Assessment on Solubility and Solid Phase Chemical Fractionation of Manganese in Hot Infusions of Green and Roasted Mate

A solid phase chemical fractionation (SPCF) of the Mn in hot infusions prepared from commercial samples of roasted (RM) and green mate (GM) using a chelating resin Chelex 100 (NH4+ form) was performed to assess the relative lability of this essential trace element (ETE). In addition, total Mn contents in the RM and GM samples and their infusions were determined by flame atomic absorption spectrometry. Total polyphenol (TP) contents and the presence of soluble melanoidins (SM) were correlated with the Mn solubility in the RM and GM infusions. From the SPCF study, it was possible to observe that the soluble Mn forms in the mate infusions were essentially associated with relatively noninert chemical species (98.4–99.7%), suggesting that they may be potentially bioavailable. In addition, the soluble Mn contents in the GM infusions were 20.5% higher than those found in the RM. Mn solubility in the (RM) infusions was highly and directly correlated (r = 0.99) with the soluble TP, while in the GM infusions, it was high and inversely correlated with soluble TP (r =  −0.87). On the other hand, Mn solubility in the RM and GM infusions was weakly correlated with the SM. It should be stressed that GM infusions can contribute with 57 and 44% more than the RM infusions to the recommended adequate intake of Mn established for females and males, respectively. Moreover, this work is the first to evaluate and compare the relative lability of Mn and its solubility in the RM and GM infusions.


Introduction
Yerba mate (Ilex paraguariensis) also known as a mate is a subtropical plant that currently has been gaining relevance in the worldwide market due to its bittersweet flavor and aroma as well as health benefits and potential use as a functional food or phytotherapeutic product.The Ilex paraguariensis plant is native to South America and is the primary material for the preparation of the roasted and green mate, which are used to elaborate the beverages known as mate tea (or red mate) and chimarrão, respectively [1,2].The main producers of mate are the regions of southern Brazil, northern Argentina, also Uruguay, and Paraguay, where the dried leaves of this herb are traditionally used for the preparation and consumption of cold (tereré) and hot infusions (mate tea and chimarrão) [3,4].Data provided by the Food and Agriculture Organization of the United Nations (FAO) has shown that between 2010 and 2017, mate was exported to several countries over the world [5].
The processing of mate to the production of its final products known as roasted mate (RM) and green mate (GM) is initialized with a bleaching (sapeco) step (± 500 °C) of fresh leaves and twigs of the mate plant, and then sequentially followed by a drying (8-24 h, ± 100 °C) for reducing approximately 20% of the mate moisture, and by a fragmentation process popularly known as cancheamento.Afterwards, an additional milling step is used to obtain the green mate in an appropriate form for the preparation of chimarrão (GM) and tereré.After that, to obtain the RM, the GM can still pass through an additional roasting step under relatively high temperatures (180-215 °C) for approximately 15 min [2,3,6].
The growing popularity of mate and its products is due to their health benefits, which can be promoted by a regular consumption of the mate beverages.Such health benefits that a regular consumption of yerba mate promotes are attributed to chemical species of mate that have high biologic activity, such as polyphenols, xanthines, methylxanthines, caffeine, saponins, and minerals, among others [7][8][9].Furthermore, the mate also stands out for being considered a rich dietary source of polyphenols, including chlorogenic acids (CGA), flavonoids, stilbenes, and lignans, which are known for their strong antioxidant activity, and can also act in the prevention of cancer, diabetes, and heart disease, and have anti-inflammatory, anti-bactericide, anti-viral, and anti-hypertensive effects [2,10,11].Polyphenols can also influence the presence of inorganic ions in mate infusions by forming complexes with metallic ionic species, altering their chemical forms and, consequently, their bioavailability [12,13].
Manganese (Mn) is an essential trace element (ETE) highly present in tea and coffee infusions [12,14,15], and it can also be found in a wide variety of foods such as vegetables, nuts, grains, seeds, seafoods, chocolates, and many others.This metal in trace concentrations has a great influence on the human body physiology, since it can activate some enzymes that act against the oxidative stress and regulate brain function, digestion, body growth, and immune response [16,17].Previous research has proved that mate can be an alternative source of Mn [18][19][20][21][22][23].However, the potential toxicity of this element at relatively high concentrations should be carefully considered, especially considering a high daily consumption of the mate beverages and products.In this context, it should be recommended the development of studies with the mate with the objective of evaluating the presence and contents of metals, as well as their potential toxic effects when in relatively high concentrations.Concerning the Mn, its toxic effects could be related to psychological and neurologic disorders, and even Parkinson's disease [17,24].
It should be emphasized that in tea and coffee infusions, Mn can be found in free forms such as aquocomplexes and/ or bound to bioligands such as polyphenols and melanoidins, among others [13,25].For a better understanding of the behavior and role of metallic ionic species in the mate infusions as well as other beverages, chemical fractionation studies have been shown to be one of the important analytical tools for this purpose.It is important to point out that the fractionation pattern of elements and the identification of their ultimate physicochemical forms, combined with the in vivo and in vitro studies, can provide a more complete understanding of the nutritional values and toxicity of the elements [26][27][28].
In general, fractionation methods are based on the use of separation procedures capable of differentiating groups of chemical species according to their physical or chemical properties by applying different analytical techniques [29][30][31].Regarding the chemical fractionation studies, solid-phase extraction (SPE) procedures have been widely used as an analytical tool to assist in the evaluation of the distribution of different groups of chemical species of elements in several types of beverages [13,29,32,33].Pohl et al. [32] conducted a study on the chemical fractionation of Ca and Mg in red beetroot juice by SPE using reverse phase and cation exchange sorption materials and proposed that these elements were mainly bound by organic compounds.On the other hand, other studies found that Mn showed a weak ability to form organic complexes in beers [34,35].As another study example, Pohl et al. [36] employed a tandem column SPE procedure based on the Amberlite XA-7HP, followed by a cation exchange column 50-W-× 8-200 to perform the chemical fractionation of Fe in wine.In that study, three different Fe groups were found, including hydrophobic species of Fe bound to polyphenol compounds, cationic species (simple Fe ions and labile Fe forms), and anionic and/ or Fe complexes formed with organic acids [36].Da Costa et al. [6] performed a chemical fractionation study using a chelating cation-exchange resin (Chelex 100) to evaluate the lability of the Al in RM and GM infusions, and they found that soluble Al was predominantly in relative inert forms in RM infusions.
In this context, the focus of this work was to evaluate and compare the solubility of the essential trace element Mn in the RM and GM infusions, correlating its solubility with polyphenols and soluble melanoidins.In addition, the relative lability of Mn in these infusions was evaluated by using a solid phase chemical fractionation procedure based on the use of a chelating resin Chelex 100 (NH 4 + form).Moreover, to the best of our knowledge, this is the first work to perform such evaluations.

