Multifunctional Probiotic and Safety Attributes Heyndrickxia coagulans Isolated from Stingless Bee Honey

doi:10.21203/rs.3.rs-4065466/v1

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Multifunctional Probiotic and Safety Attributes Heyndrickxia coagulans Isolated from Stingless Bee Honey

https://doi.org/10.21203/rs.3.rs-4065466/v1

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published 20 Jan, 2025

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Background Heyndrickxia coagulans, recognized for its probiotic attributes and resilience as an endospore-forming bacterium, is increasingly studied for health supplement applications. This study aimed to evaluate the probiotic potential and safety of novel H. coagulansstrains isolated from stingless bee honey, a new source for this bacterium, and to characterize their multifunctional probiotic properties.

Results We isolated two novel H. coagulansstrains, TBRC-18260 and TBRC-18261, and conducted comprehensive in vitroanalyses to assess their probiotic traits such as acid and bile salt tolerance, self-aggregation, and pathogen inhibition. The strains were also evaluated for safety through antibiotic susceptibility testing and hemolytic activity. Functional properties, including GABA production, antioxidant activity, were examined to establish their potential as probiotics. TBRC-18260 and TBRC-18261 exhibited core probiotic characteristics and showed excellent survivability under acidic conditions and in the presence of bile salts. They displayed strong antimicrobial activity against various pathogens and demonstrated significant GABA production and antioxidant capabilities. The safety assessments confirmed their non-hemolytic nature and susceptibility to a wide range of antibiotics.

Conclusion The novel H. coagulans strains TBRC-18260 and TBRC-18261, with their robust probiotic properties, antioxidant activities, and safety profiles, emerged as promising candidates for the development of functional foods and dietary supplements. This study enhances the biodiversity of available probiotics and supports the continuous search for novel strains with unique health-promoting characteristics.

Heyndrickxia coagulans

Multifunctional probiotic properties

Stingless bee honey

Thermotolerance

GABA production

Novel Heyndrickxia coagulans strains from stingless bee honey.
Multifunctional probiotic attributes, including antioxidation and GABA production.
Robust in various conditions: acid, bile salt tolerance, and high-temperature tolerance.
Non-hemolytic and antibiotic-sensitive, confirming safety for supplementation.

Due to the increasing demand for probiotic supplements in humans and livestock, the study of selected probiotic bacteria with appropriate properties from various sources has also received more attention. According to the World Health Organization, probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Mack, 2005). Numerous bacterial species are recognized as probiotics, including Lactobacillus, a bacteria known for producing lactic acid. Additionally, the Bacillus category includes its capacity to generate endospores, enhancing its resilience to environmental stresses and producing various digestive enzymes that confer health benefits.

As such, Heyndrickxia coagulans is a unique bacterium an attractive probiotic for several reasons because it has unique properties that shares characteristics with both Lactobacillus and Bacillus groups, and was formerly categorized under the Lactobacillaceae family. This is due to the ability to produce acid and the property of the Bacillus that can form endospores (Coelho et al., 2018; Suzuki et al., 2023). Endospore formation allow the bacteria to withstand the harsh environment in the gastrointestinal tract, and lactic acid that inhibits pathogenic bacteria in the gastrointestinal tract. Moreover, the Food and Drug Administration and the European Food Safety Authority recognize H. coagulans as generally recognized as safe (GRAS) for consumption in both human and animals (Konuray & Erginkaya, 2018).

The beneficial attributes of H. coagulans extend far beyond its lactic acid and endospore production. Numerous studies have elucidated its multifaceted probiotic properties. A recent investigation has shown that the spore-forming bacterium H. coagulans plays a role in enhancing gut motility. This research provides supporting evidence for employing

H. coagulans as a beneficial supplement for adults experiencing mild, occasional constipation, particularly those who habitually consume low amounts of fruits and vegetables (Kang et al., 2021). In another study, it was discovered that H. coagulans plays a significant role in mitigating intestinal inflammation and averting weight loss in chickens afflicted with Salmonella enteritidis (Xie et al., 2022). Moreover, the cell-free supernatant of this bacterium counteracts cytotoxic effects in HT-29 cells following S. Typhimurium infection (Kawarizadeh et al., 2019). The bacterium also showcases significant antibiofilm activity, notably against pathogens such as Staphylococcus aureus (Styková et al., 2022).

H. coagulans has been identified in various environments, ranging from fermented rice (Sreenadh et al., 2022), and soil (Xu et al., 2023), to an assortment of fruits and vegetables like potatoes, pickles, corn, and tomatoes (Konuray Altun & Erginkaya, 2021). The diversity of probiotic traits exhibited by H. coagulans across these sources suggests a significant influence of environmental conditions on its properties. This research aims to uncover and evaluate the probiotic qualities of H. coagulans isolated from stingless bee honey, an untapped source for H. coagulans presence. Stingless bees, known for their pollination activities, forage widely, gathering nectar from diverse environments. The investigation into stingless bee honey as a novel source of H. coagulans is driven by the theory that the honey's microbial diversity, which includes a wide range of microorganisms, might endow the bacteria with unique probiotic characteristics and safety for humans and livestock consumption. This diversity, stemming from the honey's rich antimicrobial and antioxidant composition, indicates that H. coagulans strains originating from this source could provide superior benefits for health promotion and disease prevention.

Bacterial isolation

The two strains of H. coagulans were isolated from stingless bee honey samples sourced from the beekeeping community enterprise in Pan Tae Subdistrict, Phatthalung Province, in March 2023. To begin the isolation process, 1 mL of fresh honey was mixed with 9 mL of sterile normal saline, and 0.1 mL was then plated onto Glucose Yeast Peptone (GYP) agar and incubated at 37°C for 24 hours. Emerging colonies were randomly selected and purified through re-streaking on GYP agar. Subsequent, the essential characteristics of the isolates, including cell morphology, Gram staining, catalase enzyme production, and endospore formation traits when culture on sporulation agar (HiMedia, India) was observed.

