Influence of thermal processing on the characteristics and chemical profile of ora- pro-nobis by PS/MS paper spray

This work aimed to evaluate the influence of dry and wet heat processing on the antioxidant profile and antioxidant activity in ora-pro-nobis leaves. The leaves were collected, washed, and separated into three groups for the treatments: application of moist heat (hydrothermal cooking at 100 oC / 4 minutes), application of dry heat (70 oC / 8 hours), and control (raw material). The characteristics were evaluated: total soluble solids (SST), total solids (ST), pH, phenolic compounds (CPT), flavonoids (FT), anthocyanins (AT), antioxidant activity (AA). To identify the chemical profile and the chemical profile, fingerprints were obtained using PS / MS paper spray. The content of SST and ST decreased with moist heat. The CFT content increased with wet heat (12.43 ± 0.98) and decreased with dry heat (4.25 ± 0.93 g EAG 100 g). There was a decrease in the FT and AT content in the leaves processed with dry heat (132.67 ± 20.28 mg 100 g and 19.70 ± 3.34 mg CG 100 g, respectively). AA decreased in both processes, which was higher in orapro-nobis leaves processed with dry heat (0.82 ± 0.0033 μmol ET g). 26 phenolic compounds were identified in the ora-pro-nob leaves by paper spray. The chemical profile, except for the substances 4,5-dimethyl-2,6-octadiene and methyl ester linolenic acid was not affected by thermal processing. Knowing the effect of food processing is important to understand the behavior of compounds that act beneficially on human health.


Introduction
Pereskia aculeata Mill., recognized regionally as food and also as a medicinal plant is considered a food alternative and cultural diversification in agricultural activity, mainly for family farming and low-income urban and rural populations (De Almeida et al., 2016). The lack of knowledge of the population concerning the properties of this vegetable cause it to be underutilized (Bacchetta et al., 2016;Menendez-Baceta et al., 2017).
Belonging to the Cactaceae family, the ora-pro-nobis, as it is best known in Brazil, has a large amount of fiber (39%) and protein (28%) in its leaves, in addition to significant amounts of iron and calcium (Kazama et al., 2012;Rocha et al., 2009;Viana et al., 2015), which are equal or even higher than what can be found in other leafy vegetables (Kinupp & Barros, 2008;Take it et al., 2009, Viana, et al., 2015. Ora-pro-nobis is not only used for fresh consumption, it can also be introduced in several other dishes such as cakes, loaves of bread, pasta, and ice cream (Martinevski et al. 2013;Mazon et al., 2020;Rosa et al., 2020;Rocha et al., 2009). Also, the mucilage provided by the leaves of the plant can be used in food formulations as a thickening agents, gelling agents, and emulsifiers, in addition to the production of biofilms (Amaral et al., 2018;Conceição et al., 2014;Junqueira, 2018;Oliveira et al., 2019;Takeiti et al., 2009).
A large amount of nutritional components present in species of the genus, Pereskia makes this group an important nutritional source combined with health, providing a better quality of life for the population that consumes them (Duarte & Hayashi, 2005;Martin et al., 2017;Pinto & Scio, 2014). Medicinally they are used for antinociceptive, anti-inflammatory, antisyphilitic and healing action (Barbalho et al. 2016;Duarte & Hayashi, 2005;Rosa & Souza, 2003;Sartor et al., 2010;Silva & Fonseca, 2016;Silva Júnior et al., 2010). The extract of the essential oil of the species presents efficient results in the antimicrobial potential in a wide spectrum (Do Carmo Pimenta et al., 2020;Souza et al., 2016).
Accordingly, the importance of better understanding the stability of these compounds in food is also increased, especially considering the processing they are submitted to before consumption. According to Torres (2009), the processing will change the matrix of the original product, consequently presenting an effect on the contents of the phytochemicals present in this. The influence of processing on bioactive compounds is still poorly studied, so it is useful to know the relationship between processing and preserving the content of natural antioxidants in food (Campos et al., 2008;Nascimento et al., 2014), especially due to the fact of the growing consumption of industrialized or processed foods in different ways, thus arousing interest in the quality of these products. The aim of the present work was to evaluate the influence of thermal processing with dry heat and moist heat on the antioxidant profile by paper spray PS / MS and antioxidant the activity of ora -pro-nobis (Pereskia aculeata Mill.).