Reagents and Solutions
All reagents used were of analytical grade, and an ultrapure water (resistivity > 18.2 MΩ cm −1 ) was obtained using a Milli-Q® system (Millipore®, USA) for preparing all solutions and infusions.Concentrated HNO 3 (Sigma-Aldrich®) and 30% w v −1 H 2 O 2 (Sigma-Aldrich®) were used for sample digestions.Before use, all glassware were soaked in 10% v v −1 HNO 3 bath for 24 h, then rinsed with ultrapure water and allowed to dry at room temperature.
A stock solution of Mn 998 mg L −1 ± 4 mg L −1 for AAS, prepared in medium of 2% w w −1 HNO 3 2%, TraceCERT® from Fluka®, was used to prepare the diluted standard solutions (0.20-4.0 mg L −1 ) in medium of 1% v v −1 HNO 3 , which were used to obtain the analytical calibration curves by the external calibration method.The Folin-Ciocalteu reagent, and gallic acid ≥ 98.0%, both from the Sigma-Aldrich®, were used for the total polyphenol determinations.A chelating-exchange resin Chelex® 100 (Sigma-Aldrich®), a styrene divinylbenzene copolymer containing paired iminodiacetate ions with a particle size of 50-100 mesh (dry), pH range 4-14, was purchased in sodium form, and then converted to a NH 4 + form before use, by treatment with a 1.0 mol L −1 NH 3 solution prepared from an ultrapure 25% w w −1 NH 3 solution (Merck®).A 0.30 mol L −1 sodium acetate-acetic acid buffer solution was used to adjust to 5.80 the pH of the standard solutions and mate infusions submitted to the batch adsorption experiments.

Instrumentation
A Varian® (Mulgrave, Australia) model SpectrAA-240FS flame atomic absorption spectrometer equipped with a deuterium (D 2 ) lamp-based background correction system was employed for the Mn determinations.The following instrumental conditions were used: wavelength of 279.5 nm; slit width of 02 nm; Mn hollow-cathode lamp, operating with a current of 8.0 mA; air/C 2 H 2 flame with an oxidizer/fuel stoichiometric ratio of 13.5 L min −1 /2.0 L min −1 .A Jung-200 hot plate (Jung®, Brazil) and a mechanical horizontal stirring system were used for performing the acid digestions and batch adsorption experiments, respectively.Filtered infusions were obtained using a vacuum filtration system (Sterifil®, Millipore®, USA).Melanoidins' presence in the filtered fresh infusions of the roasted and green mate as assessed by a UV-vis spectrophotometer (Thermo Scien-tific®, USA).The pH measurements were performed using a Lucadema® LUCA-210 pH-meter (São José do Rio Preto, Brazil) and a combined glass electrode, calibrated with pH 7.00 and 4.00 buffer solutions.An analytical balance Mark 210A (Logen Scientific®, EUA) with readability of 0.1 mg was used for all weighings, and a water bath model 550 (Fisatom®, Brazil) was used for preparing the hot infusions of mate.

Samples
Three commercial samples from different brands of the RM (packs 250 g) and GM (packs 500 g) for the preparation of the mate tea and chimarrão beverages, respectively, produced in different regions of southern Brazil (6 samples in total) were randomly purchased in the local market (Maringá, Paraná, Brazil).First, the masses of the commercial GM and RM samples were homogenized, and aliquots of 200-250 g and 350-400 g of each RM and GM sample, respectively, were taken, minced using a mortar and pestle of agate, and homogenized.After, they were sieved using a Nylon® membrane (400 μm) and randomly labeled from 1 to 3 to preserve their identities.

Acid Digestion
The conventional open flask acid digestions of the RM and GM samples were carried out using a hot plate operating at 120 °C.A 0.5000 g mass of the mate samples were digested using a mixture of 10.0 mL of a solution of concentrated HNO 3 and 2.0 mL of a solution of 30% w v −1 H 2 O 2 , and this digestion procedure was repeated continuously until a translucent solution was obtained without the presence of solid particulate matter and NO 2 fumes.After cooling, the obtained acid digested solutions were transferred to 50.0 mL graduated polypropylene flasks (Corning®), and the volume was made up to 50.0 mL using ultrapure water.All mate samples were digested in triplicate (n = 3).

