New Beauveria bassiana aerial conidia-based bioinsecticide obtained by spray-dried microencapsulation of the entomopathogenic fungi in biopolymers for crop protection

Abstract

Background

Due to their environmentally friendly character, entomopathogenic fungi (EPF) are becoming increasingly used as microbial agents in biological pest control over chemical pesticides. However, EPF are sensitive to the influence of abiotic factors, such as temperature, radiation, and humidity. To improve their efficiency as bioinsecticides, in this work, the development of a new microparticles-based formulation loading EPF conidia (B. bassiana aerial conidia) into sodium alginate/maltodextrin microparticles obtained by spray-drying was proposed. Different concentrations of both polysaccharides were tested to reach the optimal ratio and ensure a high viability of encapsulated conidia.

Results

All the produced formulations showed a moisture content < 10% and water activity (aw) < 0.4. Microparticles obtained with 2% sodium alginate and 8% maltodextrin were able to retain 89.5% of the viability of encapsulated conidia, thus being selected for further characterization. Scanning electron microscopy showed microparticles with a smooth surface, varied sizes, and irregular morphology. Microparticles retained 5.44 × 10⁸ conidia/g, presented high hygroscopicity and high suspensibility rate, yet low wettability and water activity (aw) of 0.33. The pH value ranged from 6.46 to 6.62. Microparticles were able to complete release the loaded conidia after 30 min, under constant stirring. When exposed to thermal stress (45 °C), microparticles promoted thermal protection to conidia. Enhanced pathogenicity of B. bassiana conidia against P. xylostella was also confirmed achieving 83.1 ± 5.5%, whereas non-encapsulated conidia reached only 64.8 ± 9.9%.

Conclusions

This study confirms that the encapsulation of B. bassiana fungus conidia in sodium alginate/maltodextrin microparticles by spray-drying is a promising technological approach for the biological control of agricultural pests.

Background

Because of their capacity to infect insects and mites, EPF are becoming increasingly used as microbial agents in biological pest control, by promoting fatal epizootics in their hosts (Irsad et al. 2023). Many insects and nematode species are harmful for a variety of fungal microorganisms, which can regulate these natural populations by preventing their expansion (Bava et al. 2022). Due to their potential application as insect biocontrollers, aerial conidia of EPF are produced on a large scale to meet the demands of agricultural producers, especially organic and agroecological needs, and are commercialized in different formulations, such as sprayable formulations (wettable powders) and dry formulations (contact powders) (Baker et al. 2020; Du et al. 2020; Sarma et al. 2023).

Pests’ resistance to chemicals is increasing and is causing serious public health concerns. EPF are known to be environmentally friendly and have the ability to overcome pests’ resistance, thus being a viable alternative over chemicals (Bava et al. 2022). However, the use of EPF for insect control still presents some limitations, especially related to the influence of abiotic factors, such as temperature, radiation, and humidity (Méndez-González et al. 2022). Therefore, there is a need to develop innovative approaches to improve their stability and efficiency, by preserving the viability of these microorganisms (Jaronski 2014). The microencapsulation of fungal propagules in biopolymers by spray-drying emerges as an alternative, enabling the development of dry formulations with greater fungal tolerance to abiotic conditions, with the biopolymers acting as a physical barrier (Herlinda et al. 2018).

Spray-drying is a technique commonly used in the food and pharmaceutical industries that relies on the spraying of a liquid (emulsion or suspension) through a spray nozzle followed by drying using a hot air flow, resulting in the production of dry microspheres within seconds (Dantas et al. 2024). The high temperature and drying speed employed in this technique favor the production of materials with low moisture content and low water activity, increasing their stability during storage and reducing the risks of microbial contamination (Mohammed et al. 2020). Although its use is well established in formulation development, the production of microparticles by spray-drying loading bioinsecticides is still seldomly reported, with a limited variety of polymers being described as suitable for this purpose. Currently, there are no records of microencapsulated Beauveria bassiana bioinsecticides, obtained by spray-drying, in a matrix of sodium alginate and maltodextrin. These biopolymers generally exhibit biocompatibility with EPF, are biodegradable, water-soluble, and provide thermal and osmotic protection to fungi (Martínez-Cano et al. 2022). Therefore, in this study a new formulation based on B. bassiana microencapsulated in biopolymers by spray-drying was developed, evaluating its bioinsecticidal against Plutella xylostella (Linneaeus) (Lepidoptera: Plutellidae) and heat-tolerant potential.

