Introduction

Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host1. They are currently widely used as dietary supplements, particularly to improve gastrointestinal health2. Within the intestinal microbiome, probiotics play a central role in mediating a variety of host-targeted benefits. These include defending against potential pathogens, strengthening barrier functions, regulating immune responses, and synthesizing neurotransmitters, thereby bridging the gut microbiota and the brain-gut axis as integral cellular components2,3. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) endorsed the safety assessment of probiotics through a series of experimental analyzes. These include assessments of antibiotic resistance, metabolic activities, toxin production, hemolytic activities, infectivity in immunocompromised animals, and testing of side effects and adverse reactions in humans4. Probiotic strains that are intended to benefit the host must have certain properties. These include survival in specific biological niches (such as adherence to mucosal or intestinal epithelial cells), interaction with the host’s immune mechanisms, resistance to digestive enzymes, bile or acidic conditions and the development of antimicrobial activities through competitive exclusion or the synthesis of bacteriocins and hydrogen peroxide5.

Heyndrickxia coagulans, formerly known as Bacillus coagulans or Weizmannia coagulans, is acknowledged as a safe and consumable probiotic strain6, utilized in fermentation processes and as a feed additive7,8. At the same time, H. coagulans has shown exceptional efficacy in various biological functions. Research has shown that H. coagulanscan alleviate atherosclerosis by modulating lipid metabolism, counteracting oxidative stress, and preventing endothelial damage9. Additionally, in mouse models, the mitigating effect of H. coagulanson copper-induced toxicity further demonstrates its regulatory influence on lipid metabolism. This genus is also an important reservoir of bacteriocins or bacteriocin-like substances that help to prevent the development of resistant forms of target bacteria10. Compared with the widespread Lactobacillus and Bifidobacterium, the spore-forming bacteria exhibit greater stability under unfavorable conditions. Their probiotic efficacy combined with stability during production, storage and consumption makes them ideal candidates for inclusion in functional foods and pharmaceutical preparations11,12.

The strain BC99, derived from a well-characterized collection originating from the feces of a healthy infant, has been identified and classified as H. coagulans. While previous studies have assessed its efficacy in contexts such as Helicobacter pyloritreatment, amelioration of colitis, and alleviation of constipation13, comprehensive evaluations, particularly systematic assessments of its safety and function across bioinformatics, in vitro, and in vivo studies, have been limited. Despite preliminary findings indicating no adverse reactions in animals or human subjects involved in these evaluations, a holistic safety and functional assessment of H. coagulans remains inadequately addressed in existing literature. To address these gaps, our study provides a comprehensive validation of BC99’s safety profile and biological properties, offering a robust theoretical foundation for its application in functional foods and pharmaceutical formulations.

Materials and methods

Main reagents

Soy-casein agar medium (TSA) was purchased from Guangdong Zhongshan Baiwei Microbiology Technology Co., Ltd (Zhongshan, Guangdong, China). Columbia blood agar plates were provided by Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). HT-29 cells (cell line: ACC_299) cultured in HyClone’s Dulbecco’s Modified Eagle Medium (DMEM) were supplied by Shanghai Hongshun Biotechnology Co., Ltd (Shanghai, China). Beyotime Biotechnology provided 1 × PBS and Triton X-100. The CytoTox 96®Non-Radioactive Cytotoxicity Assay Kit was provided by Promega Corporation (Madison, Wisconsin, USA). Simulated gastric and intestinal fluids (sterile, formulated according to USP14) were provided by Shanghai Yuan Ye Biotechnology Co., Ltd (Shanghai, China). DeMan-Rogosa-Sharpe (MRS) medium, starch, soy flour and yeast extract were supplied by Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). The liquid components of MRS medium, including 0.2% starch, 0.5% peptone, 1.0% soy flour, 0.1% glucose, 0.07% yeast extract, 0.02% MnSO4, were supplied by Sinopharm Chemical Reagent Co., Ltd. NaCl and bile salts were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Additionally, all other reagents were sourced from Sigma-Aldrich, Shanghai, China. The biochemical blood analyzer (Model CS-480) was purchased from Guangzhou Yutai Biotechnology Co., Ltd (Guangdong, China). The olympus microscopes (CX33) were purchased from Shanghai Linyi Biotechnology Co., Ltd (Shanghai, China).

