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Catalysis Research

(ISSN 2771-490X)

Catalysis Research is an international peer-reviewed Open Access journal published quarterly online by LIDSEN Publishing Inc. This periodical is devoted to publishing high-quality papers that describe the most significant and cutting-edge research in all areas of catalysts and catalyzed reactions. Its aim is to provide timely, authoritative introductions to current thinking, developments and research in carefully selected topics.

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Current Issue: 2026  Archive: 2025 2024 2023 2022 2021
Open Access Original Research

Stabilization Strategies of β-glucosidases Produced by Yeasts Rhodotorula oryzicola and Saccharomyces cerevisiae

Geise Camila Ribeiro 1,†, Pedro Fernandes 2,3,4,†, Floriatan Santos Costa 5,†, Sandra Aparecida de Assis 1,†,*

  1. Enzymology and Fermentation Technology Laboratory, Health Department, State University of Feira de Santana, Av. Transnordestina s/n, Novo Horizonte, 44036-900, Feira de Santana, Bahia, Brazil

  2. iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

  3. Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

  4. Lusófona University, BIORG—Bioengineering and Sustainability Research Group, Av. Campo Grande 376, Lisbon, 1749-024, Portugal

  5. Department of Chemistry, Federal University of Paraná, Curitiba, Paraná, Brazil

† These authors contributed equally to this work.

* Correspondence: Sandra Aparecida de Assis

Academic Editor: Narendra Kumar

Special Issue: Immobilized Biocatalysts: Recent Developments and Applications

Received: December 14, 2025 | Accepted: August 09, 2026 | Published: August 21, 2026

Catalysis Research 2026, Volume 6, Issue 3, doi:10.21926/cr.2603010

Recommended citation: Ribeiro GC, Fernandes P, Costa FS, de Assis SA. Stabilization Strategies of β-glucosidases Produced by Yeasts Rhodotorula oryzicola and Saccharomyces cerevisiae. Catalysis Research 2026; 6(3): 010; doi:10.21926/cr.2603010.

© 2026 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

Abstract

The rising demand for enzymes with improved properties has driven continuous developments in enzyme technology, including enzymatic formulation. Enzyme formulation primarily aims to minimize losses in enzymatic activity during transport, storage and use. One strategy to mitigate enzyme inactivation is to include stabilizing agents or metal ions that preserve enzyme structure and activity. Thus, in this work, we evaluated stabilization strategies for β-glucosidase produced by the yeasts Rhodotorula oryzicola and Saccharomyces cerevisiae using various chemical additives. The most effective additives were then applied to obtain a formulation with improved enzymatic activity. KCl, ZnSO4, EDTA, SDS, Triton X-100, Tween 20 and Tween 80 were tested as potential stabilizers for β-glucosidase. The three best stabilizers for each enzymatic sample were used in a simplex-centroid mixture design to evaluate possible synergistic effects and obtain a stable enzyme formulation. The formulations that yielded the highest enzymatic activity contained 2.5 mM of ZnSO4 and 0.375% of Tween 20 for β-glucosidase from R. oryzicola, and 0.5 mM of ZnSO4 and 2.5 mM of EDTA for the enzyme from S. cerevisiae, showing relative activities of 313.60% and 302.11%, respectively. The results obtained in our study show that both yeasts produce β-glucosidases with good tolerance to chemical additives, and that some of these additives can be used to obtain more stable enzymatic formulations with enhanced catalytic efficiency.

Graphical abstract

Click to view original image

Keywords

Chemical additives; cellulases; enzymatic formulation; enzymatic hydrolysis; optimization

1. Introduction

The emergence of new applications, alongside conventional ones that face increasing performance requirements and growing market competitiveness, has created a strong demand for enzymes with improved properties, driving continuous advances in enzymatic formulation technologies influenced by developments in biotechnology and bioinformatics [1,2].

The development of enzymatic formulations aims to preserve proper enzyme folding (the active, functional structure of the protein), thus improving catalytic efficiency under industrial conditions. Several protocols to stabilize enzymes have been developed, enabling easier handling and providing greater product versatility on the market. These advances allowed enzymes to better adapt to processing conditions and environmental requirements, resulting in significant improvements in catalytic efficiency and facilitating their broader application in industrial processes [3,4].