Methods

Origin of EPF and Plutella xylostella breeding

The EPF used in this study was an isolate of the species Beauveria bassiana (PSI strain), originated from the Entomopathogenic Fungus Bank of the Laboratory of Biotechnological Pest Control—LCBiotec (Sergipetec—Sergipe Technological Park, São Cristovão/SE). This isolate was obtained from a psyllid insect (Hemiptera: Psyllidae) collected in the state of Sergipe, Brazil, and identified through the observation of its morphological characteristics and molecular sequencing of the ITS region (unpublished data). At the LCBiotec Entomopathogenic Fungus Bank, this fungus is maintained on 5-mm disks of Potato Dextrose Agar (PDA), stored in cryogenic tubes containing a 10% glycerol solution, and refrigerated at − 20 °C.

For the breeding of P. xylostella, adult insects were kept in entomological cages (52 × 52 × 42 cm), where cabbage leaves were arranged for their oviposition and honey solution (10%) for food supply. The cabbage leaves containing the eggs were removed daily and transferred to plastic trays (20 × 4 × 15 cm). After the eggs hatched, the caterpillars were fed with new cabbage leaves and when they reached the pupal stage, the insects were transferred to an entomological cage, to continue the development of the insects. The insects were maintained in air-conditioned room (25 ± 2 °C, RH 60%, photoperiod 12–12 h).

Effect of temperature on the viability of aerial conidia of Beauveria bassiana dried using spray-drying

The fungus B. bassiana was produced on rice substrate. Briefly, the fungal conidia were separated from the rice by washing the substrate with Tween 80® solution (0.05%, m/V), and the initial concentration of the suspension was determined by direct counting of conidia using a Neubauer chamber and an optical microscope (400×). The concentration of the fungal suspension was adjusted to 1 × 10⁹ conidia/mL.

To evaluate the tolerance of B. bassiana conidia to spray-drying conditions, 100 mL of the fungal suspension was dried in a bench top spray drier (Haurok SD 1.8 L/H, Orlando, FL, USA) using two temperatures (60 and 70 °C), with the remaining equipment parameters set as follows: air flow = 2400 L/h, compressed air = 5.94 × 10⁶ L/h, and feed rate = 0.6 L/h (Braga et al. 2019). After drying, the viability of the fungal conidia was analyzed by evaluating colony-forming units (CFU). For this analysis, a mass of 0.1 g of dried conidia was transferred to Erlenmeyer flasks (5 mL) containing 10 mL of Tween 80® (0.05%, m/V) and constantly agitated on a shaker (SolabCientífica, Piracicaba, São Paulo, Brazil) for 30 min (150 rpm). Serial dilutions were then performed, and a volume of 100 μl of the dilution corresponding to a concentration of 10⁶ conidia/mL was inoculated onto the surface of Petri dishes (80 × 15 mm) containing Potato Dextrose Agar (PDA) with chloramphenicol (0.25 mg/mL) and tetracycline (0.20 mg/mL). The suspension was spread using a Drigalski loop, and the Petri dishes were sealed and incubated in a B.O.D. germination chamber (SL-225/364U, SolabCientífica, Piracicaba, São Paulo, Brazil), at 25 ± 2 °C, photoperiod 12:12-h, for 5 days.

Evaluation occurred on the fifth day of incubation, where the number of colonies contained in the culture medium was counted, and the CFU value was calculated based on the formula described by Braga et al. (2019).

where

$${\text{CFU}}\, = \,{\text{Number}}\,{\text{of}}\,{\text{fungi}}\,{\text{colonies}} \, \times \, {\text{Volume}}\,{\text{correction}}\,{\text{factor}} \, \times \,{\text{Dilution}}\,{\text{factor }}$$

Subsequently, the CFU data were transformed into a viability percentage using the formula:

$$\% \,{\text{Viability}}\, = \,\frac{{{\text{CFU}}}}{{{\text{Initial}}\,{\text{conidia}}\,{\text{concentration}}}}\, \times \,100$$

The suspension used for drying was also evaluated for its initial viability (control) by determining the CFU, using a similar methodology as described above. All the analyses were performed in triplicate.

