Araucaria angustifolia (bert.) Otto kuntze): Comparative evaluation of phenolic composition, antioxidant and antimicrobial activities of seeds cooking water Araucaria angustifólia (bert.) Otto kuntze): Avaliação comparativa da composição de fenólicos, atividade antioxidante e antimicrobiana das águas de cozimento das sementes Araucaria angustifólia (bert.) Otto kuntze): Avaluación comparativa de composición fenólica, actividad antioxidante y antimicrobiana de aguas de cocción de semillas

Araucaria angustifolia var. angustifolia and Araucaria angustifolia var. indehiscens, seeds (common pinhão and monkey pinhão) are consumed after cooking, coats and water represent waste. In this work, pinhão was submitted to different cooking conditions, the water extracts were analyzed to determine and identify their phenolic compounds content, antioxidant activity and antimicrobial potential. Cooking for 45 minutes without addition of sodium chloride resulted in residual water with highest content of phenolic compounds and antioxidant activity (DPPH and FRAP), both for the common pinhão and for the monkey pinhão. Protocatecuic acid, vanillin and coniferaldehyde were identified and quantified by HPLC-ESI-MS/MS as the most prevalent phenolic compounds. No antimicrobial potential was observed against Salmonella enterica Typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 11778).


Introduction
Araucaria angustifolia is a tree widely found in southern and southeastern Brazil, occurring in different varieties, among them, Araucaria angustifolia var. angustifolia and Araucaria angustifolia var. indehiscens, popularly known as "common" pinhão and "monkey" pinhão, respectively (Lima et al., 2007). These seeds are used for consumption, after cooking in water, which is usually considered as waste as well as the coats.
It is described that the residual water from the cooking presents a reddish tint, a bitter taste, due to the migration that occurs mainly from the seed coats to water (Koehnlein et al., 2012) and is a source of bioaccessible phenolics (Cunha et al., 2018).
Considering these residues, most of the published works value the seed (Macedo et al., 2020;Pereira et al., 2020;Thys et al., 2013) and not the husks or the water from the cooking of the pinhão. By identifying functional perspectives in cooking waters, new opportunities arise. Mainly, to establish applications to cooking water, adding commercial value, in addition, to provide socioeconomic advances, generating less waste or environmental problem due to its disposal, and also to justify the preservation of pine trees. However, to date, the cooking waters have not been studied for the purpose of justifying their application.
This work aimed to investigate the phenolic composition, antioxidant and antimicrobial activities of the seed cooking waters of the Araucaria angustifolia var. angustifolia and Araucaria angustifolia var. indehiscens.

Samples
The seeds were collected in the city of São José do Cerrito -SC, Brazil (latitude: 27° 39' 45'' South, longitude: 50° 34' 48'' West) where common pinhão was collected between June and July / 2018 and the monkey pinhão in the month of August / 2018. To obtain seed aqueous extract (residual waters), 100 g seeds were cooked in 1000 mL of distilled water (15, 30 and 45 minutes -under maximum pressure) with and without salt (NaCl -Tradicional Cisne brand) (15 g L -1 ). The procedure was done in a pressure cooker (Clock ® , São Bernando do Campo, Brazil), in triplicate.

pH, total soluble solids, minerals, soluble proteins and colorimetry
The physicochemical analyzes were performed as proposed by AOAC (2005). Total soluble solids content was determined in an Abbe refractometer, calibrated with distilled water; minerals content in a muffle at 550 °C, with prior incineration, and soluble proteins by the method of Bradford (1976). Potentiometer was used to determine pH.
CIELab parameters (L*, a* and b*) were determined using a colorimeter (Minolta Chroma Meter CR-400, Osaka, Japan), adjusted to operate with D65 illuminant and previously calibrated 2 ° viewing angle.

