Applied Biochemistry and Biotechnology (2025) 197:3271–3294

[Link]

ORIGINAL ARTICLE

Valorization of Cocoa and Peach‑Palm Wastes

for the Production of Amylases by Pleurotus pulmonarius
CCB19 and Its Application as an Additive in Commercial
Detergents

Alaiana Santos Silva1 · Karollaine Moura Neves1 · Rayssa Falcão Freitas2 ·

Thiago Pereira das Chagas3 · Luiz Carlos Salay2 · Erik Galvão Paranhos da Silva2 ·
Ana Paula Trovatti Uetanabaro1 · Andréa Miura da Costa1

Accepted: 24 December 2024 / Published online: 23 January 2025

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2025

Abstract
In the context of agribusiness, the agricultural and livestock sectors generate a consider-
able quantity of waste on a daily basis. Solid-state fermentation (SSF) represents a poten-
tial alternative for mitigating the adverse effects of residue accumulation and for produc-
ing high-value products such as enzymes. Pleurotus pulmonarius is capable of producing
a number of commercial enzymes, including amylases. Accordingly, the present study
sought to produce, characterize, and apply amylases obtained from solid-state fermentation
of cocoa and peach-palm waste by the fungus Pleurotus pulmonarius CCB19. The highest
amylase production by P. pulmonarius was observed after 3 days of solid-state fermenta-
tion of the cocoa shells, with an activity of 83.90 U/gds. The physicochemical characteriza-
tion of the crude amylase using the artificial neural network (ANN) revealed that the high-
est activity was observed at pH 9 and a temperature of 20 °C (120.7 U/gds). Furthermore,
the amylase demonstrated stability in the majority of the tested conditions, maintaining up
to 80% of its residual activity for up to 120 min of incubation. With regard to the impact
of ions and reagents on enzymatic activity, a positive effect was observed in the presence
of ­Co+ ions at concentrations of 1 and 5 mM, whereas ­Cu+ ions at 5 mM demonstrated
an inhibitory effect. The addition of SDS and EDTA reagents did not affect the observed
activity. Furthermore, the extract was tested in commercial detergent formulations and
demonstrated enhanced compatibility (110%) and efficacy (270% with boiled detergent)
in removing starch stains from fabrics with Ariel liquid detergent. In conclusion, amylase
derived from the fungus Pleurotus pulmonarius CCB19 exhibited favorable properties that
make it a suitable candidate for use as an additive in laundry detergent formulations.

Keywords Pleurotus pulmonarius · Amylase · Detergent · Agro-industrial waste ·

Fermentation

Extended author information available on the last page of the article

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3272 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

Introduction

The Brazilian agribusiness sector has been a significant contributor to the acceleration
of the national economy. However, as a consequence of these processes, a consider-
able quantity of waste is produced, which, in addition to causing adverse environmental
impacts due to inadequate accumulation and disposal, is also regarded as a waste of raw
material for biotransformation given the nutritional richness of this biomass [1]. The
waste generated by these industries includes that produced by the processing of cocoa
and peach-palm.
Theobroma cacao L. is indigenous to South and Central America, but it is cultivated
in numerous countries worldwide. Cocoa beans represent the primary ingredient uti-
lized as a raw material for the production of chocolate and its derivatives. However,
this results in a considerable amount of waste with approximately 10 tons (t) of waste
produced for every 1 t of cocoa bean, including the shells, which represent about 78% of
the total weight of the fruit [2, 3]. Hearts of palm are primarily produced and consumed
in Brazil, and the peach-palm (Bactris gasipaes Kunth.), a palm tree native to the Ama-
zon, has emerged as a promising species for commercial cultivation. The production of
this commodity results in a considerable amount of waste, with 80 to 90% of the palm
discarded without appropriate disposal methods being employed [4, 5].
In light of recent developments in biotechnology, an alternative that has attracted con-
siderable attention from numerous studies and companies, particularly those seeking to
implement more sustainable processes with reduced generation of waste and environmen-
tally toxic products, is the use of SSF with agro-industrial waste [6–9]. This represents a
viable option, with a relatively low acquisition cost and high yield. Moreover, the yield
is considerable, and the process is environmentally sustainable, as it provides a beneficial
final destination for the waste as raw material in the generation of value-added products
and the development of bioprocesses [10–12]. The principal bioproducts derived from this
process include enzymes, bioactive and phenolic compounds, sugars, bioenergy, fertilizers
and animal feed, amino acids, microbial pigments, fungi, and others [1, 13].
Among the various bioproducts, microbial enzymes are of particular interest due to
their stability characteristics under adverse conditions, which make them suitable for use
in a variety of industrial settings. This adaptability allows for utilization in a diverse array
of industrial sectors. Nevertheless, companies engaged in enzyme development may be
forced to allocate up to a third of their production costs to raw materials, which reflects
the elevated acquisition costs for industries that utilize enzymes in their processes [11].
Consequently, the search for new processes that facilitate the acquisition of these enzymes
with high yields, accompanied by a favorable cost–benefit ratio, has become a crucial and
highly regarded practice from a commercial standpoint [14]. One potential alternative raw
material that could reduce these costs is lignocellulosic materials.
Among the microorganisms employed in fermentation processes, particularly in
solid-state fermentations, fungi are the most suitable due to a number of advantageous
characteristics. These include robust growth in cultures with low water activity; fac-
ile adaptation to diverse environmental conditions; the production of a diverse range
of enzymes; the capacity to degrade complex structures such as cellulose, hemicellu-
lose, and lignin; and high yield and process efficiency [8, 15]. A number of studies have
employed fungi of the genus Pleurotus in these processes due to their capacity to adapt
to adverse environmental conditions and the absence of metabolic requirements for cul-
tivation, with relatively straightforward growth on solid lignocellulosic materials [16].
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3273

