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Article

Probiotic Potential of Weizmannia coagulans MA42, an Endospore-Forming Probiotic Bacterium Capable of Dietary Fiber Digestion

by
Punnita Pamueangmun
1,
Nang Nwet Noon Kham
1,
Apinun Kanpiengjai
2,
Chalermphong Saenjum
3,4,
Kalidas Shetty
5,
Kridsada Unban
4,6,* and
Chartchai Khanongnuch
7
1
Division of Biotechnology, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
2
Division of Biochemistry and Biochemical Innovation, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
3
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand
4
Research Center for Multidisciplinary Approaches to Miang, Chiang Mai University, Chiang Mai 50200, Thailand
5
Department of Microbiological Sciences, North Dakota State University, Fargo, ND 58102, USA
6
Division of Food Science and Technology, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
7
Center of Excellence in Agricultural Innovation for Graduate Entrepreneur, Maejo University, Chiang Mai 50290, Thailand
*
Author to whom correspondence should be addressed.
Foods 2026, 15(4), 710; https://doi.org/10.3390/foods15040710
Submission received: 17 January 2026 / Revised: 9 February 2026 / Accepted: 11 February 2026 / Published: 14 February 2026
(This article belongs to the Special Issue Probiotic Fermentation: Advances in Research on Probiotic Properties and Fermentation Techniques)
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Abstract

Weizmannia coagulans has emerged as a prominent probiotic candidate due to its resilience in extreme environments and therapeutic potential for non-gastrointestinal diseases, including obesity, bacterial vaginosis, and irritable bowel syndrome-related depression. This study comprehensively evaluated the probiotic properties, safety profile, and functional characteristics of W. coagulans strains (MA42, P13, and S5) compared with the reference strain W. coagulans ATCC 7050. All tested strains exhibited excellent gastrointestinal survival (>90% viability), superior auto-aggregation (up to 36.60%), hydrophobicity (up to 36.58%), and susceptibility to commonly used antimicrobials. Cell-free culture supernatants showed potent antimicrobial activity against pathogenic bacteria, including Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium TISTR 292, and Bacillus cereus TISTR 747, primarily through organic acid production. Notably, strain MA42 uniquely inhibited the growth of Staphylococcus aureus TISTR 746. All strains showed negative hemolytic activity, confirming their safety profile. W. coagulans MA42 distinguished itself through exceptional metabolic versatility, demonstrating vigorous growth on diverse complex dietary fibers and prebiotics, with significant production of beneficial organic acids, particularly lactic and acetic acids. This superior fermentation capacity correlated directly with elevated extracellular-enzyme activities. Furthermore, all strains maintained excellent viability (>90% recovery) after freeze-drying with maltodextrin as a cryoprotectant, indicating industrial scalability.

1. Introduction

In response to the increase in consumer health concerns and the demand for safe functional foods with high nutritional and health-promoting properties, special probiotics or probiotic-related products have been extensively produced and demonstrated their functional properties, particularly their beneficial effects on host health [1]. Currently, varieties of beneficial microorganisms including Lactobacillus, Bifidobacterium, Bacillus, and yeast are used as commercial probiotics for both humans and livestock [2,3]. Those probiotics have been extensively investigated for their functional properties in health, such as immunomodulation and the combating of harmful microbes in the host gastrointestinal tract [4,5], and some have also been demonstrated to be effective as preventive agents for colon cancer [6,7]. However, it is crucial to recognize that while probiotics offer potential benefits, they may also pose certain safety risks for humans. These concerns include the potential for bloodstream infections, the risk of acquiring and spreading genes that confer antibiotic resistance, and the risk of disrupting the natural balance and colonization patterns of microorganisms in the gut ecosystem [1]. Therefore, microorganisms must be strictly examined to assess their probiotic properties. Although probiotic products have received considerable attention, the heat sensitivity of probiotic microorganisms remains a challenge on an industrial scale [4,8]. Consequently, the concept of using thermostable probiotics in the development of functional foods has gained attention [9].
Bacillus species are ubiquitous microorganisms that thrive in diverse environments, including the atmosphere, soil, fermented foods, and the human digestive system. As probiotics, Bacillus strains possess several advantageous characteristics, including high-temperature resistance, survival at low pH, and tolerance to high bile salt concentrations. These benefits are attributed mainly to their ability to form spores. Spore formation allows Bacillus probiotics to remain viable under harsh conditions, making them exceptionally robust and versatile compared to other probiotic strains [10]. Bacillus coagulans (formerly Weizmannia coagulans) is a Gram-positive, rod-shaped, endospore-forming, lactic acid-producing bacterium that grows at high temperatures (50–55 °C) and produces essential enzymes such as amylases, proteases, β-galactosidase, and β-glucosidase [4,11,12,13]. In addition, W. coagulans has been granted Generally Recognized as Safe (GRAS) status by the U.S. Food and Drug Administration (FDA) [3]. In preclinical studies, W. coagulans has demonstrated therapeutic potential for treating gastrointestinal distress symptoms in both adults and children. It has also been investigated in human research and has been shown to be effective in controlling women’s health conditions, mood, and metabolism [9]. Furthermore, W. coagulans IS-2 has been used for the treatment and/or prevention of non-gastrointestinal diseases, such as obesity, bacterial vaginosis, and severe depression associated with irritable bowel syndrome (IBS) [9,11].
In our previous study, three strains of W. coagulans (MA42, P13, and S5) isolated from soil samples collected in Chiang Mai province, Thailand, were selected as L-lactic acid-producing strains with the specific purpose of using them in consolidated fermentation of lignocelluloses for L-lactic acid production, and with the final goal of using them in sustainable bioplastic synthesis [13]. Although W. coagulans has been extensively studied and applied in the food industry as an efficient probiotic [4,6], studies on the influencing effect of dietary fiber on the functional properties of this bacterium are scarce. The positive impact of the synbiotic formulation of soy pulp (Okara) powder mixed with B. coagulans lilac-01 on bowel movement and fecal properties has been reported [14]. Moreover, the use of a synbiotic between β-glucan and B. coagulans GBI-30 for the improvement of functional properties was also investigated [15]. However, broad-spectrum analysis demonstrates that the relationship between the W. coagulans probiotic and dietary fiber is limited. Regarding W. coagulans, which is well recognized as a probiotic bacterium with a capacity for lignocellulolytic-enzyme production, the challenges of using the newly isolated W. coagulans MA42, P13, and S5 strains to achieve health-related benefits are of interest. Therefore, this paper describes an investigation of the probiotic properties and functional characteristics of these bacterial strains for application in food products or nutraceuticals, compared with the reference strain.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

