Genomic and phenotypic safety assessment of probiotic Bacillus subtilis DC-11 isolated from traditionally fermented Idli Batter

doi:10.21203/rs.3.rs-4957452/v1

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Genomic and phenotypic safety assessment of probiotic Bacillus subtilis DC-11 isolated from traditionally fermented Idli Batter

https://doi.org/10.21203/rs.3.rs-4957452/v1

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In this study, we reported genomic and phenotypic safety assessment of probiotic Bacillus subtilis DC-11 isolated from traditionally fermented Idli Batter. The strain was evaluated for probiotic properties, biofilm formation, and antimicrobial compound production. The phenotypic safety was determined by accessing the strain’s ability to produce enterotoxins, degrade mucin, and antibiotic sensitivity. Whole genome sequencing (WGS) was performed to identify the strain and determine genetic safety by analyzing the presence of plasmids, antibiotic resistance genes, and virulence factors. In the results, B. subtilis DC-11 showed 88.98% viability in gastric juice, and 98.60% viability in intestinal juice. It showed 18.33 ± 0.44% autoaggregation, 32.53 ± 3.11% adhesion to xylene, 0.98 ± 0.05 OD unit’s adhesion to mucin (crystal violet equivalence at 550 nm), 21.2 ± 2.3% adhesion to Caco-2 cells, and − 22.3 ± 0.65 mV zeta potential. The highest co-aggregation was recorded with Escherichia coli (23.62 ± 0.70%). The strain was found negative for enterotoxin production, mucin degradation, and antibiotic resistance to the commonly used therapeutic antibiotics. It formed a good biofilm and capable of producing antimicrobial peptide subtilosin A with a molecular mass of 3400 Da. The peptide has inhibited the growth of methicillin-resistant Staphylococcus aureus (18.6 ± 0.58 mm). In genetic safety, no plasmids, antibiotic-resistant genes, and virulence factors were detected. Moreover, the strain showed close similarity with B. subtilis ATCC 6051 and proteins involved in probiotic attributes. In conclusion, B. subtilis DC-11 is safe potential probiotic candidate.

Bacillus subtilis DC-11

Probiotic

Safety

Idli Batter

MRSA

Subtilosin A

Fermentation is a well-known traditional method of transforming food components to improve nutritional qualities, shelf life, safety, and food product diversity. In India, the diversity of fermented foods is huge due to its diverse ethnicity and biological resources [1]. Cereals and legumes are the major components of Indian fermented foods and the diet of the diverse Indian population [2, 3]. Idli is one of the popular traditional fermented foods of cereal-legume. It is consumed widely as a major source of dietary nutrients (100 g portion: protein 12 g, total lipid 2 g, carbohydrate 72 g, and fiber 4 g) [4]. According to a recent metagenomic study, the phyla Firmicutes and Proteobacteria are predominant in fully fermented Idli batter, representing the genera Bacillus, Enterococcus, Enterobacter, Erwinia, Klebsiella, Lactococcus, Lactobacillus, Macrococcus, Serratia, Pseudoalteromonas, Vibrio, and Weissella [3]. These microbial communities collectively work to enhance the protein and nutrition values, and vitamin levels, and improve the digestibility, flavor, and texture of the final product Idli [5]. Overall, fermented food microorganisms have enormous potential as a future probiotic contender.

Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [6]. On commercial grounds, Lactobacillus and Bifidobacteria are well-accepted around the world. However, spore-forming Bacillus species are still undergoing enormous food safety checks, although they are present in many fermented foods [7]. Until now, B. coagulans (Heyndrickxia coagulans), B. clausii (Shouchella clausii), and B. subtilis have been available on the market for various gastrointestinal complications and in the management of diseases in humans and animals [8]. As these probiotics are in spore form, they can withstand a variety of environmental factors and hold up to the claims made on the viability label for the duration of the product's shelf life. When compared to the vegetative forms of true lactic acid bacteria, they are inexpensive in numerous capacities [8].