The bioconversion capabilities of various lignocellulosic materials derived from the

species Pleurotus pulmonarius have been documented by numerous authors in the scien-
tific literature. Examples of suitable substrates include wood sawdust [17], peach-palm
residue and cocoa bean shell [18], pineapple shell [19], banana peel, mango, and walnut
[20]. These capabilities are made possible by the enzymatic apparatus of the fungus, which
is capable of efficiently biodegrading waste to obtain carbon and energy. The enzymes lac-
case, manganese peroxidase, and protease have been identified in the literature as being
responsible for this process [19, 21, 22]. The chemical composition of peach-palm waste
and cocoa shell, which is rich in carbohydrates and micronutrients, presents nutritional
conditions that favor the growth of fungi and the production of enzymes. This has been
demonstrated in several studies [4, 7–9, 18]. Despite the documented efficiency of cocoa
and peach-palm wastes in mushroom production [18], there are no documented instances
of their use in solid-state fermentation (SSF) by the Pleurotus pulmonarius CCB19 strain
for the production of amylases.
Amylases occupy a significant position within this group due to the extensive range of
applications for which they are suitable. They represent approximately 25% of the global
enzyme market [23], which is a substantial proportion. They function by cleaving specific
regions of starch, which is a prevalent component of plant biomass. One of the primary
applications of amylases is in the production of industrial detergents. In this context, they
serve as an additive in the formulation, facilitating the removal of starch stains from fabrics
[24]. It has been demonstrated that amylase enzymes produced by microorganisms can be
utilized as detergent additives. Among these studies are investigations into the growth of
Pleurotus tuberregium on banana and pineapple peels [25], Tepidimonas fonticaldi HB23
on wheat bran [26], and Bacillus sp. BBXS-2 has been observed on wheat straw [27],
Aspergillus fumigatus NTCC1222 [5], Bacillus sp. BCC 01–50 [28], Clavispora lusitaniae
CB13 [29], and Laceyella sacchari TSI-2 [30] on commercial media.
For enzymes to perform their function adequately in detergent formulations, specific
requirements must be met. Such enzymes must also demonstrate stability across a wide
range of temperatures, thermostability, resilience to alkaline pH and anionic surfactants,
proteolytic stability with chelators and chemical oxidizing agents, bleach stability, and
broad substrate specificity [31].
The aim of this study was to assess the potential for repurposing cocoa and peach-palm
waste for the cultivation of Pleurotus pulmonarius CCB19 by SSF to produce amylases.
The crude enzymatic extract with amylolytic activity (crude enzyme) was evaluated for
compatibility and efficiency in removing starch stains from fabrics when used in conjunc-
tion with different commercial detergent formulations.

Materials and Methods

Obtaining and Preparation of Peach‑Palm and Cocoa Waste

The peach-palm waste was obtained from the company Inaceres, Uruçuca, Bahia, and the
cocoa waste was obtained from the company Mendoá, Ilhéus, Bahia. The drying process
was carried out in ovens with forced air circulation at 50 °C until a constant weight was
achieved. Subsequently, the dried residues were pulverized into small particles (5 mm) and
stored in hermetically sealed glass bottles until needed.
3274 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

Maintaining the Fungus

The fungus P. pulmonarius CCB19 was obtained from the culture collection of the Botani-
cal Institute of São Paulo. The fungus was reactivated in potato dextrose agar (Acumedia)
at 28 °C in a BOD SL-224/364 (SOLAB) incubator for 8 days or until it had reached the
entire surface of the plate. The fungus was maintained using the same medium and culture
conditions, with periodic inoculations on plates, and stored at 4 °C until needed.

Solid‑State Fermentation (SSF)

The fermentation process was conducted in 125-mL Erlenmeyer flasks utilizing two agro-
industrial waste materials: peach-palm (PP) at a quantity of 4 g and cocoa shell (CS) at a
quantity of 6 g. Potable tap water was added to achieve an initial humidity level of 80%
with respect to the dry substrate. Cocoa bean peel (CBP) was incorporated into the CS and
PP wastes at a concentration of 20%. Following sterilization in an autoclave at 121 °C for
15 min and subsequent cooling, four plugs of fungal mycelium (10 mm) previously grown
on PDA were added. The flasks were incubated at 28 °C for 3, 5, 10, and 15 days under
conditions of darkness. The experiments were conducted in triplicate.

Obtaining the Crude Enzyme Extract (CEE)

Following the fermentation period, 20 mL of distilled water was added to the cultures for
PP, and 24 mL was added for CS. The cultures were then shaken at 150 rpm in a shaker
incubator (Cientec CT-712) for 20 min at 25 °C. The extraction was performed by filtration
through gauze under a sieve. The extracts were then centrifuged at 10,000 g for 10 min in
a centrifuge (Megafuge 16R Heraeus-Thermo Scientific) at 4 °C. The supernatant obtained
was considered the crude enzyme extract (CEE) and stored for future utilization [32].

Determination of Amylase Activity

The enzymatic activity was quantified by measuring the reducing sugars using 3.5 dinitro-
salicylic acid (Sigma-Aldrich) [33]. The 1% starch substrate (Synth) was diluted in 0.1 M
sodium phosphate buffer at pH 7.0 [34]. The reaction was conducted at 50 °C, and the
absorbance was recorded at 540 nm using a spectrophotometer (BEL Photonics 2000UV).
One unit (UI) of amylase activity was defined as the amount of enzyme required to pro-
duce 1 µmol of reducing sugar per minute under the specified conditions. This value was
expressed as U/gds (grams of dry substrate).

Physicochemical Characterization of Amylase

Effects of pH and Temperature on Amylase Activity and Stability

To conduct a physicochemical characterization of the enzyme, an artificial neural network

(ANN) was employed. This is a computational algorithm that has been inspired by bio-
logical neural networks. The algorithm has the potential to yield computational models
due to its capacity for universal approximation [35]. In other words, under specific condi-
tions, this algorithm can generate precise and comprehensive computational models for a
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3275

given set of data. The ANN was implemented using the MATLAB R2018b software with
the Neural Network Toolbox. A feedforward multilayer perceptron ANN was employed,
with two inputs related to the temperature (°C) and pH of the tests, up to two hidden lay-
ers with a hyperbolic tangent activation function and number of neurons to be specified,
and an output with a linear activation function related to the enzyme activity (U/gds) of
each assay. The data is normalized by default for use by the ANN. The network is trained
using backpropagation with a cost function called mean squared error (MSE) and the Lev-
enberg–Marquardt optimization method with a stopping criterion called early stopping,
with a maximum number of 20 validation checks. The train function is employed, which
applies Bayesian regularization [36], the most suitable method among those available in
the Matlab version for training problems with limited data. The criterion for determining
the optimal network is determined based on the coefficient of determination (R2), which is
sought after with the objective of achieving the highest value and uniformity of R2 between
the training, validation, and test sets.
Based on an experimental design utilizing ANN derived from numerical simulations,
20 experiments were conducted to characterize the enzyme across a range of pH values
(3.0–11.0 in intervals of 2) and temperatures (20–80 °C, with an interval of 10°). The cen-
tral point was conducted in triplicate under standard assay conditions at a temperature of
50 °C and a pH of 7.0 (Table 1).
Stability testing was conducted over a period of up to 120 min at designated time
points, including 0, 30, 60, 90, and 120 min. The initial 10-min period was designated as
the standard test duration, referred to as time zero. The test conditions were based on the
five points of maximum activity obtained in the pH and temperature tests, which ranged
from 20 to 60 °C and pH 7 to 11. Activity analyses were conducted in accordance with the