Preparation of the Infusions
Hot infusions of the RM and GM samples were prepared in triplicate by the mixture of 0.5000 g of the samples with 40.0 mL of hot ultrapure water, which was kept at 80 °C for 3 min using a water bath.This preparation procedure was adopted as an attempt to use conditions similar to those used by the consumers of the infusions of RM (mate tea beverage) and GM (chimarrão beverage), but not identical to those described on the roasted and green mate manufacturers labels, considering that the temperature and time of infusion varies among different manufacturers.In addition, ultrapure water instead of tap water was used for the preparation of RM and GM infusions.
After, the infusions obtained were filtered through a 0.45 μm acetate cellulose membrane disc filter (∅ 47 mm) (Whatman®) using a vacuum filtration system.Subsequently, after cooling, the obtained filtered fresh infusions (soluble fraction of mate samples) were analyzed by flame atomic absorption spectrometry (F AAS) and UV-vis molecular absorption spectrophotometry (UV-vis spectrophotometry).In addition, it should be stressed that the obtained fresh infusions were 20-fold diluted before F AAS analyses to avoid the clogging of the aspiration capillary of the atomic absorption spectrometer sample introduction system.All infusions were analyzed in triplicate (n = 3).

Determination of the Total Mass Fractions
The total mass fractions of Mn in the acid digested solutions of RM and GM samples and in their filtered fresh infusions were determined by F AAS using a D 2 lamp-based background correction system.Between each aspiration of the filtered fresh infusions, it was aspirated approximately 3.0 mL of a 5.0% v v −1 HNO 3 solution for washing the spectrometer sample introduction system in order to avoid possible memory effects on the atomic absorption measurements.The reliability of the Mn determinations was evaluated by applying addition-recovery experiments.Background correction was used because spectrometric measurements were carried out at a wavelength below 330 nm.All acid digested solutions of mate samples, and their infusions were prepared and analyzed in replicates (n = 4) by F AAS.
The limits of detection (LODs) and quantification (LOQs) were estimated according to the criteria established by the International Union of Pure and Applied Chemistry (IUPAC) [37].The LOD and LOQ were defined as 3s blank /b and 10s blank /b, respectively, where s blank is the standard deviation of 10 replicates of the analytical blank solution, and b is the slope of the calibration curve.Accuracy and precision of the procedures employed for the determination of Mn in the acid digested solutions of GM and RM samples and their infusions, were evaluated by applying additionrecovery experiments at 3 different levels of Mn concentration (0.5, 1.0, and 2.0 mg L −1 ) in 3 replicates.The precision of the measurements were expressed as relative standard deviation (RSD%).

Total Polyphenols Determination
The total polyphenol (TP) determinations in the filtered fresh infusions of the RM and GM were carried out by the Folin-Ciocalteu method [38,39].This method was applied by using tenfold diluted mate infusions, according to the following procedure: addition of 1.0 mL of the Folin-Ciocalteu reagent in graduated polypropylene flasks, followed by the additions of 1.0 mL of ultrapure water and 100.0 μL of the diluted infusions.The mixture obtained was allowed to stand for 10 min, and then added 1.0 mL of a 7.5% w v −1 Na 2 CO 3 solution.The final solution was left for stabilize for 30 min, before measuring the absorbances by UV-vis spectrophotometry at 765 nm using a glass cuvette (1.0 cm optical length).All infusions were analyzed in replicates (n = 4).

Melanoidins Spectrophotometric Measurements
The presence of soluble melanoidins (SM) in the filtered fresh infusions of the RM and GM was evaluated by UV-vis spectrophotometry at 420 nm [38,39] using a glass cuvette (1.0 cm optical length).Before the spectrophotometric measurements, all mate infusions were tenfold diluted using ultrapure water.All infusions were analyzed using replicates (n = 4).

Chemical Fractionation of the Soluble Mn
A solid phase chemical fractionation (SPCF) study of the Mn contents found in the filtered fresh hot infusions of RM and GM was performed based on batch adsorption experiments using the chelating resin Chelex 100, in order to evaluate the relative lability of the Mn in these mate infusions.Before the adsorption experiments, the Chelex 100 resin was converted to a NH 4 + form.For this, the following procedure was used: the resin originally present in the sodium form was kept in contact with a 1.0 mol L −1 HNO 3 solution for 30 min.Then, it was washed several times with ultrapure water to eliminate the acid excess, and after, the resin obtained in the protonated form was kept in contact with a 1.0 mol L −1 NH 3 solution for 30 min and washed with ultrapure water until to eliminate the base excess.Finally, the Chelex 100 resin (NH 4 + form) was dried at room temperature using an evacuated desiccator.It is worth saying that each portion of the chelating resin Chelex 100 (NH 4 + form) used to carry out the adsorption studies was never reused, in order to avoid the occurrence of possible memory effects on the obtained results, which could be caused by the use of a regenerated resin.
In order to establish the suitable resin mass to be used in the batch adsorption experiments, 40.0 mL of a standard 50.0 mg L −1 Mn solution (pH 5.80), was kept in contact with different masses (0.05-0.50 g) of the Chelex 100 (NH 4 + form) for 24 h, under stirring at room temperature.Following that, 40.0 mL of the filtered fresh infusions of the RM and GM was kept in contact with the suitable mass resin previously established (0.45 g) for 2, 5, 10, 15, and 30 min on mechanical stirring at room temperature to assess the maximum removal rate of Mn.At the end of the adsorption experiments, an aliquot of each infusion was analyzed by F AAS to determine the soluble Mn content not retained on the resin surface, so that the Mn removal rate from the infusions could be assessed, as well as the relative lability of the soluble forms of Mn in the mate infusions could be evaluated.All batch adsorption experiments and analyses of the mate infusions by F AAS were performed in triplicate (n = 3).

Statistical Analysis
Data were expressed as means ± standard deviations.Results were compared at a 5% significance by applying the Tukey's test using the Statistica 10.0 software.