Microencapsulation of Beauveria bassiana aerial conidia in sodium alginate and maltodextrin by spray-drying

For the microencapsulation of B. bassiana with sodium alginate, concentrated solutions of 1, 2 and 3% (m/V) of the polysaccharide were prepared and mixed with the fungal suspension to produce 100 mL of the mixture with a final concentration of 1.0 × 10⁹ conidia/mL. The mixtures were kept under constant magnetic stirring for 30 min to ensure complete homogenization. Subsequently, the polysaccharide–fungus mixtures were subjected to spray-drying, following the drying parameters described before. The same process was repeated with maltodextrin, using the concentrations of 5%, 8%, and 10% (m/V).

The effect of combining sodium alginate solution at 2% (m/V) sodium alginate with different concentrations of maltodextrin 5, 8, and 10% (m/V) was also evaluated using the same method describe above. All formulations were dried and analyzed in triplicate. All formulations were evaluated for their viability (using the method described before).

Characterizations of the microencapsulated formulation

The microencapsulated formulation, composed of 2% sodium alginate and 8% maltodextrin, underwent morphological and size analysis, hygroscopicity, conidia concentration, moisture content, water activity, wettability, pH, and suspensibility properties following guidelines outlined by the Collaborative International Pesticides Analytical Council (CIPAC).

Scanning electron microscopy

Micrographs of the formulation were acquired utilizing a sputter coater (Denton Vacuum, Desk V) and a scanning electron microscope (SEM) (HITACHI, TM 3000, Tokyo, Japan) operating at a magnification of 2000×.

Conidia concentration and release

The concentration of conidia present in the formulation was evaluated adding 0.1 g of the formulated material in an Erlenmeyer flask (50 mL) containing 10 mL of Tween 80 (0.05%, v/V). The material remained under constant stirring in a rotary shaker (SL-225, SolabCientífica, Piracicaba, São Paulo, Brazil) at 150 rpm for 50 min. After this period, a serial dilution was performed and the amount of conidia in suspension was counted and expressed in conidia per gram. The evaluation was conducted in triplicate.

Hygroscopicity

A mass of 0.1 g of samples of pure sodium alginate, pure maltodextrin, and the formulation 2% sodium alginate (m/V) added with 8% maltodextrin (m/V) plus fungus was placed in pre-weighed Petri dishes (35 × 10 mm) and stored in a desiccator containing a saturated solution of sodium chloride, with a relative humidity of 92%, for seven days at room temperature (25 ± 2 °C). The hygroscopicity was expressed in grams of water absorbed by the sample per 100 g of dry matter.

Moisture content and water activity (aw)

Moisture content was evaluated by thermo-gravimetric method. Briefly, a mass of 0.1 g of microencapsulated conidia was placed in the Petri dishes (15 × 55 mm) and dried in a drying oven (SL-100, SolabCientífica, Piracicaba, São Paulo, Brazil) at 100 °C, until constant weight was reached. Moisture was determined as the percentage of weight lost in the sample after drying. Water activity (aw) was evaluated using a benchtop water activity analyzer (Aqualab, Series 3 TE, Pullman, WA, USA). Moisture content and water activity were evaluated in duplicate.

Wettability test

For the wettability test, a mass of 1 g of microencapsulated material was added to 250-mL beakers containing 100 mL of distilled water and soft water (water with reduced salt concentration). The time required for the sample to be completely wetted, without agitation, was recorded using a stopwatch. The evaluation was conducted in duplicate, and the result of the time required for the sample to be completely wetted was presented as the average time in seconds (s).

pH evaluation

The pH of the microencapsulated material was evaluated using 1 g of the microencapsulated material added to 250-mL glass beakers and completely dissolved in 100 mL of distilled water and soft water. The material remained under constant magnetic stirring for 1 min, and then, the pH was measured using a benchtop pH meter (KASVI, K39-1014B, Prismalab, RJ, Brazil).

Suspensibility test

The suspensibility of the formulated product was evaluated using 1 g of microencapsulated material added to 250-mL beakers containing 100 mL of distilled water and soft water, separately. The beaker was sealed and then shaken for 2 min. After this period, 150 mL of distilled water and soft water were added to their respective beakers, which were sealed again and shaken until complete dissolution of the microencapsulated material. Then, the material was left to stand for 30 min and, after this timeframe, 90% of the supernatant was removed. The residual material was transferred to pre-weighed Petri dishes and dried at a temperature of 120 °C (SL-100, SolabCientífica, Piracicaba, São Paulo, Brazil). The dried material was weighed until a constant weight was obtained. The suspensibility rate (SR) was measured based on the following equation:

$${\text{SR}}\, \left( \% \right) = \frac{{{\text{M}}_{1} - {\text{M}}_{2} }}{{{\text{M}}_{1} }} \times 90$$

where M₁ is the initial mass (g) and M₂ is the final mass (g).