Strain sequencing

Whole genome sequencing

A previously characterized bacterial strain BC99, originally isolated from the feces of a healthy infant6, was used in this study. The strain was cultivated in liquid MRS medium at 37 °C for 24 h before whole genome sequencing. The BC99 cells were harvested by centrifugation at 8000 g for 5 min, and the genomic DNA was extracted using the MGIEasy Fecal Genome Kit from MGI NE32. The extracted DNA was subjected to high-throughput sequencing on the MGI G99 platform for short-read sequencing, while long-read sequencing was performed using the Pacbio Sequel II platform. Quality control of the sequencing data was performed using fastp software, to set a minimum sequence length of 150 bp and filter out sequences with base quality lower than 99.9%15. The assembly of MGI short-read and Pacbio long-read data was performed using the Unicycler software, setting the threads of Unicycler as 5216. Used rnammer (Version: 1.2) with the parameters set for bacteria to input the genome sequence of BC99 (in FASTA format) and the General Feature Format (in GFF format) sequence information. Extracted the 16S rRNA sequence of BC99 and aligned it with the 16 S rRNA sequence of the reference strain ATCC 7050 (NCBI accession: NR_115727.1) to determine whether BC99 belongs to the species Heyndrickxia coagulans17.

Genome assembly and annotation

The sequencing data were compiled using MGAP software from BGI Genomics. Gene prediction was performed with Bakta18. Virulence factors and resistance genes were determined by analyzing the basic dataset in VFDB (Virulence Factors of Pathogenic Bacteria19, http://www.mgc.ac.cn/VFs/) and Virulencefinder (https://cge.food.dtu.dk/services/VirulenceFinder/)20,21,22. The annotation of drug-resistant genes was performed in collaboration with the CARD database (https://card.mcmaster.ca/)23and the RGI software. In addition, the NR (NonRedundant Protein Database), GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases24,25,26 were used for broader functional annotation.

Analysis of gene clusters

To identify gene clusters responsible for the synthesis of non-ribosomally synthesized peptides (NRPs) and bacteriocins, we employed the antiSMASH web tool (Version: 7.1.0)27. The bacterial sequences were submitted to antiSMASH with the detection strictness set to “relaxed” and all additional features enabled by default, facilitating a thorough analysis of relevant gene clusters. Additionally, the BC99 genome was uploaded to the BAGEL web server (http://bagel5.molgenrug.nl) with all parameters set to default to specifically analyze bacteriocins. This dual approach ensures comprehensive detection and characterization of bioactive compound-producing gene clusters in the bacterial genome28.

In vitro safety assessment

Hemolysis assay

Strain BC99 was cultured for 48 h at 37 °C on Columbia blood agar plates to determine hemolytic activity. The absence of a hemolytic ring indicated a negative hemolysis result; a grass-green ring signified α-hemolysis positivity, and a clear ring indicated β-hemolysis positivity29.

Cytotoxicity assay

HT-29 cells, a human colorectal adenocarcinoma cell line, were procured from Wuhan Procell Life Science Co., Ltd., Wuhan, China. HT-29 cells were seeded at 100 µL/well in a 96-well plate. BC99 bacterial suspensions were prepared in DMEM at concentrations of 107, 108, 109 CFU/mL. The bacterial suspension and DMEM were added to the wells (100 µL/well) and incubated at 37 °C for 4 and 8 hours. The 10x lysis solution from the CytoTox 96® kit was used to perform the maximum lactate dehydrogenase (LDH) release control. Fifty microliters from each well were transferred to a new 96-well plate, mixed with Fifty microliter of CytoTox 96® reagent and incubated for 30 min at 37 °C in the dark. 50 µL of stop solution was added to each well and the absorbance at 490 nm was measured within one hour. Cell viability % was calculated as LDH experimental release (OD490) / LDH maximum release (OD490)30.