Determination phenolic compounds
The total phenolic content (TPC) was determined as proposed by Singleton and Rossi (1965) with modifications made by Arriola et al. (2014), evaluated at 765 nm in a UV-Vis spectrophotometer (Hitachi, U-1800, Tokyo, Japan). The calibration curve used gallic acid and the total phenolic compounds content was expressed as mg of gallic acid equivalent per 100 mL sample (mg GAE 100 mL -1 ).
Phenolic compounds were identified as described by Schulz et al. (2015) with modifications. Water extract (10 mL, pH 2) were partitioned with ethyl ether and centrifuged at 4000 rpm (Hermle, Z 200A, Wehingen, Germany) for 10 minutes, three times. The supernatants were combined and vacuum concentrated at 40 °C until complete drying. They were resuspended in 1 mL of methanol and diluted 10 times for LC-ESI-MS/MS, (model 1200 Series, Agilent Technologies, Germany) analysis.
The HPLC system was coupled to a MS -triploquadrupole and linear trap ion analyzer (model Q Trap 3200, Biosystems / MDS Sciex, Canada). The ionization source was electrospray (TurboIonSpray, Applied Biosystems / MDS Sciex, Concord-ON, Canada) in negative mode. The used conditions were: capillary needle maintained at -4500V; curtain gas at 10 psi; temperature of 400 °C; gas 1 and 2 at 45 psi, and collision gas, medium. Analyst software version 1.5.1 was used to acquire and process the obtained data.
The standards (n = 43, Sigma-Aldrich, St. Louis, USA) were prepared with a stock solution (1000 mgL -1 in 100% HPLC grade methanol) and used to prepare the calibration curve by appropriate dilution of the mixture. Folin-Ciocalteu reagent, gallic acid and ultrapure phenolic standards were obtained from All solvents for chromatography analysis were of chromatographic grade.

In vitro antioxidant activity
The antioxidant activity by the DPPH method was determined as proposed by Brand-Williams et al. (1995) with modifications by Arriola et al. (2014). The absorbance of the resulting solution was evaluated at 515 nm in a UV-Vis spectrophotometer (Hitachi, U-1800, Tokyo, Japan). The calibration curve was constructed using solutions containing known concentrations of Trolox. The results were expressed in μMtrolox 100 mL -1 .
The antimicrobial potential was determined, in triplicate, by the agar diffusion method, by paper disc and in wells as recommended by the National Committee for Clinical Laboratory Standards in the M2-A8 standard.
Mueller Hinton agar (MH) was seeds, by surface spread, the disks were impregnated with CPE (4.0 g mL -1 and 2.0 g mL -1 ), MPE (2.7 g mL -1 and 1.4 g mL -1 ) and sterile distilled water, 0.05% ciprofloxacin, as controls. MH agar was also inoculated, by pour plate technique, and wells of approximately 6 mm in diameter, the wells were filled with 30 μL of the same tested solutions. The dishes were incubated for 24 hours at 35 °C.

Fluorescence microscopy
The pinhão water that presented the highest TPC was analyzed by transferring an aliquot to glass slides and viewed under fluorescence microscope (OLYMPUS ® BX4, Tokyo, Japan) employing auto fluorescence to identify the presence of structural phenolic compounds.

Statistical analysis
Statistical analysis of the data was performed using STATISTICA 13.3 (TIBCO Software Inc., Palo Alto, CA). The results were submitted to one-way analysis of variance (ANOVA), followed by Tukey's test (p < 0.05). All quantitative analyzes were performed in triplicate and the results were expressed as mean ± standard deviation (SD).