Evaluation of the thermos-protective effect of the microencapsulated formulation of Beauveria bassiana conidia

A mass of 0.1 g of the formulated material was placed in pre-weighed Petri dishes and then placed in a B.O.D. (SL-225, SolabCientífica, Piracicaba, São Paulo, Brazil) at a temperature of 45 °C for 10 intervals: 2, 4, 6, 8, 10, 12, 24, 36, 48, and 60 h. After this period, the microencapsulated formulation was transferred to Erlenmeyer flasks (50 mL) containing 10 mL of Tween 80® (0.05%, m/V) and constantly stirred on a rotary shaker at 150 rpm (SL-225, SolabCientífica, Piracicaba, São Paulo, Brazil) for 30 min. Then, serial dilution was performed, and the conidia present in the suspension were quantified by direct counting using a Neubauer chamber (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and optical microscope (400×). The suspension concentration was adjusted to 1.0 × 10⁶ conidia/mL, inoculated in Petri dishes (80 × 15 mm) containing PDA culture medium, sealed, and incubated in a B.O.D. germination chamber (SL-225/364U, SolabCientífica, Piracicaba, São Paulo, Brazil), 25 ± 2 °C, photoperiod 12:12-h, for five days. The viability analysis was determined by assessing the number of CFU, according to the methodology described above. For comparison, non-formulated B. bassiana suspension was also evaluated, following the same method. All the analyses were conducted in triplicate (Velavan et al. 2022).

Pathogenicity and virulence of the microencapsulated Beauveria bassiana against Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae)

Cabbage (Brassica oleracea L.) leaf disks (Ø 8 cm) were dipped in 20 mL of the suspensions (1.0 × 10⁶ conidia/mL) and kept at room temperature (26 ± 2 °C) to dry. Then, the cabbage disks were transferred to Petri dishes (90 × 10 mm), where P. xylostella caterpillars, which emerged at 72 h (2nd instar), were placed. The Petri dishes were sealed and placed in an air-conditioned room (26 ± 2 °C, RH 60 ± 10%, and photoperiod 12–12 h). The bioassay evaluation was carried out daily, during 7 days, and the dead insects were transferred to a humid chamber (Petri dish containing filter paper moistened with sterile distilled water) to favor the development of the fungus and confirm the death of the insect due to the action of the pathogen. The experiment was carried out with two treatments, referring to the microencapsulated and non-formulated B bassiana conidia, with eight replications and twenty P. xylostella caterpillars in each replication.

Statistical analysis

The results obtained in evaluation of conidia viability after microencapsulation in biopolymers were subjected to analysis of variance (ANOVA), Tukey test (p < 0.05). The results of the viability of unformulated fungi dried in different temperatures by spray-drying and of the wettability, pH, and sedimentation rate of the microparticles, using distilled and soft water, were analysis by T test (p < 0.05). Data from the microencapsulated fungus thermos-tolerance assay were analyzed by linear regression. The daily mortality data of P. xylostella caterpillars treated with the formulated and unformulated B. bassiana conidia were subjected to survival analysis using the Kaplan–Meier method (Log Rank: Mantel-Cox), to estimate the LT₅₀ of the treatments. All statistical analyses were performed using SPSS 22.0.0 software, and graphs were made by GraphPad Prism 8.01 software.

Results

Effect of temperature on the viability of aerial conidia of Beauveria bassiana dried using spray-drying

Different temperatures tested to dry B. bassiana conidia by spray-drying promoted significant differences in the fungi viability (T_1,4 = 17.11, p = 0.01442). The conidia viability ranged from 55.2 ± 5.27 to 39.8 ± 3.89%, using 60 and 70 °C, respectively (Table 1).

Table 1 Viability of Beauveria bassiana conidia after drying by spray-drying at different temperatures

During the drying process, it was also noted that the inlet temperature of 60 °C promoted the deposition of the conidia suspension on the internal walls of the spray-drying chamber, indicating that the inlet temperature of 60 °C was not effective for drying the conidial suspension. This deposition did not occur when using the temperature of 70 °C (Fig. 1). Furthermore, the material collected at 60 °C exhibited a viscous appearance due higher moisture content, in contrast with the drier appearance powder observed for the material dried at 70 °C.