Weizmannia coagulans MA42, P13, and S5, three bacterial strains isolated from soil samples collected in Chiang Mai, Thailand, and previously studied in [13], were chosen for probiotic-property assessment. All strains were maintained in Xylose–Yeast–Peptone (XYP) broth containing 50% (v/v) glycerol and stored at −80 °C. Five bacterial strains, including Bacillus cereus TISTR 747, Salmonella enterica serovar Typhimurium TISTR 292, Staphylococcus aureus TISTR 746, Escherichia coli ATCC 25922, and Weizmannia coagulans ATCC 7050 (TISTR 1447), were obtained from Thailand Institute of Scientific and Technological Research (TISTR) and used in antimicrobial activity evaluation as indicator and reference strains. Seed inoculum was made by transferring a single colony of W. coagulans into 5 mL of sterile modified MRS (mMRS) broth containing 10.0 g of glucose, 5.0 g of yeast extract, 10.0 g of beef extract, 10.0 g of peptone, 1.0 g of Tween 80, 2.0 g of K2HPO4, 2.0 g of (NH4)2HC6H5O7), 0.2 g of MgSO4, and 0.2 g of MnSO4 and incubated at 37 °C under static conditions for 24 h.

2.2. Resistance to Simulated Gastrointestinal Conditions

The resistance of W. coagulans to the simulated gastric and intestinal conditions was investigated using a modified method previously described by Unban et al. [16]. After being inoculated in mMRS broth and incubated at 37 °C for 24 h, W. coagulans MA42, P13, and S5, and the reference probiotic strains (ATCC 7050), were harvested by centrifugation at 10,000 rpm for 10 min at 4 °C. The cell pellets were washed twice with sterile phosphate-buffered saline (PBS) and finally resuspended in electrolyte solution containing 6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2, and 1.2 g/L NaHCO3, then resuspended in the same electrolyte solution to achieve a final concentration of viable cells of 107 CFU/mL. After adjusting the pH to 3.0, the solution was mixed in a ratio of 3:5 using a simulated gastric fluid that contained 0.3% (w/v) pepsin (Fluka Biochemika, Seelze, Germany) in the electrolyte solution. A sample was obtained to measure the live cells on an mMRS agar plate during a 1 h incubation period at 37 °C. The remaining volume was then diluted 1:4 using a simulated duodenal secretion containing 6.4 g/L NaHCO3, 0.24 g/L KCl, 1.28 g/L NaCl, 0.5% (w/v) bile salts, and 0.1% (w/v) pancreatin (Sigma-Aldrich, St. Louis, MO, USA) at pH 7.2 in order to mimic the conditions in the small intestine. The mixture was then incubated at 37 °C. Samples were taken for viable cell counts using the plate count method on mMRS agar plates after two and three hours of incubation. The survival rate percentage was calculated based on the viable cell count as follows:
S = [(Ni − Nd)/Ni] × 100
where S is the percentage survival, Ni is the initial viable cell count, and Nd is the viable cell count after the simulated gastrointestinal test.

2.3. Cell Surface Hydrophobicity

The cell surface hydrophobicity of W. coagulans was assessed by measuring bacterial cell adhesion to nonpolar solvents, based on the cells’ capacity to bind hydrocarbons [16]. Briefly, a 24 h culture of W. coagulans was harvested by centrifugation at 10,000 rpm at 4 °C for 10 min, washed twice with PBS buffer (pH 7.2), and resuspended in PBS to an optical density at 600 nm (OD600) of 1 (A0). An equal volume of toluene (RCI Labscan, Ltd., Bangkok, Thailand) was added and mixed using a vortex mixer for 5 min. After 1 h of incubation at 37 °C, the optical density of the aqueous phase was measured at 600 nm (A1). Cell surface hydrophobicity was calculated using the following equation:
Cell surface hydrophobicity (%) = [(A0 − A1)/A0] × 100

2.4. Auto-Aggregation

Auto-aggregation analysis was performed according to Unban et al. [16]. W. coagulans strains were grown for 24 h in mMRS broth at 37 °C. The bacteria were resuspended in the same buffer after being harvested by centrifugation at 10,000 rpm for 10 min at 4 °C. Cell pellets were then rinsed twice with PBS. Then, 1 mL of the W. coagulans cell suspension in PBS was measured the absorbance at 600 nm (A0) after being homogeneously vortexed for 10 s. The absorbance of the superior 1 mL fraction was measured (A1) after a 2 h incubation period without disturbance at 37 °C. The proportion of auto-aggregation was calculated using the following equation:
Auto-aggregation (%) = [(A0 − A1)/A0] × 100

2.5. Evaluation of Antimicrobial Activity Against Pathogenic Bacteria

The agar well diffusion method was used to investigate W. coagulans antagonistic activity in cell-free culture supernatant (CFCS) against several pathogenic bacteria, namely B. cereus TISTR 747, S. Typhimurium TISTR 292, E. coli ATCC 25922, and S. aureus TISTR 746, following the method described by Unban et al. [16]. Briefly, the overnight cultures of pathogenic bacteria, with cell concentrations adjusted to approximately 106–108 CFU/mL with sterile distilled water, were gently swabbed onto the surface of an NA plate. Both the un-neutralized and neutralized CFCSs, in a volume of 20 μL (neutralized to pH 7 by using 1 M NaOH), from the mMRS culture broth of W. coagulans were placed on the surface of the swabbed agar plates via sterile filter paper discs (6.0 mm diameter). A clear zone surrounding the well, showing signs of growth inhibition, was observed after 24 h of incubation at 37 °C.