Apart from probiotic properties, the safety of probiotics is one of the milestones laid out by the food safety authorities around the globe. According to their guidelines, candidate probiotics must be identified using the whole genome sequence (WGS) and checked for the presence of plasmids, antibiotic resistance genes, and virulence factors [9]. Besides this, it can be phenotypically tested for pathogenic traits and antibiotic resistance [10]. In this study, we report on the isolation of Bacillus from traditionally fermented Idli Batter, probiotic properties, antimicrobial compound production and characterization, and phenotypic and genetic safety assessment.

Isolation of bacterial strain

The traditionally fermented Idli Batter sample was collected from village Loni-Kalbhor (18.48799103 N 74.01815952 E), Pune, India, as per standard sampling protocol. The collected sample was transported under cold conditions (4 ^oC) to the Food Technology Laboratory, and diluted serially in PBS (NaCl, 8.0 g; KCl, 0.2 g; Na₂HPO₄, 1.42 g; KH₂PO₄, 0.24 g; and ultrapure water 1 L, pH 7.3). The appropriate dilutions were spread plated on nutrient agar (NA, HiMedia, India) plates and incubated at 37 ^oC for 24 h. After incubation, the colonies with different morphologies were separated, subjected to Gram staining, and catalase activity, and streaked further on HiCrome Bacillus agar (HiMedia) to differentiate Bacillus species. The selected isolate was imaged using a scanning electron microscope (SEM) EVO 18 (Carl Zeiss, Germany), and cell size was measured (ImageJ software, USA) as described by Ahire et al. [11]. The glycerol stocks (20%, v/v) were prepared and stored at – 20 ^oC.

Probiotic characterization

Gastric and intestinal juice tolerance

Gastric juice tolerance was determined as per Pedersen et al. [12]. In brief, 8 h old DC-11 cells were pelleted (8,000×g for 10 min at 4°C), washed twice with PBS (pH 7.3), and resuspended in the same. To 1 mL of this suspension, 10 mL filter sterilized [0.2 µm cellulose acetate (CA), Minisart® NML, Sartorius, Germany] synthetic gastric juice (consisting: pepsin, 0.0133 gm; lysozyme, 0.1 g; glucose, 3.5 g; bile, 0.05 g; proteose peptone, 8.3 g; KCl, 0.37 g; NaCl, 2.05 g; CaCl₂, 0.11 g; KH₂PO₄, 0.6 g; and ultrapure water 1 L, pH 2.5) was added and kept at 37 ^oC for 3 h. The bacterial viability was determined on NA (HiMedia) at the intervals of 0, 1, 2, and 3 h. The results were expressed as log₁₀ colony forming units (CFU)/mL.

Intestinal juice tolerance was performed according to Ahire [13]. A 1 mL DC-11 suspension (prepared as described in gastric juice tolerance) was added to 10 mL filter sterilized intestinal juice [consisting :1 mg/mL pancreatin (protease 100 U/mg; amylase 100 U/mg; lipase 8 U/ mg), 0.3% (w/v) bile, and 0.85% (w/v) NaCl, pH 8.0] and kept at 37 ^oC for 6 h. The bacterial viability was determined on NA (HiMedia) at intervals of 0, 2, and 6 h. The results were expressed as log₁₀ CFU/mL.

Adhesion potential

Autoaggregation

An 8 h old DC-11 cells were pelleted (8,000×g for 10 min at 4°C), washed twice with PBS (pH 7.3), and resuspended in the same to 0.5 OD₆₀₀. The 5 mL suspension was vortexed (10 s) and kept at 37°C for 1 h [14]. After incubation, the upper layer was removed and optical density was measured at 600 nm. The percentage auto-aggregation was calculated as, (OD 0.5 – OD upper layer/ OD 0.5) × 100.

Adhesion to xylene

Bacterial adhesion to xylene was performed as per the method described by Rosenberg et al. [15]. In brief, 8 h old DC-11 cells were pelleted (8,000×g for 10 min at 4°C), washed twice with PBS (pH 7.3), and diluted to 0.5 OD₆₀₀ in 0.1 M KNO₃ (pH 6.2). To 3 mL bacteria, 1 mL xylene was gently added and kept at 37 ^oC for 10 min. Both phases were mixed by vortexing (5 s) and kept at 37 ^oC for 1 h to separate. The aqueous phase was removed and optical density was measured at 600 nm. The percentage of xylene adhesion was calculated using the following formula, (OD 0.5 – OD aqueous layer / OD 0.5) × 100.