Results and Discussion
The presence of sodium chloride in the cooking of both varieties of pinhão completely changes the profile of the cooking waters. It can be explained by the salt extractive effect on the compounds of interest, in addition to the lixiviation or even the attachment of other compounds to the coats.
Common pinhão water shows a higher pH than monkey pinhão in the presence of salt. The total soluble solids as predicted increase with the addition of the cooking salt (Table 1).
The both varieties also presented different behavior regarding salt fixation, since there was less loss of it as total soluble solids. The same behavior was expected for the minerals, but both presented similar ash content in the different cooking times with and without salt.
The soluble protein fixation occurs, probably in almonds, when the pinhão is cooked with salt, compared to both samples without salt. Even so, it was observed a significant release of soluble protein, at different cooking times with and without salt, for the common pinhão. However, was not recorded soluble proteins in the cooking waters of monkey pinhão with salt at 30 and 45 minutes of cooking.
Soluble proteins contribute to the total soluble solids fraction, as well as draws attention to possible expected differences in the bioaccessibility of the phenolic compounds. In subsequent studies, the tanning effect of the pinhão phenolic Research, Society and Development, v. 10, n. 9, e8810917942, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17942 compounds on the presence and absence of salt and the consequences of this fact on the functional properties of these aqueous extracts with and without salt at different cooking times for the two pinhão varieties should be investigated.   (Table 2).
The fluorescence microscopy images of the waters of the monkey and common pinhão (Figure 1) confirm the presence of the phenolic compounds in which they are shown in agglomerates. Autofluorescence and the color variation between the red, brown and orange colors suggest the highest concentration of phenolic compounds and the bright blue color indicates the presence of lignin (structural phenolic compound).
The total phenolic content was quantified in all samples (Table 3). The effect of sodium chloride addition is clear on the release reduction of these compounds. However, the common pinhão presents significantly higher value than the monkey pinhão, at the same cooking time, without salt.   The same results were observed to antioxidant activity evaluated by DPPH and FRAP (Table 3). After cooking during 45 minutes, without sodium chloride, the samples had the highest values for antioxidant activity, with the exception of the monkey pinhão water, by the DPPH method.
The presence of phenolic compounds in the aqueous extract of common and monkey pinhões may be important for future studies on the functional properties of these cooking waters. As well as, the functional properties of the aqueous extract of Camelia sinensis tea are attributed to phenolic compounds (Al-Obaidi et al., 2015;Shannon et al., 2018).
The protocatechuic acid (3,4-Dihydroxybenzoicacid -(HO)2C6H3CO2H) was the phenolic found in all cooking water samples (4.77 mg L -1 , 3.92 mg L -1 from common pinhão, with and without NaCl and 6.49 mg L -1 , 4.05 mg L -1 from monkey pinhão with and without NaCl), which proves aqueous extraction in absence of sodium chloride for both varieties is more efficient in extracting phenolic compounds from the seeds.
Among the phenolic compounds found in pinhão extracts are proanthocyanidins, derived from catechins and epicatechins, flavonol quercetin-3-glycoside, flavanone eriodictiol hexoside and two phenolic acids, these being derivatives of protocatechuic and ferulic acids (Freitas et al., 2018;Santos et al., 2018). Due to its biological properties, such as antioxidant activity, it may be potentially associated with chemopreventive (Tanaka, Tanaka, & Tanaka, 2011), as well as other effects against chronic diseases and inflammation (D'Archivio et al., 2018).  6). a-f Lowercase letters -different in the same column and in the same variety of pinion indicates significant difference (p <0.05) between applied treatments. A-H Different capital letters on the same column indicate significant difference (p <0.05) between applied treatments and pine cone varieties concomitantly. Source: Authors.
The phenolic compounds extracted in the cooking waters are water soluble and differ from those found by , who studied the hydroalcoholic extract of the common pinhão. The presence of these compounds in the cooking waters of the pinhão is not sufficient to indicate the bioaccessibility. Therefore, da Cunha et al. (2018) evaluated bioaccessibility in simulated gastrointestinal digestion in vitro and found that both pinhão extracts showed phenolic compounds bioaccessibles.
Despite of its composition and presence of phenolic compounds, the cooking waters did not show any antimicrobial potential at the concentrations tested against the selected bacteria. It is important to consider that the lack of effect can be explained by the low concentration of functional compounds on samples.

Conclusion
The present work, opens a perspective of future applications by investigating the effects of cooking time and the presence of sodium chloride on the extraction of phenolic compounds from pinhão to cooking water. The color in the brown tint tending to red reinforces the presence of these compounds, also confirmed by the fluorescence microscopy. The phenolic compounds identified by HPLC-ESI-MS/MS should be studied for its bioaccessibility, once they present antioxidant characteristics. The soluble protein retention characteristics in cooking waters, alert us to future work to identify the chemical mechanisms responsible for this behavior. The results of this work can indicate perspectives of use for the water from the cooking of the pinhão.