The main objective of an enzymatic formulation is to minimize the loss of enzymatic activity during transport, storage, and use. Formulations for enzymatic stabilization increase stability by counteracting primary deactivation mechanisms such as denaturation, catalytic site deactivation and proteolysis. The most common strategies to prevent primary deactivation include maintaining adequate levels of essential cofactors, incorporating reversible inhibitors, eliminating reactive or oxidizing species from the formulation, and immobilizing enzymes. Additionally, the inclusion of chemical stabilizers can help preserve the three-dimensional structure of the enzyme, an approach often referred to as medium engineering [1,2,5].

By definition, enzymes bind to substrates and catalyze reactions that occur confined to an active site, also called a catalytic site [6]. Many researchers have studied how cellulases bind to their substrates. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), cellulases are classified as glycosidases (EC 3.2.1), which are enzymes that hydrolyze glycosidic bonds involving O- or S-glycosyl groups. All cellulases target the β-1,4 glycosidic bonds of the cellulose polymer, catalyzing their cleavage through either exo- or endo-acting mechanisms [7,8]. Cellulases typically hydrolyze substrates through two primary acid–base mechanisms, the retaining and inverting mechanisms, which differ in whether the anomeric configuration of the substrate is preserved or inverted during hydrolysis.

Some enzymes perform their function without the need for other chemical compounds. However, others require an additional component to enable or improve substrate binding and/or maintain structural stability under reaction conditions. These components may bind to the enzyme at an allosteric site, often near the catalytic site, thus inducing a conformational change. This conformational modification in the catalytic site can either activate or inhibit the formation of the enzyme-substrate complex and consequently affect catalysis [9]. Thus, it is essential to identify potential activators (stabilizers) and inhibitors of an enzyme of industrial interest, such as cellulases.

The effects of ion salts, chelating agents, and surfactants on cellulase activity have been studied to identify the best additives for improving the catalytic efficiency of these enzymes, as well as to predict which potential inhibitors may impair the reaction yield. However, the effect of these additives on enzymes varies, and empirical research is required to determine how each additive influences the activity of the enzyme produced by the microorganism under study [10,11,12]. Since β-glucosidase activity is a limiting factor in the enzymatic hydrolysis of lignocellulosic materials, any additive used in the enzymatic reaction should not inhibit this enzyme [13,14].

Thus, the present work aimed to evaluate stabilization strategies for β-glucosidase produced by yeasts Rhodotorula oryzicola and Saccharomyces cerevisiae using metal ions, chelating agents, and anionic and nonionic surfactants as additives. The chelating agent and surfactants used were selected based on previously published studies on this topic. The metal ion salts (KCl and ZnSO4) were selected based on preliminary results obtained in the present work.

2. Materials and Methods

2.1 Materials and Reagents

2.1.1 Reagents

The YM Agar medium was obtained from Acumedia® (Indaiatuba, São Paulo, Brazil). Bovine serum albumin, p-nitrophenyl-β-D-glucopyranoside (pNPβG) and 3,5-dinitrosalicylic acid were obtained from Sigma-Aldrich® (St. Louis, Missouri, United States). Coomassie Brilliant Blue was obtained from Merck® (Darmstadt, Germany). Acetone p.a. was obtained from Synth® (Diadema, São Paulo, Brazil).

2.1.2 Microorganism

Samples of Rhodotorula oryzicola and Saccharomyces cerevisiae were obtained from the Culture Collection of Microorganisms of Bahia (CCMB) of the State University of Feira de Santana (UEFS, Brazil). Strain identification was performed by isolating genomic DNA and amplifying the D1/D2 domain of the 26S rRNA gene using polymerase chain reaction [15].

The cultures were maintained in plates containing YM Agar medium (Acumedia®) and stored in a refrigerator (4-8°C) until use.

2.1.3 Yeast Growth

Yeast cultures were streaked on YM agar medium (Acumedia®) and incubated in a B.O.D. ABC-LAB incubator (São Bernardo do Campo, São Paulo, Brazil) at 28°C. Activation was performed through three consecutive transfers, with an interval of 24 h between each.