2.6. Antibiotic Susceptibility Test

The disc diffusion method was used to investigate the antibiotic susceptibilities of W. coagulans strains. Antibiotic discs, including 300 μg/disc of polymyxin-B, 30 μg/disc of kanamycin, 30 μg/disc of vancomycin, 15 μg/disc of erythromycin, 10 μg/disc of streptomycin, and 10 μg/disc of gentamicin, were used [9]. Approximately 50 µL of a 24 h cultured broth of W. coagulans (107–108 CFU/mL) was swabbed onto an mMRS agar plate, and antibiotic discs (HiMedia, Mumbai, India) were placed on the agar surface. After incubation at 37 °C for 24 h, the diameters of the inhibitory zone were measured. The results were assessed and classified as resistant (R), intermediate (I), or sensitive (S) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [17].

2.7. Hemolytic Activity Test

Sheep Blood Agar (SBA) (M&P IMPEX, Bangkok, Thailand) mixed with 5% (v/v) sheep blood was used to determine the hemolysis type of W. coagulans strains. After streaking the bacterial strains on a blood agar plate, all plates were incubated for 48 h at 37 °C. The indicative hemolytic clear zone surrounding colonies on blood agar was monitored and classified [9].

2.8. Digestive-Enzyme-Producing Test

A 125 mL Erlenmeyer flask containing 50 mL of mMRS broth with 10 g/L of either lactose, casein, or starch as the sole carbon source was filled with 2% (v/v) of each strain of W. coagulans. Under anaerobic conditions, the broth was covered with 5 mL of sterile paraffin oil and incubated for 24 h at 37 °C [13]. Following the incubation period, the culture was centrifuged for 10 m at 12,000 rpm and 4 °C. The clear supernatant was collected for the determination of amylase, protease, and β-galactosidase activities.

2.9. Evaluation of Dietary-Fiber Carbohydrates Fermentaion

A 250 mL Duran flask was filled with 100 mL of mMRS broth composed of 1% (w/v) of various substrates as the sole carbon source, including wheat flour (McGarrett, Bangkok, Thailand), oatmeal (McGarrett, Bangkok, Thailand), xylan (Megazyme, Wicklow, Ireland), carboxy methyl cellulose (CMC) (Nacalai Tesque, Inc., Tokyo, Japan), locust bean gum (LBG) (Sigma-Aldrich, St. Louis, MO, USA), xanthan gum (McGarrett, Bangkok, Thailand), pectin (Wako Pure Chemical, Tokyo, Japan), inulin (Wako Pure Chemical, Tokyo, Japan), xylo-oligosaccharides (XOS) (Wako Pure Chemical, Tokyo, Japan), fructo-oligosaccharides (FOS) (Wako Pure Chemical, Tokyo, Japan), galacto-oligosaccharides (GOS) (Wako Pure Chemical, Tokyo, Japan), and glucose (control). Each culture medium was inoculated with 10% (v/v) inoculum. To create and maintain the anaerobic conditions, 10 mL of sterilized paraffin oil was added to cover the culture broth surface and incubated at 37 °C for 48 h, according to the method described by Li et al. [18]. After the incubation, the culture was centrifuged at 12,000 rpm and 4 °C for 10 min. The aqueous phase of clear supernatant was collected for the determination of organic acid content, lactic acid, and short-chain fatty acids (acetic acid, propionic acid, and butyric acid) using a high-performance liquid chromatography (HPLC) system (Hitachi HighTech, Chiyoda-ku, Tokyo, Japan) equipped with a Bio-Rad organic acid column (300 mm × 7.8 mm, Aminex, Bio-Rad Laboratories, Richmond, CA, USA). The mobile phase was 0.005 M H2SO4 at a flow rate of 0.6 mL/min and 210 nm of UV detection. The activities of amylase, cellulase, β-mannanase, xylanase, β-fructofuranosidase, pectinase, and β-galactosidase in cell-free supernatant were also determined.

2.10. Enzyme Assay

Amylase, cellulase, β-mannanase, xylanase, β-fructofuranosidase, and pectinase activities were assayed using modified DNS (3,5-dinitrosalicylic acid) methods based on the release of reducing sugars from their respective substrates (starch, CMC, LBG, xylan, FOS, and pectin, respectively) [19]. Equal volumes (1 mL) of substrate and culture broth supernatant (crude enzyme) were mixed, and the reaction was incubated for 15 min at 37 °C. Then, 2 mL of DNS solution was added and heated to boiling for 5 min to terminate the reaction. The absorbance was recorded at 540 nm. Enzyme activities were calculated and expressed as units per milliliter. One unit of enzyme is defined as the amount of enzyme that catalyzes the hydrolysis of the substrate to liberate 1 µmole of reducing sugars per minute under assay conditions.
Protease activity was determined by a modified version of the procedure of Blanco et al. [20] using 0.5% (w/v) casein from bovine milk (Sigma-Aldrich, St. Louis, MO, USA) as the substrate in 0.1 M sodium phosphate buffer, pH 7.0, 0.125 mL of enzyme, and 0.125 mL of substrate for the reaction mixture. After being carried out at 37 °C for 10 min, the reaction was stopped by adding 0.125 mL of 0.4 M trichloroacetic acid (TCA). The precipitate was removed by centrifuging at 8000 rpm for 10 min at 4 °C. The supernatant (0.25 mL) was collected and mixed with 1.25 mL of 0.4 M Na2CO3 and 0.15 mL of 1N Folin–Ciocalteu reagent. Tyrosine liberation was quantified by measuring absorbance at 660 nm. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 μg of tyrosine per milliliter per minute under the described conditions.
β-Galactosidase activity was determined following the method described by Sriphannam et al. [21] using para-nitrophenol-β-D-galactopyranoside (pNPG) (Sigma-Aldrich, St. Louis, MO, USA) as a substrate. A total sample of 0.125 mL was added to 0.125 mL of 4.0 mM pNPG in 0.05 M sodium phosphate buffer, pH 7, and incubated at 37 °C for 30 min. Then, 0.25 mL of 0.05 M Na2CO3 was added to stop the reaction, and the absorbance was measured at 405 nm. The amount of pNP was calculated based on a standard plot. One unit of enzyme activity was defined as the amount of enzyme liberating 1 µmole of pNP per minute under the described conditions.