Cell adhesion assay

HT-29 cells were cultured at a density of 1 × 106 CFU/mL in a 6-well plate for 24 h. After washing with PBS and discarding the medium, 2.5 mL of BC99 bacterial suspension (1 × 109 CFU/mL) was added to each well, with DMEM added to the control wells, and incubated at 37 °C for 3 h. Unattached bacteria were washed off with PBS and the cells in each well were treated with 0.3% Triton X-100 for 10 min. The fluid from the BC99 wells was diluted to 1 × 106 CFU/mL, and 1 mL was added to MRS medium, and then incubated at 37 °C for 48 h to count colonies. HT-29 cells in control wells were also counted. Results were expressed as average number of adherent BC99 bacteria per well / average number of HT-29 cells per well31.

Antibiotic sensitivity assay

The standard disk diffusion method32 was used, whereby BC99 was applied to MRS agar plates. Antibiotic susceptibility disks were applied to the plates, including gentamicin, ampicillin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, imipenem, tetracycline, trimethoprim-sulfamethoxazole, rifampin, vancomycin, etc. After incubation at 37 °C for 24 h, the growth of the strain was observed, and the sensitivity (S), intermediate sensitivity (I) and resistance (R) indicated the antibiotic sensitivity of the strain. Criteria for determining S, I, and R were based on established MIC breakpoints for each antibiotic, which are detailed in the supplementary materials.

Artificial gastric and bile salt hydrolysis simulation experiments33

Prior to the artificial gastric and bile salt hydrolysis simulations, the BC99 bacterial culture was grown to a specific optical density, ensuring a standardized concentration of approximately 1 × 109 CFU/mL. This culture was then centrifuged at 8000 g for 5 min, and the supernatant was discarded. The bacterial pellet was resuspended in sterile 0.85% saline to a final volume of 50 mL to ensure homogeneous distribution. From this suspension, one milliliter was carefully measured and added to various testing media: MRS at pH levels of 2.5, 3.0, and 3.5, simulated gastric fluid at pH 2.5, 3.0, and 3.5, and simulated intestinal fluid as well as MRS containing 0.1% bile salts. Each of these mixtures was then incubated at 37 °C for 2 h. To determine the initial concentration of the bacterial culture before the experiment, serial dilution and plate counting were performed. Specifically, the diluted culture was plated on Columbia blood agar plates, and colony counts were recorded to calculate the initial number of viable cells (N0). After the incubation period, the number of viable cells (Ni) was again determined by the plate counting method using Columbia blood agar. The survival rate of BC99 under these conditions was calculated using the formula % = lg Ni / lg N0 × 100, where Ni represents the number of viable cells remaining after each specific treatment interval and N0is the initial count of viable cells. This method was adapted from Ting et al. (2024)34, who demonstrated its efficacy in accurately quantifying bacterial cultures.

In vivo safety experiments

Selection of animals

Healthy Sprague-Dawley (SD) rats of the specific pathogen-free (SPF) class (weight 80–100 g) and ICR mice (weight 18–22 g) were obtained from Shanghai Shengchang Biotechnology Co., Ltd. Before the experiments, the animals were acclimatized to an SPF animal facility for one week, with a 16-hour fasting period without water restriction, before being randomly divided into groups.

Acute oral toxicity experiment

The mice were randomly divided into two groups of 10 animals each. A bacterial suspension BC99 was prepared in sterile water at a concentration of 2 × 1010 CFU/kg with a gavage volume of 20 mL/kg body weight. The control group received an equal volume of sterile water. Twice daily, animals were assessed for their overall health, including monitoring for any signs of intoxication, behavioral changes, and general activity. Mortality was specifically recorded at the outset and then on days 7 and 14 of the experiment to track any adverse effects over time. Gross pathological examinations were performed on the mice that succumbed to the experiment and on those that survived until day 14 post-exposure.