Collection and preparation of plant material
Leaves of ora-pro-nobis (Pereskia aculeata Mill.) ( Figure 1) were collected from the Vegetable Bank of the Agricultural Research Corporation of Minas Gerais (EPAMIG) -Fazenda Santa Rita, located in Prudente de , from cultivation carried out without the application of pesticides.
The leaves of the vegetable were harvested at random, seeking a standardization by size.

Sample characterization
The leaves were collected in the morning and transported under refrigeration to the Food Conservation Laboratory of the Universidade Federal de São João del-Rei, Campus Sete Lagoas in Sete Lagoas / MG, where the experiment was conducted. Research, Society and Development, v. 10, n. 2, e12110212119, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12119 4 The leaves were washed in running water and separated into three parts, namely, one part was kept in natura (control) reserved for comparative analysis with the leaves submitted to thermal processing: hydrothermal heating and dry heat.

Dry heat processing
Part of the leaves (60 grams) of ora-pro-nobis were submitted to dry heat using a cabin dehydrator (Sparrow PE 60), with forced air circulation where they remained at 70 °C, for 8 hours. After dehydration, the leaves were ground manually with the aid of a gral and pistil.

Processing with application of moist heat
Part of the leaves (60 grams) were submitted to hydrothermal heating, where they were immersed in 1 liter of water at 100 °C for 4 minutes. The experiment was conducted with five replications and all analyzes were performed in triplicate.

Total soluble solids
The levels of total soluble solids were determined according to the methodology described by AOAC (2016). The samples were crushed, filtered, and placed on the prism of a Reichert, R2 Mini digital refractometer with internal temperature compensation. The results were expressed in ° Brix.

Total solids
Approximately 2 g of the homogenized samples were submitted to a temperature of 105 °C in a FANEM 515 sterilization, and drying oven for 48 hours. The percentage of total solids was obtained by the difference between the initial and final masses (after the greenhouse) of the samples, according to the AOAC protocol (2016).

Hydrogenionic potential (pH)
The pH value was determined by potentiometry with the aid of a digital pH meter Tekna T-1000, by direct immersion of the electrode in the sample homogenized with 50 milliliters of distilled water (AOAC, 2016).

Total titratable acidity
To determine the total titratable acidity, a 0.1 N NaOH solution was used as a standard and phenolphthalein as an indicator, with the assistance of a pH meter. The results were expressed in grams of citric acid per 100 grams of fresh sample (AOAC, 2016). Total titratable acidity was calculated through Equation 1.

Equation 1
In which: V = Spent volume of the NaOH solution (liters), N = Normality of the NaOH solution, F = Correction factor obtained from the standardization of NaOH, Eq. = Equivalent citric acid, and M = Mass of the sample (grams).

Total phenolic compounds
The content of total phenolic compounds was determined by the Folin-Ciocalteau method (Singleton et al., 1999) with a comparison of a calibration curve constructed with gallic acid. The absorbance was read on a FENTO 700S spectrophotometer at 740 nanometers. The results were expressed in milligrams of equivalents of gallic acid (EGA) per 100 grams of sample on a dry basis. The content of total phenolic compounds was calculated using Equation 2.

Equation 2
In which: ABS = Absorbance of the sample and EGA = Equivalent of gallic acid.

Total flavonoids
The analysis of the total flavonoid content was performed following the method of Francis (1982). The absorbance was read on a SHIMADZU UV-1800 spectrophotometer at 374 nanometers. The results were expressed in milligrams of flavonoids per 100 grams of sample on a dry basis. The content of total flavonoids was calculated using Equation 3.

Equation 3
In which: ABS = Absorbance of the sample, TF = Total flavonoids and M = Mass of the sample (grams).

Total anthocyanins
The anthocyanin content analysis was performed following the method of Francis (1982). The absorbance was read on a SHIMADZU UV-1800 spectrophotometer at 535 nanometers. The results were expressed in milligrams of cyanidin-3glucoside (CG) per 100 grams of sample on a dry basis. The total anthocyanin content was calculated using Equation 4.