Total and Soluble Contents of Mn
Using the F AAS technique, it was possible to determine the total contents of Mn in the acid digested solutions of RM and GM and also in the hot infusions of their samples.Results for the total mass fractions of Mn determined in the acid digested mate samples and in their hot infusions are shown in Table 1.Regarding the total mass fractions of Mn in GM and RM samples, they ranged from 1287 to 1463 mg kg −1 and from 1455 to 1746 mg kg −1 , respectively.
In relation to the total mass fractions of soluble, Mn leached to the hot infusions of RM and GM, they varied from 337 to 564 mg kg −1 and from 1016 to 1230 mg kg −1 , respectively.
In addition, from the results shown in Table 1, it can be observed that the total contents of Mn in the RM were, in general, significantly different from those obtained in the GM samples (p < 0.05), except for the total contents found in the RM 1 and GM 2. It is important to note that there were also significant differences for the total Mn contents found among the roasted mate samples as well as among the GM samples.The average total concentrations of Mn in the GM (1625 mg kg −1 ) were approximately 20.5% higher than those determined in the RM samples (1350 mg kg −1 ).On the other hand, in relation to the average total soluble contents of Mn of the RM and GM samples leached to their hot infusions, it can be observed that these contents in the infusions of GM (1149 mg kg −1 ) were consistently higher (approximately 144.0%) than those determined for the infusions of the RM (471 mg kg −1 ).
These results are in agreement with those found in previous studies.Rusinek-Prystupa et al. [40] analyzed six samples of RM and six of GM, and they reported that the average total concentrations determined for Mn were 1323.7 mg kg −1 and 1657.3 mg kg −1 in the RM bags and GM, respectively.Milani et al. [41] also analyzed samples of RM, and the average total Mn content determined for nine samples was 1405 mg kg −1 , and 22% of this average total concentration was extracted to the infusions.Pozebon et al. [22] analyzed 54 samples of GM produced in Argentina, Brazil, Paraguay, and Uruguay and reported that the contents of Mn ranged from 730 to 1445 mg kg −1 on average.Ulbrich et al. [42] have also determined total contents of Mn in 35 samples of GM, and it was the element that showed the highest contents (523-2475 mg kg −1 ) among all of the micronutrients assessed in those samples.In another study developed by Proch et al. [26], the Mn content determined by ICP OES in Brazilian green mate samples was 1518 mg kg −1  It is worth mentioning that the industrial process of yerba mate is among the key factors responsible for the amounts of trace metals present in tea materials.Giulian et al. [44] evaluated the relationship between the chemical composition of mate and the process required to tea production.The samples were analyzed considering three situations: harvest, roasting, and bleaching (sapeco).It was noted that the levels of metals significantly differed among the aforementioned steps, with a difference of up to 56% in the Mn contents.Furthermore, an assessment of the effect of storage time before packaging and commercialization of mate demonstrated that the levels of K, Ca, Cl, and Zn decreased substantially over time whereas the opposite was observed for Mn, Al, and Si contents.
Additionaly, the yerba mate plant shows a strong tendency to absorb Mn and store it in its leaves.This process can be strengthened depending on the soil pH and strong acidic media (pH < 5.5) favors the absorption of metallic species by the roots [19,45].However, other factors may also impact the levels of trace elements in tea plants, such as leaf age, sunlight exposure, moisture, genetics, region of cultivation, use of fertilizer and pesticides containing Mn in their composition (dithiocarbamate fungicides), pollutants, and air pollution, among others [19,21,44,46].Therefore, these mentioned factors could account for the differences in total Mn contents found in this work for the RM and GM samples and their infusions (Table 1), as well as for the differences between the concentrations previously cited in this work for Mn, which were determined for other mate samples reported in the literature [22,26,[40][41][42].
Concerning the water-soluble contents of Mn in the RM and GM samples, it can be observed from the results shown in Table S1, that 26.0-43.8%and 69.8-71.7% of the total Mn contents in the dried RM and GM samples, respectively, were extrated to their hot infusions, and the extraction percentages of Mn for the roasted and green mate infusions were significantly different (p < 0.05).It can also be observed that the average aqueous-soluble content of Mn in GM samples (70.6%) was 2 times higher than those found in the RM samples (34.9%), and this result indicates that the consumption of GM infusions provides a greater intake of this ETE in comparison to the RM infusions.
In the literature, it is reported that Proch et al. [26] found that the solubility of Mn in hot infusions of GM samples from Argentine, Brazil and Paraguay and their infusions, varied over a range of approximately 19-21%.Ulrich et al. [42] found that the average soluble content of Mn in green mate samples was approximately 53%, a result identical or similar to those found by Pozebon et al. [22] and Pohl et al.  [47], which were 53 and 54%, respectively.Furthermore, Gezgin et al. [48], Barbosa et al. [49], Dalipi et al. [50], and Elzbieta et al. [51] found that water-soluble content for Mn in green mate samples was 9, 28, and 34% and 18, 9-50, and 2%, respectively.Additionaly, Długaszek and Kaszczuk [52], determined 277.76 mg kg −1 for RM samples.Based on these results reported in the literature [47][48][49][50][51][52] and on the results obtained in this study for the contents of soluble Mn found in GM and RM samples (Table 1), it can be observed that, in general, the contents of soluble Mn found in this present work were higher.Considering that there is no standard procedure for preparing hot infusions of mate, the differences between the contents of soluble Mn found in this work and reported in the literature, may be due to the different conditions used for preparing infusions.Such conditions may envolve volume of water, mass and grinding degree of the sample, amounts of twigs and leaves and their ratios, temperature and infusion time [20,28,45].At this point, it is important to highlight that to the best of our knowledge, this is the first work to evaluate and compare the total contents of Mn in the GM and RM samples and their hot infusions.
The accuracy of the procedures used to determine the total Mn contents in the acid digested solutions of the RM and GM samples and their infusions was evaluated by performing addition-recovery experiments (Table 2).It is worth saying that the AOAC (The American Association of Official Agricultural Chemists) Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals [53] considers the use of recovery from spiked samples as an appropriate procedure for validating the accuracy of the results obtained for dietary supplements and botanicals, according to the following recommendation: minimum of nine determinations (low concentragion range × 3 replicates, medium concentragem range × 3 replicates, and high concentration range of 3 replicates).This recommendation was adopted in this study by adding three different levels of standard Mn concentrations (0.5, 1.0, and 2.0 mg L −1 ) in the acid digested solutions of mate samples and their infusions, before preparing them.According to the recovery results shown in Table 4, it can be observed that the recoveries obtained for the all the mate infusions ranged from 91.2 to 108.5%, except for the RM infusions 2 and 3 with a lower level of added concentration (0.5 mg L −1 Mn), whose recoveries were greater than 110% (111.0%) and less than 90% (86.6%), respectively.Recoveries obtained for the acid digested solutions of the samples of RM and GM 1 ranged from 90.7 to 92.8%.According to the AOAC [53], the acceptable recoveries for the range of concentrations used in this work for being added in the acid digested solutions of the mate samples and their infusions vary from 75 to 120%.Consequently, it can be considered that all recovery results (86.6-111.0%)obtained in this work can be considered adequate.Concerning the recoveries obtained for the acid digested mate samples, the addition-recovery experiments were only performed for the samples RM 1 and GM 1, considering that all the acid digested samples of RM and GM (1, 2, and 3) should have similar residual matrices.This could be due to the minimization of their organic matrix contents promoted by the application of the acid digestion procedure.On the other hand, the mate infusions were analyzed directly without submitting them the acid digestion procedure, and consequently, the additionrecovery experiments were performed for all of them, in order to evaluate the existence of matrix effects on the determination of their total contents of Mn.Moreover, all the relative standard deviation (RSD%) were lower than 5%, indicating that the precision of the Mn determinations was also good or adequate.
The analytical characteristics obtained from the calibration curves obtained for the determination of Mn in the acid digested samples of GM and RM and their infusions by F AAS, are presented in Table 3.
It is relevant to mention the values of adequate intake (AI) and maximum tolerable intake (IU) of Mn reported in the literature in order to make a preliminary assessment of the potential contribution that the GM and RM infusions prepared in this study could have on these intake values.However, additional studies should be conducted to better assess in this regard.The AIs established according to the life-stage of males are: 1.9 mg/day (9-13 years), 2.2 mg/ day (14-18 years), and 2.3 mg/day (19 years and older) and for the life-stage of females are: 1.6 mg/day (9-18 years) and 1.8 mg/day (19 years and older).In addition, the UIs stablished according to the life-stage of males and females are: 6 mg/day (9-13 years), 9 mg/day (14-18 years), and 11 mg/day (19 years and older) [54].
In this context, the assessment of the possible contribution of the prepared hot infusions of the RM and GM to the recommended values of AI for Mn was carried out considering the average masses of Mn found in the mate infusions as well as the recommended AI values established by the literature [54] (Table 4).
According to Table 4, it could be observed that the hot infusions of RM could potentially contribute on average with 39 and 31% in relation to the value established in the literature for the adequate daily intake of Mn for adult (19 years or older) females and males, respectively.In addition, it was also observed that the hot infusions of GM can potentially contribute with 96 and 75% for adult females and males, respectively.The hot infusions of GM can contribute with 57 and 44% more than hot infusions of RM.It is also observed that the hot infusions of GM and RM can contribute differently to the adequate daily intake, considering that the Mn extraction percentages to these infusions (Table S1) were significantly different (p < 0.05).The average masses of soluble Mn in the hot infusions of GM and RM were always below the value of UI established for Mn, which is 11 mg/ day for adult females and males [54].
Based on the results above, it could be suggested that the hot infusions of RM and GM can be good dietary sources of Mn, with the GM infusion being a richer dietary source of this ETE.It must be emphasized that these results regarding the percentage contribution potential of the GM and RM infusions in relation to the value established in the literature for the adequate daily intake of Mn for adult females and males are not conclusive.However, these preliminary results obtained so far can be considered important because they suggest, mainly in the case of the GM infusions, that this type of infusion, depending on the ingested volume, can promote a Mn intake higher than that recommended as adequate.Therefore, emphasis should be placed on the need for further research to evaluate the bioaccessibility and bioavailability [30] of Mn from the GM and RM infusions, which are dependent on the physicochemical forms of this element.Thus, it could be possible to know more on the actual contribution of these infusions for the average adequate daily intake for this element.
Additionaly, it is also opportune to mention some additional information reported in the literature on the absorption of Mn through its ingestion by humans.For example, Erdemir et al. [55] evaluated the bioaccessibility of Mn in different tea samples after an in vitro enzymatic digestion and found bioaccessible Mn levels ranging from 70 to 80%, 66 to 67%, and 73 to 84% in black, earl gray and green tea infusions, respectively.Milani et al. [40] assessed the bioaccessibility of trace elements in some ready-to-drink ice teas and found that most of the bioaccessible fractions of Al, Sr, Mn, and Zn were 50% of their total content.On the other hand, Alnaimat et al. [56] stated that Mn presented a low dialyzability in the tea infusions, suggesting that Mn has low bioavailability in the human organism probably because of the poor solubility of this inorganic ion in the gastrointestinal digestion process.It should be stressed that absorption of Mn is relatively low, and for adult humans, dietary Mn ranges between 1 and 5% and is dependent on the amount and form of Mn as weel as on other dietary components [57].Therefore, this may indicate that yerba mate infusions, as well as other foods that have total Mn levels greater than those recommended for an adequate daily intake, may not be toxic to adult humans.In the case of neonates, infants, and children, the recommended daily intake of Mn can be much higher than for adults, based on the greater demand for this element due to a less developed regulatory system in the early stages of development [58].Thus, toxic effects of Mn may be more important to occur in newborn infants, considering the higher absorption of Mn in neonates [59].