Table 1  Matrix generated by Experiment Temperature (°C) pH

artificial neural network (ANN)
with different combinations of
1 20 7.0
temperature and pH
2 20 9.0
3 20 11.0
4 30 7.0
5 40 3.0
6 40 5.0
7 40 9.0
8 50 3.0
9 50 7.0
10 50 7.0
11 50 7.0
12 50 11.0
13 60 5.0
14 60 5.0
15 60 7.0
16 60 11.0
17 70 7.0
18 80 3.0
19 80 5.0
20 80 7.0
3276 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

previously described methodology. The residual activity was expressed as a percentage,

with the initial activity of the enzyme for each condition serving as the baseline for com-
parison (100%).

Effects of Ions and Chemical Reagent on Amylase Activity

The impact of cations, chemical reagents, and detergents on amylase activity was exam-
ined. The enzyme extract was incubated in the presence of different ionic salts at concentra-
tions of 1 mM or 5 mM. The following chemical reagents were utilized in this experiment:
calcium chloride (­CaCl2), cobalt chloride (­CoCl2), manganese sulfate (­MnSO4), magne-
sium sulfate ­(MgSO4), zinc sulfate ­(ZnSO4), sodium carbonate ­(Na2CO3), copper sulfate
­(CuSO4), nickel sulfate (­NiSO4), aluminum nitrate (Al(NO3)3), lithium sulfate (­Li2SO4),
and other reagents, including EDTA (ethylenediaminetetraacetic acid), β-mercaptoethanol,
and SDS (sodium dodecyl sulfate), were incubated at the same concentrations for 15 min at
room temperature (28 ± 2 °C).
Subsequently, the enzymatic reaction was conducted under standard conditions, and the
outcomes were compared with those of the control (no incorporation of ions or reagents)
for the purpose of evaluating relative activity. The residual activity was quantified using
standard enzymatic assay conditions and expressed as a percentage [37].

Fourier Transform Infrared Spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) in attenuated total reflection (ATR) mode
was employed to characterize the residue prior to and following fermentation with the fun-
gus P. pulmonarius CCB19. The measurements were conducted within the spectral range
of 650 to 4000 ­cm−1 using a Spectrum 400 FT-IR/FT-NIR spectrometer (PerkinElmer).
The spectra were obtained by averaging 10 scans, obtained with a resolution of 4 ­cm−1,
and subsequent processing using Spectrum software (PerkinElmer). This characterization
facilitated the identification of the functional groups present in cocoa residues subjected to
disparate treatment types.

Application of Crude Amylase as an Additive to Commercial Detergent

Compatibility with Commercial Detergent

The crude enzymatic extract with amylolytic activity was subjected to testing with a range
of commercial detergents from different brands, including Omo, Ariel, Brilhante, and Teiú
(Brazil). Detergent solutions with a concentration of 5 g/L were prepared and subsequently
boiled for 15 min to inactivate the enzymes present in the formulations. Subsequently, the
enzyme extract (50%) was added and incubated at 40 °C for 30 min. The reducing sugars
were then quantified by the DNS technique [33] to assess the residual activity of the con-
trol, which was 100%. Tap water was employed as a control for the dilution of the extract
and was incubated under the same conditions as previously described [37].

Washing Performance Analysis

The tests were conducted on white fabrics measuring 5 × 5 cm which were artificially
stained with 0.5 mL of a starch solution (0.5%) and then dried in an oven at 50 °C for
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3277

30 min. Subsequently, the tissues were immersed in 5 mL of different solutions, which

consisted of the following: tap water, crude enzyme extract, detergent, and detergent plus
crude enzyme extract. All samples were subjected to rotation at 100 rpm for 20 min at
40 °C in an orbital shaker (Cientec CT-712), followed by tissue twisting and analysis of
the washing solution. The reducing sugars present in solution were quantified using the
DNS reagent as described by Miller [33]. The control sample was prepared by replacing
the solution with the crude extract.
Subsequently, tests were conducted using starch-stained fabrics in accordance with the
procedures described above but employing four distinct commercial detergents. The deter-
gents were tested in solutions with a final concentration of 5 g/L, both independently and
in combination with the same detergents mixed with the crude enzymatic extract (5 g of
detergent per liter of crude enzymatic extract). The detergents were applied in two distinct
conditions: in their original state, as supplied by the industry (traditional), and after boil-
ing for 15 min to inactivate the enzymes present in the formulation (boiled). The objective
of this experiment was to assess the efficacy of the enzymes present in the P. pulmonarius
CCB19 crude enzymatic extract in removing starch stains without interference from the
enzymes present in the commercial formulation.
The efficacy of the starch removal process was assessed in accordance with the method-
ology described by Simair et al. (28), as follows:
100 × A × 0.9
Ef f iciency (%) =
B
where A represents the quantity of sugar produced by hydrolysis of the starch in the fabric
(g/mL) while B denotes the amount of starch applied to stain the fabric.

Statistical Analyses

The analyses were conducted in triplicate, with standard deviation, and the difference
between cocoa and peach-palm waste was statistically evaluated by analysis of variance
(ANOVA) and Turkey’s test using Statistica software, with a confidence level of 95%.

