Maintaining the firmness of minimally processed papaya using pectin methylesterase and calcium lactate

The objective of this work was to evaluate the effect of vacuum infusion of pectin methylesterase (PME) and calcium lactate (C6H10CaO6) in maintaining the firmness of minimally processed papaya, in order to maintain the quality and cellular integrity of the fruit. After minimal processing, the treatments used were: fruit without infusion (control), with H2O infusion, with PME infusion, with C6H10CaO6 infusion and with PME+C6H10CaO6 infusion. At zero times, four and eight days of storage, analyzes of total galacturonic acid, methanol, cell integrity, vitamin C, pH, acidity, soluble solids, damage, freshness and contamination were performed. Papaya treated with the PME+calcium combination showed an increase in firmness (5.8 N) on the eighth day of storage, differing from the control treatment (1.3 N), reporting the least leakage of electrolytes. On the fourth day, the fruit treated with PME+C6H10CaO6 showed the highest content of galacturonic acid and on the eighth day the highest content of methanol, indicating an effective action of the enzyme PME and calcium in this period. The PME+C6H10CaO6 treatment was effective in maintaining and improving the quality of papaya while preserving freshness, soluble solids content, acidity and pH throughout storage.


Introduction
Papaya (Carica papaya L.) is one of the main fruit trees present in tropical and subtropical regions of the planet, with varieties classified into two groups, Solo and Formosa (Dantas et al., 2013). The fruits of the Formosa group are well accepted by consumers due to the quality of their pulp, however they are not convenient for individual use, as they are large and need preparation before consumption. In this way, minimal processing becomes a marketing alternative, providing a more practical consumption, adding value to the final product (Teixeira et al., 2001).
When a fruit is harvested, it continues to breathe and undergo a series of endogenous transformations, which will reflect changes in its characteristics (Batista, 2015), mainly changes in texture and firmness. The ripening process is accompanied by a loss of firmness, caused by changes in the polysaccharides that make up the cell wall, such as cellulose, hemicellulose and pectin. These changes result from the performance of endogenous enzymes Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 4 in the fruit (Zhang et al., 2019).
The decrease in pulp firmness during ripening is attributed to the action of polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) (Kohli et al., 2015). PME catalyzes the demethylation of pectin, damaging the horizontal calcium links of the acid polysaccharide chain, leading to cell separation, resulting in a greater number of carboxylic acid groups, which facilitates the action of polygalacturonase. PG acts by hydrolyzing pectin acid, causing pectin degradation, cell wall dissolution and fruit softening (Zhang et al., 2019).
The demethylation of pectin caused by the action of PME can improve the texture of fruits and vegetables, since the carboxylic groups resulting from this process when interacting with divalent ions such as exogenous Ca +2 , form a network maintaining the stability of pectin and the cell wall structure (Guillemin et al., 2008;Kohli et al., 2015). In the cell wall of plants, calcium suppresses the decline in quality, preserves integrity and reduces membrane permeability during storage (Aguayo et al., 2015).
Calcium applications have been used for producing beneficial effects on the texture of fresh fruits . The process of de-esterification of pectin by the PME and subsequent association with calcium forms the complex known as "egg box", which acts as a cement, providing firmness to vegetables (Aghdam et al., 2012). The technique that has been used for the application of calcium and exogenous PME to fruits is vacuum impregnation, which consists of exchanging native gases and liquids with an impregnation solution under the action of the hydrodynamic mechanism (Derossi et al., 2013).
Thus, the combined use of the enzyme pectin methylesterase with calcium becomes important to avoid post-harvest losses related to decreased firmness of MP fruits, being an alternative for maintaining the sensory characteristics, prolonging the shelf life of these products. The objective of this work was to evaluate the effect of the infusion of pectin methylesterase (PME) and calcium in maintaining the firmness of the minimally processed papaya in order to maintain the quality and cellular integrity, as well as to increase the useful life of the fruit. Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.

Minimal processing
Papaya of the Formosa variety were selected according to their degree of ripeness (½ ripe fruit, 25 to 50% of the yellowish peel surface), according to the classification by Ceagesp (2015).
After reception, the fruits were washed in running water and sanitized in an aqueous chlorine solution at a concentration of 200 mg L -1 at a temperature of 5±1°C for 10 minutes.
Then, peel removal and manual cutting into cubes was promoted. The cubes were sanitized in an aqueous chlorine solution at 5±1°C at a concentration of 5 mg L -1 for 3 minutes.