2.11. Viability of Probiotic Strains After Freeze-Drying

A 24 h culture of W. coagulans strains in mMRS broth at 37 °C was harvested by centrifugation at 10,000 rpm for 10 min at 4 °C, then washed twice with PBS. The washed cell pellets were resuspended in maltodextrin (10%, w/v) to approximately 107 CFU/mL. The cell suspensions were frozen at −20 °C for 24 h, after which they were transferred to a laboratory-scale freeze-dryer (DW-12N Vertical Freeze Dryer, Chongqing, China) operated at 1 Pa and −35 °C for 24 h. The survival percentage was calculated as follows:
Survival rate (%) = (N/N0) × 100
where N represents the number of viable cells of dry matter after drying, and N0 is the number of viable cells of dry matter in the bacterial suspension before drying [16].

2.12. Statistical Analysis

All experiments were performed in triplicate, and the results were calculated as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to study significant differences between means, with a significance level of p < 0.05, using SPSS statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Survival Against Simulated Gastrointestinal Conditions

An in vitro model of simulated gastrointestinal conditions was used to evaluate bacterial survival of W. coagulans MA42, P13, and S5, along with the probiotic reference strain, W. coagulans ATCC 7050. The influence of gastric and duodenal juice conditions on all tested W. coagulans strains is demonstrated in Figure 1. At the end of the treatments, statistical analysis of bacterial viability showed that the survival of the MA42, P13, and S5 strains was equivalent to that of the ATCC 7050 probiotic strain [22]. All strains showed tolerance and a good survival rate of more than 90% after being tested under the stress of simulated gastrointestinal conditions. The MA42 and P13 candidates demonstrated a greater capacity for survival under in vitro gastrointestinal conditions than the ATCC 7050 probiotic strain. In addition, Mazhar et al. [9] reported that W. coagulans CGI314 spores have the ability to endure passage through the upper digestive tract, a property superior to that of traditional Lactobacillus probiotic strains.

3.2. Auto-Aggregation and Hydrophobicity

Auto-aggregation and hydrophobicity are key functional traits of probiotic strains. Auto-aggregation is typically associated with hydrophobicity and is also indicative of the bacteria’s ability to survive and endure in the digestive system [23]. The auto-aggregation of all W. coagulans strains varied from 19.68 to 36.60% after 2 h of incubation (Figure 2A). The MA42, P13, and S5 strains showed moderate auto-aggregation with values of 36.60%, 33.11%, and 27.20%, respectively, and the reference strain ATCC 7050 had only 19.68% activity. In contrast, the auto-aggregative values of pathogenic bacteria such as E. coli ATCC 25922, E. faecalis, and S. Typhimurium were 11.8%, 5.5%, and 12.2%, respectively, after incubation at 37 °C for 2 h [24]. According to reports, increased colonization of probiotic strains in the gastrointestinal tract is associated with greater auto-aggregation. Auto-aggregation values are strain-specific and have previously been reported for B. coagulans at 25.8% and 29.0% [9,25].
Similarly to auto-aggregation, all strains showed a moderate level of hydrophobicity towards toluene, ranging from 16.19 to 36.58% after a 1 h incubation period (Figure 2B). W. coagulans candidates showed better hydrophobicity compared to the reference strain ATCC 7050. However, no statistically significant difference was observed in the hydrophobicity values of the W. coagulans strains MA42, P13, and ATCC 7050. Previous studies have reported a high degree of variation in hydrophobicity among W. coagulans strains. W. coagulans has shown cell surface hydrophobicity of 29% and 43% with chloroform and xylene, respectively [25,26]. Probiotic strains with high hydrophobicity are more likely to interact effectively with gastrointestinal tract cells. This enhanced interaction suggests a greater ability to prevent pathogen colonization. Variations in hydrophobicity among bacterial species can be attributed to differences in protein expression on their cell surfaces [16].

3.3. Antimicrobial Activity Against Pathogenic Bacteria

W. coagulans strains were evaluated to assess their antimicrobial activities against the indicator microorganisms E. coli ATCC 25922, S. Typhimurium TISTR 292, B. cereus TISTR 747, and S. aureus TISTR 746 using the disc diffusion method. The un-neutralized CFCS from W. coagulans MA42, P13, and S5, and the reference strain (ATCC 7050), exhibited clear inhibitory zones against all tested indicator microorganisms, except for S. aureus TISTR 746, which was inhibited only by the MA42 strain. The results revealed that all W. coagulans strains exhibited inhibition zones ranging from 7.50 to 17.50 mm. W. coagulans MA42 was the most effective in inhibiting target pathogens, with 17.50 ± 1.50, 16.00 ± 1.00, 14.50 ± 0.50, and 8.50 ± 0.50 mm clear zones against E. coli ATCC 25922, S. Typhimurium TISTR 292, B. cereus TISTR 747, and S. aureus TISTR 746, respectively, showing an inhibition zone better than W. coagulans ATCC 7050, as shown in Figure 3A. The neutralized CFCS from all W. coagulans strains failed to produce inhibition zones (Figure 3B). This contrasts with un-neutralized CFCS, which typically exhibits antimicrobial activity due to its lactic and acetic acids, creating an environment unfavorable to most pathogens. Furthermore, the difference in inhibitory activity of W. coagulans MA42 and the other tested strains could have been caused by the lactic acid concentration, as it was found that the strain MA42 produce lactic acid at the highest level (8.96 ± 0.19 g/L), which was significant different from the levels in W. coagulans P13, S5, and ATCC 7050 (5.36 ± 0.29, 4.77 ± 0.28, and 6.04 ± 0.19, respectively). When CFCS is neutralized, any observed inhibitory effects are generally attributed to the presence of bacteriocins or similar antimicrobial compounds, rather than pH [27]. The study by Mazhar et al. [9] found that W. coagulans CGI314 exhibited antimicrobial activity against intestinal (E. coli ATCC 25922), oral (S. sobrinus DSM 20742), and common skin (S. aureus RF122) pathogens. In addition, previous studies using other methods with W. coagulans members demonstrated an effective antimicrobial profile [3,11].