Adhesion to mucin

The mucin (100 µg/mL, porcine stomach, Type II, Sigma- Aldrich, USA) was coated to the wells of a 96-well plate (ThermoFisher, USA) as described by Ahire et al. [16]. A 250 µL DC-11 suspension (0.5 OD₆₀₀) prepared in PBS (pH 7.3) containing 0.05% (w/v) Tween 20 was added to mucin-coated wells (n = 15) and kept at 4 ^oC for 24 h. After incubation, the cells that did not adhere to the mucin were carefully decanted and washed with PBS + Tween 20 (0.05% w/v; pH 7.3). The wells were air-dried and bacterial adhesion was determined using the crystal violet staining method [17].

Adhesion to Caco-2 cells

Human colorectal adenocarcinoma (Caco-2) cell adhesion was performed as per the method described by Tuomola and Salminen [18]. In brief, Caco-2 cells (1 × 10⁵ / mL) were cultivated for 2 weeks on glass coverslips placed at the bottom of 24-well tissue culture plates (ThermoFisher) containing Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (ThermoFisher). During the 2-week cultivation phase, the DMEM was changed every 24 h and 1 h before the bacterial adhesion. A 400 µL, 8 h old DC-11 cells (10⁹ cells per mL) diluted in PBS (pH 7.3) were added into Caco-2 cells. The plates were incubated at 37 ^oC for 2 h to facilitate bacterial adhesion. After incubation, the cells were washed thrice with PBS (pH 7.3) and adhered bacterial cells were enumerated under the microscope.

Zeta potential

An 8 h old DC-11 cells were pelleted (8,000×g for 10 min at 4°C), washed twice with PBS (pH 7.3), and suspended in the same to 0.5 OD₆₀₀. This suspension was filled in a DTS1070 capillary cell and zeta potential was measured using a Nano-ZS Zetasizer (Malvern, UK) [11].

Phenotypic safety

Detection of hemolytic and non-hemolytic enterotoxins

A 100 µL, 18 h old DC-11 culture was inoculated in a circular sample port of Duopath® Cereus Enterotoxins immune-chromatographic rapid test kit (Merck, Germany) and observed for development of colored lines at C (control), HBL (hemolysin BL), and NHE (non-hemolytic enterotoxin) as instructed by the manufacturer. Bacillus cereus ATCC 10876 was used as a positive control.

Gelatinase activity

The gelatin-nutrient agar (0.8% gelatin (w/v) in 2.3% nutrient agar) plates were streaked with DC-11 cells and incubated at 37°C for 24 h. After incubation, 5% (w/v) trichloroacetic acid (TCA) solution was gently poured on top of the agar surface and observed for clear zones surrounding the growth.

Mucin degradation

Mucin degradation was performed as per the method described by Ahire et al. [16]. In brief, minimal agarose medium (consisting: K₂HPO₄, 3.0 g; KH₂PO₄, 0.5 g; MgSO₄, 0.5 g; NaCl, 5.0 g; meat extract, 5.0 g; yeast extract, 3.0 g; tryptone, 7.5 g; pancreatic enzymatic digest of casein, 7.5 g; cysteine HCl, 0.5 g; agarose, 15 g, and ultrapure water 1L; pH 7.2 ± 0.2) was prepared with or without 3% (w/v) glucose and supplemented with 0.3% (w/v) mucin (porcine stomach, Type II, Sigma- Aldrich). The plates were surface-dried and inoculated with 5 µL suspension of DC-11. All the plates were incubated at 37°C for 48–72 h and stained with coomassie blue (0.1%, w/v). The bacterial growth surrounded by a discolored halo indicates mucin lysis.

