Optimizing Culture Conditions for Bacillus subtilis Strains from Indigenous Chicken Feces as Antibiotic Replacements in Poultry FarmingABSTRACT
The extensive use of antibiotics in poultry farming has contributed to the rise of antibiotic-resistant bacteria, which poses serious risks to both animal and human health. As an alternative, probiotics such as Bacillus subtilis have shown potential in suppressing pathogenic bacteria and enhancing gut health, making them promising replacements for antibiotics in poultry production. This study focused on isolating and characterizing B. subtilis strains from indigenous chicken feces, specifically targeting strains with strong antibacterial activity against Escherichia coli and Salmonella typhimurium, which are common pathogens causing diarrhea in poultry. Key parameters for optimizing spore production of selected B. subtilis strain were examined, including carbon source, nitrogen source, mineral salts, inoculum age, inoculum size, medium volume, and initial pH. From an initial pool of 168 Bacillus spp. strains isolated from the feces of free-range indigenous chickens, B. subtilis BA07 was selected. The optimal culture medium for spore production was identified as 20 g/L glucose, 30 g/L soybean meal, and 1 g/L K2HPO4. Ideal fermentation conditions included a temperature of 37 °C, 12 hours of incubation, an initial pH of 7.0, an inoculum size of 2%, and a medium volume of 20 mL in a 250 mL flask. Under these optimized conditions, the spore yield of B. subtilis BA07 increased by 10.28 times compared to the initial medium, with a spore formation rate exceeding 90%. This study provides a basis for the large-scale production of B. subtilis BA07 biopreparations, reducing antibiotic dependence in poultry farming.
Keywords:
Antibacterial activity; antibiotic alternative; Bacillus subtilis BA07; poultry farming; spore production
INTRODUCTION
The rising antibiotic resistance in animals, which can also transfer to human pathogens, prompted the European Union (EU) to ban antibiotics as growth promoters (AGPs) in 2006. Vietnam followed suit with Directive No. 468/CN-TACN issued on March 31, 2017 by the Department of Livestock Production, mandating the cessation of AGP imports in livestock feed. To eliminate AGPs in poultry production, producers have adopted various strategies, including (i) incorporating live microbial supplements (probiotics) that benefit the host by enhancing gut microbial balance, and (ii) modifying management practices to maintain livestock productivity (Teillant and Laxminarayan, 2015). Among these, bacterial spores from the genus Bacillus, particularly Bacillus subtilis, have emerged as promising alternatives due to their heat resistance and extended shelf life (Permpoonpattana et al., 2012), enabling them to endure feed pelleting processes and reach the animal gut.
Previous studies have demonstrated that B. subtilis can inhabit the intestines of various mammalian species, highlighting its potential to enhance gut health (Guo et al., 2006; Fakhry et al., 2008). While there are numerous reports on the use of B. subtilis spores as feed additives, detailed characteristics of specific B. subtilis strains are often not disclosed. Incomplete characterization can lead to the misidentification of probiotic strains on product labels and may inadvertently introduce strains with virulence factors, toxins, antibiotic resistance, or harmful metabolites.
Additionally, many researchers emphasize that probiotics isolated from the host can produce more effective biopreparations compared to bacteria isolated from other sources, as they provide better survival opportunities and reduce challenges associated with introducing foreign bacteria (Chiang et al., 2015; Dowarah et al., 2017). Optimizing the fermentation environment is a critical step in enhancing probiotic yield. Appropriate media formulation and cultivation conditions can significantly increase strain biomass or spore yield, while reducing production costs. Furthermore, optimizing the environment offers valuable insights for large-scale microbial product manufacturing. Based on the initial culture media, optimal fermentation conditions for B. subtilis were determined through single-factor and orthogonal experiments, laying the groundwork for large-scale applications.
This study is the first to focus on isolating and selecting potential B. subtilis strains from indigenous chicken feces to combat Gram-negative pathogens (E. coli and Salmonella spp.) responsible for diarrhea in broilers. Subsequently, the optimal submerged fermentation conditions for producing biomass of potential B. subtilis strains were established for probiotic formulation in livestock farming. In this research, some components of the traditional culture medium were replaced with more economically viable, locally available alternatives, aiming at scalability in industrial production.