Results and Discussion

P. pulmonarius CCB19 Cultivation Conditions for Amylase Production

The growth of Pleurotus sp. on different substrates depends on its capacity to synthesize
enzymes that are capable of acting on the lignocellulosic components that are present. As
with other microorganisms, this production is influenced by variables that affect its devel-
opment [38, 39]. In their study, Belettini and colleagues [40] identified several factors that
influence the growth and enzyme production of the basidiomycete Pleurotus sp. These
include substrate composition and availability, carbon and nitrogen content, temperature,
water activity, pH, and the dependence or otherwise on cofactors or coenzymes. By study-
ing the impact of these variables on fungal growth and enzyme secretion, it is possible to
identify the optimal conditions for achieving the desired outcomes.
The present study analyzed the following variables: fermentation time and the types of
substrates utilized for cultivation with a focus on their impact on enzyme production. The
results of the analyses indicated that the highest amylase activity was observed after 3 days
3278 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

of fermentation at 28 °C in static cultivation, using a mixture of cocoa shell residues with

the addition of 20% cocoa bean peel (83.9 U/gds) at 80% humidity. This is demonstrated in
Fig. 1.
Inácio and colleagues [32] assessed the efficacy of P. pulmonarius in amylase produc-
tion using orange residues through solid-state fermentation, resulting in a maximum activ-
ity of 1.16 U/mL. The basidiomycete fungus Tuber maculatum mycelium exhibited 1.41
U/mL of amylase [41] in submerged fermentation using a medium prepared at pH 7 and
22 °C, both after 7 days of cultivation. Arora et al. [42] observed a maximum glucoamylase
activity of 10.29 U/mL from the growth of Penicillium sp. using rice bran after 7 days of
solid-state fermentation. In comparison to previously published studies, it is evident that
the present study yielded greater activity in a shorter period of time.
Kumar et al. [43] observed that the enzymatic activities remained comparable over a
longer fermentation period. In the study, Fusarium solani NAIMCC-F-02956 was culti-
vated using mango seeds as a substrate in submerged fermentation, resulting in the highest
α-amylase activity of 88.9 U/gds, pH 4, 30 °C, and 9-day incubation period. Moreover,
Bhatt et al. [44] achieved α-amylase production from Bacillus velezensis KB2216 by sub-
merged fermentation using a supplemented growth medium, resulting in a maximum activ-
ity of 17.066 U/mL in 3 days of fermentation. The results were then compared to those
obtained in the present study based on fermentation activity and time.
It was observed that the activity in the present study exhibited a magnitude approxi-
mately 20 times higher than that observed in some previously presented studies, and in a
shorter cultivation time. Moreover, this study represents the inaugural investigation into the
efficacy of utilizing cocoa shells (CS) in conjunction with cocoa bean peel as a supplemen-
tary nitrogen source to produce amylase by Pleurotus pulmonarius CCB19. Accordingly,
cocoa shells (CS) were employed as the substrate for the subsequent stages of the process.

Fig. 1  The amylase production by P. pulmonarius CCB19 in solid-state fermentation (SSF) using CS
(cocoa shell) and PP (peach-palm) wastes as substrates, with the addition of 20% CBS (cocoa bean peel) at
28 °C. The fermentation process was conducted over a period of 3 to 15 days
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3279

Influence of pH and Temperature on Enzyme Activity and Stability Using ANN

(Artificial Neural Network)

The ANN computational model, resulting from the application of the methodology
described above, exhibits two hidden layers, comprising 27 and 2 artificial neurons in the
first and second hidden layers, respectively. The initial layer, comprising 27 neurons, per-
forms a transformation on the data set. However, the high number of neurons in this layer
renders the network susceptible to overfitting. The second layer, comprising two neurons,
serves to enhance the capacity of the network for generalization. This two-layer architec-
tural configuration has been demonstrated to yield superior outcomes and is consistent
with the findings of Thomas et al. [45] and Chester’s proposal [46].
It is notable that analogous methodologies, which are also susceptible to data leakage
[47], have been demonstrated to be effective in other applications [48–51]. Moreover, they
are founded upon an exhaustive search for the optimal combination of network structure,
weight initialization, and data partitioning between training, validation, and test sets.
The data set, comprising the assays utilized for the training of the artificial neural net-
work (ANN), is presented in Table 2. Assays 9, 10, and 11 represent the triplicates of the
central point of the experimental design. The mean value employed for training the ANN is
provided in the enzyme activity column.
Moreover, the table presents the estimates obtained with the trained model, the residual
and total MSE, the R2, and the training validation and test sets.
Table 3 presents supplementary measures that are utilized to qualify the model. These
include the mean and standard deviation of the residual as well as the mean and standard
deviation of R2 for all possible combinations of three data sets for the test set.
This combination of measures is intended to demonstrate that the model maintains its
approximation quality regardless of the organization of the available data.
As illustrated in Fig. SM 1 (supplementary material), the linear regression plots yield
results that are suitable for generalization. Table 3 illustrates that for any combination of
three tests as a test set, there is a high R2. As a consequence of the restricted data set, the
reuse of data for testing represents an additional means of evaluating the models. As the
validation and test data were not employed to adjust the weights of the selected model, the
performance averages associated with these data are representative of those that would be
expected for new experimental data.
Figure 2 shows a three-dimensional representation of the computational model of the
experiment which correlates temperature (°C), pH, and enzyme activity (U/gds). The appli-
cation Bayesian regularization results in the training process yield an interpolation model
with the objective of generalization. Therefore, the model is not anticipated to capture data
exhibiting high scatter. Nevertheless, should the model fail to attain the maximum enzyme
activity observed in test 2 at 20 °C and pH 9.0, the maximum value for the interpolation
model is obtained for the same temperature and pH values.
The study of physicochemical characterization in relation to pH and temperature of the
reaction represents a crucial area of inquiry, as these factors exert a profound influence on
enzymatic activity. Given that pH can act on the enzyme structure among amino acids in
the active site, promoting the denaturation of the enzyme, and that temperature can act on
biological activation by denaturing the enzyme or inhibiting it [52], it is imperative that
this factor be considered when studying enzymatic activity.
It can be observed that the maximum enzyme production is obtained at low tempera-
tures (20 to 40 °C), with a subsequent decrease from 50 °C and an increase in activity with
Table 2  Assays varying temperature and pH of amylase produced by P. pulmonarius CCB19, values estimated by artificial neural network (ANN) model, residual and assay
3280

performance measures
Experiment Temperature pH Enzymatic activity (EA) (U/gds) Estimated enzymatic activity Residue (EA-EEA) MSE R2
(°C) (EEA) ANN (U/gds)