Bacterial Identification

The identification method was performed with three different principal methods. Initial identification focused on bacteria displaying characteristics of being gram-positive, catalase-positive, and endospore formation at the distal end of cell. Bacteria fitting these criteria were further verification. Confirmatory tests comprised 16S rRNA gene sequencing, MALDI-TOF analysis, and evaluations using API-50 CHB/API-20NE kit (bioMérieux, France).

16S rRNA gene amplification

Genomic DNA from pure H. coagulans samples were isolated following the guidelines provided with the GF-1 Bacterial DNA Extraction Kit (Vivantis Technologies, Malaysia). The spectrophotometer (NanoDrop Lite, Thermo Scientific, USA) quantified and verified the DNA's quality. For PCR amplification, the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Lan et al., 2004) was used. Amplification was conducted in a 50 µL using the MultiGene™ Mini Personal Cycler (Labnet, USA). The PCR regimen was: 3 minutes at 95°C, followed by 30 cycles of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, concluding with an extension at 72°C for 5 minutes. The PCR products, approximately 1,525 bp, underwent agarose gel electrophoresis analysis and were purified with the GF-1 AmbiClean Kit (Gel and PCR from Vivantis Technologies, Malaysia). The purified PCR products were analyzed (1st BASE DNA Sequencing Services, Singapore). Sequence alignment was performed using MEGA-X and cross-referenced via BLAST against sequences in the GenBank database.

Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF)

A fresh colony sample was analyzed by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) characterization using the MALDI Biotyper system (Bruker, Daltonik GmbH, Germany) at the Office of Scientific Instrument and Testing, Prince of Songkla University, Thailand.

Carbohydrate utilization

The API-50 CHB strip together with API-20 NE strip (bioMérieux, France), were used for bacterial identification. The method for assay following to the protocol provided by the manufacturer.

Probiotics properties of H. coagulans

Antipathogenic bacteria activity

The agar well diffusion method assessed the antimicrobial properties of isolated

H. coagulans against various pathogenic bacteria. H. coagulans cultures were grown on GYP broth and incubated at 37°C for 24 hours. The tested pathogens included Enterohemorrhagic Escherichia coli (EHEC), Enteropathogenic Escherichia coli (EPEC), S. Typhimurium (ESBL-producing isolated strain) (Lertworapreecha et al., 2016), Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis (ATCC 29212), Staphylococcus epidermidis, Aeromonas veronii, Aeromonas hydrophila, Bacillus cereus, ESBL-producing Klebsiella pneumoniae (ATCC 700603), and Streptococcus agalactiae. Each pathogen was cultured on trypticase soy agar (TSA) (HiMedia, India) and adjusted to a turbidity equivalent to McFarland No. 0.5. Each test bacterium was spread onto Mueller-Hinton agar (MHA) (HiMedia, India), and a 6 mm Cork Borer was used to punch wells in the agar plate. Then, 100 µL of cell-free supernatant from H. coagulans was added to these inoculated plates. The inhibition zones were measured after incubating at 37°C for 16–18 hours.

Acid and bile salt tolerance

The resistance of the bacteria to acid and bile salt was measured using a method adapted from a previous study (Jin et al., 1998) H. coagulans was prepared to a concentration about

10⁸ CFU/mL. To evaluate endurance in an acidic environment, 0.5 mL of the bacterial mix was combined with 4.5 mL GYP broth, the pH of which was adjusted to 3 using 1M HCl. In contrast, bile salt resilience was tested by mixing 0.5 mL of the bacterial mix with 4.5 mL GYP broth infused with 1% (w/v) bile salt (Hi-media, India). Both mixtures were left at 37°C, with readings taken at the start (0 h) and after 3 hours (3 h). The Lactobacillus plantarum (B17) from previous studies that were found to be able to withstand acids and bile salts at 100% is positive control (Tuyarum et al., 2021). The bacterial survival rates at these two time points were determined through the single plate-serial dilution spotting technique (SP-SDS) (Thomas et al., 2015). The survival rate was calculated as this followed formula:

Survival rate = $\left[\left({\text{N}}_{1}/{\text{N}}_{0}\right)\right]\times 100$ (1)

N₀ represents the initial bacterial concentration

N₁ represents the count of the bacteria that endured

Temperature Tolerance of H. coagulans

Thermotolerance growth tests of H. coagulans were conducted at six different temperatures: 37°C, 40°C, 45°C, 50°C, 55°C, and 60°C. An initial vegetative cell culture concentration was adjusted to achieve 105 CFU/mL. From this culture, 0.1 mL aliquots were spread onto GYP agar plates. The inoculated plates were then incubated at above temperatures for 24 hours.

Screening of acid production

H. coagulans were grown in Modified GYP-PBS medium. A 1,000 mL of medium was prepared by dissolving 20 g of glucose, 10 g of yeast extract, 10 g of tryptic peptone, 10 g of sodium acetate, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, 0.245 g of KH₂PO₄, and 0.01% (w/v) bromocresol purple as indicator. The pH of the media was adjusted to 7.4 to ensure optimal bacterial growth conditions before sterilization by autoclaving at 121°C for 15 minutes. The sterilized medium was then cooled to 50°C. Fresh cell of H. coagulans (18–24 hours) were introduced into the medium to achieve a final concentration of 10⁶ CFU/mL, followed by thorough mixing. The medium was then aliquoted 200 µL into 1.5 mL microcentrifuge tubes, which were stored at 4°C overnight in a refrigerator. Acid production was monitored the next day by incubating these tubes at 37°C for 8 hours.