Vacuum infusion and treatments
The vacuum infusion was performed using 85 g of minimally processed papaya arranged in 250 mL beakers with 125 mL of aqueous solution. A pressure of 33 KPa or 250 mmHg was applied for 10 minutes using a desiccator with a pressure gauge attached to a vacuum pump. When the pressure was reached, the system was turned off.
The enzyme used in the experiments was pectin methylesterase (NovoShape, Novozymes, Denmark) diluted to achieve the activity recommended by the manufacturer, 10 PEU/mL, that is, 1 mL PME/kg of fruit. The concentration of calcium lactate (C6H10CaO6), at 1% was determined based on the experimental results obtained by (Batista, 2015;Carnelossi et al., 2018).
After infusion, the minimally processed papayas (85 g) were drained and then packed in polypropylene packages and stored in a vertical display with air circulation (Springer) at a temperature of 5°C range 1 for 8 days, under relative humidity of 78-82%. Samples were collected at times 0, 4 and 8 days for biochemical and physical-chemical analysis, previously crushed and homogenized with the aid of the Sorvall Ominimixer equipment and stored at

Determination of firmness
The determination of firmness was performed on the papaya MP with the aid of the Brookfield CT3 Texture Analyzer equipment using the TexturePro CT V1.2 Build 9 program with application of the TPA test. The test was carried out with an aluminum probe of 5.0 mm in diameter at a speed of 0.5 mm/s. The strain distance used was 3.0 mm and the firing load 0.06 N. Triplicate readings were performed and the result was expressed in Newton (N).

Cell integrity
The analysis of cell integrity was determined using the methodology of Villalta and Sargent (2004). For this, a 2 g sample of the papaya MP cut in cubes of 5mm-2mm was used.
After weighing, the sample was washed with distilled water on filter paper, placed in centrifuge tubes (50 mL) and added with 35 mL of 0.6 M isotonic mannitol solution at 23°C for 4 hours. After this time, the conductivity was determined and the samples were frozen (24 h at -20°C). After 24 h, the samples were thawed and heated in boiling water for 30 minutes.
After cooling, the conductivity was again determined, with the electrolytic flow expressed as a percentage of the total tissue electrolytes.

Determination of methanol
The analysis of methanol determination was performed according to the methodology described by Wood and Siddiqui (1971), with adaptations. For analysis, 250 mg of the sample were suspended in 2 mL of distilled water and taken to an ultrasonic bath for 10 minutes.
Then 0.8 mL of 2M NaOH were added for deesterification and the sample was incubated at 20°C with occasional shaking. Then, neutralization was carried out with the addition of 0.8 mL of 2M HCl, equilibrated at a temperature of 25°C in a water bath for 15 minutes. Soon after, the sample was centrifuged at 8000 rpm for 10 minutes at 25°C.
The quantification of methanol was determined using a 1 mL aliquot of the sample preparation collected and placed in a test tube with 1 mL of 1.0 N H2SO4. The test tubes were cooled in an ice water bath and adding 0.2 mL of 0.2% potassium permanganate. It was mixed and kept in an ice bath for 15 minutes. Subsequently, 0.2 mL of 0.5 M sodium arsenate solution in 0.12N H2SO4 was added, followed by the addition of 0.6 mL of distilled water, remaining for 1 hour at room temperature. Soon after, 2 mL of the 0.02 M acetylacetone solution dissolved in a solution containing 2M ammonium acetate and 0.05M acetic acid were Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 7 added, the tubes were shaken, capped with "marbles" and heated in a water bath the temperature of 58-60°C for 15 minutes. After heating, the sample was centrifuged at 10,000 rpm for 10 minutes, for deposition of the precipitate, followed by the absorbance reading in a spectrophotometer at 412 nm.

Determination of total galacturonic acid
The contents of galacturonic acid were determined according to the methodology of Ahmed and Labavitch (1977), with adaptations. For sample preparation, 100 mg was weighed in a 20 mL beaker containing a magnetic stir bar. 2 mL of refrigerated sulfuric acid and 0.5 mL of cold distilled water were added, promoting gentle agitation for 5 minutes. Another 0.5 mL of cold distilled water was added, followed by another 10 minutes of stirring. 17 mL of cold distilled water was added to complete the volume of the beaker.
The total galacturonic acid was determined by collecting a 0.5 mL aliquot of the sample contained in the beaker, placing it in test tubes previously cooled in an ice water bath.
Afterwards, 3.6 mL of the 0.0125 M sodium tetraborate solution in refrigerated sulfuric acid was added and the mixture was promoted. The tubes were heated in a boiling water bath for 5 minutes and cooled under running water. After being cooled, 60 μL of the 0.15% mhydroxydiphenyl solution in 0.5% NaOH was added. A rapid homogenization was carried out and the absorbance was read on a spectrophotometer at 520 nm.

pH, soluble solids and titratable acidity
The pH of the samples was determined in a digital pH meter model DLA-PH, previously calibrated and the soluble solids were quantified by adding drops of the sample previously homogenized on the prism of the digital refractometer (Hanna Instruments).
Titratable acidity was determined using the 1% phenolphthalein indicator and 0.1 M sodium hydroxide solution, the acidity being expressed as a percentage of citric acid (IAL, 2008).