Polyphenolic Compounds and Melanoidins
It was carried out the evaluation of the TP contents and SM`s presence in the hot infusions of RM and GM (Table 5), which are known as complex forming compounds with metal ions.Concerning the TP contents, it could be observed that these contents in the hot infusions of GM were significantly different (p < 0.05) and consistently higher (2.2 to 3.5 times) when compared to those found in the RM infusions.The lower polyphenol contents found in the hot infusions of RM may be due to the losses of thermolabile compounds such as polyphenols, which can occur during the additional roasting step used in its industrial process [1,60].Melanoidins are dark-colored polymeric compounds with high molecular weight, which are formed in the final steps of the Maillard reaction (MR), and they are associated with color, taste, and aroma in thermally processed foods, such as bakery products, cocoa, teas, wine, and coffee.The MR products also have a strong bioactive activity related to antioxidant, antimicrobial, antihypertensive, and anti-inflammatory activities [61][62][63].
Melanoidins' structure is not completely elucidated; however, it is well established that they are nitrogen-containing polymers formed by the reaction between reducing sugars with proteins or amino acids.It is important to notice that, during the roasting step, phenolic compounds can be incorporated into the high molecular weight melanoidins (HMWM) skeletons, and these phenolics are predominantly chlorogenic acids (CGA) [61,64].Smrke et al. [65] reported a negative correlation between the content of CGA and the roasting time of coffee beans, and the CGA contents in the samples decreased during the occurrence of the roasting process.According to Coelho et al. [66], the incorporation of phenolic acids into HMWM is an important pathway of the degradation of CGAs during the roasting process.The MR products have a structure that is hydrophilic, anionic, and able to form bonds with positively charged substances with low molecular weight, and consequently, they can form stable complexes with cationic species such as Mn 2+ [62,63].According to Welna et al. [28] in a chemical fractionation study of Mn and other elements in brewed grounds and soluble coffees by tandem solid phase extraction with reversephase and strong cation-exchange extraction tubes, it was found that the Mn was predominantly in the form of simple ionic species or stable cationic complexes.
According to the Table 5, it can also be observed that the absorbance measurements at 420 nm for the SM indicated that the presence of them in the RM infusions was 2.1 to 4.5 times higher than those observed in the GM infusions.Thus, this result is in opposition to that observed for TP in the hot infusions of RM and GM, in which the TP contents were higher in the GM infusions.In addition, the higher presence of SMs in the RM infusions may be explained due to the use of phenolic compounds for their formation by the occurrence of the MR during the roasting process [64,65].
Considering that the TP and SM can form complexes with inorganic ions, it was evaluated the existence of correlation between the TP contents and SM with the soluble Mn contents in the hot infusions of RM and GM.In addition, it is important to point out that our research group was the first to assess the existence of correlation between the SM contents and the soluble metals contents in mate infusions [6], in a study that evaluated the fractionation of a potential toxic element, aluminum (Al), in GM and RM and their infusions.In that work, it was found that the SM contents together with the TP contents had influence on the Al extracted to the RM infusions.From the results shown in Table 1 and Table 4, as well as based on their relationships established by the Pearson correlation coefficients, it was possible to observe that high correlations were obtained between the TP contents and soluble Mn in the hot infusions of both RM and GM.Furthermore, this correlation was direct for the RM infusions (r = 0.99) and inverse for the GM infusions (r = − 0.87).It is worth saying that positive Pearson's correlation coefficients (direct correlations) indicate that the higher the contents of TP and SM; the higher are the contents of soluble Mn.On the other hand, negative Pearson's coefficient correlations (inverse correlations) indicate that the higher the contents TP and SM, the lower are the contents of soluble Mn.Therefore, higher TP contents found in RM infusions (Table 5) had influence over the higher soluble Mn contents in this type of infusion (Table 1), but the same correlation was not observed for the GM infusions, because the higher the TP contents (Table 5), the lower was the soluble Mn contents (Table 1).The inverse correlations between the TP contents and the soluble Mn contents in the GM infusions as found in this work are in agreement with the results found by Wróbel et al. [67] who found that the lower the tannin content in GM samples, the higher the soluble contents of Mn and other metals (Al, Cu, and Fe).
The highly positive correlation (r = 0.99) observed for hot infusions of RM may indicate that the soluble polyphenols in the RM infusions probably contribute to the extraction of the Mn from the RM samples to their hot infusions, in the forms of aqueous-soluble polyphenol-Mn complexes.Differently, the high negative correlation (r = − 0.87) found for the GM infusions may suggest that probably the content of Mn in this type of infusion interacts more with non-soluble polyphenols than with soluble polyphenols.Despite this, the consistently higher soluble TP contents in the hot infusions of the GM in comparison to those found in the RM infusions (Table 5), may account for the higher soluble Mn contents found in the hot infusions of the GM.Concerning the relationship between the SM's presence and soluble Mn contents in the hot infusions of RM and GM, a small negative correlation (r = -0.43)was observed for the RM infusions and a small positive correlation (r = 0.50) was observed for the GM infusions.Consequently, based on these correlations, it may be suggested that the SM present in the RM and GM samples has a minor influence on the extractability of the Mn from these mate samples to their hot infusions.In another study developed by Ribeiro et al. [38], the correlation between the solubility of some potential toxic elements (PTEs) with the SM contents in GM and RM infusions was evaluated.As a result, it was observed that there were direct correlations (high to very high) between the soluble Cd, Cr, and Pb contents with the SM contents in GM infusions.Therefore, the results found by Ribeiro et al. [38] for GM infusions indicated the SMs had a strong influence on the extractability of Cd, Cr, and Cu from these mate samples to their infusions, differently from their weak influence on the extractability of Mn in both the infusions of GM and RM, as found in this study.
The molecular weight of MR products increases as the browning reaction proceeds, resulting in less soluble compounds such as the HMWM.Consequently, the Mn bound to HMWM may have its solubility decreased in the hot infusions of the RM depending on the time and temperature heating used in the roasting process [62].Therefore, the probable presence of the insoluble HMWM in the RM samples may have contributed for the lower contents of soluble Mn found in the RM infusions (Table 1).Pohl et al. [14] observed in a physical fractionation study of Mn in ground coffee infusions that 61-68% of the soluble Mn was found associated to low molecular weight ligands (< 5 kDa), 8-38% to high molecular weight ligands (5-10 kDa) such as melanoidins and proteins, and a very small part related to moderate and high molecular weight ligands .
It is important to highlight that the soluble Mn contents that showed a positive (direct) correlation with the TP contents in the hot infusions of RM analyzed in this work, showed a negative (inverse) correlation with the TP contents in the GM infusions.Concerning the relationship between soluble Mn and SM in the hot infusions of the RM and GM, the soluble Mn showed a negative (inverse) correlation with SM in the RM and showed a positive (direct) correlation with the SM in the GM infusions.It is worth mentioning that there is room for further research to investigate the correlations of soluble forms of Mn with the TP contents and SM in hot infusions of RM and GM.