Equation 4
In which: ABS = Absorbance of the sample, TA = Total anthocyanins and M = Mass (grams).

Evaluation of antioxidant activity
The antioxidant activity was determined by the DPPH method (2,2-defenyl-1-picryl-hydrazil), based on the scavenging of free radicals by antioxidant, according to the method of Brand-Williams et al. (1995).
Research, Society and Development, v. 10, n. 2, e12110212119, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12119 6 A quantity of 0.2 milligrams of sample was weighed and 15 milliliters of methanol acidified with 1% HCL were added, then they were shaken in the Shaker Novatécnica incubator at 150 rpm for 2 hours. Subsequently, 10 milliliters of this content was transferred to a Falcon tube and it was centrifuged at 3000 rpm for 15 minutes. A 0.1 milliliter aliquot of the supernatant was transferred to a test tube, where 2.9 milliliters of the working radical were added. After the reaction of the solutions in the dark (30 minutes), the reading was performed on a spectrophotometer at 515 nanometers. The results were calculated using Equation 5 and expressed in µmol of trolox equivalent per gram of sample on a dry basis (µmol ET / g sample).
Equation 5 In which: ∆ABS = White absorbance -absorbance of the sample, b = linear coefficient, a = angular coefficient, V = Volume used in the extraction (liters), M = Mass of the sample (grams), and D = Dilution.

Qualitative assessment of the chemical profile by paper spray
The analysis of the chemical profile of the samples was performed using an LCQ Fleet mass spectrometer (Thermo Scientific, San José, CA, USA) equipped with a source of ionization by paper spray. The analysis was performed in triplicate in positive and negative ionization modes, according to Silva et al. (2019).
In this analysis, the chromatographic paper was cut into an equilateral triangle shape (1.5 centimeters) and positioned in front of the mass spectrometer entrance. This paper was supported by a metal connector and positioned 0.5 centimeters apart with the aid of a mobile platform (XYZ).
This device was connected to a high voltage source of the spectrometer through a copper wire. Finally, 2.0 µL of the sample methanolic extract was applied to the edge of the triangles, 40.0 µL of methanol transferred to the chromatographic paper was added and the voltage source was connected for data acquisition. For the analysis, the instrument was operated at a voltage of -3.0 kV (negative ionization mode); capillary voltage of 40 V; transfer tube temperature 275 °C; the voltage of 120 V tube lenses; and mass range from 100 to 1000 m/z (negative ionization mode). The ions and their fragments obtained in this analysis were identified based on the data described in the literature. Collision energies used to fragment the compounds ranged from 15 to 40 V.
The analysis of each sample was performed in triplicate and the presence of the compound was taken into account when it appeared at least twice, the absence when it did not appear or when it appeared only once in the triplicate.

Data analysis
For the statistical analysis, a completely randomized design (CRD) was performed, with 5 replications. The evaluations were carried out in triplicate. The verification of the assumptions of normality and homogeneity of the variances was performed by the Shapiro-Wilk and Levene tests. The data from all analyzes were submitted to analysis of variance (ANOVA) and the means compared by Tukey's test with 5% probability (p < 0.05), using the SISVAR 5.6 software. Table 1 shows the values for total soluble solids, total solids, pH and total titratable acidity of ora-pro-nobis in natura leaves and after thermal processing. Table 1. Average values of the parameters of total soluble solids, total solids, pH and total titratable acidity of ora-pro-nobis in natura leaves and post-processing (mean ± standard deviation). SST= total soluble solids (° Brix), ST= total solids (%), U = moisture (%) ATT= total titratable acidity (citric acid 100 g -1 fresh sample), N. r. = Unrealized e CV = Coefficient of variation. Averages followed by the same lowercase letter in the columns do not differ significantly from each other, at the significance level of 5% probability by the Tukey test. Source: Authors.