Vitamin C
To quantify the vitamin C content, 5 g of the sample were weighed in a beaker and homogenized with an extraction solution, 2% oxalic acid. Subsequently, the sample was filtered with the aid of filter paper into a 50 mL volumetric flask and the flask volume was completed with extraction solution. Then, 7 mL of the flask solution were collected, transferred to a 50 mL conical flask and titrated with the 2,6-dichlorophenolindophenol solution (DCPIP), until the formation of a persistent pink color. The results were expressed in Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 8 mg of ascorbic acid/100g of fresh matter (AOAC, 1992).

Damage, freshness and contamination
The damage analysis consisted of counting the number of minimally processed papaya cubes that presented some type of post-harvest mechanical damage, the result being expressed as a percentage of the total papaya cubes present in the packaging. The freshness of the fruit was assessed using the following scale: 9 = excellent (completely fresh appearance, high gloss); 7 = good (still looks fresh, still shiny); 5 = fair (does not have a fresh appearance, low gloss, liquidity limit); 3 = poor (dull, usability limit); 1 = extremely poor (withered appearance), the result being expressed as the average of repetitions per treatment. The contamination consisted of counting the papaya cubes that had an incidence of post-harvest rot and/or the presence of visible fungi, the results being expressed as a percentage (Jacomino et al., 2011).

Statistical analysis
A completely randomized design with a 5 x 3 factorial scheme (5 treatments and 3 times) was used, with three replications per treatment. The data collected were submitted to analysis of variance (ANOVA) and the means compared by the Tukey test (p<0.05), expressed as mean ± standard deviation, with the aid of the Sisvar 5.7 computer program (Ferreira, 2011).