MATERIALS AND METHODS
Pathogenic Bacteria
The study used Escherichia coli FG31-1 and Salmonella typhimurium FC13827 (Genbank ID: CP142680.1 and MN704402.1), which possess the virulence genes invA and stn. These strains were isolated from the feces of indigenous chickens with suspected infections of E. coli or Salmonella spp., and are maintained at the Microbiology Laboratory, Faculty of Animal Science and Veterinary Medicine, Hue University of Agriculture and Forestry, Vietnam.
Culture Media
Yeast Soluble Peptone (YSP) with 20 g/L sucrose, 10 g/L yeast extract, 10 g/L tryptone, and 5 g/L NaCl at pH 7.0-7.2; Luria-Bertani (LB) containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl at pH 7.0-7.2; Nutrient Agar (NA) composed of 20 g/L glucose, 5 g/L tryptone, and 3 g/L beef extract at pH 7.0-7.2; and Nutrient Broth (NB) with 10 g/L tryptone, 3 g/L beef extract, and 5 g/L NaCl at pH 7.0-7.2.
Isolation and Identification of Bacillus subtilis
Isolation of Bacillus spp.
Bacillus spp. were isolated from the feces of healthy, semi-free-range indigenous broilers (Ga Kien) that had not received digestive enzyme supplements. Spore enumeration was conducted by heat-treating the fermented broth at 80 °C for 15 minutes to eliminate all vegetative cells, as described by Cazorla et al. (2007). The heat-treated sample was then serially diluted in a saline solution, and 0.1 mL of the appropriate dilutions was spread onto Nutrient Agar plates (Merck, Germany). The plates were incubated at 37 °C for 24 hours. The resulting colonies, representing viable spores, were counted and expressed as Colony-forming units per milliliter (CFU/mL). The spore formation rate was calculated using the formula: Spore rate (%) = (Spore count / Total viable count) × 100. Morphologically distinct single colonies were transferred to fresh plates until consistent monocultures were achieved after three rounds of subculturing. Different colony morphologies were purified and examined using Gram staining, followed by biochemical tests such as lactose, glucose, mannitol, and xylose fermentation, starch hydrolysis, gas production, motility, indole, urease, catalase, H2S production, and Voges-Proskauer, as described in the Manual of Systematic Bacteriology (Claus, 1986). Pure cultures were then propagated in LB broth, with cultures preserved in Eppendorf tubes supplemented with 40% glycerol and stored at -30 °C.
Species Identification of B. subtilis Using API 50 CHB Kit
The API 50 CHB kit (bioMérieux, France), comprising 50 biochemical reactions, was used to identify Bacillus to the species level. After incubation, the bacteria were transferred into the kit wells. After 24-48 hours of incubation, results were interpreted based on color changes: positive (red to yellow) or negative (no color change). The results of the 50 reactions were input into the API web software to determine the Bacillus species and similarity percentage.
Identification of Suspected B. subtilis Strains Via Gene Sequencing
The identification was performed using 16S RNA sequence homology. Genomic DNA was extracted from a single pure colony of each isolated strain using the Bacterial DNA Kit TM (Zymo Research, Cat. No. D6005, USA). A 500 bp fragment of the 16S rRNA gene was amplified for bacterial identification. The 16S rRNA gene was amplified using universal primers with sequences: 27F: (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R: (5’-TACGGYTACCTTGTTACGACTT-3’) (Saminathan and Narayanan, 2015). Thermal cycling conditions included: 1 cycle at 94 °C for 5 minutes; 35 cycles at 94 °C for 1 minute, 55 °C for 1 minute, 72 °C for 1 minute; and a final extension at 72 °C for 15 minutes. PCR products were purified using the ZR-96 DNA Sequencing Clean-up Kit (Zymo Research, USA) as per the manufacturer’s instructions and sequenced on the CLC 7 sequencing system (QIAGEN, Germany). After Sanger sequencing and BLAST analysis, the data were compared with the GenBank database at NCBI for species identification.
Evaluation of Antibacterial Activity Against Gastrointestinal Pathogens
Pathogenic Bacteria
The study used Escherichia coli FG31-1 and Salmonella typhimurium FC13827 (Genbank IDs: CP142680.1 and MN704402.1), which carry the virulence genes invA and stn. These strains were isolated from the feces of indigenous chickens with suspected infections of E. coli or Salmonella spp., and are maintained at the Microbiology Laboratory, Faculty of Animal Science and Veterinary Medicine, Hue University of Agriculture and Forestry.