3.4. Antibiotic Susceptibility

A critical consideration in probiotic use is their safety profile, particularly regarding antibiotic resistance. The primary concern is that these antibiotic-resistant probiotics may transfer their resistance genes to pathogenic bacteria. Consequently, probiotics should be sensitive to low concentrations of prescribed antibiotics [25]. The results of antibiotic susceptibility testing (Table 1) showed that the W. coagulans candidates were sensitive to all tested antibiotics (streptomycin, erythromycin, gentamicin, kanamycin, vancomycin, and polymyxin B), with inhibition diameters ranging from 23.0 to 44.0 mm. Previous reports have stated that W. coagulans is susceptible to antibiotics [9,25,26].

3.5. Hemolytic Activity

Hemolytic activity is generally caused by pathogenic bacteria, including many Gram-positive bacteria, and therefore, the absence of hemolytic activity is an important safety criterion for probiotic selection [28]. Our findings showed that W. coagulans strains exhibited γ-hemolysis (no hemolysis) when cultured on SBA (Figure 4), while S. aureus, used as the positive control, clearly induced β-hemolysis. Similarly, several studies have demonstrated that different W. coagulans strains lack hemolytic activity [25], supporting their safety profile for probiotic applications. With regard to safety concerns, W. coagulans has been shown to be safe against antibiotic-resistant strains and to lack a toxic gene cluster in its whole genome [29,30]. Moreover, the commercial products from B. coagulans strains, such as LactoSpore and GBI-30, have been listed as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) since 2012 [28].

3.6. Digestive-Enzyme Activity

All W. coagulans strains secreted amylase in amounts ranging from 631.00 to 856.48 mU/mL and released protease in amounts ranging from 20.57 to 97.33 mU/mL, while the activities of β-galactosidase ranged from 7.94 to 21.36 mU/mL (Table 2). However, W. coagulans MA42, P13, and S5 showed significantly higher enzyme activities than the reference strain. Regarding its ability to produce the main human digestive enzymes in anaerobic conditions, such as amylase and protease, this probiotic bacterium demonstrates an additional advantage to the host after its consumption. Amylase and protease acquired by W. coagulans MA42 are expected to assist the host digestive system in the conversion of starch and proteins into simple saccharides and free amino acids, respectively. Protease also helps to regulate amino acid metabolism in various body regions [4]. Furthermore, W. coagulans’s capacity to release β-galactosidase is also able to improve the digestion of lactose found in milk and is possibly helpful for reducing the problems associated with lactose intolerance [31].