Solid Phase Chemical Fractionation
A SPCF study based on a batch adsorption procedure using the chelating resin Chelex 100 (NH 4 + form) was performed for evaluating the relative lability of the soluble Mn found in filtered fresh infusions of the RM and GM samples.In this chemical fractionation study, it was considered that relatively labile species of Mn in their free or less strongly complexed forms were adsorbed by the adsorption sites of chelating resin, whereas the relatively inert or strongly complexed species remained in the infusions of GM and RM.
Firstly, the amount of the Chelex 100 resin sufficient to promote complete adsorption of a Mn 2+ concentration higher than those found in the hot infusions of the RM (17.0 mg L −1 ) and GM (36.6 mg L −1 ) and also higher than those reported in the literature for mate infusions [23,[40][41][42][43] was evaluated.The use of the resin in the NH 4 + form instead of the protonated (H + ) form allowed keeping the pH of the infusions submitted to each of the adsorption experiments constant.This was possible because during the ion exchange process that occurs during the adsorption experiment, the NH 4 + ions (weak conjugate acid) released from the surface of the resin for the aqueous infusions are not capable of significantly altering their pH.Changing the pH of the infusions obtained after their preparation can alter the chemical forms of the soluble Mn present in them, which may cause changes in their labilities, making it impossible to carry out a chemical fractionation study to evaluate the relative lability of the Mn present in the GM and RM infusions as originally obtained and prepared.Preliminary experiments to compare the effect of using the chelating resin Chelex 100 in both forms, NH 4 + and H + , on the change in pH of the infusions submitted to the adsorption experiments were carried out, and the change in their pH was only observed by using the protonated form of the resin.
The suitable amount of the resin was determined by keeping a 40.0 mL of a 50.0 mg L −−1 Mn standard solution with pH adjusted to pH 5.80 with a 0.30 mol L −1 sodium acetateacetic acid buffer solution, in contact with different masses of Chelex 100 for 24 h [6].It was necessary to adjust the pH value of the Mn standard solutions to 5.80 for keeping it within the range of pH values found for the infusions of the RM and GM, which ranged from 5.71 to 5.97.
From the results shown in Fig. 1, it can be observed that a 0.45 g mass of the chelating resin was sufficient to promote the complete adsorption of the total Mn 2+ concentration present in the standard solution.
After that, batch adsorption experiments using different adsorption times and the adequate mass of the resin Chelex 100 (NH 4 + form) were performed to evaluate the rate of removal of Mn from the hot infusions of the RM and GM, so that the relative lability of the soluble forms of Mn in these infusions could be assessed.Based on the results obtained by performing the batch adsorption experiments for the infusions of the RM 1, 2, and 3 (Fig. 2), it was observed that the adsorption equilibrium of Mn from these infusions on the Chelex 100 resin was reached after 5 min.From this adsorption equilibrium time, the average Mn removals for the infusions of the RM ranged from 98.4 ± 0.8 to 99.4 ± 0.2%, which were significantly different (p < 0.05) from the average Mn removal percentages obtained in the first evaluated adsorption time (2 min).These results indicated that the total soluble Mn content from the RM infusions, essentially corresponded to relatively labile (noninert) chemical forms.Such noninert chemical forms of Mn in the RM infusions may be related to aquocomplexes of Mn ions (free Mn 2+ ) and other Mn complexes with low relative stability formed with other ligands including polyphenols, melanoidins, among others from the matrix of the RM, which are able to be dissociated by interaction with the active sites on the resin surface.Retained Mn (%) In relation to the results obtained for the batch adsorption experiments applied to the infusions of the GM samples (Fig. 3), it could be observed that the adsorption equilibrium of the Mn from the GM infusions 2 and 3 was reached after 5 min, and the average Mn removal percentages ranged from 99.0 ± 0.2 to 99.7 ± 0.1%.These Mn removal percentages were significantly different (p < 0.05) from those obtained for these same GM infusions in the adsorption time of 2 min (98.1 ± 0.1 to 98.3 ± 0.1%).On the other hand, the adsorption equilibrium occurred faster (2 min) for the soluble Mn from the GM infusion 1 and from this equilibrium time, the observed removal ranged from 99.8 ± 0.3 to 100 ± 0.1%.Consequently, the results obtained from these adsorption experiments showed that the adsorption behavior for the Mn from the infusions of the RM and GM was similar, indicating that the soluble forms of Mn in these infusions were essentially related to the relatively labile or noninert soluble chemical forms, suggesting that these soluble Mn species have potential to be bioavailable.These results are in disagreement with the relative lability found for Al in RM infusions according to what was found in a previous study carried by our research group [6], by using SPE with the same type of resin.In that work, differently than was found for Mn in the present study, soluble Al in RM infusions was found predominantly in relative inert forms (50-70%).Moreover, based on the average Mn removal percentages found for the infusions of the RM and GM, it could also be observed that the removal percentages obtained for the GM infusion 1 showed a tendency to be higher than those obtained for all other infusions, although they were not significantly different from each other (p < 0.05).These results may suggest that the Mn soluble forms from the GM infusion 1 seem to be slightly more labile or noninert than those found in all other infusions analyzed, due to their shorter adsorption equilibrium time.Differences among the contents of the relatively labile Mn soluble forms in the hot infusions of the RM and GM could be due not only to the different heating degrees of their industrial processing, but also due to the different growing and harvesting conditions of the mate plants.It is important to mention that there are no reports in the literature that evaluated and/or compared the relative lability of the Mn in the of RM or GM infusions.Additional studies should be conducted in this regard to better understand the influence of polyphenols, melanoidins, and other ligands on the chemical forms of Mn by conducting additional studies on lability as well as on the bioavailability and bioaccessibility of this ETE in mate infusions.