The antibacterial activity of the Bacillus strains against these pathogens was evaluated using the agar well diffusion method, as described by Aujoulat et al. (2011). Muller Hinton Agar (MHA) plates were overlaid with a bacterial suspension of 0.5 OD630. After allowing the suspension to absorb for 15-20 minutes, six wells (spaced 30 mm apart) were made on the MHA plates. Each well received 100 μL of overnight LB broth culture of the selected Bacillus strains, adjusted to 0.5 OD630. The plates were incubated at 4˚C for one hour to allow diffusion, then incubated at 37˚C for 24 hours. The inhibition zone (diameter, mm) was calculated as: Inhibition Zone Diameter = Diameter of clear zone - Diameter of well. Antibacterial activity was considered significant if the inhibition zone was ≥10 mm (Georgieva et al., 2015).
Optimization of Nutritional and Fermentation Conditions for B. subtilis Strains
Carbon Source Selection
Using YSP medium as the base, sucrose was replaced with alternative carbon sources (glucose, corn flour, glycerol, lactose, maltose, and soluble starch) at concentrations of 10 to 30 g/L. Following flask fermentation, bacterial suspensions were heat-treated at 80˚C for 15 minutes.
Nitrogen Source Selection
After determining the optimal carbon source, tryptone was substituted with various alternative nitrogen sources (including soybean, fish meal, NO3NH4, peanut, and (NH4)2SO4) at concentrations of 10 to 40 g/L. Fermentation and heat treatment followed the same procedure as above.
Inorganic Salt Source Selection
The culture medium was supplemented with CaCO3, FeSO4, MgSO4, ZnSO4, and K2HPO4 at concentrations of 0.1 to 0.9 g/L. Flask fermentation and heat treatment were conducted under similar conditions.
Optimization of Fermentation Conditions
Different fermentation conditions were tested, including incubation times of 8, 10, 12, 14, and 16 hours; inoculum sizes ranging from 1 to 5%; initial pH values of 6.0, 6.5, 7.0, 7.5, and 8.0; and culture volumes of 20, 30, 40, 50 and 60 ml in 250 ml flasks. These conditions were assessed for their impact on the biomass yield of BA07.
Statistical Analysis
Statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). All data were analyzed using analysis of variance (ANOVA) to assess significant differences among treatments. Single-factor experiments were arranged in a completely randomized design with three replicates per treatment. Means were compared using Duncan’s multiple range test at a significance level of p<0.05. For optimization experiments, a three-level orthogonal experiment was designed, focusing on glucose, soybean meal, and K2HPO4, which significantly influenced BA07 fermentation. Inoculum size, initial pH, and culture volume were also optimized across three levels (Table 1). This facilitated the refinement of the culture medium formulation and fermentation conditions. The relative importance of each factor was assessed using range (R) and F-values from ANOVA.
RESULTS
Isolation of Bacillus spp.
Bacterial strains were isolated from 35 fecal samples collected from free-range indigenous chickens not previously treated with probiotics. The samples underwent heat treatment, serial dilution, spread plating, and incubation at 30ºC for 24 hours. After incubation, colonies were classified based on morphology, and each bacterial strain was repeatedly sub-cultured until purity was achieved. A total of 168 bacterial strains resembling Bacillus subtilis were obtained, based on morphological characteristics described by Gingichashvili et al. (2019). These characteristics included Gram-positive bacilli with an opaque white appearance, dry texture, irregular colony margins, cell size greater than 3 µm, central spores, and no alteration in cell shape.
Biochemical Identification
From the 168 strains, biochemical tests were conducted in the following sequence: negative for lecithinase, positive for catalase, Voges-Proskauer (VP), amylase, cellulase, and growth at 50ºC (Phillips, 1993). After excluding non-conforming strains, 121 strains matched the six biochemical characteristics typical of B. subtilis and were further identified using the API CH50B kit. The results indicated that 100% of the B. subtilis strains tested positive for catalase; 58.7% (71/121) were positive for the VP test; 73.2% (52/71) exhibited amylase activity; 52.5% (40/52) grew at 50ºC; and 52.5% (21/40) tested positive for cellulase. These 21 strains were selected for further analysis.
Identification Using API CH50B Kit
Following biochemical characterization, 20 strains exhibited traits consistent with B. subtilis. To confirm species-level identification, additional specific tests were performed using the API CH50 kit (Table 2). The results revealed that 16 strains were identified as B. subtilis, with a high similarity rate (>90%).