Cytotoxicity

Cytotoxicity analysis was performed as per the method described by Tjandrawinata et al. [19]. In brief, human hepatocarcinoma (HepG-2) and Caco-2 cells were each seeded separately (1 × 10⁴ cells / well) in wells of a 96-well plate (ThermoFisher) and inoculated with 200 µL, 8 h old DC-11 cells (10⁹ cells per mL) diluted in MEM-alpha and or MEM. The plates were incubated statically at 37 ^oC for 24 h and cell viability was determined using Cell Proliferation Kit-MTT based (Merck, USA). The viability (%) was calculated as, absorbance @570 nm _{treated cells}/absorbance @570 nm _{untreated cells} × 100.

Antibiotic sensitivity

Antibiotic sensitivity was determined according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [20] and the Clinical and Laboratory Standards Institute [21]. In brief, 100 µL (~ 10⁶ cells per mL) of 8 h old DC-11 cells were aseptically added into 20 mL molten NA, mixed to homogeneity, and poured into the plates. The plates were surface-dried, and standard antibiotic disks (Table 1, HiMedia) were placed and kept at 4 ^oC for 20 min. After radial diffusion of antibiotics at 4 ^oC, the plates were incubated at 37 ^oC for 24 h. The clear zones surrounding the antibiotic disks were measured in millimeters (mm).

Minimum inhibitory concentrations (MICs) were determined as per the method described by Andrews [22]. The selected antibiotics (Table 2) were each diluted (0.06 to 128 mg/L) in molten NA and poured into the plates. The plates were allowed to solidify and surface-dried. A 5 µL suspension of DC-11 cells (10⁶ cells/mL) was spot inoculated and plates were incubated at 37 ^oC for 24 h. The MIC was defined as the lowest antibiotic concentration in the agar medium that prevented the growth of DC-11 cells. The results were compared with European Food Safety Authority (EFSA) MIC cut-off values given for Bacillus spp. [23].

Antimicrobial activity

Screening for antimicrobial activity

An 18 h old colony of DC-11 was inoculated in 10 mL NB and incubated by shaking (120 rpm) for 8 h at 37 ^oC. After incubation, 1 mL culture was transferred to 100 mL fresh sterile NB and incubated as described above for 24 h. The cells were separated by centrifugation (8,000×g for 10 min at 4°C) and the supernatant was collected. The pH of the supernatant was adjusted to 7.0, filter sterilized using 0.2 µm CA filter (Sartorius), and checked for antimicrobial activity against Micrococcus luteus MTCC 106^T as described above. A100 µL M. luteus culture previously cultivated at 30 ^oC for 18 h (shaking 120 rpm) was inoculated in 20 mL molten Mueller-Hinton (MH) agar (HiMedia) and poured into a sterile Petri plate. The agar was allowed to solidify and 9 mm wells were created using a sterile 1 mL tip into the agar slab. A 25 µL supernatant was dispensed into the well and allowed to diffuse at 4 ^oC for 15 min. Later, the plates were incubated at 30 ^oC for 24 h. Sterile NB served as a control. The zone of growth clearance around the well was measured in mm [11].

Production and isolation of antimicrobial compounds (AMC)

The production and isolation of AMC was performed as per the method described by Ahire et al. [24]. In brief, the Amberlite® XAD16N (Sigma-Aldrich, USA) beads required for NB clarification and AMC isolation were activated by immersing beads into 80% isopropanol (IPA) (ThermoFisher) containing 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich) (v/v/v) for 30 min shaking (120 rpm) at 28°C. The activated beads were rinsed 4 to 5 times with ultrapure water and collected. These beads were sterilized at 121 ^oC, 15 lbs pressure for 15 min, for their need in sterile applications [24]. The NB required for the cultivation of DC-11 was clarified using activated Amberlite® XAD16N (Sigma-Aldrich) beads (10 g activated beads to 1 L broth) to avoid interference of media ingredient (up to 40 kDa) during the purification process as described by Ahire et al. [11].