Conclusions
From the solid phase chemical fractionation study, it was possible to observe that the soluble forms of the essential trace element Mn in the roasted and green mate infusions were essentially associated to relatively labile or noninert chemical forms.Therefore, it could be suggested that the almost all (98.4-99.7%) of the soluble Mn content from these mate infusions may be related to chemical forms potentially bioavailable.In addition, the soluble Mn contents in the hot green mate infusions were consistently higher than those contents found in the hot infusions of roasted mate.Furthermore, it could also be observed that the soluble Mn contents in the hot infusions of the roasted and green mate were highly correlated with their soluble polyphenol contents.However, although these correlations have been high for both the types of mate infusions, they were positive or direct (r = 0.99) for the roasted mate infusions and negative or inverse (r = −0.87)for the green mate infusions.On the other hand, the soluble Mn contents in both the hot infusions of roasted and green mate were weakly correlated with the soluble melanoidins' presence.Besides, the prepared hot infusions of green mate can contribute with 56.7 and Retained Mn (%) 44.4% more than the roasted mate infusions to the adequate Mn intake recommended for adult females and males [54].Consequently, the green mate infusion can be considered as a better dietary source of Mn in comparison to the roasted mate infusions.It should also be emphasized that this work is the first to evaluate and compare both the relative lability and solubility of Mn in the hot infusions of roasted and green mate infusions.Moreover, there is still room for further research to better understand the lability and the solubility of this and other essential trace elements in these two types of mate infusions.