3.7. Fermentation of Dietary-Fiber Carbohydrates

A comparison study was conducted on the fermentation capacity of complex dietary components/ingredients and prebiotics in the culture broth as the sole carbon source between W. coagulans MA42 and W. coagulans ATCC 7050. As the key parameter of microbial fermentation is the growth of the microbe, the growth of both microbial strains was assessed at 12, 24, and 48 h of fermentation, and is presented as the bacterial viable cell number in Figure 5. Of the eleven carbohydrate sources and one control substrate (glucose) tested under anaerobic fermentation, W. coagulans MA42 demonstrated excellent growth in wheat flour, oatmeal, xylan, carboxymethylcellulose (CMC), locust bean gum (LBG), xanthan gum, pectin, inulin, xylo-oligosaccharides (XOS), fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and glucose, with maximum growth mostly observed at 24 h (growth range: 7.68 ± 0.06 to 8.49 ± 0.01 Log CFU/mL). Conversely, the reference strain ATCC 7050 clearly showed growth only in basal medium containing glucose as a carbon source, particularly at 12 h of fermentation (Figure 5A). In contrast, only a slight increase in bacterial growth was observed in other substrates, including xylan, inulin, XOS, GOS, wheat flour, oatmeal, CMC, LBG, xanthan gum, and pectin (Figure 5B–K). A decline in bacterial growth in most substrates was clearly observed after the fermentation time was prolonged to 48 h. This evidence indicates that a higher growth rate of W. coagulans MA42 was found compared to the growth of the reference strain.
The organic acid production ability of W. coagulans MA42 and W. coagulans ATCC 7050 is presented in Table 3. Both strains revealed the ability to efficiently produce lactic acid, but W. coagulans MA42 exhibited a higher capacity to produce lactic acid from various carbon sources, with the following concentrations observed: wheat flour (2.24 ± 0.13 g/L), oatmeal (2.23 ± 0.14 g/L), xylan (2.42 ± 0.11 g/L), CMC (2.44 ± 0.10 g/L), LBG (2.31 ± 0.10 g/L), xanthan gum (2.70 ± 0.12 g/L), pectin (2.86 ± 0.03 g/L), inulin (2.22 ± 0.14 g/L), XOS (2.34 ± 0.12 g/L), FOS (2.97 ± 0.15 g/L), GOS (2.41 ± 0.11), and glucose (7.57 ± 0.15 g/L). This lactic acid accounted for an estimated 50–92% of the sugar consumed. In contrast, ATCC 7050 also produced lactic acid across the tested carbon sources, ranging from 0.54 ± 0.08 to 1.29 ± 0.06 g/L, with glucose yielding a significantly higher concentration of 6.32 ± 0.12 g/L. This result confirms the advantage of W. coagulans MA42 over the reference strain, highlighting the merit of W. coagulans MA42, which was specifically isolated for use in consolidated bioprocessing (CBP) to convert lignocellulose into L-lactic acid [13]. Furthermore, both MA42 and ATCC 7050 exhibited the capacity to synthesize short-chain fatty acids (SCFAs). SCFAs, such as acetic acid, have been associated with various health-promoting effects in the host, such as anti-inflammatory, immunoregulatory, anti-obesity, anti-diabetic, anticancer, cardioprotective, hepatoprotective, and neuroprotective activities [9]. Generally, W. coagulans is able to produce lactic acid, a significant antimicrobial substance in the human gut, due to its ability to grow under anaerobic conditions, similar to those in the human gastrointestinal tract (GIT) [11]. In addition, lactic acid and its derivatives have been reported to accelerate the development of intestinal-stem-cell-derived epithelial tissue in rats [32]. However, propionic acid and butyric acid were not detected in the culture broth of either strain. This result corresponds to the previous results on solid-state fermentation of soybean residue (Okara) by Bacillus coagulans IS-2 and 123 [33].
Additionally, both bacterial strains produced various extracellular enzymes in varying quantities depending on the type of dietary-fiber substrate (Figure 6). Overall, W. coagulans MA42 possessed superior extracellular polysaccharide-degrading enzymes (cellulase, xylanase, β-mannanase, pectinase, β-fructofuranosidase, amylase, and β-galactosidase) when grown on all dietary-fiber substrates, particularly wheat flour and oatmeal, and these activities directly correlated with growth performance and organic acid production. However, higher xylanase activity was found in the culture of W. coagulans ATCC 7050 grown with xanthan gum (Figure 6F). W. coagulans is well recognized as a thermophilic, lactic acid-producing bacterium capable of lignocellulose-degrading-enzyme production [13,31,34,35,36]. The ability of W. coagulans to produce extracellular dietary-fiber-degrading enzymes directly indicates the involvement of genes encoding enzymes in the genome of both strains [37,38].
Dietary fiber comprises a diverse array of complex plant polysaccharides that largely resist digestion in the small intestine and subsequently move to the colon. Extensive research has explored the impact of dietary fiber on human health, generating various hypotheses regarding the beneficial effects on bacterial fermentation of carbohydrate fibers in the intestinal tract. These proposed benefits include the provision of short-chain fatty acids to colonic epithelial cells, suppression of microbial protein metabolism, and inhibition of other potentially harmful bacterial processes [39,40]. Consequently, the capacity to either ferment or degrade dietary-fiber polysaccharides derived from cereals, vegetables, or other food components represents one of the beneficial properties of probiotics. According to our previous study, W. coagulans MA42 is the most suitable strain compared to W. coagulans strains P15 and S5, as they had the highest capacity to produce lignocellulolytic enzymes, including cellulase, xylanase, and β-mannanase, which is an important factor facilitating the consolidated bioprocessing (CBP) of L-lactic acid from lignocelluloses [13]. These enzymes are expected to play a crucial role in the beneficial breakdown of complex lignocellulosic polysaccharides and other antinutritional components present in dietary fibers. In addition, the results of this study concerning the safety of W. coagulans, in combination with previous reports on whole-genome analysis [29,41], support the application of this microbe as a therapeutic agent in humans. These comprehensive findings support advancing the use of W. coagulans MA42 as a probiotic candidate.

3.8. Viability of Probiotic Strains Against Freeze-Drying

The effect of freeze-drying on the viability of the W. coagulans strains is shown in Table 4. The viability of W. coagulans MA42, P13, S5, and ATCC 7050 (reference strain) coated with maltodextrin was 99.20% (7.60 log CFU), 94.89% (6.92 log CFU), 95.06% (6.98 log CFU), and 97.03% (7.54 log CFU), respectively. The recovery yield of all strains was greater than 90% after freeze-drying. Freeze-drying is a favored technique for preserving bacterial cells because it uses low temperatures and pressures. These conditions help maintain the cells’ original structure, biochemical characteristics, and functions. In some studies, to reduce cellular damage during freeze-drying and enhance cell survival, the protective agent maltodextrin has been added to the biomaterial prior to drying, thereby improving its oxidative stability and glassy barrier properties [42,43]. In fact, the commonly used probiotic cryoprotectants include disaccharides (saccharose, lactose, trehalose), polyols (mannitol, sorbitol), and polysaccharides (maltodextrin, dextran, inulin) [44]. The criteria for selection of cryoprotectants include availability, commercial feasibility, non-animal origin, and being Generally Recognized as Safe (GRAS) [42]. In our previous report, spray-drying maltodextrin showed an ability to efficiently protect Lactobacilli probiotic bacteria from cell damage after the powdering process [14]. Therefore, for the preliminary drying process in this study, we decided to use 10% maltodextrin following our previous work. Although this preliminary result of freeze-drying indicated the merits and feasibility of these W. coagulans for industrial-scale application, a comparative study on the most suitable cryoprotectants and their protective mechanism must be conducted.

4. Conclusions

This study presented an extensive overview of the potential probiotic properties of the novel strains of W. coagulans MA42, P13, and S5, including safety evaluation approaches, resistance to gastric and intestinal conditions, and quantitative analysis. All three novel strains demonstrated good probiotic properties, including the ability to inhibit pathogens, be non-hemolytic, be sensitive to all antibiotics, and survive under simulated gastrointestinal conditions. In addition, they showed moderate auto-aggregation and hydrophobicity, produced lactic acid and SCFA, and exhibited digestive-enzyme activity (amylase, protease, and β-galactosidase). These W. coagulans strains also showed tolerance against the freeze-drying process. Overall, the results from all tests showed that MA42, P13, and S5 had relatively more prominent probiotic properties compared to the probiotic strains (ATCC 7050). Beyond its ability to produce L-lactic acid directly from lignocellulose, these research results strongly demonstrate that the novel strain of W. coagulans MA42 has the most promising probiotic potential, which indicates that this strain is an excellent candidate probiotic for food products.