Subacute oral toxicity experiment

This 28-day studies were conducted in accordance with GB/T 15193.22–2014. Forty SPF-grade Sprague-Dawley rats, 5 weeks old, weighing 80–100 g, with an equal number of males and females, were obtained from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd. (Certificate No: SCXK [Zhe] 2019-0001) and housed in an SPF-grade animal facility (Certificate No: SYXK [Shanghai] 2019-0033) at a controlled temperature of 20–22 °C, relative humidity of 45–65%, with a 12-hour light/dark cycle and ventilation rate > 15 times/hour. The rats were divided into four groups of 10 animals each. A bacterial suspension of strain BC99 was prepared in sterile water and administered to the rats at three dose levels: low (0.125 × 1010 CFU/kg/day), medium (0.5 × 1010 CFU/kg/day), and high (2 × 1010 CFU/kg/day), representing the actual daily doses administered per kilogram of body weight. The control group received sterile water. Oral gavage was performed once daily for 28 consecutive days, with the administered volume set at 20 mL/kg body weight, adjusted according to each rat’s weight. Throughout the study, the rats were monitored daily for signs of toxicity, including behavioral changes, physical appearance alterations, and any adverse effects. After the final administration, blood samples were collected for comprehensive hematological and biochemical analysis to assess the impact of BC99 on various physiological parameters. The following parameters were analyzed: (1) Hematological indices: white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and percentages of neutrophils (NEUT%), lymphocytes (LYM%), monocytes (MONO%), and eosinophils (EOS%). (2) Coagulation profiles: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FBG). (3) Biochemical markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), globulins (GLO), glucose (GLU), blood urea (BU), creatinine (CREA), cholesterol (CHOL), and triglycerides (TG). Upon completion of the testing period, the animals were humanely euthanized, and their organs (liver, kidney, heart, spleen, and lungs) were harvested, weighed, and examined for any morphological changes to further assess potential toxicity.

Ethical considerations and euthanasia method

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Shengchang Biotechnology Co., Ltd. All methods were carried out in accordance with relevant guidelines and regulations, and are reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). Euthanasia was performed in a humane manner using carbon dioxide inhalation, followed by cervical dislocation to ensure death, as recommended by the IACUC guidelines.

Statistical analysis

Data are presented as mean ± standard deviation, based on at least three independent experiments. Student’s t-test was used for comparisons between two groups, while one-way ANOVA and Fisher’s LSD multiple comparison test were used for more than two groups. All statistical analyzes were performed using GraphPad Prism software (version 8.0.1).

Results

Morphological analysis of BC99

Microscopic observation revealed that the isolated and purified colonies of BC99 were pale yellow, round, opaque, with a moist surface and clean edges. Under the microscope, the bacteria appeared rod-shaped, about 0.4–0.6 μm × 1.5–4.0 μm in size, typically arranged singly or in pairs, and were Gram-positive.

Whole genome sequencing

The complete genome sequence of Heyndrickxia coagulans BC99 consists of a single circular chromosome totaling 3,655,496 base pairs, with an average GC content of 46.23%, and no detectable plasmids. The genomic layout, including gene distribution and GC content variations, is effectively illustrated in a Circos plot (Fig. 1A). The gene prediction analysis performed by Bakta for BC99 revealed a total of 3,466 genes. Among them, there were 3,357 coding sequences (CDSs), which comprised 3,273 protein-coding sequences, accounting for 97.5% of the total CDSs. In addition, there were 109 genes coding for RNA, including 27 rRNA, 77 tRNA and 5 ncRNA (Table 1). The genome sequence of BC99 was deposited into NCBI under accession number CP092692.1. The alignment results of the 16 S rRNA sequences showed that BC99 has a 99.06% similarity with the 16 S rRNA sequence of ATCC 7050, exceeding the 97% threshold35, thereby confirming that BC99 belongs to the species Heyndrickxia coagulans. By comparison with databases of virulence factors and resistance genes, no relevant resistance genes were found in BC99. With a similarity of > 70% and a sequence coverage of > 70% as filter criteria, 7 potentially virulence-related genes were found, consisting of gnd, clpP, clpC, tufA, groEL, bpsC and bpsD.

Fig. 1
figure 1

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99. Whole genome sequencing and analysis of gene clusters of BC99. (A) Circos plot of BC99; (B) Gene Ontology (GO) annotation of BC99. The x-axis represents the number of genes, while the y-axis represents the GO functions, which are divided into three modules: biological process, cellular component and molecular function; (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of BC99. The x-axis shows the number of genes, while the y-axis represents the KEGG pathways.

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Table 1 Characteristics of the genome of Heyndrickxia coagulans BC99.
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Figure 1B illustrated the GO annotation results, showing that in the biological process category, most genes were involved in biological regulation and metabolic processes. In the cellular component category, the functions of the genes were concentrated on proteinaceous complexes and cellular anatomical units. In the molecular function category, most genes were involved in binding and catalytic activity.