Fig. 1

Visual appearance of the spray-drying chamber after drying process of Beauveria bassiana conidia, at two different inlet temperatures of 60 °C (A) and 70 °C (B)

Microencapsulation of Beauveria bassiana aerial conidia in Sodium alginate and maltodextrin by spray-drying

Different concentrations of sodium alginate tested for the encapsulation of B. bassiana significantly affected the conidia viability subject to the spray-drying process, when comparing to the drying of unformulated conidia (F_3,8 = 148.923, p = 0.000) (Fig. 2). The use of 2% sodium alginate improved the conidia preservation, with the highest viability of 51.6 ± 0.75%, whereas 3% sodium alginate reduces the viability of B. bassiana conidia down to 16.6 ± 6.13%.

Fig. 2

Conidia viability (%) of Beauveria bassiana drying by spray-drying process with the biopolymer Sodium alginate. Conidia = unformulated conidia, C+Alg 1% = conidia encapsulated with the combination of the 1% Sodium alginate, C+Alg 2% = conidia encapsulated with the combination of the 2% Sodium alginate, C+Alg 3% = conidia encapsulated with the combination of the 3% Sodium alginate. All the treatments were dried at 70 °C

The use of different concentrations of maltodextrin for the encapsulation of B. bassiana also significantly affected the conidia viability subjected to the spray-drying process, when comparing with the drying of unformulated conidia (F_3,8 = 32.951, p = 0.000) (Fig. 3). The use of maltodextrin improved the conidia preservation, with viability of 54.37 ± 2.65%, 62.61 ± 3.22%, and 57.61 ± 1.43%, for the use of 5%, 8%, and 10% of maltodextrin, respectively. These results demonstrate that increasing the concentration of maltodextrin does not necessarily increase the conidia viability in spray-drying process microencapsulation, having better protection effectiveness (upper limit) with the use of 8% of this sugar.

Fig. 3

Conidia viability (%) of Beauveria bassiana drying by spray-drying process with the biopolymer maltodextrin. Conidia = unformulated conidia, C+Malt 5% = conidia encapsulated with the combination of the 5% maltodextrin, C+Malt 8% = conidia encapsulated with the combination of the 8% maltodextrin, C+Malt 10% = conidia encapsulated with the combination of the 10% maltodextrin. All the treatments were dried at 70 °C

The use of sodium alginate and maltodextrin as encapsulating agents for the microencapsulation of B. bassiana conidia by spray-drying showed promising results in preserving conidia viability. From these results, tests were performed with the combination of 2% sodium alginate and concentrations of maltodextrin (5, 8 and 10%) to increase the conidia viability after the drying process. The combination of different concentrations of maltodextrin with 2% sodium alginate resulted in a significative effect in conidial viability (F_3,8 = 62.201, p = 0.000); however, the formulation composed of 2% sodium alginate and 8% maltodextrin presented the highest viability value (89.5 ± 4.92%). The use of 2% sodium alginate +5% maltodextrin and 2% sodium alginate +10% maltodextrin promoted 58.6 ± 4.54 and 72.8 ± 4.06% of conidial viability, respectively (Fig. 4).

Fig. 4

Conidia viability (%) of Beauveria bassiana drying by spray-drying process with the biopolymers sodium alginate and maltodextrin. Conidia = unformulated conidia, C+Alg 2% + Malt 5% = conidia encapsulated with the combination of the 2% Sodium alginate and 5% maltodextrin, C+Alg 2% + Malt 8% = conidia encapsulated with the combination of the 2% Sodium alginate and 8% maltodextrin, C+Alg 2% + Malt 10% = conidia encapsulated with the combination of the 2% sodium alginate and 10% maltodextrin. All the treatments were dried at 70 °C

Characterization of the microencapsulated formulation

The micrographs of the non-formulated B. bassiana conidia (not subjected to the encapsulation process) and B. bassiana conidia microencapsulated in 2% sodium alginate and 8% maltodextrin by spray-drying are presented in Fig. 5.

