Trends Sci. 2026; 23(3): 11347

Introduction

Nitrogen is the 2 ^nd most important macronutrient required by plants, accounting for approximately 1% - 5% of total plant dry matter. It plays a vital role in the formation of nucleic acids, such as DNA and RNA, and is essential for the synthesis of proteins and enzymes involved in plant growth and metabolism [18]. Despite its critical role, the current agricultural model heavily relies on synthetic nitrogen fertilizers to meet crop nutrient demands. While these fertilizers can enhance yields, their extensive use has resulted in escalating production costs, declining soil health, and negative environmental impacts, such as water contamination and greenhouse gas emissions [1,2]. Although inorganic fertilizers contain high nutrient concentrations and provide immediate nutrient availability, their long-term use has contributed to the deterioration of soil structure, reduced microbial diversity, and the disruption of natural biogeochemical cycles [3]. Furthermore, the efficiency of nitrogen use in current systems remains low, with a significant proportion lost through leaching, volatilization, or denitrification [4]. To address these challenges, there is a growing interest in sustainable nutrient management strategies that integrate organic amendments and biofertilizers, aiming to improve soil fertility, reduce chemical inputs, and maintain crop productivity [5].

Endophytic nitrogen-fixing bacteria (ENFB) have emerged as a promising tool in sustainable agriculture. These bacteria inhabit the internal tissues of plants without causing harm and can fix atmospheric nitrogen into forms usable by plants, such as ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ⁻ ). By encoding nitrogenase enzymes, ENFB convert atmospheric nitrogen (N ₂ ) into biologically available nitrogen, contributing significantly to the nitrogen needs of crops [6,7]. In maize ( Zea mays L. ), ENFB have been reported to supply up to 30% of the plant’s nitrogen requirement under field conditions [8,9]. The symbiotic relationship between ENFB and maize not only supports nitrogen uptake but also enhances plant growth through various mechanisms, including phytohormone production, phosphate and potassium solubilization, siderophore secretion, and pathogen suppression [10,11]. Several genera of ENFB, such as Bacillus , Enterobacter , and Azospirillum , have been successfully isolated from maize roots and rhizosphere environments [12,13]. Among them, Bacillus megaterium is particularly notable due to its large cell size, diverse metabolic capabilities, and ability to adapt to various environmental conditions [14].

Bacillus megaterium has been extensively studied for its role as a biofertilizer. It is capable of fixing nitrogen, solubilizing phosphorus and potassium, and producing plant growth-promoting substances such as indole acetic acid (IAA) and siderophores [15,16]. These traits enable it to enhance root growth, improve nutrient uptake, and increase crop resilience to abiotic stresses. Recent studies have demonstrated that Bacillus megaterium can enhance yields of crops like cucumber and corn while simultaneously improving the bioavailability of phosphorus and potassium in the soil [17,3]. A field trial combining varying doses of P and K fertilizers with Bacillus megaterium inoculation reported an 11.8% to 15.2% increase in cucumber yield and a 27.5% to 46.1% rise in nutrient accumulation in plant tissues. Furthermore, Bacillus megaterium inoculation has been shown to increase microbial diversity in the rhizosphere, including beneficial bacterial and fungal taxa, which play critical roles in nutrient cycling and plant health [18,19]. These findings underscore the potential of Bacillus megaterium not only as a nitrogen fixer but also as a comprehensive plant growth-promoting rhizobacterium (PGPR) capable of improving soil health and crop productivity.

Despite these promising results, there remains limited research focusing on the dual role of Bacillus megaterium as both an ENFB and a broader PGPR species in maize cultivation. Most existing studies treat nitrogen fixation and other growth-promoting mechanisms separately. This compartmental approach overlooks the potential synergistic effects that multifunctional bacteria like Bacillus megaterium can offer. Therefore, investigating the integrated nitrogen-fixing ability, phosphate and potassium solubilization, and plant growth-promoting traits of Bacillus megaterium is crucial to fully exploit its agricultural benefits [21].