The KEGG annotation results are displayed in Fig. 1C. Most of the 20 pathways with the highest number of genes were involved in metabolism, especially on the synthesis of multiple amino acids. The metabolic pathway with the most genes were the ABC transporter, while other pathways include the two-component system, the phosphotransferase system (PTS), quorum sensing, and those pathways regulating self-assembly of flagella, such as flagellar assembly.

Gene clusters of BC99

AntiSMASH analysis revealed multiple gene clusters in BC99, including T3PKS and two ribosomally synthesized and post-translationally modified peptides (RiPP)-like gene clusters. Of these, one remained unidentified, while the other displayed 50% similarity to amylocyclicin, as shown in Fig. 2. Furthermore, a NRP, fengycin, was also detected in this analysis. BAGEL4 analysis, detailed in Table 2, confirmed the presence of two cyclic peptides, circularin_A and amylocyclicin.

Fig. 2
figure 2

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99. To analyze and visualize the gene clusters of Heyndrickxia coagulans BC99, genomic data were annotated using the antiSMASH and BAGEL4 web tools. The annotation diagram of the strain BC99 gene cluster. (A) T3PKS; (B) amylocyclicin; (C) betalactone; (D) RiPP-like.

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Table 2 BAGEL4 results of Heyndrickxia coagulans BC99.
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Tolerance assessment of BC99

The tolerance of the BC99 strain to acidic and bile environments was rigorously evaluated in simulated gastrointestinal fluid conditions. These conditions included simulated gastric fluid, simulated intestinal fluid, and media supplemented with bile salts, as detailed in Table 3. BC99 was incubated for 2 h in various media such as MRS adjusted to pH levels of 2.5, 3.0, and 3.5, simulated gastric fluid at pH 2.5, 3.0, and 3.5, simulated intestinal fluid, and MRS containing 0.1% bile salts. The strain consistently demonstrated survival rates over 90% across all conditions tested, achieving 97.45% at pH 2.5 and up to 99.83% at pH 3.5 in MRS; 97.44–99.88% in simulated gastric fluid; and 99.74% in simulated intestinal fluid. Notably, even in the challenging environment of 0.1% bile salts, BC99 maintained a survival rate of 92.85%. These results affirm BC99’s robust adaptability and potential as an effective probiotic capable of withstanding the harsh conditions of the gastrointestinal tract.

Table 3 Tolerance experiment of Heyndrickxia coagulans BC99.
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In vitro safety profile of BC99

The hemolysis test showed no hemolytic activity around BC99 colonies cultured on Columbia blood agar, indicating a negative hemolytic reaction (Fig. 3A). In antibiotic susceptibility tests, BC99 showed sensitivity to a comprehensive spectrum of antibiotics, including gentamicin, ampicillin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, imipenem, tetracycline, trimethoprim-sulfamethoxazole, rifampin and vancomycin, with MIC values consistently below 2 µg/mL (Table 4), indicating a favorable antibiotic susceptibility profile. Cytotoxicity assays performed with different concentrations of BC99 bacterial suspension in co-culture with HT-29 cells resulted in cell viability values predominantly above 100%, with the exception of 100% viability observed at a concentration of 107 CFU/mL after 8 h, indicating negligible cytotoxicity (Fig. 3B). The cell adhesion assay highlighted the remarkable adhesion ability of BC99 to HT-29 cells with an average of (40 ± 11) CFU per 100 cells, supporting its credibility in terms of in vitro safety.

Fig. 3
figure 3

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99. In vitro safety assessment of BC99. (A) Growth of BC99 on Columbia Blood Agar; (B) Cell viability of HT-29 cells after treatment with BC99.

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Table 4 Minimum inhibitory concentration values and results of the antibiotic susceptibility test for Heyndrickxia coagulans BC99.
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In vivo safety evaluation of BC99

Use the 1 × 109 CFU/mL BC99 seed solution was inoculated in 50 mL MRS Liquid medium for 24 h, cultured at 10,000 rpm, centrifuged for 15 min, and the bacterial precipitate count was diluted in 0.85% normal saline solution to obtain the liquid with a concentration of 8 × 108 CFU/mL BC99. During the acute oral toxicity study, both male and female mice showed a consistent increase in body weight similar to that of the control group, with no deaths over a 14-day period, indicating that BC99 is non-toxic at a concentration of 8 × 108 CFU/mL (Fig. 4A-B).