Selection of Potential B. subtilis Strains to Replace Antibiotics
Antagonistic Activity Against Two Gram-Negative Enteropathogens
The antagonistic activity of 16 isolated Bacillus subtilis strains was tested against two Gram-negative pathogens, E. coli FG31-1 and S. typhimurium FC13827. As shown in Table 3, 6 of the B. subtilis strains (37.5%) demonstrated inhibitory effects against both pathogens (zone of inhibition ≥ 10 mm). Among the remaining strains, 2 strains (12.5%) inhibited only one of the pathogens, while 8 strains (50%) showed no inhibitory effect against either pathogen (data not shown).
Overall, for E. coli FG31-1, strain BA07 exhibited the highest antibacterial activity with a zone of inhibition of 18.67mm, which was significantly larger (p<0.05) compared to strains BA38 (13.67mm) and BA16 (11.01 mm). In contrast, for S. typhimurium FC13827, strains BA79 and BA81 displayed the highest antibacterial activity, with zones of inhibition of 17.33 mm and 17.0 mm, respectively, both significantly larger (p<0.05) than those of strains BA38 (15.21mm) and BA29 (12.45mm).
Among the strains that showed inhibitory activity against both Gram-negative pathogens, strain BA07 demonstrated superior antibacterial potential and was thus selected for further studies.
Identification of BA07 by Gene Sequencing
The most promising strain, BA07, was selected for 16S rRNA gene sequencing. Phylogenetic analysis revealed that strain BA07 shared 100% similarity with B. subtilis strain BSn5 (GenBank Accession No. CP002468.1) (Figure 1).
A. Phylogenetic tree based on the 16S rRNA gene sequences of B. subtilis strain BSn5 and other strains within the B. subtilis genus. Bootstrap values (from 1000 replicates) are presented at the nodes. The scale bar represents 0.02 nucleotide substitutions per position. B. agarose gel electrophoresis of the PCR-amplified 16S rRNA product: Lane M (1kb marker) and positive lane of B. subtilis strain BSn5 (BA07) at 1500bp.
Effect of Initial Media on Probiotic Biomass Production
The spore biomass of BA07 after fermentation in four different initial media is shown in Figure 2. The highest spore yield was obtained in the YSP medium (14.07 × 108 CFU/mL), which was significantly higher (p>0.05) than in the other three media. As a result, the YSP medium was chosen as the initial fermentation medium for BA07.
Effect of different initial culture media on spore yield of BA07. Values with different letters (a-d) indicate statistically significant differences (p<0.05).
Effect of Carbon Sources and Their Concentrations on Biomass Production
The impact of various carbon sources on the biomass production of BA07 is shown in Figure 3. Among the seven organic carbon sources tested, glucose significantly enhanced the biomass of BA07 compared to other sources, followed by sucrose, soluble starch, lactose, glycerol, and maltose, while cornmeal resulted in the lowest biomass yield. Given the much lower cost of glucose compared to sucrose, glucose was selected as the carbon source for optimizing the culture process.
Effect of different carbon sources and glucose concentrations on spore yield of BA07. Values with different letters (a-d) indicate statistically significant differences (p<0.05).
With increasing glucose concentration, BA07 biomass initially increased but subsequently decreased, reaching its peak at a glucose concentration of 25 g/L, when the biomass of BA07 was significantly higher than at lower concentrations (p<0.05).
Effect of Nitrogen Sources and Their Concentrations on Biomass Production
The evaluation of various nitrogen sources showed that BA07 preferred organic nitrogen sources over inorganic ones (Figure 4). Specifically, soybean meal and fish meal significantly supported higher biomass production compared to other tested organic nitrogen sources. In contrast, inorganic nitrogen sources, such as (NH4)2SO4 and NH4NO3, were less effective and did not promote spore formation. Among the different concentrations of soybean meal evaluated, 30 g/L was found to be optimal for biomass production. Increasing concentrations above 30 g/L did not further enhance biomass, suggesting that higher concentrations could negatively impact oxygen transfer due to increased medium viscosity.
Effects of different nitrogen sources and different concentrations of soybean meal on spore yield of BA07. Values with different letters a-d indicate statistically significant differences (p<0.05).