Maize, one of the world’s most important cereal crops, plays a central role in global food security and agricultural economies, especially in developing countries. Its high nutrient demand, particularly for nitrogen, phosphorus, and potassium, makes it an ideal model for evaluating the effectiveness of biofertilizers like Bacillus megaterium [2,22]. Enhancing nutrient use efficiency in maize through microbial inoculants can reduce the dependency on synthetic fertilizers and contribute to more resilient agroecosystems. Considering the above considerations, this study was conducted to isolate and identify 4 strains of Bacillus megaterium from maize roots and to evaluate their nitrogen-fixing capacity alongside other beneficial traits such as phosphate and potassium solubilization and plant growth-promoting potential. The goal is to determine the potential application of these strains as biofertilizers to support sustainable maize production, reduce chemical fertilizer inputs, and promote ecological balance in cropping systems.

Materials and methods

Collection and treatment of corn roots

The corn root samples were obtained from corn fields in the Cho Moi district, An Giang, Vietnam. The roots were carefully uprooted, placed in sterile plastic bags, and transported to the laboratory. Before isolation, maize roots were sterilized by surface of 1 ^st thoroughly washing them with tap water to remove soil and debris. The roots were then immersed in 70% ethanol for 3 min for partial sterilization, followed by treatment with 2.5% sodium hypochlorite for 5 min to eliminate most surface bacteria. Finally, the roots were rinsed again with 70% ethanol and several times with sterile distilled water to remove any remaining sterilizing agents. To verify the effectiveness of surface sterilization, sterilized root segments were placed on agar plates. The absence of bacterial growth after incubation indicated successful sterilization [23,24].

Isolation and morphological observation of 4 Bacillus megaterium strains

The maize root samples were finely ground and diluted to 10 ^{− 8} . Then, 0.1 mL from per dilution was spread on YMA plates to isolate ENFB, and the petri dishes were incubated at 32 °C for 60 h. Among the pure colonies that were Gram-stained and observed at 100× magnification, 10 colonies showing several traits like Bacillus megaterium were selected for subsequent molecular identification; these pure colonies exhibited sizes of 3 - 4 mm with regular margins, and all 10 selected colonies were large, rod-shaped, endospore-forming, and Gram-positive [25].

Identification of 10 selected colonies

The 10 selected colonies were identified with their 16S rRNA genes by sequencing. Gene amplification was carried out using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') [26,27]. The obtained sequences were compared to the GenBank database using BLAST, and multiple sequence alignment was performed using alignment software (https://www.ncbi.nlm.nih.gov/). The evolutionary relationships were determined based on the method outlined by Saitou and Nei [28], and Tamura et al . [29], with a bootstrap tree generated from 1000 replicates (Felsenstein, 1985). All ambiguous sites were excluded using the pairwise deletion option, phylogenetic and molecular evolutionary analyses were carried out with MEGA 11 [30]. The sequence data for the 4 strains consisted of Bacillus megaterium KL-197, Bacillus megaterium KSW-114, Bacillus megaterium ROA024 and Bacillus megaterium KSW-113, which were thoroughly compared with reference sequences in GenBank via BLAST, and given that the identification thresholds were 100%, these sequences were subsequently submitted to the NCBI GenBank database ( Figure 1 ).

Assessing thermal adaptation

The heat adaptation of the 4 Bacillus megaterium strains was assessed in 2 stages. First, 4 test tubes containing YMA nutrient medium were prepared for each strain. Second, the strains were streaked onto YMA agar medium in test tubes and incubated at temperatures of 30, 35, 40, and 45 °C. Each species was tested with 4 replicates, and colony growth was monitored for 1 week [31].

Assessing salt adaptation

The salt adaptation of the 4 selected trains was evaluated through the following procedure: Initially, test tubes containing YMA agar medium were prepared for each train. Next, NaCl solution was added to reach final concentrations of 1.0%, 2%, 3%, 4%, and 5%. Lastly, selected colonies were separately inoculated for each selected strain and incubated at 28 °C with 4 replicates. The results were recorded after 1 week [31,32].

Ammonia production

The ammonia production capability of the identified strains was assessed qualitatively. Each colony was inoculated into peptone water and incubated on YMA medium at 30 °C for 60 to 80 h. The ammonia production was indicated by a color change from brown to yellow after adding Nessler’s reagent [33] .

Nitrogenase activity

The nitrogenase activity was assessed using the acetylene reduction assay. Four selected and identified strains were grown in YMA liquid medium and incubated for 24 h. The resulting suspension was then used to inoculate a nitrogen-free liquid medium. A control sample was prepared using the N-free liquid medium without the addition of 4 selected Bacillus megaterium strains. The cell density of the selected Bacillus megaterium suspension, determined using spectrophotometry at a wavelength of 600 nm, reached 0.8 under incubation conditions of 30 °C and 160 rpm [34].