Fig. 4
figure 4

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99. In vivo safety evaluation of BC99 with acute oral toxicity test. (A) Body weight changes in male rats; (B) Body weight changes in female rats. The notation ‘ns’ indicates that the changes were not statistically significant, illustrating that BC99 did not significantly affect the body weights compared to controls. Statistical symbols used include: ‘*’ for p < 0.05, indicating statistically significant differences.

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In the 28-day subacute oral toxicity study, rats were administered varying doses of BC99, and their body weights, organ indices, hematological parameters, and coagulation profiles were monitored. Initially, all groups showed similar weights; however, from day 14, the groups receiving higher doses (0.5 × 1010 and 2 × 1010 CFU/kg) exhibited reduced weight gains, a trend that continued until day 28, indicating a dose-dependent effect on body weight (Table 5). Notably, in male rats, significant reductions in liver weights were observed at the higher doses, suggesting a specific impact of BC99 on liver mass, with statistical significance noted (p < 0.05) (Table 6). Hematological assessments remained stable across all groups, with minor, non-significant variations in platelet counts and white blood cell percentages, indicating no hematological disturbances from BC99 administration (Table 7). Coagulation profiles showed only slight variations in PT and APTT, with fibrinogen levels slightly elevated in the highest dose female groups, indicating a minimal influence of BC99 on coagulation processes (Table 8). The biochemical analysis revealed no significant differences across most parameters in the dosage groups, except for AST levels and liver organ indices in the higher dose male groups, which were statistically significant (p < 0.05), suggesting a localized liver response without general systemic effects (Fig. 5A-C and Table S1). In conclusion, by comprehensively evaluating the toxicological and physiological responses in rats at administered doses, we established the No Observed Adverse Effect Level (NOAEL) for BC99 at 0.5 × 1010 CFU/kg body weight/day, confirming the safety of BC99 at tested doses and supporting its use under the evaluated conditions.

Table 5 Effect of Heyndrickxia coagulans BC99 on rat body weight.
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Table 6 Organ weights and organ indices of the organs in rats treated with Heyndrickxia coagulans BC99.
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Table 7 Effect of Heyndrickxia coagulans BC99 on rats’ hematological indexes.
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Table 8 Effect of Heyndrickxia coagulans BC99 on rat blood coagulation indexes.
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Fig. 5
figure 5

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99. In vivo safety evaluation of BC99, subacute oral toxicity test. (A) AST (aspartate aminotransferase) values; (B) ALT (alanine aminotransferase) values; (C) ALP (alkaline phosphatase) values. (ns: not significant, * p < 0.05, ** p < 0.01.

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Discussion

Proof of safety is a fundamental criterion, whether for pharmaceutical or nutraceutical applications. The safety of new probiotic strains must also be thoroughly evaluated36. According to the guidelines of authoritative bodies such as ICMR-DBT (Indian Council of Medical Research and Department of Biotechnology) and ISAPP (International Scientific Association for Probiotics and Prebiotics), safe probiotic strains should be subjected to detailed identification at the genus, species and strain levels, complemented with a series of in vitro and in vivo tests37. Our study adhered to these guidelines and confirmed the acid and bile salt tolerance and antibiotic susceptibility of Heyndrickxia coagulans BC99 in vitro - key properties for the survival and functionality of probiotics in the gut environment.

