH.
The CO 2 , produced by the fruits, was measured by titration and after 24 h, the NaOH was titrated with 1 N HCl with results expressed as mg of CO 2 kg −1 h −1 . The respiratory rate was estimated by the equation: mgCO 2 Kg −1 fresh matter = (B − L) × C/MF where B = volume in mL spent for titration of the "control" (container without fruit, only with NaOH); L = volume spent to neutralize NaOH; C = correction factor (0.98); MF = fresh fruit mass. The hourly respiratory rate was determined with the formula mg CO 2 Kg −1 h −1 = mg CO 2 g −1 fresh matter × 1000/IT; IT = time interval between titrations (24 h).
Ascorbic acid. ascorbic acid content was determined by titration with a 0.02% 2,6-diclophenol-indophenol experimental design and data analysis. The experiment was carried out in a completely randomized design with eight treatments and four replications. Each experimental unit had one banana per tray. The results were submitted to variance analysis by the F test and the means compared by the Tukey test (P < 0.05) with the program Assistat version 7.7.