Fig. 5

Scanning electron micrographs (2000 ×) of non-formulated Beauveria bassiana conidia (A) and microparticles of Beauveria bassiana conidia loaded in 2% sodium alginate and 8% maltodextrin by spray-drying (B). The arrow indicates the presence of Beauveria bassiana conidia close to the surface of the microparticle (B)

Conidia release was monitored over a 50-min period, peaking at 30 min, and remaining constant thereafter (Fig. 6). Within the first 10 min, conidia release reached 44% of the maximum capacity, totaling 5.60 × 10⁷ conidia/mL. By the 20-min’ mark, release increased to 67% of the maximum capacity, reaching 8.56 × 10⁷ conidia/mL, ultimately achieving total release of 1.27 × 10⁸ conidia/mL reaching 100% released.

Fig. 6

Release time of B. bassiana conidia loaded into 2% sodium alginate and 8% maltodextrin microparticles

The results of the analysis of hygroscopicity, conidia concentration, moisture content, water activity, wettability, pH, and suspensibility rate are shown in Table 2. The B. bassiana conidia microencapsulated showed hygroscopicity of 20.08 g/100 g. Hygroscopicity is the capacity to absorb water from the atmosphere and can affect the stability of bioinsecticide formulations, stimulating the conidia germination in storage period. Materials with a moisture absorption exceeding 15% are considered very hygroscopic (Murikipudi et al. 2013). Both polymers used and the formulation are highly hygroscopic; however, evaluations indicate that formulations with lower moisture content were more likely of exhibit greater hygroscopicity. Consequently, it can be asserted that both the employed polymers and the resulting formulation can be categorized as highly hygroscopic materials.

Table 2 Physicochemical properties of microcapsules of sodium alginate and maltodextrin with Beauveria bassiana conidia

Evaluation of the thermoprotective effect of the formulation on encapsulated conidia

The results of the thermotolerance analysis of the B. bassiana conidia formulated and unformulated indicate that the combination of sodium alginate and maltodextrin polymers is effective in promoting thermal protection to B. bassiana conidia exposed to 45 °C (Fig. 7). After being exposed to 45 °C for 60 h, the conidia encapsulated in 2% sodium alginate and 8% maltodextrin showed 45.02 ± 4.91% of conidial viability, while the unformulated suspension completely lost its viability after 4 h.

Fig. 7

Conidia viability of B. bassiana conidia formulated (microencapsulated in 2% sodium alginate and 8% maltodextrin) and unformulated, exposed to 45 °C for 60 h

Pathogenicity and virulence of the microencapsulated B. bassiana against Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae)

The formulated and non-formulated B. bassiana conidia showed significant differences in increasing mortality in P. xylostella caterpillars (T = − 3.870, df = 9, p = 0.004), resulting in values of 83.1 ± 5.5% and 64.8 ± 9.9%, respectively (Fig. 7). The average time required to induce mortality in 50% of the insects (LT₅₀) was also calculated for the treatments. No significant difference was observed in the estimated LT₅₀ values of the treatments, with LT₅₀ values of 5.889 days (5.540–6.238), for the unformulated conidia, and 5.504 days (5.220–5.788) for the formulated conidia (Fig. 8).

Fig. 8

Evaluation of potential insecticide of microencapsulated B. bassiana conidia by pathogenicity assay (left) and survival analysis (right). For comparison, unformulated conidia were also used in the evaluations. For the control in survival analysis, the caterpillars of P. xylostella were treated with Tween 80® solution (0.05%, v/v)

Discussion

The effect of exposure to high temperatures on the aerial conidia of B. bassiana showed a negative impact on viability, with a significant decrease observed between the temperatures of 60 °C and 70 °C. The visual characteristics of the spray-drying chambers indicate differences in moisture content between the two tested temperatures. The high moisture content in the conidia can cause instability during storage and in the microencapsulation process of the EPF (Ziaee et al. 2019). Therefore, the temperature of 70 °C was selected for the drying in the subsequent stages of formulation development.

The thermal protective capacity of sodium alginate as a wall material was observed in the microencapsulation of other microorganisms by spray-drying. The bacterium Proteus penneri showed 93.88% of viability before dried at 125 °C by spray-drying with use of the 1% sodium alginate (Noor et al. 2022). The encapsulation of Lactobacillus zae in 1% sodium alginate by spray-drying resulted in a viability of 81.98 ± 4.45% (Liu et al. 2018). The results observed in the present study for the microencapsulation of B. bassiana conidia in sodium alginate by spray-drying indicate that increasing the concentration of sodium alginate as a wall material in the microparticles diminishes the impact of temperature on conidia viability during the drying process. However, it was also observed that there was a higher concentration threshold that promotes effectiveness in protecting conidia, which in our study was the concentration of 2% sodium alginate (51.6 ± 0.75%). When exceeding this threshold, the alginate reduced its protective effectiveness, as observed in our study with the use of 3% sodium alginate (16.6 ± 6.13%).