Results and Discussion
After the vacuum infusion process, all treatments showed a decrease in firmness when compared to the control sample, with the exception of minimally processed papaya treated only with PME ( Figure 1). The decrease in firmness, in this first moment, can be explained by the pressure difference to which the papaya cubes are submitted when the vacuum is applied, this change in pressure may have weakened the cellular structures, resulting in the loss of firmness soon after impregnation vacuum (Guillemin et al., 2008;Yang et al., 2017). Treatments: Control, without infusion; H20, water infusion; Ca2+, infusion with calcium lactate; PME, infusion with the enzyme pectin methylesterase; and PME+Ca2+, infusion with the enzyme pectin methylesterase and calcium lactate. Three repetitions were performed. The bars indicate the standard error of the mean. Source: Author's own compilation (2020).
On the other hand, the momentary increase in firmness in treatment only with PME may have happened due to the action of the exogenous PME associated with the endogenous calcium present in the fruit matrix. However, as endogenous calcium ions are insufficient to bind to all free carboxyl groups released by the action of the PME, after the momentary increase in firmness, papaya subsequently softens (Lara et al., 2004).
It was found that papaya treated with the PME+calcium combination showed an increase in firmness on the eighth day of storage compared to the other treatments, differing significantly (p<0.05) from the control treatment ( Figure 1). This result demonstrates that the PME+C6H10CaO6 treatment was effective in maintaining and increasing papaya firmness during storage.
The increase in firmness occurs once the enzyme PME breaks the chains of galacturonic acid, the methanol clusters of the pectin are hydrolyzed and then the calcium is bound with the groups of carboxylic acids, maintaining the structure of the wall, making it the firmer (Guillemin et al., 2008;Batista, 2015).
The control sample showed a gradual decrease in firmness, differing significantly (p<0.05) over time. This behavior may be associated with the action of the polygalacturonase enzyme, which can be elevated in freshly cut papayas, when compared to intact fruit, due to the increase in ethylene production and damage caused by cuts, as observed by Karakurt and Huber (2003). Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 10 The treatment with C6H10CaO6 maintained the firmness of the minimally processed papaya achieved after vacuum impregnation during storage; however the combined treatment with the PME enzyme proved to be more efficient because in addition to maintaining, it increased firmness over the 8 days. According to Aghadam et al. (2012), calcium lactate can inhibit endogenous PME and/or promote greater "egg box" conformation, maintaining the firmness of the plant matrix, which may have occurred in the present study.
Similar results were found in the study by Yang et al. (2017) using vacuum impregnation of calcium lactate and pectin methylesterase in fresh papayas cut from the cultivar "Sekaki" at 5 KPa. It was observed that the hardness of all papayas treated with vacuum impregnation dropped shortly after the process and that the samples subjected to treatment with the enzyme and Ca 2+ had high hardness during storage.  Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 Figure 2. A, methanol content; and B, galacturonic acid content in papaya minimally processed after infusion (day 0) and during storage (4 and 8 days).
Treatments: Control, without infusion; H20, water infusion; Ca2+, infusion with calcium lactate; PME, infusion with the enzyme pectin methylesterase; and PME+Ca2+, infusion with the enzyme pectin methylesterase and calcium lactate. Three repetitions were performed. The bars indicate the standard error of the mean. Source: Author's own compilation (2020).
The leakage of electrolytes from the minimally processed papaya mesocarp increased significantly (p<0.05) as a function of time for all treatments applied, with the exception of the PME+C6H10CaO6 treatment, for which there was also an increase, but without significant difference (Table 1). Ion leakage is directly related to the integrity of cell membranes because high electrolyte leakage rates indicate changes in membrane permeability (Villalta & Sargent, 2004 Although there was no significant difference between treatments, on the eighth day it was possible to observe that the PME+C6H10CaO6 treatment had the lowest percentage of electrolyte leakage, indicating that the use of these substances can help to maintain the structural integrity of papaya tissues. The calcium and pectin complex, formed from the action of the PME, acts as cement, providing firmness to the plant tissue, contributing to the maintenance of cellular integrity, promoting the delay of maturation and senescence of the fruit (Aghdam et al., 2012).
The rapid softening of freshly cut papaya can be a limiting factor in its useful life, as the rupture of the cell wall and the consequent loss of integrity are inevitable, since cutting the fruit releases pectinolytic enzymes that act in the degradation of pectin and wall dissolution (Toivonen & Brummell, 2008;Yang et al., 2017). Therefore, the application of PME+ calcium is an alternative to maintain and improve the firmness of minimally processed papaya, delaying the rupture of the cellular structure, the leakage of electrolytes and the loss of integrity.
The levels of vitamin C present in the minimally processed papaya cubes increased on the fourth day when compared to day 0, in all treatments (Table 2). This increase may be associated with ascorbic acid synthesis under the conditions in which the fruit was (stored at 5°C in polypropylene packaging) or the loss of water by the vegetable tissue (Dea et al., 2010). When some citrus fruits or vegetables are stored, they may show retention or increase in the vitamin C content (Batista, 2015).
Between the fourth and the eighth day, in turn, the levels of vitamin C decreased in all treatments, differing significantly (p<0.05) in the treatments with H2O infusion and with C6H10CaO6 infusion (Table 2). Vitamin C is an antioxidant component present in the plant matrix, which can act as a reducing and chelating agent in the elimination of free radicals, decomposing during storage to prevent oxidation, leading to a decrease in its content (Zhang et al., 2019). Therefore, the decrease in vitamin C content observed in papaya MP between the fourth and eighth day may be associated with its performance as an antioxidant in response to oxidative reactions that occur due to ripening, preventing oxidation of the fruit. Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 Table 2. Vitamin C (mg/100g), titratable acidity (%), pH, soluble solids (°BRIX), freshness and damage (%) in minimally processed papaya after infusion (day 0) and during storage (4 and 8 days).  The means followed by the same letter, lower case in the column and upper case in the line, do not differ by the Tukey test at the 5% level of significance. Treatments: Control, without infusion; H20, water infusion; C6H10CaO6, infusion with calcium lactate; PME, infusion with the enzyme pectin methylesterase and PME+C6H10CaO6, infusion with the enzyme pectin methylesterase and calcium lactate. Three repetitions were performed. Source: Author's own compilation (2020).