The strain DC-11 was cultivated as per the process described in ‘Screening for antimicrobial activity’, except NB, the clarified-NB was used. A 20 mL culture was mixed with 20 g activated (sterilized) XAD16N beads and distributed evenly on the surface of a sterile clarified-nutrient agar plate (200 mm × 30 mm, Borosil, India). All the plates were sealed with parafilm and incubated statically at 37 ^oC for 96 h. After incubation, beads were collected and attached bacteria were washed off with ultrapure water 5–6 times. The beads were then subjected to 30% (v/v) ethanol wash followed by 5–6 times ultrapure water wash. All the beads were collected and immersed in 80% isopropanol (IPA) (ThermoFisher) containing 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich) (v/v/v) for 30 min shaking (80 rpm) at 28°C to extract AMC. The extracted AMC was filtered through 0.2 µm CA membrane and tested against M. luteus MTCC 106^T in an agar well plate inhibition assay [11]. The AMC was further concentrated using Rotavapor (R-300, Buchi, Switzerland) and subjected for reversed-phase Sep-Pak® Vac 35 cc (10 g) C18 cartridge (Waters, USA) purification.

Purification of AMC

The reversed-phase C18 cartridge was preconditioned as per the manufacturer's instructions and loaded with AMC concentrate. The cartridge was washed with 5 column volumes of ultrapure water and AMC was eluted using IPA gradients (10–90%) containing 0.1% TFA (v/v/v) with 10% increments at a flow of 1 mL/ min [11]. The collected fractions were checked for antimicrobial activity against M. luteus MTCC 106^T. The fractions positive against M. luteus were mixed and concentrated using Rotavapor followed by freeze-drying (Vertis, USA). The freeze-dried AMC was stored at – 18 ^oC freezer until further use.

Characterization of AMC

The protein content in AMC was determined by using Pierce™ bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, USA). The UV-visible spectra were obtained using a Thermo- spectrophotometer (Thermo Scientific). Fourier transform infrared (FTIR) analysis was performed on Bruker FTIR-spectrometer (Bruker, USA). Proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectra were obtained using Bruker NMR-spectrometer (Bruker, USA). The deconvoluted mass and mass to charge (m/z) were determined using an Agilent quadrupole time-of-flight (QTOF) (Agilent Technologies, Inc., USA).

The antimicrobial activity of purified AMC was performed as per the method described previously in the section "Screening for antimicrobial activity”. In brief, M. luteus, methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA1720™, Staphylococcus aureus MTCC 737, Pseudomonas aeruginosa MTCC 1688, and Escherichia coli MTCC 1687 were each grown 18 h in their specified growth media and incubation conditions. A 100 µL culture was inoculated in 20 mL molten MH agar (HiMedia) and poured into a sterile Petri plate. The wells were created and 25 µL AMC (1 mg dissolved in 80% IPA containing 0.1%TFA) was dispensed into the well and allowed to diffuse at 4 ^oC for 15 min. All the plates were incubated at specified growth temperatures of test cultures for 24 h. An 80% IPA containing 0.1%TFA served as a control. The zone of growth clearance around the well was measured in mm.

The strain DC-11 whole genome sequence (WGS) was analyzed for the presence of secondary metabolites gene clusters, bacteriocins, ribosomally synthesized post-translationally modified peptides using antiSMASH-DB 4.0 [25] and BAGEL 4 [26]. The physical and chemical properties of AMC were determined using Expasy ProtParam [27].

Co-aggregation with pathogens

Co-aggregation with pathogens was determined as per the method described by Ahire et al. [16]. Bacterial cultures [probiotic: B. subtilis DC-11 (vegetative form); pathogens: Proteus mirabilis MTCC 425, E. coli MTCC 1687, and Candida albicans ATCC 14053) were each grown 18 h in their specified growth media at 37 ^oC. The cell pellets were harvested, washed twice with PBS (pH 7.3), and diluted to 0.5 OD_{600 nm} in PBS. A 2 mL probiotic suspension was mixed gently (10 s vortexing) with 2 mL pathogen and incubated statically for 1 h at 37 ^oC. After incubation, the upper layer was removed and OD_{600 nm} was measured. The optical density readings of the upper layer of individual culture suspension (4 mL) served as an individual aggregation density. The percentage co-aggregation was calculated using following formula, [(OD₆₀₀ x + OD₆₀₀ y) / 2] – OD₆₀₀ [x + y] / OD₆₀₀ x + OD₆₀₀ y / 2 × 100.

where OD₆₀₀ x and OD₆₀₀ y are individual aggregation densities of test cultures. OD₆₀₀ (x + y) are co-aggregation densities.