2.1.4 Obtaining β-Glucosidase by Submerged Fermentation of Saccharomyces cerevisiae and R. oryzicola

The stock culture was maintained on YM agar plates at 4°C until use. S. cerevisiae CCMB 520 and R. oryzicola were grown on YM Agar (Acumedia®, Indaiatuba, São Paulo, Brazil) previously sterilized in a vertical autoclave (AVM Marte Científica, São Paulo, Brazil) at 121°C for 15 min. The yeast was activated on YM agar through three consecutive subcultures, incubated at 28°C for 24 h (BOD Incubator ABC-LAB, São Caetano do Sul, São Paulo, Brazil).

Yeast cells were pre-inoculated in sterile saline solution (0.45% w/v NaCl) that had been autoclaved at 121°C for 15 min. A 10% (v v-1) aliquot of the resulting pre-inoculum (108 CFU mL-1) was then transferred to the appropriate liquid induction medium. For R. oryzicola, YP broth was used, supplemented with 1% (w v-1) yeast extract, 2% (w v-1) peptone, and 2% (w v-1) cellobiose as the inducer [16]. For S. cerevisiae, the induction medium consisted of YPD broth containing 1% (w v-1) yeast extract, 2% (w v-1) peptone, and 2% (w v-1) D-glucose as the inducer [17].

The enzyme-rich supernatant was collected by centrifugation (14,000 × g, 15 min at 4°C) using a Hettich® Universal 320R centrifuge (Tuttlingen, Germany). The supernatant was filtered through filter paper (Whatman n. 1) and stored at 0°C until use. This cell-free supernatant was used as a crude extract with no further purification; therefore, it contained β-glucosidase and likely also other secreted proteins and residual medium components. Accordingly, the observed stabilization effects correspond to the crude enzyme preparation.

2.1.5 Enzymatic Assays

β-glucosidase activity in the supernatant was determined using a chromogenic substrate, 4-nitrophenyl-β-D-glucopyranoside (ρNPβG), in a reaction mixture containing 1 mL of 4 mM ρNPβG solution in 0.1 M citrate-phosphate buffer pH 5.0 and 0.25 mL of enzyme sample incubated at 50°C for 20 min. The reaction was subsequently stopped by adding 1.25 mL of 100 mM sodium carbonate solution [18,19].

The enzymatic activity (U) was calculated using a ρ-nitrophenol calibration curve (Sigma-Aldrich®). One β-glucosidase activity unit was defined as the amount of enzyme required to release 1 µmol of ρ-nitrophenol per minute under the reaction conditions. The released ρ-nitrophenol was measured at 420 nm using a UV/Vis spectrophotometer (Cary 50 UV-Visible spectrophotometer, Varian Inc., São Paulo, São Paulo, Brazil).

Total protein content (mg) was determined by the Bradford method using a bovine serum albumin (Sigma-Aldrich®) calibration curve in a concentration range of 0 to 100 µg/mL [20]. The specific activity (U/mg) was calculated as the ratio of β-glucosidase activity to total protein concentration. All experiments were performed in triplicate.

2.1.6 Stabilization of β-Glucosidase by Chemical Additives

For the stabilization of β-glucosidase, the salts KCl and ZnSO4, the chelating agent EDTA, and SDS (sodium dodecyl sulfate) were used at concentrations ranging from 1 to 20 mM. The surfactants Triton X-100, Tween 20 and Tween 80 were tested at concentrations ranging from 0.10-1.0% (v v-1). All additives were solubilized in 0.1 M phosphate buffer, pH 6 [21,22,23]. The enzyme was mixed with the respective buffers containing the additives at different concentrations and incubated at 25°C for 24 h in a shaking incubator at 100 rpm. Enzymatic activity was expressed as relative activity (%) using the β-glucosidase activity of the enzyme without stabilizing treatment as the control [24].

2.1.7 Optimization of Enzyme Stabilization

The three most effective stabilizers previously identified for each enzymatic sample were selected to evaluate their combined ability to retain β-glucosidase activity, including potential synergistic effects. A simplex-centroid mixture design was used to assess the effect of the additives on retention of enzymatic activity, by varying the volume (mL) of each additive in the assays. Each enzymatic extract was diluted 1:4 (extract:water), and in all experiments, 4 mL of water, 1 mL of enzymatic extract, and a total of 1 mL of additives were used. The proportion of each additive within the mixture varied from 0 to 1 mL (central point, 0.33 mL), according to the experimental design. The study was conducted for 72 hours at 25°C, providing a measure of short-term formulation stability under the tested conditions.