Training set
5 40 3 87.02 89.04 − 2.02 5.30 0.96
6 40 5 99.68 97.99 1.69
7 40 9 114.41 112.38 2.03
8 50 3 104.74 103.26 1.48
9, 10, 11 (central point) 50 7 87.71 (Central point median) 89.99 − 1.95
87.02
88.40 88.04
12 50 11 99.45 96.96 2.49
14 60 5 84.71 87.63 − 2.92
15 60 7 107.74 103.81 3.93
17 70 7 91.16 91.51 − 0.35
18 80 3 98.53 95.05 3.48
19 80 5 66.76 68.54 − 1.78
20 80 7 94.84 93.88 0.96
Validation set
1 20 7 112.57 112.70 − 0.13 7.98 0.94
2 20 9 120.17 115.37 4.80
16 60 11 92.08 91.12 0.96
Test set
3 20 11 112.00 114.87 − 2.87 4.30 0.96
4 30 7 108.66 108.45 0.21
13 60 5 89.78 87.63 2.15
Total dataset 5.58 0.97

EA enzyme activity, EEA estimated value obtained by the ANN model

Applied Biochemistry and Biotechnology (2025) 197:3271–3294
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Table 3  Residual and R2 of Residue Combined test set R2

all possible measurements of
combinations of the test set Average Standard deviation Average Standard deviation

0.68 2.33 0.98 0.11

Fig. 2  Three-dimensional graph obtained from the computational model of the experiment relating temper-
ature (°C), pH, and enzyme activity (U/gds). The circles (○) represent the experimental data used to obtain
the model, available in Table 2

an increase in pH from 7.0 to 11.0 (Table 2). The enzyme displays optimal performance at
a pH of 9.0 and a temperature of 20 °C, exhibiting an activity of 120.17 U/gds. The exten-
sive range of enzyme activity with respect to pH and temperature indicates that the poten-
tial presence of two or more subtypes of amylases within the crude extract is relatively low.
This characteristic is consistent with the results obtained from the correlation coefficient
between temperature (°C) and enzymatic activity (U/gds) of − 0.70, as well as between pH
and enzymatic activity (U/gds) of 0.40. It is evident that there is a strong negative correla-
tion between temperature and enzymatic activity as well as between pH and enzymatic
activity. Moreover, pH values between 8.0 and 9.0 are associated with elevated enzymatic
activity, even in the presence of elevated temperatures.
Other studies have yielded comparable results, with optimal outcomes observed at
pH 9.0. Arora et al. [42] observed outcomes under these pH conditions for glucoamyl-
ase produced by Penicillium in agro-industrial residues by solid-state fermentation (SSF).
3282 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

Additionally, Simair et al. [28] observed comparable outcomes for α-amylase produced by
Bacillus in synthetic media.
As previously observed by Costa et al. [15], the application of ANN in predictive mode-
ling represents an effective approach for identifying the physicochemical factors that influ-
ence enzymatic activity. This comprehension of the optimal conditions for the process ena-
bles its more efficient execution, thereby reducing both time and cost in its development.

Enzyme Stability at Different pH and Temperatures

In order to ascertain the stability of the enzyme with respect to pH and temperature, five
points were subjected to analysis with different combinations of these variables, as illus-
trated in Fig. 3. The amylases present in the extract exhibited high stability in the majority
of the tested conditions, displaying a neutral to basic character. The enzymes demonstrated
the capacity to maintain over 80% of their activity for up to 60 min of incubation in a wide
temperature range from 20 to 60 °C.
The stability demonstrated by the enzyme in the present study with respect to pH and
temperature exhibits industrially important properties that are desirable for application in
various processes. As previously observed by Far et al. [53], amylases with activity at low
temperatures and alkaline pH are the primary characteristics necessary for application in
the removal of starch stains in the detergent industry. As demonstrated by the findings of
this study and those of other researchers, such as Simair et al. [28], the inclusion of crude
alkaline amylase derived from Bacillus can enhance the efficacy of detergents.
The present study demonstrated that the enzyme exhibited stability across a range of
pH and temperature conditions. At pH 7.0 and temperatures of 50 °C and 60 °C, pH 9.0
at 20 °C, and pH 11.0 at 20 °C, the enzyme exhibited over 80% of its activity for up to
120 min of incubation under these conditions. Other studies have yielded comparable
results, including those of Bedade et al. [41], which demonstrated the stability of amylase
at 50 °C, with 98% of the maximum activity retained after 100 min of reaction. Singh et al.
[54] observed that the α-amylase from Bacillus spp. exhibited optimal stability between
pH 7.0 and 8.0 and significant activity at temperatures between 30 and 60 °C. The enzyme
exhibited a high degree of stability, retaining over 80% of its maximum activity for up to
24 h of incubation.
In the present study, residual activity above 50% (65, 74%) was observed up to 120 min
at 40 °C, pH 9.0 for the crude extract with amylolytic activity. These findings are corrobo-
rated by the observations of other researchers, such as Fincan et al. [34], who reported the
stability of 94.4% of α-amylase from Bacillus licheniformis at 40 °C for up to 120 min.
The results obtained under the specified conditions tested indicated that the enzyme
activity remained stable at levels exceeding 80% for up to 120 min. These findings illus-
trate the enzyme’s notable stability across a broad pH and temperature range, which makes
it a compelling subject for further investigation in diverse fields where amylase with these
attributes could be utilized.

Influence of Ions and Chemical Reagents

The impact of ions and chemical reagents represents a phenomenon that can occur in
the natural environment and interact with enzymes in industrial application processes
[41]. Table 4 presents the results of testing different cations and reagents on the amylase
activity of P. pulmonarius CCB19. The results demonstrated that ­Co2+ exerted the most
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3283

Fig. 3  Amylase stability evalu-

ated at different temperatures (30
to 60 °C) and pH (7.0, 9.0, and
11.0) produced by Pleurotus pul-
monarius CCB19. The residual
activity (%) was determined from
the activity obtained under stand-
ard test conditions, 50 °C, pH
7.0. a Stability carried out at pH
9.0, and a temperature of 40 °C.
b Stability achieved at pH 7.0 at
temperatures of 50 and 60 °C. c
Stability achieved at pH 9.0 and
11.0 and temperature of 20 °C

pronounced effect on amylase activity, with an increase of 66.98% at a concentration

of 5 mM and 30.21% at 1 mM. A subsequent, albeit smaller, increase was observed
­ l3+ (34.39%), ­Mn2+ (15.39%), ­Ca2+
at 5 mM for the ions. The results indicated that A
3284 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