Processing
The leaves of ora-pro-nobis showed to possess a large amount of soluble solids, being higher than in their fruits, in which, Silva et al. (2018) analyzed the SST content at different stages of maturation and pointed out higher levels for ripe fruits (5.65 ° Brix). After the processing of ora-pro-nobis leaves with moist heat, the content of soluble solids decreased by 1.5 ° Brix, being still, in higher levels than in the ripe fruits described by Silva et al. (2018).
When performing a chemical characterization of two species of the genus Pereskia, De Almeida (2014) reported the 12.46% of total solids for leaves of Pereskia aculeata Mill., being slightly lower than the content reported in this work for in natura leaves.
The processing of ora-pro-nobis leaves with dry heat decreased the moisture content of the sample since the water present in the leaf tissues of the vegetable evaporated due to the processing conditions. On the other hand, ora-pro-nobis leaves submitted to moist heat processing showed a significantly higher water loss compared to fresh vegetable leaves (5.59%), as can be observed in Table 1, however, still keeping water as its main component.

Bioactive compounds and antioxidant activity
The ora-pro-nobis leaves submitted to moist heat processing showed an increase of 29.84% in their contents of total phenolic compounds, reaching 12.43 ± 0.98 g EGA 100 g-1 dry sample. The ora-pro-nobis leaves submitted to processing using dry heat, presented a reduction of just over half of their total phenolic compounds, being detected a total of 4.25 ± 0.93 g EGA 100 g-1 dry sample (Table 2).
There was a retention of 94.78% of the total flavonoids and 76.81% of the total anthocyanins in the leaves submitted to moist heat, and 10.91% of the total flavonoids and 16.80% of the total anthocyanins in the leaves submitted to dry heat, which shows that leaves submitted to heat for a longer period of time have a large part of their constituents lost.
Research, Society and Development, v. 10, n. 2, e12110212119, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12119 8 Tsantili et al. (2010), stated that with the damage suffered by vegetables submitted to processing, the cell membranes are broken, which leads to the leakage of their contents, consequently leading to a decrease in the content of bioactive compounds in the leaves of the processed vegetable. Table 2. Average values of the contents of total phenolic compounds, total flavonoids and total anthocyanins (of ora-pro-nobis in natura leaves and post processing (mean ± standard deviation). TPC= total phenolic compounds (mg EGA 100 g -1 sample on dry basis), TF= total flavonoids (mg 100 g -1 sample on dry basis), TA= total anthocyanins (mg cyanidin-3-glycoside 100 g -1 dry sample), CV = Coefficient of variation. Averages followed by the same lowercase letter in the columns do not differ significantly from each other, at the significance level of 5% probability by the Tukey test. Source: Authors. Sultana et al. (2008), studying the influence of different cooking methods on the total phenolic content and antioxidant activity of spinach, carrots, turnips, cabbage, peas, and cauliflower show that the contents of phenolic compounds had a decrease after cooking, reaching up to 70% losses. The leaves of ora-pro-nobis in natura and those submitted to hydrothermal heating did not show any difference in the contents of total flavonoids ( Table 2).

Processing
The cooking process is reported to destabilize the matrix of compounds presents inside plant cells, with losses due to leaching. Boiling water may cause losses of these compounds to the cooking water due to the polarity of the molecules, which may explain the decrease in the contents of the bioactive compounds evaluated and also in the total soluble solids (Table 1) in the leaves of ora-pro-nobis submitted to hydrothermal processing (Lima et al., 2017;Palermo et al. 2014).
Both processes affected the free radical scavenging activity negatively. The activity was drastically reduced in leaves processed with dry heat, whereas in leaves processed with moist heat, the reduction was smaller (Table 3). Likewise, there was less retention of the bioactive compounds in leaves submitted to dry heat, compared to in natura leaves (Table 2) and this directly interfered with the antioxidant capacity.
The contents of bioactive compounds showed greater retention when submitted to moist heat, reflecting on its antioxidant activity. Even using a higher temperature (100 ºC) during hydrothermal heating, the leaves of ora-pro-nobis were submitted to heat for less time (4 minutes). The leaves processed with dry heat were exposed for 8 hours at a lower temperature (70ºC). Therefore, preparations involving hydrothermal processing lead to lower losses. Table 3. Average values of antioxidant activity of ora-pro-nobis in natura leaves and post-processing (mean ± standard deviation).