Biofilm formation

An 18 h old DC-11 colony was inoculated in 10 mL nutrient broth and incubated shaking (120 rpm) at 37 ^oC for 8 h. After incubation, cells were separated and diluted in 30 mL fresh sterile nutrient broth. Each 200 µL (2 × 10⁴ CFU/mL) of this suspension was carefully transferred into 6 wells of 96-well plate (ThermoFisher). The plate was incubated statically at 37 ^oC for 24 h. The total biofilm mass, free cells, and viable biofilm cells were investigated as per the method described by Ahire and Dicks [17].

Complete genome sequence analysis

DNA extraction

An 18 h old colony of strain DC-11 was inoculated in 10 mL nutrient broth and incubated shaking (120 rpm) at 37 ^oC for 18 h. After incubation, the cells were separated and genomic DNA was extracted using MO BIO’s genomic DNA extraction kit (Carlsbad, CA, USA). The extracted DNA was subjected to 0.8% (w/v) agarose gel and the Qubit dsDNA HS assay kit (ThermoFisher) for quantitative and qualitative estimation.

Library preparation and sequencing

Nextera DNA Flex kit (Illumina, San Diego, CA, USA) was used for DNA fragmentation and library preparation as instructed by the manufacturer. The library was further sequenced on the Illumina MiSeq platform (Illumina Inc., USA) by using 2 × 250 bp chemistry. A 5% PhiX spike-in was used during library sequencing. NGS-QC tool was used to remove low-quality sequences (http://www.nipgr.res.in/ngsqctoolkit.html) [28].

Genome BLAST Distance Phylogeny (GBDP) analysis

The genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available at https://tygs.dsmz.de, for a whole genome-based taxonomic analysis [29]. The analysis also made use of recently introduced methodological updates and features [30]. Information on nomenclature, synonymy, and associated taxonomic literature was provided by TYGS's sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at https://lpsn.dsmz.de) [30]. Digital DNA-DNA Hybridization (dDDH) values and confidence intervals were calculated using the recommended settings of GGDC 4.0 [30]. The results were provided by the TYGS on 2024-08-02. Orthologus Average Nucleotide Identity (OrthoANI) was determined using the OrthoANIu tool (EzBioCloud, USA) [31].

Genome assembly, annotation, and safety

DC-11 genome was assembled using SPAdes-3.11.1 assembler (St. Petersburg, Russia) and quality was determined using the Quality Assessment Tool. RNAmmer 1.2 (USA) was used to identify a number of rRNAs. The genome was annotated by Rapid Annotations using Subsystems Technology (RAST), UniProt/SwissProt, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. tRNA_scan-SE, and RNAmmer were used to predict genes for tRNA, and rRNA. Protein-coding genes were determined by using Glimmer version 3.02. SignalP finder was used to identify signal peptides. AntiSMASH was used to identify secondary metabolites [25], the Comprehensive Antibiotic Resistance Database (CARD) for antibiotic resistance genes [32], Virulence Factor Database (VFDB) for virulence factors [33], IslandViewer [34], and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Finder [35] for insertion and bacteriophage associated sequences.

In silico assessment of genome features contributing to probiotic properties (dup: abstract ?)

Bacillus subtilis DC-11 WGS was investigated using RAST (Rapid Annotation using Subsystem Technology) [36] and SEED [37] viewer comparative blast search tool along with NCBI standard protein BLAST to explore the genes or specific domains responsible for acid and bile tolerance, adhesion to the gut, and environmental stress resistance.