Co-aggregation, auto-aggregation, and co-existence assay

Auto-aggregation and co-aggregation were derived from the protocol previous published (Dias et al., 2013). In brief, H. coagulans strains were grown in GYP media at a temperature of 37°C for 48 hours. Post-culturing, they were rinsed twice using PBS with a pH value of 7.4, and then their concentration was standardized to an OD600 of 0.6. A sample of 5 mL was vigorously vertexing for 2 minutes and then left to stand at ambient temperature for 4 hours. Subsequently, 10 mL of the top layer of the suspension was gently moved to a 96-well plate already containing 190 mL of PBS. The decrease in light absorbance was measured at 600 nm. The percentage of auto-aggregation of each bacterial isolate was computed as given by:

$$1-\left(\frac{\text{O}\text{D} \text{o}\text{f} \text{t}\text{h}\text{e} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\text{d} \text{l}\text{a}\text{y}\text{e}\text{r}}{\text{O}\text{D} \text{o}\text{f} \text{t}\text{h}\text{e} \text{c}\text{o}\text{m}\text{p}\text{l}\text{e}\text{t}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}\text{l} \text{m}\text{i}\text{x}\text{e}\text{d}}\right)\times 100$$

Pathogenic bacteria like S. Typhimurium, EHEC, and EPEC were used for co-aggregation assessment. An equivalent volume of each H. coagulans and pathogenic strain, standardized to an OD600 of 0.6, was mixed (Konuray Altun & Erginkaya, 2021). After a 4-hour standing period, the absorbance at 600 nm was retaken, and the co-aggregation percentage was derived using a similar formula:

% co-aggregation = $\left[\left({\text{A}}_{\text{p}\text{a}\text{t}} +{\text{A}}_{\text{p}\text{r}\text{o}}\right)-\frac{\left({\text{A}}_{\text{m}\text{i}\text{x}}\right)}{{\text{A}}_{\text{p}\text{a}\text{t}} +{\text{A}}_{\text{p}\text{r}\text{o}}}\right]\times 100$ (3)

A_pat and A_pro represent absorbance of the controls.

A_mix represents the absorbance of the mixed bacteria.

Adhesion to Caco2 cells

Adhesion of bacteria to Caco2 cells was evaluated using a procedure based on previous method (Morita et al., 2002). Briefly, Caco2 human colorectal adenocarcinoma cells were grown at a density of 1.2×10⁵ cells/mL in 24 well plates using DMEM enriched with 10% fetal bovine serum. These cultures were kept at 37°C in a CO₂ incubator until they reached confluence. The cultured cells were then rinsed twice with PBS (pH 7.4) and exposed to 1 mL of the H. coagulans (adjusted to 0.5 McFarland standards) in DMEM devoid of serum. After incubation at 37°C in a CO₂ atmosphere for 90 minutes, the medium was discarded, and the cells were rinsed thrice with PBS. Subsequently, each well received 1 mL of 0.05% Triton X-100, followed by a 10 minutes incubation at ambient temperature. This was then diluted by a factor of ten using sterile saline and plated on GYP agar. After a 48 hours incubation at 37°C, H. coagulans colonies were enumerated. The adhesion rate was determined using the equation:

% adhesion = $\frac{\text{B}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}\text{l} \text{c}\text{e}\text{l}\text{l}\text{s} \text{b}\text{o}\text{u}\text{n}\text{d} \text{t}\text{o} \text{C}\text{a}\text{c}\text{o}2 \text{c}\text{e}\text{l}\text{l}\text{s}}{\text{t}\text{o}\text{t}\text{a}\text{l} \text{C}\text{a}\text{c}\text{o}2 \text{c}\text{e}\text{l}\text{l} \text{c}\text{o}\text{u}\text{n}\text{t}} \times 100$ (4)

Safety assessment

Hemolytic activity

H. coagulans was cultured on GYP agar and incubated at 37°C for 24 hours. Post-incubation, the bacterial strains were streaked onto 5% sheep blood agar plates. These plates were then incubated at 37°C for another 24 hours. Following incubation, the reactions on the blood agar were analyzed. Based on the hemolytic pattern, the reactions were categorized as Beta (β) hemolysis, Alpha (α) hemolysis, or Gamma (γ) hemolysis.

Antimicrobial susceptibility testing by disc diffusion

To assess the antimicrobial susceptibility of H. coagulans, it was cultured on GYP agar. After achieving growth, the bacterial concentration was adjusted to approximately 10⁸ CFU/mL. A sterile cotton swab was used to uniformly inoculate the adjusted bacterial culture onto GYP agar plates. Twelve antibiotic discs—ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), vancomycin (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), cephalothin (30 µg), streptomycin (10 µg), nalidixic acid (30 µg), and cotrimoxazole (25 µg), norfloxacin (10 µg), and tetracycline (30 µg) (Oxoid, UK)—were subsequently placed on the agar surface. The plates were incubated at 37°C for 24 hours. After incubation, the diameter of the clear inhibition zones around each antibiotic disc was recorded. The results were interpreted based on the size of the inhibition zones as per the guidelines from the Clinical and Laboratory Standards Institute (CLSI)(Weinstein & Lewis, 2020). H. coagulans ' susceptibility to each antibiotic was categorized as either resistant (R), moderately susceptible (MS), or susceptible (S).

Minimal inhibitory concentration (MIC)

The minimal inhibitory concentration (MIC) of H. coagulans was ascertained using the broth microdilution technique, adhering to the CLSI 2020 standards (CLSI 2020). Twelve antimicrobial agents, including ampicillin, chloramphenicol, erythromycin, vancomycin, ciprofloxacin, gentamycin, cephalothin, streptomycin, nalidixic acid, cotrimoxazole, norfloxacin and tetracycline, were subjected to serial two-fold dilutions. Concentrations ranged from 0.25 to 512 µg/mL, with an exception for cotrimoxazole, which varied between 3040/160 to 0.742/0.078 µg/mL. These dilutions were prepared in Mueller Hinton broth (HiMedia, India). Within the microplates, each well contained a bacterial concentration of approximately 5×10⁵ CFU/mL in a 200 µL volume. Following inoculation, plates were incubated at 35°C for a period of 16 to 20 hours. Subsequent analysis of susceptibility patterns was conducted with the WHONET 2023 software (Agarwal et al., 2009).

Antioxidant activity

Antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

H. coagulans was cultivated in GYP broth and incubated at 37°C for 24 hours. Following incubation, the culture was centrifuged at 10,000 rpm for 5 minutes. The supernatant was then subjected to antioxidant evaluation using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.