Effect of Mineral Salts and Their Concentrations on Biomass Production
The assessment of different mineral salts indicated that K2HPO4 had the most pronounced stimulatory effect on BA07 biomass (Figure 5). Therefore, K2HPO4 was chosen as the inorganic salt for subsequent experiments. An optimal concentration of 1 g/L K2HPO4 was determined, which produced significantly higher biomass than lower (0.5 to 1.5 g/L) or higher concentrations (above 2 g/L).
Effects of K2HPO4 and mineral salts on spore yield of BA07. Values with different letters a-d indicate statistically significant differences (p<0.05).
Orthogonal Experiment for Culture Condition Optimization
Based on the effects of varying concentrations of glucose, soybean meal, and K2HPO4 on BA07 biomass, optimal concentrations of these three components were selected for an orthogonal test. As seen from the range values in Table 4, the most important factor influencing BA07 biomass was glucose (A) and soybean meal (B), with K2HPO4 (C) having the least impact. According to the mean value results, the best combination was A2:B3:C1. Thus, the optimal culture conditions were: glucose 20 g/L, soybean meal 30 g/L, and K2HPO4·3H2O 0.5 g/L. The orthogonal test results were analyzed using analysis of variance (ANOVA) (Table 5), and the significance of each factor was evaluated based on the F-value. The order of importance was A: glucose > B: soybean meal > C: K2HPO4, similar to the range analysis. The optimized BA07 production reached 128.00 × 108 CFU/mL.
Optimization of Fermentation Conditions
Results of Figure 6 show that under different strain ages, BA07 biomass initially increased and then decreased. Biomass was highest at 12 hours compared to other stages, indicating that BA07 was strongest during the logarithmic growth phase at 12 hours. Therefore, in optimizing fermentation conditions, a three-factor, three-level orthogonal test for filling volume, and initial pH was conducted, as these had significant effects on fermentation, alongside temperature and strain age. While there was no statistically significant difference between the 2%, 3%, and 4% inoculum sizes; the selection of 2% as optimal was made based on economic considerations.
The orthogonal test results (Table 6) were analyzed using range analysis and F-value assessment. The relative importance of factors influencing BA07 biomass was ranked as follows: inoculum size ~ initial pH > filling volume. The optimal fermentation conditions were determined to be: strain age of 12 hours, inoculum size of 2%, initial pH of 7.0, and a filling volume of 20 mL/250 mL. Under these conditions, fermentation was carried out for 48 hours at 240 rpm and 37˚C. Spore yield reached 141.42 × 108 CFU/mL with a spore formation rate exceeding 90%, which was 10.26 times higher than the initial medium yield.
DISCUSSION
The gut microbiota of indigenous free-range chicken breeds differs significantly from that of commercial breeds due to various factors (Paul et al., 2021). The diverse natural diet available to free-range chickens can enrich their gut microbiota, resulting in higher immunity compared to commercial breeds. Consequently, these chickens are better equipped to combat various infectious diseases (Kannaki et al., 2021). Bacillus represents a major component of the chicken gut microbiota, with concentrations reaching up to 109 g-1 in the caeca (Barnes, 1979). Spores of Bacillus species, particularly B. subtilis, have been identified as promising probiotics candidates due to their heat tolerance and extended shelf life (Permpoonpattana et al., 2012). These characteristics allow them to withstand pellet processing and reach the animal’s gut. Previous studies have shown that B. subtilis can reside in the gut of various mammals, indicating its potential to enhance gut health (Guo et al., 2006; Fakhry et al., 2008).
In this study, 16 B. subtilis strains were selected from a pool of 168 bacterial isolates obtained from fecal samples of indigenous free-range chickens. Among them, 6 strains demonstrated high antibacterial activity against two Gram-negative pathogens that cause gastrointestinal diseases in chickens. These findings highlight the potential of specific B. subtilis strains as natural antibiotic substitutes, aligning with previous research that emphasizes the efficacy of Bacillus strains in inhibiting pathogenic bacteria (Penaloza-Vazquez et al., 2019). The superior performance of strain BA07, selected for its high inhibitory potential, was further confirmed by 16S rRNA gene sequencing, showing 100% similarity to B. subtilis strain BA07. This confirms its identity and potential as an effective probiotic candidate for antibiotic substitution (Golnari et al., 2024).