Table 4  Effect of ions and Relative activity (%)

reagents on the amylase activity
of P. pulmonarius CCB19 Reagents 1 mM 5 mM

Control* 100 100

Li ­(Li2SO4) 118.72 ± 0.014 109.50 ± 0.013
Ni ­(NiSO4) 125.95 ± 0.005 105.88 ± 0.001
Co ­(CoCl2) 130,21 ± 0.003 166.98 ± 0.006
Ca ­(CaCl2) 98.73 ± 0,006 110.85 ± 0.012
Cu ­(CuSO4) 75.31 ± 0,011 9.04 ± 0.003
Mn ­(MnSO4) 103.83 ± 0.010 115.39 ± 0.004
MgCl2 101.69 ± 0.007 74.65 ± 0.006
Na ­(Na2CO3) 86.80 ± 0.006 62.89 ± 0.001
Al ­(AlNO3) 104.25 ± 0.006 134.39 ± 0.019
Zn ­(ZnSO4) 115.74 ± 0.001 96.83 ± 0.000
SDS 95.74 ± 0.010 80.99 ± 0.016
EDTA 84.25 ± 0.006 80.54 ± 0.001
β–mercaptoethanol 105.53 ± 0.003 104.53 ± 0.000

*Control: without ion and reagent

Value of 100% activity in 1 mM (90.05 U/gds) and 5 mM (84.69 U/
gds)

(10.85%) and at 1 mM by N ­ i2+ (25.95%), L

­ i+ (18.72%), and Z­ n2+ (15.74%) ions exhib-
ited the greatest increases in amylase activity.
Similarly, our findings Dey and Banerjee [55] align with those of observed a pro-
nounced activation of the enzymatic activity of α-amylase from Aspergillus oryzae by
the ­Co2+ ion resulting in a 58% increase. Similarly, Wang et al. [56] who also observed
an increase in the enzymatic activity of α-amylase from Rhizomucor miehei with the
ions ­Li+ (113.9%), N ­ i2+ (101.9%), M­ n2+ (101.8%), and C ­ o2+ (108.9%). Additionally,
Gois et al. [57] observed that amylase activity was activated in the presence of ­Co2+ and
­Ca2+ ions produced by Bacillus through solid-state fermentation using cassava bagasse,
while activity was inhibited in the presence of C ­ u2+ and ­Zn2+ ions. Conversely, Kumar
et al. [43] observed that the production of enzyme increased in the presence of ­Ca2+
and ­Mg2+ while the activity of the enzyme decreased in the presence of M ­ n2+ and C­ u2+.
These observations were made with α-amylase produced by Fusarium solani NAIMCC-
F-02956 under submerged fermentation using mango seeds.
Bedade et al. [41] observed a reduction in the activity of the amylase enzyme pro-
duced by Tuber maculatum when the organism was exposed to ­Ca2+, ­Mg2+, and ­Zn2+
ions at a concentration of 1 mM. Similarly, Fican et al. [34] observed inhibition of
α-amylase activity in a study with Bacillus licheniformis in the presence of C ­ u2+ ions
(26%) and the chelating agent EDTA (35%). In the present study, the amylase from P.
pulmonarius CCB19 also exhibited a notable reduction in enzymatic activity (90.96%)
in the presence of ­Cu2+ at 5 mM, followed by ­Na2+ ions (37.11%) and ­Mg2+ (25.35%).
As stated by Farooq et al. [58], the loss of enzymatic activity in the presence of these
ions is attributed to their binding to the active site of the enzyme. With regard to the
chemical reagents, a slight reduction in activity was observed in the presence of the sur-
factant SDS (19.01%) and the chelating agent EDTA (19.46%), as illustrated in Table 4.
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3285

In order to comprehend the inhibition reaction with some of these reagents, it is essen-
tial to recognize that surfactants such as SDS are constituted of hydrophilic and hydro-
phobic segments that facilitate the solubilization of proteins by disrupting non-covalent
bonds, which can result in the loss of activity or even denaturation [59, 60]. This provides a
rationale for the reduction of enzyme activity under the conditions tested. EDTA, which is
a chelating agent, can react with various metal ions present in the extract, such as calcium
ions, forming complexes with them and affecting the activity of metalloenzymes [55].
β-Mercaptoethanol functions as a denaturing agent, reducing the disulfide bonds
between amino acids present in protein, thereby destabilizing their tertiary structure [61].
However, in this study, a subtle activation of amylase was observed when in contact with
this chemical reagent, with an increase in activity from 4.53 to 5.53%. In a study conducted
by Siddiqui et al. [62], it was observed that when α-amylase produced by Pseudoalter-
omonas haloplanktis reacted with β-mercaptoethanol, all disulfide bonds were removed,
resulting in alterations to the enzyme’s stability, particularly in the active site. This resulted
in the enzyme becoming susceptible to ionic interactions at the active site.
The results of this study demonstrate that the reducing agent did not have a deleterious
effect on the disulfide bridges present in the amylase. It is conceivable that the reducing
agent may have protected the amylase from undergoing alterations to its native structure,
thereby enhancing its stability and protecting it from the deleterious effects of 2-mercap-
toethanol [15]. However, the amylase also exhibited other types of influence, including
ionic interactions in the active site and thermal, which resulted in an increase in catalytic
activity.

Fourier Transform Infrared Spectroscopy (FTIR) of Cocoa Shell Waste

Following Fourier transform infrared (FTIR) analysis of the fermented residues, it was
observed that the profile of molecules formed prior to the growth of the fungus Pleurotus
pulmonarius CCB19 was distinct after 3 days of fungal growth on the residues (Table SM
1 (supplementary material), Fig. 4). It can be postulated that the formation of these dispa-
rate molecules is a consequence of the enzymatic activity of the fungus on these wastes,
which were generated with the objective of degrading lignocellulosic materials, predomi-
nantly composed of cellulose, lignin, and hemicellulose, to obtain the nutrients essential
for their growth. This results in molecules derived from these reactions [40].
The sample analyzed consisted of cocoa shell and cocoa bean peel. The composition of
these materials included lignin, cellulose, hemicellulose, proteins, carbohydrates, and ash
[2]. The infrared spectra of the cocoa samples without and with extraction exhibited a nota-
ble distinction, indicating the efficacy of the enzymatic extraction process (Fig. 5). All the
fundamental vibrations observed in the region between 4000 and 2500 ­cm−1 can be attrib-
uted to X–H stretching. In the spectral range between 3614.28 and 3838.57 ­cm−1, bands
are observed that can be attributed to molecular vibrations of hydroxyl groups (O–H) found
in carbohydrates, carboxylic acids, protein species, lignin, cellulose, and water molecules.
Following the application of the extraction process to the cocoa residue sample, a band
was observed at 3439.29 ­cm−1. This wavenumber value is typically attributed to N–H
stretching, which is commonly observed between 3400 and 3300 ­cm−1. In the samples
(with and without extraction, as well as the control sample), bands of weak and medium
intensity are observed in the range of 3000–2850 ­cm−1, which are attributed to C-H stretch-
ing of aliphatic compounds. Wavenumber values within the range of 2500–2000 ­cm−1
are attributed to the stretching of triple bonds. Covalent triple bonds have been observed
3286 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