A standard ascorbic acid solution was prepared by dissolving 0.01 g of ascorbic acid in 50 ml of distilled water to yield a concentration of 200 mg/L. This solution was further diluted to concentrations ranging from 10–100 mg/L using distilled water. Separately, a DPPH solution was prepared at a concentration of 0.6 mmol by dissolving 0.0236 g of DPPH in 100 ml of methanol. For the assay, 100 µL of the sample was added to a 96-well plate, followed by 100 µL of the 0.6 mmol DPPH solution. The mixture was shaken for uniformity and then incubated in the dark for 30 minutes. Absorbance was measured using a microplate reader at wavelengths between 515–517 nanometers (Xing et al., 2015). The presence of antioxidants was indicated by a color shift from purple to yellow. Antioxidant activity was quantified as %DPPH free-radical inhibition using the following equation:

% DPPH free-radical inhibition = $\left[\left({\text{A}}_{\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}} -{\text{A}}_{\text{D}\text{P}\text{P}\text{H}}\right)/{\text{A}}_{\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}\right]\times 100$ (5)

A_sample represents the absorbance of the sample.

A_DPPH denotes the absorbance of the DPPH solution without the inclusion of the sample

Antioxidant activity by 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay

In the study, H. coagulans was cultured in GYP medium and allowed to grow at 37ºC for 24-hours. The pre-grown cultures were then subjected to centrifugation at 10,000 rpm for 5 minutes. The resulting supernatant was then evaluated for antioxidant presence using the 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay (Ilyasov et al., 2020).

For the ABTS assay, an ABTS solution with a concentration of 7 mM was prepared, combined with a 2.45 mM potassium persulfate solution at a 1:1 (v/v) ratio. This mixture was set aside to stand at ambient temperature for a full day. Before its application, the ABTS mixture was diluted with water to achieve an absorbance reading within 0.7 ± 0.005 when measured at 734 nm. Subsequently, 50 µL of the sample was combined with 100 µL of the diluted ABTS mixture. This solution was mixed thoroughly and kept away from light for 10 minutes. Absorbance readings were then taken using a microplate reader at the wavelength of 734 nm. Presence of antioxidants in the sample would manifest as a change in color from green to clear. The absorbance data was calculated in term of the antioxidant percentage with the following formula as % scavenging rate:

Scavenging rate (%) = $\left[\left({\text{A}}_{\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}} -{\text{A}}_{\text{A}\text{B}\text{T}\text{S}}\right)/{\text{A}}_{\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}\right]\times 100$ (6)

A_sample represents the absorbance of the sample.

A_ABTS denotes the absorbance of the ABTS solution without the inclusion of the sample

Detection of gamma-aminobutyric acid (GABA) Production by HPLC

To analyze the gamma-aminobutyric acid (GABA) content, High Performance Liquid Chromatography (HPLC) was performed. Firstly, H. coagulans was cultivated in GYP broth enriched with 5% monosodium glutamate (MSG). The culture was then incubated at 37°C for 24 hours. Following incubation, the sample was centrifuged at 10,000 rpm for 5 minutes. The cell-free supernatant was then filtered using a 13 mm Nylon membrane syringe filter with a pore size of 0.22 µm (One PURE). The filtrate was analyzed using an Agilent 1260 HPLC system fitted with a Hypersil Gold C-18 column (250 × 4.6 mm I.D., 5µm particle size; Thermo Scientific, Meadow, UK). The mobile phase composition was as follows: Solution A: 60% (100.02 mM sodium acetate, 3.59 mM triethylamine, and 12.49 mM acetic acid in 1000 mL of water), pH adjusted to 5.8. Solution B: 28% deionized water. Solution C: 12% acetonitrile. The mobile phase was pumped at a flow rate of 0.6 ml/min and maintained at a temperature of 26°C. Detection was carried out using a UV detector set at 254 nm (Wan-Mohtar et al., 2020). Analysis of the concentration of GABA production was done by comparing with the standard curve of 4-aminobutanoic acid substance (Sigma-Aldrich).

Statistical analysis

All the data were presented as the mean value ± SD. All data was analyzed statistically with GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA). Analysis of variance (one-way ANOVA) performed by Dunnett’s multiple comparisons test was used to compare more than two groups, and a paired t-test was used to compare the means of two groups. The P value less than 0.05 (p < 0.05) was considered statistically significant.

Bacillus isolation and Identification

In the present study, a Gram-positive and catalase-positive bacterial strains were isolated. Of these, only two isolates exhibited sporulation in their distal regions (Fig. 1A). Consequently, these isolates underwent detailed classification using techniques such as 16S rRNA sequencing, MALDI-TOF, and API-50 CHB/API-20 NE analyses. The nucleotide sequences for these strains, designated as TBRC-18260 and TBRC-18261, have been deposited in the GenBank database with accession No. OR636959 and OR624758, respectively. Sequence comparison against type strains in GenBank revealed that TBRC-18260 and TBRC-18261 share 98.13% and 98.98% sequence identity with H. coagulans NR_041523 (Fig. 1B). Further validation through MALDI-TOF confirmed congruent genus and species classification, while API-50 CHB/API-20 NE analysis also identified the strains as

H. coagulans (Fig. 1C and Table 1). Additionally, both strains have been successfully deposited at the Thailand Bioresource Research Center (TBRC).