Processing AA
In natura 20,00 ± 1,29 a Moist heat 12,03 ± 0,41 b Dry heat 0,82 ± 0,0033 c CV (%) 13,56 AA = antioxidant activity (µmol ET / g dry sample) e CV = Coefficient of variation. Averages followed by the same lowercase letter in the columns do not differ significantly from each other, at the significance level of 5% probability by the Tukey test. Source: Authors.
Heat treatment can present an effect on the antioxidant capacity since it promotes the destruction and/or oxidation of compounds with such potential (Mazzeo et al., 2011, Rajauria et al., 2010. In addition, both the increase in temperature and the residence time to which the leaves are submitted can cause a certain destabilization of plant cells, causing the availability of these compounds to be altered (Gliszczyńska-Świglo et al., 2006). Garcia et al. (2019) reported that the extract of ora-pronobis leaves showed high activity when evaluated by different methods (DPPH and ABTS). Rigueira et al. (2016) evaluated the ability to neutralize free radicals from conventionally and organically grown kale leaves submitted to dry and moist heat treatment and reported 34.8% higher antioxidant activity in lettuce leaves grown in conventional systems, lettuce leaves grown in an organic system showed antioxidant capacity 36% higher in leaves submitted to dry heat compared to leaves submitted to moist heat. Table 4 shows the qualitative analysis of the chemical profile of ora-pro-nobis in natura leaves and post-processing.

Chemical profile by paper spray
The presence of 56 compounds of different chemical classes in the ora-pro-nobis leaves was detected. Many of these compounds are being reported for the first time in the literature for the leaves of Pesreskia aculeata. Research, Society and Development, v. 10, n. 2, e12110212119, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12119 10   Table 4. Qualification of compounds in ora-pro-nobis in natura leaves and post-processing with dry heat and moist heat identified by PS. The letter X in the body of the Although the content of total phenolic compounds changed after thermal processing (Table 2), there was no variation in the profile of the constituents of this class of compounds in those processed with both dry and moist heat in relation to in natura leaves (Table 4). Thus, the processing may have affected quantitatively some of the compounds belonging to this class, which were qualified in this study.
Concerning other classes of compounds, changes in the chemical profile were observed. For example, the substance 4,5-Dimethyl-2,6-octadiene was detected only in natura leaves, the compound Linolenic acid methyl ester was not detected in the leaves processed with dry heat. Garcia et al. (2019) identified ten phenolic compounds in ora-pro-nobis leaves from southern Brazil, namely, two phenolic acids (derived from caffeic acid) and eight flavonoids (quercetin, kaempferol, and derivatives of the glycoside isorhamnetin). They also reported that caffeic acid was the main phenolic constituent of the extract, accounting for more than 49% of the phenolic content, followed by quercetin-3-O-rutinoside (14.99%) and isorhamnetin-O-pentoside-O-rutinoside (9.56%), nevertheless the stability of these compounds in ora-pro-nobis leaves after processing with dry heat and moist heat was not known. All the compounds previously mentioned were not affected by any of the processing methods used in this work.
Using ultra-high performance liquid chromatography ( (Porto Alegre). The former has the potential to treat lung cancer (Islam et al., 2018).
Analyzing the chemical profile and the contents of bioactive compounds present in food are extremely important so that such information can be taken into account in decisions concerning the type of processing to which the food will be submitted. Despite the presence of compounds with high antioxidant capacities in the fresh form, it is important to clarify the behavior of these components under processing conditions.

Conclusion
The content of bioactive compounds present in ora-pro-nobis leaves processed with dry heat was drastically reduced, which consequently affected the scavenging activity of free radicals, which showed a decrease of 93.18% when compared to the processing of ora-pro-nobis leaves with moist heat, and 95.90% when compared to unprocessed leaves.
The chemical profile of the leaves was also affected by processing, in which the compound 4,5-Dimethyl-2,6octadiene was detected only in natura leaves, the compound Linolenic acid methyl ester was not detected in the leaves processed with dry heat. The profile of phenolic compounds in ora-pro-nobis leaves was not affected by thermal processing.
The acknowledgement of the effect of processing in food is extremely important in order to understand the behavior of compounds that act in a beneficial way in human health, and thus identify the best way to process and consume them, being also extremely important in the pharmaceutical industry, which uses these compounds for the production of drugs.

Aknowledgements
To FAPEMIG for the financial support and EPAMIG for providing the vegetables used in this work.