Spore formation in Bacillus species is influenced by nutritional sources and environmental factors. To achieve high yields in Bacillus preparations, optimizing liquid fermentation conditions to enhance biomass and spore production is essential. This study analyzed the effects of culture conditions - including carbon sources, nitrogen sources, and inorganic salts-as well as fermentation parameters such as strain age, inoculum size, filling volume, and initial pH on the production yield of B. subtilis BA07 through single-factor experiments and orthogonal testing. Spores in Bacillus preparations are critical due to their high stress resistance. Thus, spore-forming ability and yield are crucial for potential applications.
Research has shown that spore formation is influenced by both nutritional and environmental factors. Optimizing liquid fermentation conditions to increase biomass and spore yield is essential for producing high-yield Bacillus preparations (Li et al., 2019). This study examined the impact of culture conditions (carbon source, inorganic salts, nitrogen source) and fermentation conditions (inoculum size, strain age, filling volume, initial pH) on Bacillus spp. yields using single-factor and orthogonal tests. Carbon is a primary component of the microbial environment, providing essential nutrients and energy for microbial growth and reproduction. Among the different carbon sources tested, glucose was identified as optimal for BA07 growth, aligning with Li et al. (2019), who found glucose to be the most efficient carbon source for B. subtilis (Das et al., 2021).
Nitrogen sources provide essential structural materials and nitrogen for microbial metabolism and can serve as an energy source when carbon is not readily available. Organic nitrogen sources contain not only soluble proteins, peptides, and free amino acids, but also sugars, inorganic salts, fats, vitamins, and metabolic precursors. This study demonstrates that organic nitrogen sources are better suited for Bacillus spp’s growth. Inorganic salts play a crucial role in supporting microbial growth and metabolite production. They promote microbial growth and metabolism at lower concentrations, while at higher concentrations, they exhibit a clear inhibitory effect. This study shows that K2HPO4·3H2O is beneficial for Bacillus spp. preparations. In addition to essential microbial nutrients, the medium must maintain an appropriate ratio of components to ensure maximum yield.
Current common medium optimization methods include Orthogonal Design, Full Factorial Design, Uniform Design, Plackett-Burman Design, and Response Surface Methodology based on single-factor level testing. Ye et al. (2017) used orthogonal design experiments to optimize a medium based on yeast extract and glucose as the carbon and nitrogen sources, respectively, resulting in a 37.3% improvement in antibacterial activity in B. amyloliquefaciens. This study successfully optimized the culture and fermentation conditions for Bacillus spp. preparations. The optimized medium contained soybean 30.0 g/L, glucose 20.0 g/L, and K2HPO4·3H2O 1.0 g/L, with fermentation conditions including a strain age of 12 hours, inoculum size of 2%, temperature of 37˚C, initial pH of 7.0, and a filling volume of 20 mL/250 mL. Under these optimal fermentation conditions, the spore yield of Bacillus spp. reached 141.42 × 108 CFU/mL, representing a 10.26-fold increase compared to the initial medium, with a spore formation rate exceeding 90%.
CONCLUSION
From 35 fecal samples of indigenous free-range chickens, 168 bacterial strains were isolated and screened based on morphological and biochemical characteristics, ultimately selecting 16 B. subtilis strains. Further evaluation of their antibacterial activity against E. coli FG31-1 and S. typhimurium FC13827, both of which cause gastrointestinal diseases in chickens, identified strain BA07 as the most effective. 16S rRNA gene sequencing confirmed that strain BA07 has 100% similarity with B. subtilis strain BA07. Subsequently, this study successfully optimized the culture medium and fermentation conditions for producing B. subtilis BA07 spores. The optimal medium included glucose, soybean meal, and K2HPO4, alongside specific fermentation conditions involving temperature, incubation time, initial pH, inoculum size, and liquid volume. Under these optimal conditions, the spore yield of BA07 significantly increased (over 10-fold compared to the initial medium), with a spore formation rate exceeding 90%.
ACKNOWLEDGEMENTS
The authors appreciate and acknowledge the laboratory and technical assistance provided by the Faculty of Animal Sciences, Universtiy of Agriculture and Forestry, Hue University.
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FUNDING
The research was funded by the Vietnam Ministry of Education and Training (project code: B2023-DHH-24).
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DATA AVAILABILITY STATEMENT
Data supporting the findings of this study are available from the corresponding author under the Project funding, upon reasonable request.
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DISCLAIMER/PUBLISHER’S NOTE
The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
Data supporting the findings of this study are available from the corresponding author under the Project funding, upon reasonable request.