Author Contributions

Conceptualization, K.U. and C.K.; methodology and formal analysis, P.P., N.N.N.K., A.K., K.U. and C.K.; investigation, P.P. and K.U.; writing—original draft preparation, P.P. and K.U.; writing—review and editing, P.P., A.K., C.S., K.S., K.U. and C.K.; supervision, K.U. and C.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Royal Golden Jubilee Ph.D. Programme (RGJPHD 21) (Grant No. PHD/0219/2561) and was also supported by the Fundamental Fund 2024 (Grant No. FF082/2567), Chiang Mai University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw data supporting the conclusions of this article can be made available by the authors on request.

Acknowledgments

The authors gratefully acknowledge the Faculty of Agro-Industry, Chiang Mai University, for providing access to essential research facilities.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Survival of W. coagulans ATCC 7050, MA42, P13, and S5 under simulated gastrointestinal conditions. Black and white arrows indicate the addition of simulated gastric juice and simulated duodenal juice, respectively.
Figure 1. Survival of W. coagulans ATCC 7050, MA42, P13, and S5 under simulated gastrointestinal conditions. Black and white arrows indicate the addition of simulated gastric juice and simulated duodenal juice, respectively.
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Figure 2. Auto-aggregation (A) and cell surface hydrophobicity (B) of W. coagulans ATCC 7050, MA42, P13, and S5. Different letters (A–C) indicate significant differences in the values (p < 0.05).
Figure 2. Auto-aggregation (A) and cell surface hydrophobicity (B) of W. coagulans ATCC 7050, MA42, P13, and S5. Different letters (A–C) indicate significant differences in the values (p < 0.05).
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Figure 3. Disc diffusion assay of cell-free culture supernatants (CFCSs), obtained from W. coagulans ATCC 7050, W. coagulans MA42, W. coagulans S5, and W. coagulans P13 cultured in mMRS broth at 37 °C under static conditions for 24 h, against E. coli ATCC 25922, S. Typhimurium TISTR 292, B. cereus TISTR 747, and S. aureus TISTR 746. Control (M): uninoculated mMRS broth. Un-neutralized CFCS (A) and neutralized CFCS (pH 7) (B).
Figure 3. Disc diffusion assay of cell-free culture supernatants (CFCSs), obtained from W. coagulans ATCC 7050, W. coagulans MA42, W. coagulans S5, and W. coagulans P13 cultured in mMRS broth at 37 °C under static conditions for 24 h, against E. coli ATCC 25922, S. Typhimurium TISTR 292, B. cereus TISTR 747, and S. aureus TISTR 746. Control (M): uninoculated mMRS broth. Un-neutralized CFCS (A) and neutralized CFCS (pH 7) (B).
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Figure 4. Hemolytic activity of W. coagulans ATCC 7050, MA42, P13, and S5 on blood agar at 37 °C for 48 h. Staphylococcus aureus was used as the positive control.
Figure 4. Hemolytic activity of W. coagulans ATCC 7050, MA42, P13, and S5 on blood agar at 37 °C for 48 h. Staphylococcus aureus was used as the positive control.
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Figure 5. Viable cell count of W. coagulans MA42 and ATCC 7050 on modified MRS medium supplemented with glucose (A), wheat flour (B), oatmeal (C), xylan (D), carboxymethyl cellulose (CMC) (E), locust bean gum (LBG) (F), xanthan gum (G), pectin (H), inulin (I), xylo-oligosaccharides (XOS) (J), fructo-oligosaccharides (FOS) (K), and galacto-oligosaccharides (GOS) (L) as the sole carbon source. *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively.
Figure 5. Viable cell count of W. coagulans MA42 and ATCC 7050 on modified MRS medium supplemented with glucose (A), wheat flour (B), oatmeal (C), xylan (D), carboxymethyl cellulose (CMC) (E), locust bean gum (LBG) (F), xanthan gum (G), pectin (H), inulin (I), xylo-oligosaccharides (XOS) (J), fructo-oligosaccharides (FOS) (K), and galacto-oligosaccharides (GOS) (L) as the sole carbon source. *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively.
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Figure 6. Enzyme activity profile of W. coagulans MA42 and W. coagulans ATCC 7050 cultivated in modified MRS medium supplemented with wheat flour (A), oatmeal (B), xylan (C), carboxymethyl cellulose (CMC) (D), locust bean gum (LBG) (E), xanthan gum (F), pectin (G), and inulin (H) as the sole carbon source at 37 °C for 48 h. * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively.
Figure 6. Enzyme activity profile of W. coagulans MA42 and W. coagulans ATCC 7050 cultivated in modified MRS medium supplemented with wheat flour (A), oatmeal (B), xylan (C), carboxymethyl cellulose (CMC) (D), locust bean gum (LBG) (E), xanthan gum (F), pectin (G), and inulin (H) as the sole carbon source at 37 °C for 48 h. * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively.
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Table 1. Antibiotic susceptibility of W. coagulans.