Table 1

Biochemical profile of the API-50 CH/API-20 NE kit (bioMérieux) of the two representatives *H. coagulans* isolates
Test	Biochemical test API-50 CHB	TBRC 18261	TBRC 18260	Test	Biochemical test API-20 NE	TBRC 18261	TBRC 18260
1	Glycerol	+	+	1	ONPG: ß-galactosidase (Ortho NitroPhenyl-ßDGalactopyranosidase)	-	-
2	Erythritol	-	-	2	ADH: Arginine DiHydrolase	-	-
3	D-Arabinose	-	-	3	LDC: Lysine DeCarboxylase	-	-
4	L-Arabinose	+	+	4	ODC: Ornithine DeCarboxylase	-	-
5	Ribose	+	+	5	CIT: CITrate utilization	-	-
6	D- Xylose	+	-	6	H2S: H2S production	-	-
7	L-Xylose	-	-	7	URE: UREase	-	-
8	Adonithol	-	-	8	TDA: Tryptophane DeAminase	+	+
9	Methyl xyloside	-	-	9	IND: INDole production	-	-
10	Galactose	+	+	10	VP: acetoin production (Voges Proskauer)	+	+
11	D-Glucose	+	+	11	GE: GELatinase	-	-
12	D-Fructose	+	+	12	GLU: fermentation / oxidation (GLUcose)	-	-
13	D-mannose	+	-	13	MAN: fermentation / oxidation (MANnitol)	-	-
14	Sorbose	-	+	14	INO: fermentation / oxidation (INOsitol)	-	-
15	Rhamnose	+	-	15	SOR: fermentation / oxidation (SORbitol)	-	-
16	Dulcitol	-	-	16	RHA: fermentation / oxidation (RHAmnose)	-	-
17	Inositol	-	-	17	SAC: fermentation / oxidation (SACcharose)	-	-
18	Mannitol	-	-	18	MEL: fermentation / oxidation (MELibiose)	-	-
19	Sorbitol	+	+	19	AMY: fermentation / oxidation (AMYgdalin)	-	-
20	Methyl-D-mannoside	+	+	20	ARA.: fermentation / oxidation (ARAbinose)	-	-
21	Methyl-D-glucoside	-	+	21	NO₂: Reduction of nitrates to nitrites	-	-
22	N-acetyl-glucosamine	+	+
23	Amygdalin	+	+
24	Arbutin	+	+
25	Esculine	+	+
26	Salicin	+	+
27	Cellobiose	+	+
28	Maltose	+	+
29	Lactose	+	+
30	Melibiose	+	+
31	Sucrose	+	+
32	Trehalose	+	+
33	Inulin	-	-
34	Melizitose	-	-
35	D-raffinose	-	+
36	Starch	+	+
37	Glycogen	-	-
38	Xylitol	-	-
39	Gentibiose	+	+
40	Turanose	+	+
41	Lyxose	-	-
42	Tagatose	-	-
43	D-fucose	-	-
44	L-fucose	-	-
45	D-Arabitol	-	-
46	L-Arabitol	-	-
47	Gluconate	+	+
48	2, Keto-gluconate	+	-
49	5, keto-gluconate	+	-

Antipathogens activity

Inhibitory effects against pathogenic bacteria are considered indicative of potential probiotic properties. In this study, the two H. coagulans strains demonstrated varying degrees of inhibition against a range of pathogens. Both strains exhibited antimicrobial activity against all tested pathogens. TBRC-18260 generally showed a stronger inhibitory effect than TBRC-18261, especially against S. Typhimurium (ST), S. epidermidis (SE), and A. veronii (AV), with inhibition zones measuring 15 mm, 24 mm, and 24 mm, respectively.

The highest antimicrobial activity observed for TBRC-18261 was against A. veronii (AV) with an inhibition zone of 25 mm. The strains exhibited similar levels of efficacy against Enteropathogenic E. coli (EPEC), P. aeruginosa (PA), and E. faecalis (EF), with inhibition zones ranging from 10 to 15 mm for TBRC-18261 and 12 to 15 mm for TBRC-18260 (Table 2).

Acid and bile salt tolerance

The two H. coagulans strains exhibited a remarkable ability to withstand acidic conditions, with survival rates surging to 300% at pH 3.0 over 3 hours, compared to the control strain, L. plantarum (B-17) isolated from a previous study (Tuyarum et al., 2021). This indicates their aptitude to thrive in low-pH environments. Despite not experiencing the same increase in bacterial numbers as seen in the acidic conditions, both strains demonstrated significant tolerance to 1% bile salt over 3 hours. The viable bacterial count remained relatively stable, with only a 10–30% reduction from the start. The statistical analysis revealed that both isolates exhibited significantly higher acid resistance than L. plantarum. However, no significant difference was observed in their tolerance to bile salts (Fig. 2A).

Acid production

Both strains of H. coagulans were able to produce acid in the Modify GYP-PBS medium, as evidenced by the change in color of the bromocresol purple indicator from purple to yellow. The dark yellow color after the 12-hour incubation period suggests a strong acid production by both bacterial strains, indicating a significant drop in pH. This change happening in approximately 8 hours aligns with the bacteria's characteristics of being potent acid producers (Fig. 2B).

Thermotolerance of H. coagulans

Temperature resistance assays of both isolated H. coagulans were conducted at 40°C to 60°C. Both isolates exhibited average growth between 40°C and 55°C, with the growth pattern comparable to that observed at the control temperature of 37°C in terms of both colony size and number. The study results found that the optimum temperature for growth of both isolates range from 37–40°C, with isolate TBRC-18260 showing a higher growth rate trend than TBRC-18261 at these temperatures. The growth of both isolates decreased in number and size of colonies when the temperature was increased to 50–60°C. No statistical differences were observed in the growth of TBRC-18261 at various temperatures. However, TBRC-18260 showed significant differences in growth between temperatures of 55°C and 60°C (Fig. 2C).

Co-aggregation, auto-aggregation,

After 4 hours of incubation, both isolate showed co-aggregation with the pathogenic strains. The co-aggregation abilities of TBRC-18260 and TBRC-18261 were at a similar level. Specifically, TBRC-18261 co-aggregated with S. Typhimurium, EHEC, and EPEC at rates of 39.96%, 39.99%, and 39.92%, respectively. In comparison, TBRC-18260 co-aggregated with S. Typhimurium, EHEC, and EPEC at rates of 36.91%, 36.88%, and 36.89%, respectively (Table 2).

Both H. coagulans strains demonstrated the capability to auto-aggregate over time. For the BPW-4TBRC-18261 strain, auto-aggregation commenced at 4 hours, showing 23.63% aggregation, which rose to 43.78% by the 8-hour mark. On the other hand, the TBRC-18260 strain began auto-aggregating at 2 hours, with an initial rate of 17.16%, increasing to 50.94% after 8 hours (Table 2). No statistical differences were observed in the co-aggregation, auto-aggregation between TBRC-18260 and TBRC-18261.