Titratable acidity had a significant reduction (p<0.05) in treatments with C6H10CaO6 infusion and with PME infusion between the day of vacuum impregnation (day 0) and the eighth day ( Table 2). The control and infusion samples PME+C6H10CaO6 did not differ in acidity content over time, demonstrating that the combination of the enzyme PME with calcium was effective in maintaining acidity during storage, without changing its content when compared to the control sample.
Although the control, water and PME+C6H10CaO6 treatments did not differ significantly (p<0.05) as a function of time, all treatments had reduced acidity during storage, which may be related to the fact that organic acids are the first compounds consumed during breathing (Chitarra & Chitarra, 2005).
After vacuum impregnation, it was possible to observe that papaya treated only with PME had the highest percentage of acidity in citric acid (Table 2). This behavior can be explained by an increase in the concentration of acids resulting from the performance of exogenous PME and other pectic enzymes (Pinto et al., 2011).
As for pH, it was possible to observe that there were no significant differences (p<0.05) as a function of time, with the exception of the PME treatment for which the values differed between day zero and day eight. At zero time, this treatment reported the lowest pH value, which can be explained by the same reason that led to an increase in the acidity of this treatment at zero time, that is, after vacuum impregnation there is a greater performance of pectic enzymes and consequent release of organic acids, making the pH more acidic. It was also possible to observe a reduction in the levels of soluble solids for all treatments (Table 2), and the samples with H2O, C6H10CaO6, PME+C6H10CaO6 infusion differed significantly (p<0.05) from the control sample.
There were no significant differences (p<0.05) in the content of soluble solids as a function of storage time, but it was observed that in the treatment with water there was an increase in the content on the fourth day. An increase in the content of soluble solids was also found in the study by Paixão et al. (2020) to evaluate the postharvest behavior of green peppers after application of PME and calcium, with an increase in the content of soluble solids from the sixth to the ninth day of storage from 3.1 to 5.0ºBrix, in peppers treated with H2O infusion.
For the PME+C6H10CaO6 treatment, it was observed that the soluble solids content practically did not vary over time ( As for freshness, at time zero, all treatments received a score of 9 (Table 2), indicating that samples had a fresh appearance and high gloss, that is, they had the same degree of freshness. On the fourth day, only papaya treated with the PME enzyme differed significantly (p<0.05) when compared to day zero, presenting an appearance with low freshness and low Research, Society and Development, v. 9, n. 11, e62091110205, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10205 15 brightness. The other treatments had a freshness rating ≥ 7, classified as "good", keeping them fresh and shiny.
Papayas treated with PME+C6H10CaO6 and only with C6H10CaO6 did not differ (p<0.05) in freshness over time. On the last day, the combined treatment of PME+calcium presented the highest score for assessing freshness.
The minimal processing of papaya is convenient, but the accelerated catabolism of cell wall components and the loss of fluid, result in decreased firmness and loss of freshness (Karakurt & Huber, 2003;Yang et al., 2017). The reduction of freshness is associated with factors such as water loss, mechanical damage and contamination (Batista, 2015). Therefore, the vacuum impregnation of PME+Ca 2+ proves to be an effective alternative to prevent deterioration, maintaining freshness and improving firmness during storage.
As for the damage, on the fourth day the papaya treated with PME showed the highest percentage of damage to the structure of the plant tissue (Table 2). This behavior is explained because the PME acts on the fraction of the pectin that makes up the cell wall, catalyzing its demethylation, leading to cell separation and decreased firmness, increasing the percentage of damage to the minimally processed product (Zhang et al., 2019).
The treatments with C6H10CaO6 and PME+ C6H10CaO6 had similar behavior, both reduced the appearance of damage during storage. This can be explained by the fact that Ca 2+ is able to inhibit the action of endogenous PME and thereby maintain the structure of the cell wall, but it cannot maintain or increase the firmness of the fruit (Yamamoto et al., 2011;Batista, 2015). On the other hand, when vacuum impregnation of PME+C6H10CaO6 occurs, the formation of calcium pectate occurs, which improves and maintains the firmness of the fruit, thus preventing the appearance of damage to its structure (Carnelossi et al., 2018).
During the storage period, no incidence of contamination was observed in papaya, that is, there was no occurrence of rot and/or the growth of visible fungi.

Final Considerations
The use of vacuum infusion of PME and calcium lactate in minimally processed papaya is an alternative to preserve the firmness of the fruit of the Formosa variety. In addition to increasing and maintaining papaya firmness until the eighth day, the combination PME+calcium lactate promoted the least leakage of electrolytes in the papaya's vegetable tissues, demonstrating that there was preservation of the cellular structure.
The vacuum impregnation process of PME+Ca 2+ , did not influence the quality parameters of the product, since the levels of vitamin C, soluble solids, pH and acidity were maintained after processing and throughout storage.
There was also an increase in the useful life of the minimally processed papaya, by maintaining freshness, reducing damage and the absence of contamination in the product.
The use of vacuum infusion of PME and calcium lactate preserved the quality and firmness of minimally processed papaya, it is suggested that future work be carried out in order to explore the impact of this technique on the acceptability of minimally processed papaya.