Fig. 4  FTIR spectra of cocoa residues. Control (without fermentation—RCTRL), cocoa residue 3 days after
fermentation, without enzyme extraction (RC3SE), cocoa residue after 3 days of fermentation, with enzyme
extraction (RC3CE)

Fig. 5  Compatibility analysis of different commercial detergents with crude amylase at 40 °C for 30 min at
100 rpm. The control was the use of the enzyme without the addition of commercial soaps
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3287

to absorb between 2300 and 2050 ­cm−1 while C≡N nitrile groups have been observed to
exhibit absorption between 2300 and 2200 ­cm−1. It is essential to note that while the wave-
number values for the cocoa residues without and with extraction, as well as for the control
sample, are situated within these spectral bands, the values assigned are distinct in each
case. This provides further evidence that the extraction process induces structural and/or
conformational alterations.
The wavenumber values of 1642.85 ­cm−1 and 1545.81 ­cm−1 for cocoa residues with
extraction and 1697.14 ­cm−1 and 1527.86 ­cm−1 for cocoa residues without extraction
are indicative of C = C and C = O stretching, respectively. Similarly, the control sam-
ple exhibited spectral bands with wavenumber values of 1815.71 ­cm−1, 1702.86 ­cm−1,
1700.81 ­cm−1, and 1533.57 ­cm−1. As previously observed, there are notable inconsisten-
cies in the wavenumber values when the samples with and without treatment are compared.
These inconsistencies can be attributed to the extraction process applied to the cocoa resi-
dues. Wavenumber values within the range of 1500–600 ­cm−1 preclude the assignment of
individual absorption bands relative to the infrared spectrum. This region is referred to
as the fingerprint region, as it is distinguished by the presence of numerous similar mol-
ecules that exhibit disparate absorption patterns at these frequencies [63]. The wave num-
bers of 1461.43 ­cm−1, 1243.57 ­cm−1, and 1067.86 ­cm−1 were observed for the cacao resi-
dues subjected to the extraction process, while the wave numbers of 1231.43 ­cm−1 and
1062.14 ­cm−1 were observed for the cacao residues not subjected to the extraction process.
In the case of the control sample, the observed wave numbers of the spectral bands were
1455.00 ­cm−1, 1237.14 ­cm−1, and 1067.86 ­cm−1. Moreover, discrepancies are apparent
between the two spectral regions, including differences in the wavenumber values and the
transmittance intensity.

Compatibility Analysis and Washing Performance of Crude Amylase

with Commercial Detergents

The neutral-basic properties of the crude amylase present in the extract at moderate tem-
peratures were found to be conducive to testing in the detergent sector. According to
Gurkok [64], for the enzyme to be utilized in detergents, it must demonstrate optimal func-
tionally at alkaline pH and across a broad range of temperatures, in addition to exhibiting
compatibility with the other ingredients present in the formulation.
Therefore, the crude enzymatic extract with amylolytic activity obtained from the fer-
mentation of cocoa residues by Pleurotus pulmonarius CCB19 was evaluated for its com-
patibility with a range of commercially available boiled detergents. The crude amylase
exhibited a favorable interaction with the three detergent brands, namely Ariel, Brilhante,
and Teiú. The greatest effect was observed with the Ariel brand of detergent. The enzyme
exhibited an 11.53% increase in activity relative to the control (Fig. 5).
The Omo brand, when combined with crude enzymes, exhibited a 52.3% reduction in
activity following a 30-min incubation period. This result may be attributed to the incom-
patibility of the enzyme with specific components present in the detergent formulation.
Herrera-Marquéz and colleagues [65] evaluated the stability of the amylase enzyme in the
presence of two nonionic surfactants (amine oxide and alkyl polyglycoside) which are com-
monly present in detergent formulations. The findings revealed that the amine oxide had a
deactivating impact on amylase, emphasizing the necessity of employing stable enzymes in
formulations where these components are present. Furthermore, Correa et al. [66] observed
3288 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

a reduction in α-amylase activity produced by Bacillus sp. strain S ­ MIA−2 in the presence
of OMO, with a 22% reduction in activity.
To evaluate the potential of crude amylase to enhance the cleansing properties of deter-
gents, the enzymatic extract was incorporated into a detergent formulation to assess its
capacity to improve the removal of starch stains. As illustrated in Table 5, the crude amyl-
ase demonstrated efficacy in the hydrolysis of the starch present in the fabrics. Approxi-
mately 80% of detergents contain one or more enzymes in their composition. The primary
categories of enzymes utilized in detergents are amylases, with α-amylases representing a
significant area of interest, as evidenced by numerous patents filed for their application in
the detergent industry [67]. This industrial sector has experienced consistent growth, and
with the advent of biotechnology, the utilization of enzymes to augment their cleansing
efficacy has been expanded [65]. Among these are the amylase enzymes, which play a sig-
nificant role in the processing of starch from a variety of food sources, including potatoes,
meat sauces, ice cream, chocolate, and others [24].
The findings revealed that the combination of detergents with crude amylase yielded the
most optimal outcomes, both in the traditional state and with the boiled formula. The com-
bination of detergents with crude amylase demonstrated up to 200 times greater efficiency
in starch removal compared to the test with detergent alone (Table 5).
The use of crude amylase as an additive in detergents has been documented in the
scientific literature, with encouraging results indicating the efficacy of these enzymes in
such processes [5]. The findings of this study were derived from the utilization of a crude
enzyme extract (crude enzyme), which offers distinct advantages over purified enzymes.
The use of crude enzymes eliminates the need for costly purification processes. Moreover,
the presence of additional enzymes in crude enzyme solutions may contribute to enhanced
outcomes.
The amylase produced by the fermentation of apple waste has been tested for the
removal of starch stains from fabrics and has demonstrated promising results in combi-
nation with detergents [25]. Simair and colleagues [28] tested crude amylase from Bacil-
lus sp. BCC 01–50 and observed favorable outcomes in terms of efficiency in cooperation
with the detergent in removing starch stains when added to the crude amylase. Moreover,