Strain deposition and WGS accession number

Bacillus subtilis strain DC-11 whole genome shotgun sequencing project is available at https://www.ncbi.nlm.nih.gov/nuccore/2318375570 with accession number JAOWBV000000000. The strain has been deposited in culture collection at the National Centre for Microbial Resource, Pune, and is available under accession number MCC4788.

Statistical analysis

The data was analyzed using GraphPad Prism (Trial version 10, USA). The statistical significance was determined using Tukey’s multiple comparison test (one-way ANOVA) and t-test. The data were presented as the mean ± standard deviation. The p-value of less than 0.05 was considered significant.

Isolation of bacterial strain

Traditionally fermented Idli Batter sample produced two different dominant colony types on NA viz. white pin-pointed small colonies (1 − 2 mm) and fuzzy white larger colonies (2 − 3 mm). The bacteria that produced small colonies are Gram-positive, cocci-shaped, non-spore former, and catalase-negative. However, the bacteria that produced large colonies are Gram-positive, rod-shaped, spore former, and catalase-positive. In this study, we report on the probiotic characterization of bacteria that produced a large colony and were designated as DC-11. The DC-11 colony was circular, rough, opaque, fuzzy white, and 2–3 mm in diameter. On HiCrome Bacillus agar, DC-11 produced yellowish-green to green colonies (characteristic of Bacillus subtilis). The rods occurred singly or in pairs, and measured about 1.4 ± 0.1 µm long and 0.6 ± 0.1 µm wide (Fig. 1a). The cells are capable of fermenting glucose, fructose, mannitol, starch, and glycogen. The indole, methyl red, and urease tests are negative, whereas Voges Proskauer and nitrate reduction were positive for B. subtilis.

Probiotic characterization

Gastric and intestinal juice tolerance

In gastric juice, the viability of DC-11 was significantly (p 0.0001) reduced from 7.99 ± 0.003 log₁₀ CFU/mL (0 h) to 7.58 ± 0.01 (1 h), 7.28 ± 0.003 (2 h) and 7.11 ± 0.001 log₁₀ CFU/mL at 3 h (Fig. 1b). The percentage of bacterial viability reduction from 0 h to 3 h was 11.01%.

The viability of DC-11 was significantly (p 0.0001) reduced in the intestinal juice. The initial 7.89 ± 0.001 log₁₀ CFU/mL cells of DC-11 were reduced to 7.83 ± 0.004 at 3 h and 7.78 ± 0.008 at 6 h (Fig. 1c). The percentage of bacterial viability reduction from 0 h to 6 h was 1.39%.

Adhesion potential

The strain DC-11 showed 18.33 ± 0.44% autoaggregation, 32.53 ± 3.11% adhesion to xylene, 0.98 ± 0.05 OD unit’s adhesion to mucin (crystal violet equivalence at 550 nm), and 21.2 ± 2.3% adhesion to Caco-2 cells (Fig. 2a, b). The zeta potential for 2 Billion CFU/mL DC-11 cells dissolved in PBS buffer (pH 7.3) was − 22.3 ± 0.65 mV.

Phenotypic safety

No colored lines were developed at HBL, and NHE when DC-11 culture broth was added in the sample port as compared to control B. cereus ATCC 10876 (Fig. 2c). On the gelatin-nutrient agar plate, the surrounding area of DC-11 growth showed a clear zone when treated with TCA (Fig. 2c). In mucin degradation, after coomassie blue treatment, no discolored halo was observed surrounding the bacterial growth (with or without glucose) (Fig. 2c). The Caco-2 and HepG-2 cells exhibited 64.0 ± 0.27% and 68.01 ± 0.31% viability in presence of DC-11 cells (Fig. 2d).

The strain DC-11 was sensitive to most of the antibiotics tested, except cefdinir, rifampicin, and antifungal drugs nystatin, itraconazole, and fluconazole (Table 1). The MIC cut-off values for chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, tetracycline, and vancomycin are below and within the limits of MIC cut-off values provided by EFSA for Bacillus spp., (Table 2).