Adhesion to Caco2 cell

Both H. coagulans strains, demonstrate distinct adhesion characteristics. The data suggests that TBRC-18261 has a remarkably higher efficiency in adhering to Caco-2 cells compared to TBRC-18260. Specifically, the adhesion percentage for TBRC-18261 was calculated to be 32.15%, while for TBRC-18260, it was substantially lower at 7.13% (Tabel 2).

Safety assessment

In the evaluation of the safety profile of H. coagulans, two distinct isolates were subjected to tests assessing their potential to lyse red blood cells and their susceptibility to a panel of 12 antimicrobial agents. Notably, neither of the isolates exhibited hemolytic activity (γ-hemolysis) and both demonstrated susceptibility to all the antimicrobial agents evaluated (Tables 3 and 4).

Antioxidant activity

The antioxidant capabilities of two H. coagulans isolates were assessed using both DPPH and ABTS assays, comparing their performance against a 5mg/mL ascorbic acid standard. Both isolates exhibited marked antioxidant activities that were on par with ascorbic acid. Specifically, the TBRC-18261 isolate demonstrated scavenging activity values of 78.36% and 68.79% for DPPH and ABTS respectively. Conversely, the TBRC-18260 isolate presented with 80.32% activity in the DPPH assay and 86.82% in the ABTS assay (Fig. 3). There were no statistically significant differences found between the antioxidant values of the two isolates and ascorbic acid using both the DPPH and ABTS methods.

Table 2

Antibacterial activity and adhesion properties of *H. coagulans*
strains	Antimicrobial activity (Diameter of inhibition clear zone in mm.)											Co-aggregation (%)			Auto-aggregation (%)		Adhesion to Caco2 cells (%)
strains	EHEC	EPEC	ST	SA	PA	EF	SE	AV	AH	BC	KP	ST	EHEC	EPEC	4 hours	8 hours	Adhesion to Caco2 cells (%)
TBRC-18261	10	11	11	10	15	15	20	25	18	17	12	39.96 ± 0.45^a	39.99 ± 0.24 ^a	39.92 ± 0.55 ^a	23.63 ± 0.24 ^a	43.78 ± 0.24 ^a	32.15
TBRC-18260	10	12	15	14	15	15	24	24	17	15	10	36.91 ± 0.47 ^a	36.88 ± 0.68 ^a	36.89 ± 0.36 ^a	17.16 ± 0.69 ^a	50.94 ± 0.33 ^a	7.13
Values are mean ± SD of three independent determinations (n = 3) of each sample with the same letter in the same column are not significantly different. EHEC; Enterohemorrhagic Escherichia coli, EPEC; Enteropathogenic Escherichia coli,ST; Salmonella Typhimurium (ESBL strain), SA; Staphylococcus aureus (MRSA strain, DMST 20637), PA; Pseudomonas aeruginosa (ATCC 27853), EF; Enterococcus faecalis ATCC 29212, SE; S. epidermidis (isolated strain), AV; Aeromonas veronii (isolated strain), AH; Aeromonas hydrophila, BC; Bacillus cereus (isolated strain), KP; Klebsiella pneumoniae (ATCC 700603)

Table 3

Antimicrobial susceptibility testing by agar disc diffusion and hemolytic activity
strains	Antibiotics susceptibility by the disc diffusion agar method (zone dimeter in mm.)												Hemolytic activity
strains	Amp (10 µg)	Chl (30 µg)	Ery (15 µg)	Van (30 µg)	Cip (5 µg)	Gen (10 µg	Cep (30 µg)	Str (10 µg)	Na (30 µg)	Co-tri (25 µg)	Nor (10 µg)	Tet (30 µg)	Hemolytic activity
TBRC-18261	28 (s)	30 (s)	30 (s)	32 (s)	28 (s)	30 (s)	35 (s)	33 (s)	23 (s)	23 (s)	29 (s)	30 (s)	γ
TBRC-18260	31 (s)	31 (s)	31 (s)	32 (s)	37 (s)	25 (s)	33 (s)	26 (s)	31 (s)	33 (s)	29 (s)	39 (s)	γ
Amp; ampicillin, Chl; chloramphenicol, Ery; erythromycin, Van; vancomycin, Cip; ciprofloxacin, Gen; gentamycin, Cep; cephalothin, Str; streptomycin, Na; nalidixic acid and Co-tri; cotrimoxazole, Nor; Norfloxacin, Tet; Tetracycline, S = susceptible

Table 4

Antimicrobial susceptibility testing microbroth dilution technique
strains	Antibiotics susceptibility by the microbroth dilution technique (MIC)
strains	Amp	Chl	Ery	Van	Cip	Gen	Cep	Str	Nal	Co-tri	Nor	Tet
TBRC-18261	$\le$0.25	2	$\le$0.25	$\le$0.25	$\le$0.25	$\le$0.25	$\le$0.25	$\le$0.25	4	$\le 1.48/0.$156	$\le$0.25	$\le$0.25
TBRC-18260	$\le$0.25	8	$\le$0.25	$\le$0.25	$\le$0.25	$\le$0.25	$\le$0.25	$\le$0.25	4	$\le 1.48/0.$156	$\le$0.25	$\le$0.25

Amp; ampicillin, Chl; chloramphenicol, Ery; erythromycin, Van; vancomycin, Cip; ciprofloxacin, Gen; gentamycin, Cep; cephalothin, Str; streptomycin, Nal; nalidixic acid and

Co-tri; cotrimoxazole, Nor; Norfloxacin, Tet; Tetracycline.

Concentrations ranged from 0.25 to 512 µg/mL, with an exception for cotrimoxazole, which varied between 3040/160 to 0.742/0.078 µg/mL.

GABA production

GABA, a valuable compound, has been identified as a product of lactic acid bacteria. In the present investigation, the capacity of two H. coagulans isolates to synthesize GABA was evaluated. Results by HPLC technique indicated that both isolates were efficient GABA producers: TBRC-18261 generated 87.48mg/mL of GABA and TBRC-18260 yielded 84.08mg/mL of GABA (Fig. 4).