Nitrogen concentration determination

The selected strains were cultured in an N-free medium with 0.05% malate as the primary carbon source and incubated at 30 °C. After centrifugation at 3000 rpm for 1 min, the supernatant was collected for nitrogen determination following the method of Li et al . [35].

Density augmentation of selected strains

The selected strains were inoculated into sterile YMA medium and incubated at 32 °C for 60 h. Following incubation, the cultures were centrifuged at 6,000 rpm for 5 min, and the supernatant was discarded. The cells were then washed with sterile saline to achieve a concentration of 10⁸ CFU mL⁻¹. The resulting suspensions of the 5 selected strains were prepared as a 1.0% (v/v) inoculum to assess factors affecting plant growth. In the field experiment, 100 mL of this cell suspension was applied to peanut seeds prior to planting [36]. Given their strong nitrogen-fixing capacity and consistent enhancement of maize growth and yield under field conditions, the 4 Bacillus megaterium strains (KL-197, KSW-144, ROA024, and KSW-113) have been preserved under cold storage for future formulation and application as biofertilizers. Their potential use as sustainable alternatives to chemical nitrogen inputs highlights their value in promoting eco-friendly maize production and long-term soil fertility.

Study area

The field experiment was conducted in Cho Moi Commune, An Giang Province, Vietnam. This area is situated in the Mekong Delta region, characterized by high agricultural intensity and tropical monsoon climate conditions. The estimated geographical coordinates of the study site are approximately 10.50° N latitude and 105.40° E longitude. The region is representative of intensive maize cultivation systems, making it a suitable location for evaluating the agronomic potential of plant growth-promoting bacterial strains.

Experimental design

An arranged treatments of field experiment was in a completely randomized design with 5 treatments and 4 replications. The 5 treatments included C0, C1, C2, C3, and C4, with the arrangements shown in Table 1 . Inorganic fertilizers used were single fertilizers such as urea, superphosphate, and potassium chloride. All fertilizer quantities were converted to N, P, and K weight per hectare, applied at a rate of 150 kg Urea [CO(NH ₂ ) ₂ ] ha ⁻ ¹ + 80 kg P ₂ O ₅ ha ⁻ ¹ + 80 kg KCl ha ⁻ ¹ for all experimental treatments ( Table 1 ). The area of arranged treatments was 400 m² (2.0 m×10 m×5 treatments×4 replications) and the distance between 2 plots was 0.5 m. T he hybrid corn variety F1 LION 10 from Hai Mui Ten Do Company, Vietnam were used during the experiment, which were planted in single rows with a spacing of 30 cm (2 seeds per hole). The corn seeds ( F1 LION 10) were manually sown from November 2024 to February 2025. The corn was harvested 90 days after sowing (DAS). One healthy plant was kept in each hole to monitor throughout the experiment.

Table 1 The layout of the experimental treatments under field conditions.

Data analysis

Data were processed using Statgraphite XVI software to determine the mean and perform variance analysis. Statistical analysis was carried out, and comparisons were made using Duncan's test, considering a significant level of p ≤ 0.05.

Results and discussion

Morphological and biochemical testing of 4 selected colonies

The 4 colonies were selected from the initial 10 isolates based on molecular identification results. All 4 strains were 4 Bacillus megaterium strains, they included Bacillus megaterium KL-197, Bacillus megaterium KSW-114, Bacillus megaterium ROA 024, and Bacillus megaterium KSW-113, which were confirmed to exhibit genetic similarity (Table 2). In the next step, their morphology was examined to assess their nitrogen-fixing potential before conducting field experiments. Four strains, including Bacillus megaterium KL-197, Bacillus megaterium KSW-144, Bacillus megaterium ROA 024, and Bacillus megaterium KSW-113, were noted by BM1, BM2, and BM4, respectively.

Table 2 Similar percentage of 4 strains and code number.

Table 3 The physical and chemical characterization of 4 Bacillus megaterium strains was determined using the VITEK 2 analyzer.

Notes: (–): Negative reaction; (+) Weak reaction; (++) Strong reaction.

Figure 1 The phylogenetic tree for the 16S rRNA sequence of 4 Bacillus megaterium strains was constructed using the neighbor-joining method.