Table 5  Washing performance Washing agent % starch content in the wash

with water, crude amylase,
and commercial detergents
Tap water as control -
(traditional and boiled) with
crude amylase produced by P. Crude amylase 50% (20.22 U/mL) 126.72
pulmonarius CCB19 Traditional detergent brand 5 g/L Detergent Deter-
gent + amyl-
ase
Omo 29.61 136.57
Teiú 0.391 249.07
Ariel 0.819 240.07
Brilhante 17.93 285.07
Boiled detergent 5 g/L
Omo 26.82 173.52
Teiú 1.13 213.12
Ariel 3.29 270.72
Brilhante 20.52 211.32
Applied Biochemistry and Biotechnology (2025) 197:3271–3294 3289

Qureshi et al. [27] observed promising outcomes regarding the efficiency of starch removal
from stained fabrics with both the crude enzyme from Bacillus sp. BBXS-2 and the enzyme
combined with detergents, demonstrating a threefold improvement in starch removal.
Allala et al. [26] evaluated the efficacy of the tested crude alkaline α-amylase from Tepidi-
monas fonticaldi HB23 in combination with diverse detergent formulations. The findings
revealed that the enzyme exhibited high compatibility and stability with the majority of
components and demonstrated the capacity to remove the stain with greater intensity than
the commercial enzyme Termamyl® 300 L.
It is of paramount importance to underscore that the test conducted with boiled deter-
gents also exhibited remarkable washing performance when crude enzymes were incor-
porated. Given that the purpose of boiling is to inactivate enzymes present in commercial
detergents, this action serves to highlight the activity of the crude enzyme added to the for-
mulation. Additionally, Correa et al. [66] evaluated the efficacy of α-amylase in conjunc-
tion with a range of detergents following boiling, and their findings indicated a significant
improvement in fabric washing performance. Lahmar et al. [68] assessed the compatibility
and washing efficacy of purified amylase derived from the leaves and stems of Pergularia
tomentosa when combined with detergents after boiling. The results of this study indicate
that enzymes may provide a valuable contribution to the detergent industry.
Given the inactivation of the enzymes present in the commercial detergent formulations
used in these experiments after boiling and the favorable outcomes observed in the experi-
ments conducted, it can be posited that the crude amylase produced by Pleurotus pulmo-
narius CCB19 played an important role in enhancing the efficacy of washing starch stains
on fabrics under the specified conditions.

Conclusion

The fungus Pleurotus pulmonarius CCB19 has been demonstrated to be an extremely

effective producer of amylase. Cocoa husks and peach-palm wastes were identified as
effective inducers for the production of this enzyme. Cocoa waste, husks, and cocoa bean
peel were identified as the most effective substrates for SSF when fermented for 3 days at
28 °C. The ANN computational model proved to be a valuable tool for the characterization
of the crude enzyme, which demonstrated high activity over a wide range of pH and tem-
perature conditions, with optimal and favorable results observed at pH 9 and a temperature
of 20 °C. The enzyme exhibited a pronounced increase in activity in the presence of ­Co2+
and a reduction in the presence of ­Cu2+, SDS, and EDTA.
With regard to potential biotechnological applications, the results obtained from test-
ing the crude enzyme as an additive in four distinct formulations of commercial laundry
detergents demonstrated notable efficacy in the removal of starch stains on fabrics. This
renders it a viable option for use as an additive in commercial formulations. Consequently,
this study proposes the utilization of solid fermentation of cocoa residues by Pleurotus
pulmonarius CCB19 as a cost-effective and sustainable alternative for the utilization of
these agro-industrial residues with the objective of obtaining crude amylase with favorable
properties for application in industrial production of detergents.
Supplementary Information The online version contains supplementary material available at [Link]
org/​10.​1007/​s12010-​024-​05147-3.

Author Contribution All the authors contributed to the conception and design of the study. Alaiana
Santos Silva and Karollaine Moura Neves: conceptualization, methodology, validation, investigation,
3290 Applied Biochemistry and Biotechnology (2025) 197:3271–3294

writing—original draft preparation. Raissa Falcão: methodology, investigation and editing. Ana Paula Tro-
vatti Uetanabaro: funding acquisition, writing—review and editing. Andréa Miura da Costa: conceptualiza-
tion, validation, writing—review and editing, supervision, funding acquisition. Luiz Carlos Salay: formal
analysis, writing—review and editing, investigation. Erik Galvão Paranhos da Silva: formal analysis, writ-
ing—review and editing, data curation. Tiago Pereira das Chagas: formal analysis, writing—review and
editing. All the authors have read and approved the final manuscript.

Funding This work was supported by the Coordenação de Aperfeiçoamento Pessoal de Nível Superior
(CAPES) with the award of Alaiana Santos Silva doctoral scholarship (grant number 88882.451467/2019–
01) and the Universidade Estadual de Santa Cruz (UESC) (073.11014.2020.0003766–48 and
073.11014.2022.0003271–26).

Data Availability Data will be made available on request.

Declarations
Ethics Approval The present research study does not involve any human participants, their data, or biological.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Competing Interests The authors declare no competing interests.

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Authors and Affiliations

Alaiana Santos Silva1 · Karollaine Moura Neves1 · Rayssa Falcão Freitas2 ·

Thiago Pereira das Chagas3 · Luiz Carlos Salay2 · Erik Galvão Paranhos da Silva2 ·
Ana Paula Trovatti Uetanabaro1 · Andréa Miura da Costa1

* Andréa Miura da Costa

amcosta@[Link]
1
Department of Biological Sciences, UESC – Universidade Estadual de Santa Cruz, Rodovia Jorge
Amado, Km 16, Ilhéus, BA 45662‑900, Brazil
2
Department of Exact Sciences, UESC – Universidade Estadual de Santa Cruz, Rodovia Jorge
Amado, Km 16 Salobrinho, Ilhéus, BA 45662‑900, Brazil
3
Department of Engineering and Computing, UESC - Universidade Estadual de Santa Cruz,
Rodovia Jorge Amado, Km 16, Ilhéus, BA 45662‑900, Brazil