2.1.8 Statistical Analysis

The results obtained were statistically evaluated using analysis of variance (ANOVA) and the means were compared using the F-test (p < 0.05) with Statistica software, version 7.0.

3. Results

3.1 Stabilization of β-Glucosidase

The yeasts were cultivated by submerged fermentation using cellobiose (R. oryzicola) and glucose (S. cerevisiae) as inducers, as described in previous publications [16,17]. The crude, non-purified β-glucosidase preparations showed initial enzymatic activity of 2.17 ± 0.04 U for R. oryzicola (BGL oryzicola) and 1.41 ± 0.03 U for S. cerevisiae (BGL cerevisae). Table 1 presents the relative activity (%) of both enzymes after 24 h of treatment with different additives.

Table 1 Relative activity of β-glucosidase from R. oryzicola (BGL oryzicola) and S. cerevisiae (BGL cerevisiae) in the presence of various chemical additives.

BGL oryzicola displayed high tolerance to KCl at all tested concentrations, showing an increase in enzymatic activity of up to 202.39% at 1 mM. Moreover, activity remained high (177-190%) across the remaining concentration range [24]. In the presence of ZnSO4 and SDS, enzymatic activity increased at low concentrations (1-5 mM) but decreased as the concentration of these additives increased. EDTA increased enzymatic activity only at the lowest concentration, 1 mM.

In contrast, BGL cerevisae displayed improved activity in the presence of 1 mM ZnSO4 (108.16%), 5 mM EDTA (116.02%), and 0.25% Tween 20 (127.61%). All concentrations of KCl, SDS and Triton X-100 inhibited enzymatic activity, reducing it by more than 40% relative to the control.

The synergistic effects of the most successful additives for BGL oryzicola, KCl, ZnSO4 and Tween 20, were further assessed using a simplex-centroid mixture design. Different proportions of these additives, at initial concentrations of 1 mM (KCl), 5 mM (ZnSO4), and 0.75% (Tween 20) were evaluated, and their effects on relative activity are shown in Table 2. The highest activity was obtained using the formulation containing 2.5 mM ZnSO4 and 0.375% Tween 20, which yielded a relative activity of 313.60 ± 9.59%.

Table 2 Simplex-centroid mixture design and enzymatic activity obtained for BGL oryzicola.

Figure 1 shows the Pareto chart summarizing the linear (L) and quadratic (Q) effects of the studied variables on enzyme activity after 72 h of treatment with additives. The corresponding surface plot retrieved from the cubic model is shown in Figure 2.

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Figure 1 Pareto plot showing the influence of linear (L) and quadratic (Q) effects on the optimization of BGL oryzicola activity, illustrating the interaction among the different additives in the enzyme formulation.

Click to view original image

Figure 2 Response surface plot for the optimization of BGL oryzicola activity showing the interaction among the different additives of the enzyme formulation.

The Pareto chart shows that the three selected additives influenced enzyme activity. The pronounced ZnSO4–Tween 20 interaction observed in the response surface (Figure 2) aligns with a mechanism suggested here by analogy with previous reports and not directly tested in this work, in which Zn2+ would enhance structural stability. At the same time, Tween 20 would reduce interfacial denaturation and improve substrate accessibility. The negative cubic term in Equation (1) indicates that excessive additive concentrations may disrupt enzyme conformation, highlighting the relevance of balanced proportions [25,26,27]. Table 3 shows the Analysis of Variance (ANOVA) for the studied model. The F test yielded a value of 647.87, which is higher than the tabulated F-value (9.01), with an R2 of 0.99 at a 95% confidence level, showing that the model fits the experimental data well.

Table 3 Analysis of variance for BGL oryzicola activity optimization with various enzyme formulation additives.