The reclassification of the genus Bacillusin 2020 by Gupta et al38. led to a comprehensive reclassification and renaming of many Bacillus species. Our strain, isolated and sequenced from infant feces, is classified under the genus Heyndrickxia, based on official phylogenomic and comparative genomic analyses39, and is designated as H. coagulans BC996. When compared to probiotic strains such as Lactobacillus reuteri IDCC 3701, Bifidobacterium bifidum BGN4, and Bifidobacterium longum BORI, BC99 exhibits distinct genomic features that confer unique advantages4,40. Unlike these strains, which predominantly focus on lactic acid production, BC99’s genome encodes for a broader spectrum of functionalities, including enhanced stress response capabilities and a diverse array of bacteriocins like circularin_A and amylocyclicin. These features potentially grant BC99 superior resilience and efficacy in competitive gut environments, where it can inhibit pathogenic bacteria more effectively while supporting the growth of beneficial microbiota. Recently, BC99 was the subject of a clinical trial focusing on constipation. This randomized, double-blind, placebo-controlled trial, conducted over four weeks with weekly follow-ups, instructed participants to adhere to their regular diets while avoiding yogurt, probiotic foods, and any products that could influence the gastrointestinal microbiota. The trial confirmed BC99 as an effective and safe therapeutic agent for the alleviation of adult chronic constipation, enhancing gut microbiota balance and impacting key metabolic pathways13. Importantly, this study confirmed that the virulence gene is not transferable. Genetic analysis further corroborated the safety of BC99, revealing only a small number of potential virulence-related genes (e.g., gnd, clpP, clpC, tufA, groEL, bpsC, and bpsD), which are primarily associated with bacterial survival, stress response, and metabolic regulation rather than direct pathogenicity41,42,43,44,45. The gndgene, integral to the pentose phosphate pathway, enhances oxidative stress resistance and supports nucleic acid synthesis, critical for BC99’s survival in the stressful gastrointestinal environment42. Proteins encoded by clpP and clpCare essential for protein quality control, removing misfolded or damaged proteins and enabling the strain to cope with environmental stresses within the gut41,43,44. The tufA gene, crucial for protein synthesis, facilitates bacterial adaptation and fosters interactions with intestinal epithelial cells, enhancing colonization46,47. GroEL, involved in protein folding, is vital for managing stress responses during transit through the acidic stomach48,49. Our comprehensive evaluation using VirulenceFinder 2.0 and PathogenFinder 1.1 further supports BC99’s non-pathogenic status. Hosted by the CGE, these phenotype prediction tools underscored the absence of acquired virulence genes and indicated a low probability of pathogenicity toward human hosts21,50. Moreover, the lack of mobile genetic elements capable of mediating horizontal gene transfer of virulence factors substantiates BC99’s safety for probiotic use. Collectively, the pathogenic phenotype predictions and bioinformatic assessments are consistent with the established safety profile of B. coagulans strains, which are listed on EFSA’s Qualified Presumption of Safety (QPS) list and have been recognized as Generally Recognized as Safe (GRAS) by the U.S. FDA. These analyses confirm that BC99 does not possess pathogenic potential, endorsing its use in dietary supplements and food products.