The reduction in conidia viability observed with the use of 3% sodium alginate may have occurred due to the formation of particles with heterogeneous sizes or because of cell shear induced by heightened viscosity of the formulation. This could result in inadequate distribution of the liquid through the atomizing nozzle of the equipment, thereby causing variations in particle morphology and exposing conidia to the inlet temperature of the apparatus, compromising the preservation of the actives. Furthermore, high concentrations of alginate may have caused osmotic stress, resulting in the formation of a hyperosmotic environment in which the cell is more prone to dehydration, which can lead to cell death (Tapia et al. 2015). Therefore, it is possible to conclude that sodium alginate provides thermal protection in B. bassiana conidia during microencapsulation by spray-drying; however, its protective capacity is directly related to the concentration used.

However, as happened with sodium alginate, there is a threshold on the concentration of maltodextrin to be effective in the microencapsulation of B. bassiana conidia, showing a reduction in the conidia viability with the use of 10% maltodextrin (57.61 ± 1.43%). Previous studies indicate that the maltodextrin concentration necessary for the effective encapsulation of microorganisms by spray-drying is directly related with the microorganism species used (Bagdat et al. 2024). The results obtained in the present study reinforce the hypothesis that there was a threshold for the use of polymers, such as maltodextrin, as encapsulating agent and this is especially governed by the characteristics of the active and the selected polymer.

The combined use of maltodextrin with other biopolymers is reported to be effective in preserving EPF and other microorganisms encapsulated by spray-drying.

In the present study, the combination of 2% sodium alginate and 8% maltodextrin increased the viability of B. bassiana conidia when compared to the isolate effect of these polymers in the conidia viability. Maltodextrin and sodium alginate exhibit properties that favor the preservation of cells and biomolecules in encapsulated systems. Maltodextrin is a biopolymer osmotically inactive, with low viscosity that allows to fill in the space between the cells, strengthening the encapsulating matrix. Sodium alginate promotes high mechanical stability, improving the mechanical resistance of the formed microparticles. Furthermore, both polymers contribute to the thermal protection of the encapsulated agent (Agudelo-Chaparro et al. 2022; Iñiguez-Moreno et al. 2021). Therefore, the enhanced viability results of the B. bassiana, encapsulated with the combination of sodium alginate and maltodextrin, indicate the existence of a synergistic effect between the polymers in the thermal protection of the encapsulated conidium by spray-drying.

The non-formulated B. bassiana conidia image in SEM showed circular morphology, with an average diameter of 2 μm, which is aligned with the literature. The microparticles produced with 2% sodium alginate and 8% maltodextrin as wall material showed a smooth surface, irregular morphology, and varying sizes (diameter ranged from 2.1 to 188.4 μm). No conidia adhered to the surface of the microparticles was observed. Microparticles produced with biopolymers by spray-drying are not uncommon to show smooth surfaces (Sakoui et al. 2022) but irregular morphology and size variation depending on the polymers and parameters used in formulation process (López-Cruz et al. 2020). These results suggest that irregular microparticle morphology is an inherent characteristic of microparticles obtained by spray-drying sodium alginate and maltodextrin as biopolymers.

The release test determines the duration needed for complete release of the active ingredient from the microparticles. For bioinsecticide formulations, this test provides information about the functionality and behavior of the microencapsulated product after application, with the gradual release of the active ingredient being common in microencapsulated systems. The results indicate that a minimum of 30 min of vigorous agitation is necessary for complete release of the microencapsulated products to ensure optimal effectiveness upon resuspension.

The concentration analysis of microencapsulated B. bassiana conidia showed a concentration value of 5.44 × 10⁸ conidia/g. This value is higher than the concentration minimum established and recommended to products based on commercially distributed EPF (1.00 × 10⁸ conidia/g) (Mascarini and Paulo 2010).