Equation 1 shows the correlation between the variables of the model under study, where A is the volume of 1 mM KCl, B is the volume of 5 mM ZnSO4, and C is the volume of 0.75% Tween 20.

\[ \begin{aligned} Relative\,\,Activity\,\,(\%) =&129.95\times A+154.59\times B+167.31\times C \\+&393.57\times A\times B+101.26\times A\times C+610.57\times B\times C \\-&3451.84\times A\times B\times C \end{aligned} \tag{1} \]

The synergistic effect of the most successful additives for BGL cerevisiae, ZnSO4, EDTA, and Tween 20, was further assessed using a simplex-centroid mixture design. Different proportions of these additives, with initial concentrations of 1 mM (ZnSO4), 5 mM (EDTA), and 0.25% (Tween 20), were tested, and the impact on relative activities shown in Table 4. The highest activity was obtained with the formulation containing 0.5 mM ZnSO4 and 2.5 mM EDTA, yielding 4.36 ± 0.17 U, roughly three times the initial enzymatic activity (1.41 ± 0.03 U), with a relative activity of 302.11 ± 3.67%.

Table 4 Simplex-centroid mixture design and enzymatic activity obtained for BGL cerevisae.

Figure 3 shows the Pareto chart and the influence of linear (L) and quadratic (Q) effects of the variables under study on enzyme activity after 72 h of treatment with additives. The surface plot of the cubic model under study is shown in Figure 4.

Click to view original image

Figure 3 Pareto plot and the influence of linear (L) and quadratic (Q) effects on the optimization of BGL cerevisae activity showing the interaction among the different additives of the enzyme formulation.

Click to view original image

Figure 4 Response surface plot for the optimization of BGL cerevisae activity showing the interaction among the different additives of the enzyme formulation.

Equation 2 shows the correlation between the variables of the model under study, where A is the volume of ZnSO4, B is the volume of EDTA, and C is the volume of Tween 20.

\[ \begin{aligned} Relative\,\,Activity\,\,(\%) =&253.36\times A+151.09\times B+102.85\times C \\\quad+&399.56\times A\times B-370.09\times A\times C+36.50\times B\times C \\\quad+&1755.47\times A\times B\times C \end{aligned} \tag{2} \]

The positive ZnSO4–EDTA interaction (Equation 2) suggests a complementary dual mechanism, again proposed by analogy with the literature, rather than confirmed experimentally. Thus, while EDTA would remove inhibitory metal contaminants, Zn2+ would stabilize the active conformation of the enzyme. Tween 20 had a smaller influence, possibly enhancing solubility while also promoting partial enzyme unfolding at higher concentrations, again highlighting the relevance of the balanced proportions noted previously for the BGL oryzicola formulation [25,26,27].

Table 5 shows the Analysis of Variance (ANOVA) for the model under study. The F test yielded a value of 41161.81, which is higher than the tabulated F-value (9.01), with an R2 of 0.99 at a 95% confidence level, showing that the model fits the experimental data well. The Pareto graphs showed that the three selected additives influenced enzyme activity.

Table 5 Analysis of variance for BGL cerevisiae activity optimization with various enzyme formulation additives.

The results obtained in our study show that R. oryzicola produced an enzyme with greater stability in the presence of additives, maintaining or increasing its enzymatic activity in most of the tested formulations. The divergence in activity between the two enzymatic samples was expected, since the amino acids sequence present in the catalytic site of the enzyme may vary depending on the species used to obtain the enzymatic extract. These amino acids may or may not contain chemical groups that interact differently with the additives under study.

Dikshit and Tallapragada [28] studied the effect of 5 mM KCl on the β-glucosidase activity of Monascus sanguineus, reporting a 69% inhibition of enzymatic activity. Conversely, Silva et al. [29] studied the influence of 5 mM KCl on the β-glucosidase activity of Pichia guilliermondii G1.2, reporting a positive modulating effect, with a 7% increase in activity. Similarly, when assessing the effect of KCl and ZnSO4 (1-20 mM) on the β-glucosidase activity of Trichoderma reesei, Obeng et al. [23] found that the relative activities peaked at 15 mM KCl (112.2%) and 20 mM ZnSO4 (102.8%). Aligned with these observations, Li et al. [30] reported that the activity of a β-glucosidase isolated from Acidothermus cellulolyticus increased more than twofold in the presence of 5 mM KCl.