The acidic environment in the host’s stomach and the high bile salt concentrations near the intestine are the greatest challenges for the viability of the strain in the host51. Interest in spore-forming probiotics has increased due to their resilience under adverse conditions52. For instance, Bacillus coagulans strains demonstrate remarkable stability under extreme conditions, including high temperatures, acidity, and bile salts53, facilitating their survival and beneficial activities within the host gastrointestinal tract54. In our study, BC99 exhibited exceptional survival rates in simulated gastrointestinal environments, validating its robustness in the human gastrointestinal tract. Genomic analysis revealed the presence of genes that regulate flagellar assembly and quorum sensing, potentially enhancing the motility and colonization capabilities of BC99 within the gastrointestinal tract. Furthermore, KEGG pathway analysis showed BC99’s involvement in key metabolic pathways essential for its function as a probiotic. Notably, pathways involved in the synthesis of multiple amino acids were prominent, highlighting BC99’s ability to produce essential compounds that may be beneficial for both bacterial survival and host health. These amino acids play vital roles in various physiological processes including tissue repair, immune response, and neurotransmitter production, which can significantly influence the host’s gut health55. Also the analysis revealed an extensive role of ABC transporter systems in BC99. These systems are crucial for the efficient transport of a wide array of substances across cellular membranes, including nutrients and metabolic byproducts56. This capability not only facilitates the strain’s adaptation to the nutrient fluxes within the gastrointestinal tract but also enhances its defensive mechanisms against gut pathogens by effectively managing toxin expulsion57. These insights into the metabolic functions of BC99, as revealed by KEGG pathway analysis, provide a clearer picture of how the strain sustains its viability and activity in the gut, contributing to its probiotic effects. This detailed understanding of metabolic pathways supports the potential of BC99 in enhancing gut microbiota balance, improving nutrient absorption, and promoting overall gastrointestinal health58. Moreover, the identification of gene clusters in BC99 responsible for producing non-ribosomal peptides such as fengycin, and cyclic peptides like amylocyclicin, highlights their critical roles in enhancing the strain’s probiotic efficacy. These peptides are not only help in directly suppressing pathogenic bacteria but also play a pivotal role in maintaining a balanced gut microbiota. By disrupting the membrane integrity of harmful bacteria, these peptides ensure the suppression of undesirable pathogens, thereby promoting an environment that favors the growth of beneficial gut flora. This selective antibacterial activity is crucial for maintaining gut health, enhancing nutrient absorption, and modulating the immune system. The presence of these peptide-producing gene clusters thus substantiates BC99’s potential as a powerful probiotic, capable of actively contributing to the host’s gastrointestinal health and overall well-being59,60,61,62. Furthermore, BC99’s lack of hemolytic activity, confirmed by the negative hemolysis test, highlights its biocompatibility, indicating its safe interaction with human cells without inducing harm or immune responses, a critical aspect of its in vitro safety evaluation63. Probiotics have been recognized for their role in modulating systemic inflammation, cellular apoptosis, and proliferation, and in preventing the excessive growth of cancer cells64. The human colon cancer cell line HT-29, which represents mature colon cells, serves as an exemplary model for further exploration into the effects of fermented milk products on colon cancer65. In our in vitro assays, BC99 showed minimal cytotoxicity and strong adhesion to HT-29 cells, essential for gut mucosa colonization. These adhesion properties help stabilize the gut microbiota and enhance resistance to intestinal clearance, while its low translocation potential reduces the risk of systemic infection. Besides, Probiotics that are sensitive to antibiotics do not transfer resistance traits to other pathogenic bacteria in the same ecological niche, making their resistance essentially non-transferable63. This specificity necessitates a comprehensive sensitivity analysis66. In our study, BC99 showed sensitivity to a wide range of common antibiotics, which is an important finding in the context of preventing the development of antibiotic resistance.

Oral toxicity experiments are of fundamental importance for the evaluation of the safety of probiotics and have been used in numerous safety assessment studies67. Single-dose acute toxicity studies provide preliminary toxicologic findings that inform appropriate dosing for subsequent repeated-dose toxicity studies68. According to the globally harmonized system (GHS) of classification, the LD50threshold for single oral administration of BC99 in animals is > 5000 mg/kg body weight and is classified under GHS Category 5, which identifies substances with relatively low acute toxicity risks69. The ‘No Observed Adverse Effect Level (NOAEL)’ derived from the 28-day repeated dose toxicity study is 5 billion CFU/kg body weight/day. In our study, significant differences in liver weights, both relative and absolute, were noted. We have expanded our discussion to include that these changes in liver weights, particularly noted in the absence of concurrent clinical, biochemical, or histopathological abnormalities, may reflect physiological adaptations rather than toxic effects. Such adaptations are typical in non-terminal studies and are not necessarily indicative of biological adversity. This interpretation aligns with the current understanding in toxicology that changes in organ weights can often represent adaptive, non-adverse responses to dietary or environmental changes70. Furthermore, it is noteworthy that strain BC99 has undergone clinical trials without any reported adverse events during the intervention, thereby supporting its safety profile13. The absence of adverse events in clinical settings complements our findings from animal studies, confirming that the observed liver weight changes are likely non-adverse and reflective of an adaptive physiological response rather than toxicity.

Considering that probiotics are frequently used in healthy food additives and medicinal formulations, the safety assessment of microorganisms selected as probiotics and of commercial probiotic products is essential71. In summary, BC99 not only exhibits basic probiotic properties such as tolerance, adhesion ability and antibiotic sensitivity, but also shows commendable in vitro and in vivo safety. These properties make it a valuable probiotic candidate for the food and pharmaceutical sectors.

Conclusion

Comprehensive genomic sequencing analysis, physiological and biochemical experiments and in vitro and in vivo safety studies have validated H. coagulans BC99 as a probiotic product with essential safety characteristics suitable for use as an ingredient in food and pharmaceutical products. However, to fully establish its safety and functionality, future research should include a broader range of basic safety investigations and functional validation trials.