Fig. 1 Fig. 2
Fig. 1 Effect of different masses of the chelating resin Chelex 100 (NH 4 + form) on the retention of Mn using a batch adsorption procedure performed under the following conditions: 40.0 mL of a 50.0 mg L −1 Mn standard solution (pH 5.80)

Fig. 3
Fig. 3 Effect of different adsorption times on the soluble Mn removal from the infusions of the green mate using a batch adsorption procedure with Chelex 100 resin (NH 4 + form).Conditions: 0.45 g of resin; 40.0 mL of the filtered fresh infusion of green mate

Table 1
Total mass fractions of Mn in the acid digested solutions of roasted and green mate samples and in their hot nfusions determined by F AAS (mean ± standard deviation, n = 4) e 1, 2, and 3 correspond to different brands of commercial samples of roasted and green mate f infusions prepared by the mixture of minced and sieved mate samples with hot ultrapure water (80 °C for 3 min); different superscript letters (a, b, c, and d) presented in the same column indicate significant difference at 95% by the Tukey's test (p < 0.05); RM and GM are the abbreviations used for roasted mate and green mate, respectively.

Table 2
Recovery (%) (RSD*) for the Mn determinations in the acid digested solutions of the samples of roasted and green mate and their infusions by F AAS (n = 3)

Table 4
Average masses of Mn in the hot infusions of roasted and green mate (mean ± standard deviation, n = 3), and their potential contribution to the recommended adequate intake for adult females and males [54]fusions prepared by the mixture of minced and sieved mate samples with 40.0 mL of hot ultrapure water (80 °C for 3 min).bAdequateintake of Mn stablished for adult (19 years and older) females (1.8 mg Mn/day) and males (2.3 mg Mn/day)[54]RM and GM are the abbreviations used for roasted mate and green mate, respectively; different supercript letters (a, b, c, and d) presented in the same column indicate signicant difference at 95% by the Tukey's test (p < 0.05).

Table 5
Total polyphenols contents and the detection of the presence of soluble melanoidins in the hot infusions of roasted and green mate (mean ± standard deviation, n = 4)