Salts that contain strongly hydrated kosmotropic anions such as SO42-, and, to a much lesser extent, Cl-, can alter water structure and protein hydration, while increasing solution viscosity and decreasing fluidity. In many aqueous systems, this tends to favor protein compaction, stabilize the native folded state relative to the unfolded counterpart, and promote salting-out [31,32].

These characteristics may or may not favor enzyme-substrate binding, provided that the structural modifications do not alter the catalytic site or cause steric hindrance to enzyme-substrate interaction. At lower salt concentrations, the anions' polarizability is reduced, which tends to favor the reaction. At low concentrations (<0.1 M), ions affect protein stability and enzymatic activity primarily through electrostatic interactions. At higher salt concentrations (usually >0.1-0.3 M, but not extremely concentrated, up to about 3.0 M), ion dispersion forces exceed the electrostatic forces [32].

The effects of SDS and EDTA on the β-glucosidase activity of M. sanguineus were studied, both compounds caused more than 60% inhibition of enzyme activity [28].

When evaluating the effect of non-ionic surfactant agents, all samples in the present study showed increased β-glucosidase activity in the presence of Tween 20, Tween 80 and Triton X-100, except for the sample containing 1% Triton X-100. The greatest increase in activity was observed with 0.75% Tween 20, yielding an enzymatic activity of 4.83 ± 0.04 U, twice the initial activity.

Tween 20 and Tween 80, also known as polysorbate 20 and polysorbate 80, are nonionic, amphipathic surfactants, consisting of polyoxyethylene sorbitan esters of fatty acids. Tween 20 is the laurate derivative of polyoxyethylene (20) sorbitan, whereas Tween 80 is the monooleate derivative, and both are commonly used in biotechnological applications [33,34]. Polysorbates are effective at low concentrations, exhibit relatively low toxicity, are relatively inert when mixed with proteins and can strongly inhibit protein surface adsorption [35].

Similarly, Obeng et al. [23] reported a positive effect of surfactants on β-glucosidase activity of Trichoderma reesei, with the highest relative activity (145.6%) observed in samples containing 0.5% Tween 20. The surfactant concentration strongly influences enzyme solubilization and catalytic efficiency, with higher surfactant concentrations leading to decreased catalytic activity, as under such environments, surfactant–protein interactions promote partial unfolding, active‑site disruption, or aggregation [28,36,37].

4. Conclusion

Comparing the results obtained for the two yeasts under study with those from previous studies, the β-glucosidase from R. oryzicola showed a favorable modulation in response to most additives across the tested concentrations. Conversely, the enzyme produced by S. cerevisiae showed lower tolerance and retention of activity when exposed to the same additives. These differences likely result from distinct interactions between additive molecules and amino acid residues at the enzyme surface, leading to conformational changes that may either favor or hinder substrate binding and catalytic efficiency. Among the additives assessed, ZnSO4 at low concentration (<5 mM) proved to be a good chemical additive for both enzymes. Tween 20 also showed positive effects on both enzymes, both when considered individually and when combined with other additives, suggesting its potential in enzyme formulations, particularly for applications in the hydrolysis of lignocellulosic biomass, since this surfactant decreases the non-productive adsorption of cellulases to lignin. The observed synergistic effects, namely between ZnSO4 and Tween 20 for R. oryzicola, and between ZnSO4 and EDTA for S. cerevisiae, highlight the importance of rational additive combinations in enhancing enzyme performance.

Overall, these formulations improved catalytic activity and suggested practical use in developing more efficient and stable enzyme systems for industrial bioconversion processes.

Acknowledgments

The authors are grateful to the UEFS Postgraduate Programme in Biotechnology for support and scholarship.

The authors are grateful to the Brazilian agencies FAPESB (Fundação de Amparo à Pesquisa do Estado da Bahia), FINEP, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for their financial support.

Author Contributions

Geise Camila Ribeiro: methodology, writing – original draft. Floriatan Santos Costa: methodology, statistical analyses, writing – original draft. Pedro Fernandes: writing – review and editing. Sandra Aparecida de Assis: Conceptualization, methodology, writing – review and editing. All authors have read and approved the published version of the manuscript.

Competing Interests

The authors have declared that no competing interests exist.

Data Availability Statement

Data sharing will be provided by requested.

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