Heyndrickxia coagulans, formerly known as Bacillus coagulans, is a probiotic bacterium garnering significant interest for development as a dietary supplement for both humans and livestock. In this study, two isolates of H. coagulans, namely TBRC-18260 and TBRC-18261, were isolated. Due to significant species diversity in genera like Bacillus, this method alone may not always ensure precise species identification. This is particularly true for genera with high intraspecies genomic variability (Celandroni et al., 2019). In this study, the presence of spores at the distal ends of the bacterial cells, a key trait of H. coagulans, along with consistent results from 16S rRNA sequencing, MALDI-TOF, and API-50 CHB/API-20 NE tests, solidifies the accurate classification of our isolates as H. coagulans.

Evaluating the inhibitory effect of probiotic strains on pathogenic bacteria is essential for assessing their probiotic capabilities. In our investigation, the two H. coagulans strains exhibited diverse inhibitory activities against several pathogens. Notably, both strains were equally effective in inhibiting pathogenic bacteria, particularly showing a significant ability to suppress A. veronii and A. hydrophila. This aligns with prior studies that have demonstrated

H. coagulans proficiency in combating pathogens such as S. Typhimurium, S. Enteritidis and Clostridium perfringens (Kawarizadeh et al., 2019; Xie et al., 2022). An intriguing finding from our study is the strain TBRC-18260's effectiveness against Gram-positive cocci, an aspect not previously reported. Furthermore, its antagonistic impacts on A. veronii and A. hydrophila underscore its promising potential for use in aquaculture. This suggested that both isolates may produce antimicrobial agents including organic acids that are highly effective in inhibiting bacteria. It was reported that whole genome sequencing of Weizmannia coagulans (PL-W), the former name of H. coagulans have a bacteriocin synthesis gene cluster in their whole genome (Wang et al., 2023).

The ability to resist acid and bile salt is crucial for probiotic bacteria, enabling them to withstand harsh conditions and successfully inhabit the digestive system. This findings study underscores the exceptional ability of the two H. coagulans strains to endure acidic environments. Their survival rates surged to 300% at pH 3.0 over three hours. This increase is not just an indicator of their tolerance but suggests an actual proliferation in acidic conditions, a trait that could have vast implications for various applications. Several reports have shown that H. coagulans survive in acidic conditions and maintain a 100% survival rate. Furthermore, some studies have even reported an increase in bacterial numbers (Chaudhari et al., 2021; Ritter et al., 2018; Sui et al., 2020). A previous study (Chaudhari et al., 2021), shows that H. coagulans are also resistant to high bile salts, with a 70–100% survival rate. The displayed acid and bile salt tolerance has potential industrial applications, given the need for probiotics to survive the stomach's highly acidic environment and colonize within the intestine. The capacity to withstand acidic and saline environments is vital for specific probiotic bacteria, allowing them to endure extreme conditions and establish themselves within the gastrointestinal tract (Tuyarum et al., 2021). These processes facilitate bacterial respiration by transporting protons, indicating that F0F1-ATPase may elevate the cell's pH in acidic conditions through gene expression regulation. Conversely, the ability to tolerate bile salts arises from the expulsion of bile and its breakdown (Ruiz et al., 2013).

For temperature tolerance study of H. coagulans, the observations highlight that both H. coagulans isolates can grow moderately at 50–60°C. TBRC18260, in particular, is sensitive to temperature increases within the higher range, evidenced by the significant growth differences observed. In the literature, it has been established that B. coagulans possesses the ability to withstand and proliferate under high-temperature conditions. Nonetheless, the extent of this thermotolerance is influenced by the specific strain and the environmental context from which the bacterium was isolated. For instance, B. coagulans isolated from wastewater at a cassava processing facility exhibited optimal growth at 42°C (Tongpim & Sakai, 2021). Another study reports that the B. coagulans strain 36D1 can grow and efficiently ferment lignocellulosic biomass at 50°C (Ou et al., 2011). The mechanism involved in withstanding high temperatures is unknown. However, it may be because the bacteria's enzymes used to digest carbohydrates are heat-resistant, allowing them to survive in high temperatures (Rhee et al., 2011). The advantage of heat-tolerant cultures is that they can be used in the fermentation industry, which requires high temperatures.

The safety evaluation of both isolates was fascinating. We found that neither isolate was resistant to any of the 12 drugs tested, which gave consistent results for both disc diffusion, and micro broth dilution. Moreover, both isolates also showed g-hemolysis characteristics, indicating that they were non-virulence strains. Safety is critical in developing probiotics as dietary supplements for humans and livestock. This suggests that the discovery of H. coagulans from flower honey samples may be a source without antimicrobial exposure.

In addition to the general properties of probiotics We found a special feature that makes both isolates more interesting, namely their tolerance to high temperatures, antioxidant activity and gamma-aminobutyric acid (GABA) production.

It was found that the vegetative form of both isolates can grow at temperatures of 60°C. It was found that its ability to withstand high temperatures is superior to B. coagulans isolates from other reports that were found to be able to withstand heat in the range of 42-55°C (Rhee et al., 2011; Tongpim & Sakai, 2021). This indicates that both isolates are thermotolerant strains that will be of great benefit in the development of probiotic products that require heat treatment such as spray drying or the production of animal feed pellets.

Antioxidant activities in bacteria, particularly lactic acid bacteria, have increased attention due to their potential therapeutic applications and benefits in food preservation (Hu et al., 2023; Zapaśnik et al., 2022). In this study, the antioxidant capacities of two H. coagulans isolates, TBRC-18261 and TBRC-18260, were thoroughly evaluated using two well-established radical scavenging assays: DPPH and ABTS. The antioxidant capabilities of the H. coagulans strains TBRC-18261 and TBRC-18260 are notably significant, as they demonstrated scavenging solid activities and paralleled the antioxidant prowess of a 5 mg/mL ascorbic acid standard antioxidant reference (Sricharoen & Chanthai, 2015).