The VITEK 2 analysis system (BioMérieux, France) that provided a 92% probability and were confirmed using Illumina gene analysis, was utilized to identify the morphological and biochemical characteristics of 4 selected strains. Morphologically, before identification was sent out the identification, the 4 colonies of Bacillus megaterium appeared cream-yellow, 3 mm in diameter, non-pigmented, and ranged from round to irregular with smooth to wavy edges on YMA medium. All 4 Bacillus megaterium strains were Gram-positive, motile rod-shaped bacteria, catalase-positive, and grew aerobically at temperatures of 20 - 40 °C, with optimal growth at pH 4 - 7, a temperature range of 20 - 50 °C, and tolerance to salt concentrations up to 5%. Four Bacillus megaterium strains were mainly identified through the API 50 BCL system, and some of the results from the VITEK 2 analyzer are presented in Table 3. Furthermore, the species phylogenetic analysis based on the 16S rRNA sequence with a total branch length of 0.01 confirmed the isolated strains as Bacillus megaterium KL-197, Bacillus megaterium KSW-144, Bacillus megaterium ROA 024, and Bacillus megaterium KSW-113, with the respective accession numbers AY030338.1, OR 514194.1, MT510154.1 and OR514193.1, all showing 100% similarity with the NCBI reference sequence as shown in Figure 1.

Determination of ammonia activity and concentration

The reduction process of C ₂ H ₂ to C ₂ H ₄ using the C ₂ H ₂ gas injection system initially showed a delay after introducing C ₂ H ₂ into the system and then became stable over a period of 0 to 72 h. The rate of C ₂ H ₂ to C ₂ H ₄ reduction in the experimental setups, conducted with 4 replications in the laboratory, was measured by tracking the increase in C ₂ H ₄ concentration during the stable period, which lasted around 72 h.

Results of Figure 2 show that 4 Bacillus megaterium strains were monitored for acetylene reduction tests to evaluate the nitrogenase activity. Figure 2 shows that Bacillus megaterium ROA024 (BM3) obtained the highest value of ethylene (330 nmol C ₂ H ₄ /h/mL), followed by Bacillus megaterium KSW-113 (BM4: 270 nmol C ₂ H ₄ /h/mL) and Bacillus megaterium KSW-114 (BM2: 213 nmol C ₂ H ₄ /h/mL), while Bacillus megaterium KL-197 (BM1:198 nm/h/mL) had the lowest value. The Kjeldahl method is commonly used to estimate nitrogen concentration. The research results from Figure 2 indicate that Bacillus megaterium strains actively participate in the nitrogen fixation [13] . The amount of nitrogen was produced by the 4 strains ranging from 164 to 312 mg N/100 mL. Bacillus megaterium ROA 024 produced the highest amount of nitrogen (312 mg N/100 mL), followed by Bacillus megaterium KL-197 (187 mg N/100 mL), while Bacillus megaterium KSW-113 and Bacillus megaterium KSW-114 had the lowest result (164 and 165 mg N/100 mL), with a statistically significant difference at the 1% level.

Figure 2 Nitrogenase activity of 4 Bacillus megaterium strains measured by the acetylene reduction assay, along with corresponding nitrogen concentrations. Data are presented as mean ± standard deviation. Different lowercase letters (a, b, c, and d) indicate statistically significant differences among treatments, as determined by one-way ANOVA followed by Duncan’s multiple range test at p < 0.01.

However, the results from Figure 2 show that all 4 strains can produce high nitrogen concentrations after just 72 h of experimentation. In this research, 4 novel Bacillus megaterium strains were isolated from maize roots using YMA nutrient medium. Phylogenetic analysis of their 16S rRNA gene sequences confirmed that they belong to the Bacillus genus and show close genetic relationships to known megaterium species and strains, which consisted of KL-197, KSW-144, ROA024 and KWS-113 ( Table 3 ). Through biochemical tests, it showed that all 4 strains could produce ammonia, synthesize the nitrogenase enzyme, and possess nitrogen-related genes involved in nitrogen fixation. Their growth capability using ammonia gas as the sole nitrogen source and acetylene-reducing activity were successfully demonstrated for these 4 newly isolated strains [2,37].