Table 1. Antibiotic susceptibility of W. coagulans.
AntibioticInhibition Zone Diameters (mm)
ATCC 7050MA42P13S5
Streptomycin (10 μg)33.00 ± 1.00 b23.00 ± 0.00 d37.50 ± 1.50 a27.00 ± 0.00 c
Zone diameter interpretive criteriaSSSS
Erythromycin (15 μg)44.00 ± 1.00 a39.50 ± 0.50 bc37.50 ± 1.50 c41.00 ± 0.00 b
Zone diameter interpretive criteriaSSSS
Kanamycin (30 μg)31.00 ± 1.00 b26.50 ± 0.50 c40.00 ± 0.00 a30.50 ± 0.50 b
Zone diameter interpretive criteriaSSSS
Vancomycin (30 μg) 32.50 ± 0.50 b26.50 ± 0.50 c31.00 ± 1.00 b35.00 ± 0.00 a
Zone diameter interpretive criteria SSSS
Gentamicin (10 μg)34.50 ± 0.50 a26.00 ± 1.00 b35.00 ± 0.00 a32.50 ± 1.50 a
Zone diameter interpretive criteria SSSS
Polymyxin-B (300 units)35.00 ± 0.00 b26.50 ± 0.50 c39.50 ± 0.50 a39.00 ± 1.00 a
Zone diameter interpretive criteria SSSS
Note: The results are expressed as sensitive, S; intermediate, I; or resistant, R as described by the Clinical and Laboratory Standards Institute (CLSI). Means in columns with different superscripts are statistically different at p < 0.05.
Table 2. Digestive-enzyme activity exhibited extracellularly by selected strains of W. coagulans.
Table 2. Digestive-enzyme activity exhibited extracellularly by selected strains of W. coagulans.
StrainsActivity of Enzymes (mU/mL)
AmylaseProteaseβ-Galactosidase
W. coagulans MA42613.00 ± 37.15 c20.57 ± 1.51 c7.94 ± 0.92 c
W. coagulans P13856.48 ± 24.31 a97.33 ± 3.04 a13.69 ± 0.55 b
W. coagulans S5712.45 ± 31.72 b50.17 ± 2.20 b8.22 ± 0.55 c
W. coagulans ATCC 7050631.00 ± 15.19 c92.82 ± 1.57 a21.36 ± 0.83 a
Note: Means in columns with different superscripts are statistically different at p < 0.05.
Table 3. Lactic acid and short-chain fatty acid production by W. coagulans MA42 in comparison to W. coagulans ATCC 7050.
Table 3. Lactic acid and short-chain fatty acid production by W. coagulans MA42 in comparison to W. coagulans ATCC 7050.
SubstrateW. coagulans MA42W. coagulans ATCC 7050
Lactic Acid
(g/L)		Acetic Acid
(g/L)		Lactic Acid
(g/L)		Acetic Acid
(g/L)
Complex Dietary component/ingredient
Wheat flour2.24 ± 0.1 f0.19 ± 0.1 ab0.80 ± 0.0 ij0.19 ± 0.1 ab
Oatmeal2.23 ± 0.1 f0.22 ± 0.0 ab0.96 ± 0.0 hi0.33 ± 0.0 a
Xylan2.42 ± 0.1 f0.21 ± 0.1 ab0.84 ± 0.0 ij0.21 ± 0.1 ab
CMC2.44 ± 0.1 ef0.23 ± 0.1 a0.92 ± 0.0 hi0.26 ± 0.1 a
LBG2.31 ± 0.1 f0.02 ± 0.0 c0.54 ± 0.1 j0.01 ± 0.0 c
Xanthan gum2.70 ± 0.1 de0.06 ± 0.0 bc0.81 ± 0.0 ij0.02 ± 0.0 c
Pectin2.86 ± 0.0 cd0.14 ± 0.1 abc1.29 ± 0.0 g0.24 ± 0.1 a
Inulin2.22 ± 0.1 f0.13 ± 0.0 abc1.14 ± 0.0 gh0.20 ± 0.1 ab
Prebiotic
XOS2.34 ± 0.1 f0.14 ± 0.0 abc0.99 ± 0.1 hi0.20 ± 0.1 ab
FOS2.97 ± 0.1 c0.25 ± 0.1 a1.00 ± 0.1 hi0.29 ± 0.0 a
GOS2.41 ± 0.1 f0.21 ± 0.1 ab0.90 ± 0.0 hi0.21 ± 0.1 ab
Control
Glucose7.57 ± 0.1 a0.17 ± 0.0 abc6.32 ± 0.1 b0.29 ± 0.0 a
Note: Means in columns for each acid with different superscripts are statistically different at p < 0.05.
Table 4. Survival and recovery yield of W. coagulans after freeze-drying with maltodextrin as the protectant.
Table 4. Survival and recovery yield of W. coagulans after freeze-drying with maltodextrin as the protectant.
StrainsViable Cell (Log CFU)Survival Rate (%)Recovery Yield (%)
Before Freeze-DryingAfter Freeze-Drying
W. coagulans MA427.77 ± 0.197.54 ± 0.1297.03 ± 0.13 b90.12 ± 0.96 b
W. coagulans P137.67 ± 0.157.60 ± 0.0799.20 ± 0.12 a93.80 ± 0.24 a
W. coagulans S57.29 ± 0.306.92 ± 0.2694.89 ± 0.14 c90.51 ± 0.96 b
W. coagulans ATCC 70507.58 ± 0.136.98 ± 0.3295.06 ± 0.14 c91.00 ± 0.90 b
Note: Means in columns with different superscripts are statistically different at p < 0.05.
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Pamueangmun, P.; Kham, N.N.N.; Kanpiengjai, A.; Saenjum, C.; Shetty, K.; Unban, K.; Khanongnuch, C. Probiotic Potential of Weizmannia coagulans MA42, an Endospore-Forming Probiotic Bacterium Capable of Dietary Fiber Digestion. Foods 2026, 15, 710. https://doi.org/10.3390/foods15040710

AMA Style

Pamueangmun P, Kham NNN, Kanpiengjai A, Saenjum C, Shetty K, Unban K, Khanongnuch C. Probiotic Potential of Weizmannia coagulans MA42, an Endospore-Forming Probiotic Bacterium Capable of Dietary Fiber Digestion. Foods. 2026; 15(4):710. https://doi.org/10.3390/foods15040710

Chicago/Turabian Style

Pamueangmun, Punnita, Nang Nwet Noon Kham, Apinun Kanpiengjai, Chalermphong Saenjum, Kalidas Shetty, Kridsada Unban, and Chartchai Khanongnuch. 2026. "Probiotic Potential of Weizmannia coagulans MA42, an Endospore-Forming Probiotic Bacterium Capable of Dietary Fiber Digestion" Foods 15, no. 4: 710. https://doi.org/10.3390/foods15040710

APA Style

Pamueangmun, P., Kham, N. N. N., Kanpiengjai, A., Saenjum, C., Shetty, K., Unban, K., & Khanongnuch, C. (2026). Probiotic Potential of Weizmannia coagulans MA42, an Endospore-Forming Probiotic Bacterium Capable of Dietary Fiber Digestion. Foods, 15(4), 710. https://doi.org/10.3390/foods15040710

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