Antioxidant Activiti between TBRC-18261 and TBRC-18260 was different especially in the ABTS assay. This variation may be attributed to distinct metabolic pathways or the presence of specific antioxidant molecules inherent to each strain. However, it's essential to highlight that both strains exhibited consistent scavenging activities above 65%, indicating their substantial antioxidant potential. When juxtaposed with prior research, the antioxidant capacities of different H. coagulans strains vary significantly. A previous study in B. coagulans with high lactase-producing activity showed a high capacity for scavenging DPPH free radicals 35.0%, hydroxyl radicals 39.0%, and superoxide anion radicals 14.8%, and good reducing power 58.5 µmol/L ascorbic acid equivalent (Sui et al., 2020). Important antioxidant mechanisms reported in B. coagulans was that the B. coagulans boosts the concentration of proteins related to the Nrf2/Keap1 pathway (Nrf2, Keap1, heme oxygenase-1 (HO-1)), along with enhancing the function of enzymes that combat oxidative stress (glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD)). Furthermore, it reduced the levels of malondialdehyde (MDA) and diminished the expression of proinflammatory cytokines especially TNF-α (Gao et al., 2022).

While the production of GABA is predominantly associated with Lactobacillus strains, its synthesis in Bacillus strains remains relatively less explored. Even within the Bacillus genus, reports on GABA production by H. coagulans are rare. The findings of this study, which document substantial GABA yields from H. coagulans isolates, therefore, present a significant addition to the existing body of literature. The levels of GABA produced by the TBRC-18261 and TBRC-18260 isolates notably position H. coagulans as a potent GABA producer among bacteria.

The majority of research emphasizes the ability of lactobacilli, particularly L. brevis, to synthesize significant GABA quantities, with certain strains achieving concentrations of up to 176.04 g/L (Cha et al., 2023). Likewise, a few Bacillus strains, such as B. subtilis, have been documented to yield GABA levels around 12.5 g/L (Asun et al., 2022). Intriguingly, prior studies have not highlighted GABA production in H. coagulans. The findings from this investigation not only place H. coagulans on the GABA production but also suggest its superior synthesis capabilities compared to several Lactobacillus and Bacillus strains. These results underscore the potential of the examined H. coagulans isolates for probiotic applications.

In light of these results, it would be prudent to explore the potential of these probiotics in scenarios where controlled NO production is desirable, such as in the treatment of infections where an immune response needs to be modulated. Additionally, the differential effects observed between supernatant and pellet forms could be harnessed to tailor the therapeutic properties of these strains for specific applications.

In conclusion, this research has successfully isolated and characterized two novel strains of H. coagulans, TBRC-18260 and TBRC-18261, from stingless bee honey, marking a significant contribution to probiotics in humans and livestock. These strains exhibit multifunctional probiotic properties, including robustness in various environmental conditions, high tolerance to acid and bile salts, and notable adhesion capabilities to epithelial cell lines. They demonstrated antimicrobial activity against various pathogens. Of particular interest is the ability of these strains to grow in high temperatures (60°C) and produce GABA coupled with their antioxidant activities. These features enhance their probiotic profile and open avenues for their application in functional foods and dietary supplements. The comprehensive safety assessments, including hemolytic activity and antimicrobial susceptibility, affirm their potential for safe consumption. The findings of this study align with the growing interest in leveraging natural sources for novel probiotics and highlight the importance of continuous exploration in this domain. Characterizing this novel H. coagulants strain presents a promising prospect for their application in enhancing human and livestock wellness.

Ethics approval and consent to participate

This work did not require ethical approval under the research governance guidelines operating during the research.

Consent for publication

All the authors have approved the manuscript that is enclosed.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare no competing interests.

Funding

This work was financially supported by the National Higher Education, Science, Research and Innovation Policy Council, Thaksin University (Research project grant no. TSU-66A105000003) Fiscal Year 2023.

Authors' contributions

Benyapa Prakit & Rungravee Chaiyod: Methodology, Investigation, Formal analysis, Editing. Kittiya Khongkool: Investigation, Formal analysis, Writing - Original Draft. Wankuson Chanasit: Supervision, Writing - Review & Editing. Monthon Lertworapreecha: Funding acquisition, Conceptualization, Supervision, Visualization, Writing - Review & Editing. All authors contributed significantly to the discussion of the results, reviewed the manuscript, provided critical feedback, and approved the final version for publication.

Acknowledgments

We would like to thanks to the Microbial Technology for Agriculture, Food, and Environment Research Center, Faculty of Science and Digital Innovation, Thaksin University, Thailand, for the generosity and laboratory research tools.

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strains	Antibiotics susceptibility by the microbroth dilution technique (MIC)
strains	Amp	Chl	Ery	Van	Cip	Gen	Cep	Str	Nal	Co-tri	Nor	Tet
TBRC-18261	\(\le\)0.25	2	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	4	\(\le 1.48/0.\)156	\(\le\)0.25	\(\le\)0.25
TBRC-18260	\(\le\)0.25	8	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	\(\le\)0.25	4	\(\le 1.48/0.\)156	\(\le\)0.25	\(\le\)0.25

Multifunctional Probiotic and Safety Attributes Heyndrickxia coagulans Isolated from Stingless Bee Honey

Status:

Journal Publication

Version 1

Abstract

Figures

Highlights

Background

Methods

Bacterial isolation

Bacterial Identification

16S rRNA gene amplification

Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF)

Carbohydrate utilization

Antipathogenic bacteria activity

Acid and bile salt tolerance

Screening of acid production

Co-aggregation, auto-aggregation, and co-existence assay

Adhesion to Caco2 cells

Safety assessment

Hemolytic activity

Antimicrobial susceptibility testing by disc diffusion

Minimal inhibitory concentration (MIC)

Antioxidant activity

Antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

Antioxidant activity by 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay

Detection of gamma-aminobutyric acid (GABA) Production by HPLC

Statistical analysis

Results

Antipathogens activity

Acid and bile salt tolerance

Acid production

Adhesion to Caco2 cell

Safety assessment

Antioxidant activity

GABA production

Discussion

Conclusion

Declarations

References

Status:

Journal Publication

Version 1