The aim of this study is not only to isolate and identify new Bacillus megaterium strains from maize roots, but more importantly, to select strains with strong plant growth-promoting potential. These selected endophytic strains were further evaluated for their effectiveness as biofertilizers through field experiments on maize. Nitrogen fixation of ENFB is an ecologically important role, which contributed to a natural N source as an input of nitrogen into the environment and represents the nitrogen-fixing capability of the Bacillus megaterium strains [38,39]. All 4 strains exhibited high nitrogen-fixing mechanisms, making them suitable for use as biofertilizers ( Table 3 and Figure 2 ). The production of nitrogenase enzymes plays a crucial role in nitrogen fixation, raising the natural N content in the soil, reducing chemical nitrogen usage, increasing available nutrients, and promoting plant growth, especially in nitrogen-deficient soils [40].

Effect of 4 Bacillus megaterium strains on corn growth traits

All 4 Bacillus megaterium strains promoted corn agronomy traits compared to those of the control ( Figure 3 ). Analyzing ANOVA to compare each strain to the uninoculated treatment, 4 strains significantly increased 3 agronomy parameters: Leaf number, chlorophyll content, and plant height. Bacillus megaterium ROA024 (C3) exhibited the highest stimulation of these 3 parameters, followed by Bacillus megaterium KSW-113 (C4), Bacillus megaterium KSW-144 (C2), and Bacillus megaterium KL-197 (C1). Meanwhile, no strain had the lowest values for all 3 parameters, and all differences were highly significant ( p < 0.01). Bacterial treatments had significant influences on leaf number, chlorophyll content, and plant height. Bacillus megaterium strains observed during the field experiment demonstrated the potential for plant growth promotion in corn experiments. Bacillus megaterium has been shown to have broad-spectrum activity, as it has been effective in promoting the growth of soybean and wheat. The plant growth stimulation by Bacillus megaterium species observed in this research aligns with prior studies indicating that Bacillus megaterium strains effectively enhanced growth across various crop species [41]. According to Akinrinlola et al . [42], corn exhibits a stronger response to Bacillus megaterium strains compared to soybean or wheat. Similarly, Lee et al . [43], assessed the plant growth-promoting abilities of thirty bacterial strains on wheat seedlings and found that only 4 Bacillus megaterium strains were effective. Furthermore, Bacillus megaterium strains demonstrated the highest growth-promoting effects on corn compared

to other strains [44].

Figure 3 Effects of 4 Bacillus megaterium strains on maize agronomic traits. Values are expressed as mean ± standard deviation. Different lowercase letters (a, b, c and d) denote significant differences among treatments based on 1-way ANOVA with Duncan’s multiple range test at a significance level of p < 0.01 .

Effects of 4 Bacillus megaterium strains on yield component, productivity and nutrient compositions

The addition of the 4 Bacillus megaterium strains resulted in higher values for all yield parameters, including fruit length and diameter, the number of rows per fruit, the number of seeds per row, and plant biomass, compared to the control without strains. Among them, treatment C3 ( Bacillus megaterium ROA024) and C4 ( Bacillus megaterium KSW-113) showed the highest improvement in yield components, followed by Bacillus megaterium KSW-144 (C2), and Bacillus megaterium KL-197 (C1), while the control (C0) had the lowest values among the 5 treatments ( Table 4 ).

The ENFB application as biofertilizers to regulate rhizosphere microbial ecosystems and boost nutrient absorption is a potential strategy for enhancing crop development in nutrient-deficient soils [45,46]. In a recent study, A successfully isolated a novel Bacillus megaterium strain with plant growth-promoting capabilities. The findings revealed that Bacillus megaterium OQ560352 influenced the fungal and bacterial communities in the maize rhizosphere and was positively associated with phosphorus solubilization activity in alkaline soil. The combination of NPK fertilizer with Bacillus megaterium resulted in a 4% increase in shoot biomass, a 6% rise in shoot phosphorus concentration, a 10% boost in soil phosphorus concentration, and a 13% enhancement in phosphorus solubilization activity. In conclusion, this study emphasizes the potential of the newly isolated Bacillus S megaterium strain as an effective plant growth-promoting rhizobacterium capable of enhancing maize growth and yield through modulation of the microbial community [47,48]. Similarly, Li et al . [49], recently identified 3 endophytic bacterial strains, consisting of Bacillus megaterium , Bacillus flexus , and Bacillus subtilis , which were isolated from maize roots and applied as bio-stimulants to assess their impact on maize development.

Table 4 The effect of 4 Bacillus megaterium strains on corn yield traits.