The microencapsulated B. bassiana conidia showed values of 6.19 ± 0.07% and 0.33 ± 0.00, for the moisture content and water activity (aw), respectively. Moisture and water activity are parameters that influence the maintenance of conidia viability during shelf storage. Moisture content is a measure of the amount of water present in a product; water activity (a_w) is a measure that expresses the amount of water available for chemical reactions and biological activities in each environment (Syamaladevi et al. 2016). The moisture and a_w values of the formulation of microencapsulated B. bassiana conidia in sodium alginate and maltodextrin are below of 10% and 0.4, respectively, ideal values for maintaining the viability of the fungus in storage.

The wettability data obtained for the analysis of microencapsulated B. bassiana conidia using soft and distilled water did not show a significant difference. The average wettability time for the formulated material was 827 s for distilled water and 465 s for soft water. Wettability affects the dispersion of the active ingredient in liquids and its availability on the applied surface. Wettable powders typically exhibit a wettability time of around 120 s (2 min) (Diao et al. 2021).

The results indicate a longer wetting time for the sample, suggesting it lacks wettable powder characteristics. The need for prolonged wetting time may stem from two factors: the formulation's composition and the absence of wetting adjuvant. Regarding composition, it is composed of sodium alginate and maltodextrin; while maltodextrin is known for having high solubility in aqueous media, sodium alginate is known for forming gels due to its viscosity, a factor that may have contributed to the prolonged water absorption time (Chen and Bonaccurso 2014; Zhong et al. 2010).

The pH values of the microencapsulated formulation resuspended in different types of water did not show significant differences, with pH values of 6.62 ± 0.13 and 6.46 ± 0.12, with the use of distilled water and soft water, respectively. pH values can impact the viability and development of B. bassiana, which normally have an ideal pH for their development between 6 and 7 (Dhar et al. 2016), as observed in our study.

The suspensibility rate of formulation showed no significant difference when resuspended in distilled water and soft water, with sedimentation rates of 88.2 ± 1.55% and 85.8 ± 2.89%, respectively. The suspensibility test evaluates a material's ability to stay suspended when mixed with liquids (Nasir et al. 2023). According to Collaborative International Pesticides Analytical Council (CIPAC) guidelines, uniform pesticide distribution during application requires a high suspensibility rate in suspension, with excellent suspensibility at rates above 70% (Tsujo et al. 1983). The presence of little sedimented material after 30 min in both water types indicates the formulation's ability to remain suspended post-solubilization, favoring the maintenance of suspension homogeneity during application.

The development of EPF-based formulations aims to provide improvements in the storage, application, and effectiveness of these microorganisms in the field, e.g., crop protection (de Jesus Oliveira et al. 2024). The temperature presented during the storage period can affect the viability of EPF conidia formulations, reducing the quality of these formulations.

To solve this problem, evaluations have been carried out to assess the thermos-protective capacity of the EPF formulations.

The thermal protection capacity of the formulation developed in this study is attributed to the polymers used in the encapsulation of B. bassiana conidia. Sodium alginate and maltodextrin act synergistically as a wall material, providing greater thermal resistance to the encapsulated conidium when exposed to high temperatures. Therefore, the combination of the sodium alginate and maltodextrin used for microencapsulation of B. bassiana conidia promoted thermal protection for the fungus.

Pathogenicity analysis and LT₅₀ assessment provide a comprehensive view of bioinsecticide efficacy. While the pathogenicity test indicates the lethal capacity of the formulation, the estimated LT₅₀ shows the virulence of the pathogen. The high mortality observed in the P. xylostella caterpillars exposed to the formulation, when compared to the unformulated conidia application, may be attributed to the production of fungal metabolites induced by heat stress promoted by the drying process. The heat stress can affect the fungal physiology, inducing the production of heat shock proteins, which can increase the pathogenicity and virulence of the fungus. However, the estimated LT₅₀ of the microencapsulated formulation does not present a significant difference with the LT₅₀ of non-formulated conidia, not affecting the virulence of the fungus.

Conclusions

The current investigation demonstrates advancements in utilizing the spray-drying technique for the microencapsulation of B. bassiana conidia. The increase in temperature in the drying process reduces the viability of aerial conidia of B. bassiana by spray-drying. However, the microencapsulation of the conidia with sodium alginate and maltodextrin increases the preservation of conidial viability and improves the thermotolerance of the fungus. Furthermore, the microencapsulated B. bassiana conidia present insecticide efficacy against P. xylostella caterpillars. Therefore, the use of polymers in microencapsulated systems for the development of B. bassiana based formulations may be an advantageous alternative for the preservation and application of these microorganisms in biological control of agricultural pests.