Notes: ±: The standard error of the mean is based on 4 replicates per treatment. Different superscript letters (a, b, and c) in the same column denote significant differences among treatments according to Duncan’s multiple range test at p < 0.01 (**), and ns indicates no significant difference .

Table 5 The effect of 4 Bacillus megaterium strains on yield traits, yield and seed-nutrient composition.

Notes: ±: The standard error of the mean is based on 4 replicates per treatment. Different superscript letters (a, b, and c) in the same column denote significant differences among treatments according to Duncan’s multiple range test at p < 0.01 (**), and ns indicates no significant difference.

Four Bacillus megaterium strains demonstrated favorable outcomes across multiple parameters during the experiment. During the harvest season, corn ears and seeds were harvested to assess the weight of 1,000 seeds, ear yield, and the percentage of lipid and protein content in the seeds. As shown in Table 5 , the weight of 1,000-seeds and fresh ear yield. The treatment C2, C3, and C4 were higher than those in C0 and C1. While lipid concentration showed no significant difference, protein content was highest in C3 and C4, followed by C2 and C1, with the lowest in C0. Compared to the control (C0), the average yield increase in treatments of Bacillus megaterium addition reached up to 12%, with a statistically significant difference at the 1% level ( Table 5 ).

In recent years, there has been growing interest in examining the influence of various Bacillus megaterium strains on different crop species. According to a recent study of Oliveira-Paiva et al . [50] used 40 ENFB species to assess their impact on maize and sorghum growth. Among these species, Bacillus megaterium exhibited a greater increase in plant height, shoot dry weight, and chlorophyll content compared to the control group without Bacillus megaterium . This aligns with our findings, as the 4 Bacillus megaterium strains also demonstrated significant improvements in multiple parameters: 1) Agronomic traits ( Figure 3 ), including plant height, leaf number, chlorophyll concentration, and plant biomass; 2) Yield components such as 1000-seeds weight and fresh ear yield; 3) Grain nutritional composition including percentage of lipid and protein content percentages ( Table 5 ). These values were significantly higher in maize plants inoculated with Bacillus megaterium compared to the non-inoculated control. This consistency in results further confirms that Bacillus megaterium strains exhibit high performance and have a positive impact on maize growth [50].

Conclusions

This study successfully isolated and identified 4 indigenous Bacillus megaterium strains (KL-197, KSW-144, ROA024, and KSW-113) from maize root tissues cultivated in intensive farming areas of Cho Moi commune, An Giang, Vietnam. These strains demonstrated strong nitrogen-fixing capabilities under laboratory conditions, with notable ammonia production and nitrogen-fixing activity. Field trials further validated their agronomic potential, where all 4 strains significantly enhanced maize growth, yield, and seed protein content compared to the uninoculated control. Among them, strains ROA024 and KSW-113 consistently outperformed the others, contributing to a yield increase of up to 12%. The isolated strains also improved key yield components, including ear length, seed number, biomass, and 1000-seed weight, while maintaining stable lipid content. The findings highlight the dual function of these native strains as both endophytic nitrogen-fixers and plant growth-promoting rhizobacteria. This research introduces novel strains adapted to local agro-ecological conditions, offering an eco-friendly and sustainable alternative to chemical nitrogen fertilizers. The promising performance of these isolates supports their application in biofertilizer development, particularly for improving maize productivity in tropical agroecosystems.

Acknowledgements

The authors would like to thank An Giang University, Vietnam, for providing laboratory equipment and research facilities that enabled us to complete this study.

Declaration of Generative AI in Scientific Writing

The authors declare that generative AI tools were used solely to enhance the readability and language clarity of the manuscript. These tools were applied under full human oversight and control. The authors take complete responsibility for all scientific content, analyses, and conclusions presented in the article. No AI tool was credited as an author or co-author in this work.

CRediT author statement

Nguyen Van Thuan, Tran Le Kim Tri, Tran Thanh Liem, Trinh Van Tuan Em, and Nguyen Ngoc Phuong Trang contributed to the study design, conducted in vivo and in vitro experiments, and were involved in data collection, data analysis, result interpretation, and preparation of figures and tables. Nguyen Van Chuong provided critica l revisions to the manuscript, contributed to the interpretation of results within the physiological context, supervised the overall research process, coordinated team activities, and finalized the manuscript.

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