Antimicrobial activity, production, purification, and characterization of AMC

A 24-hour-old cell-free supernatant of DC-11 inhibited the growth of M. luteus. The zone of inhibition measured for supernatant pH 7.0 (adjusted) was 10.33 ± 0.58 mm and pH 6.8 (as such) was 10.0 ± 1.0 mm (p 0.6499). The rotavapor concentrate of AMC extracted from XADN16 beads with IPA (80% containing 0.1% TFA, v/v/v) showed 15.33 ± 0.58 mm inhibition of M. luteus. The C18 cartridge fractions eluted with 60 and 70% IPA containing 0.1% TFA (v/v/v) had an activity of 17.0 ± 1.0 mm and 19.0 ± 1.0 mm. The overall purification process yielded 152 mg (per 100 g XAD16 beads) of AMC.

The estimated protein content in 1000 µg AMC was 402.1 ± 3.2 µg. In UV-visible spectroscopic analysis, the compound showed a broad peak in the range of 270 to 310 nm (Fig. 3a). In FTIR analysis, C-I stretching was recorded at wavenumber 522 /cm, C-C bending at 593/cm, C-H bending at 800/cm and stretching at 2700 and 2800/cm, C-O stretching at 1101/cm, O-H bending at 1345/cm and stretching at 3700/cm, N-O stretching at 1484/cm, C = C stretching at 1632/cm, C = O stretching at 1700/cm, C = C = C stretching at 1956/cm, C = C = N stretching at 2013/cm, C ≡ C stretching at 2104/cm, N = C = O stretching at 2248/cm, S-H stretching at 2591/cm, and N-H stretching at 3400/cm (Fig. 3b). The ¹H and ¹³C NMR showed the signals at following ppm scale. ¹H (400 MHz, DMSO-d6): δ ppm range 0.9-1.0, 1.2–1.7, 1.5-2.0, 1–3, 2.3-3.0, 1–5, 3.7–6.5 (Fig. 4a), 5–9, 6.0-8.7, and 10–13 (Fig. 4b). ¹³C NMR (100 MHz, DMSO-d6): δ ppm range 10–30, 35–45, 105–145, 155–165, and 165–175 (Fig. 4c). The deconvoluted mass spectra showed the intense peak of 3400 Da (Fig. 5a, b) and mass to charge (m/z) peaks at 1700.96 (2+), 1134.29 (3+), and 850.98 (4+) m/z (Fig. 5c).

The purified AMC (1 mg/mL) showed significantly (p 0.0001) highest growth inhibition zones against S. aureus (MRSA) (18.6 ± 0.58 mm), and S. aureus (17.6 ± 0.58 mm), as compared to M. luteus (11.0 ± 1.0 mm) (Fig. 5d). The activity difference recorded between MRSA and S. aureus was insignificant (p 0.2789). No activity was recorded against P. aeruginosa and E. coli (Fig. 5d).

WGS mining of DC-11 showed the presence of a biosynthetic gene for sactipeptide subtilosin A (cds118043_118175). It has an amino acid sequence of MKKAVIVENK (leader) GCATCSIGAACLVDGPIPDFEIAGATGLFGLWG (Core) and showed 100% similarity with NC_000964.3 BSU_37350_sactipeptide. The core peptide analysis by using Expasy ProtParam showed an instability index of 21.0, an aliphatic index of 94.85, and a grand average hydropathicity of 0.958.

Co-aggregation

A significantly (p 0.0001) higher percentage of co-aggregation was recorded with E. coli (23.62 ± 0.70%) as compared to C. albicans (5.47 ± 2.06%) and P. mirabilis (3.89 ± 0.32%) (Fig. 6a). The difference between C. albicans and P. mirabilis co-aggregation was insignificant (p 0.3417).

Biofilm formation

DC-11 biofilm formed after 24 h of incubation showed a total biofilm mass of 2.03 ± 0.05 OD units of crystal violet staining and 7.75 × 10⁸ viable cells (per mL) (Fig. 6b). The free/planktonic cells determined were 4.48 × 10⁸ cells per mL (Fig. 6b). No statically significant (p 0.2371) difference was recorded between free and biofilm cells.

Complete genome sequence analysis

The strain DC-11 whole genome assembly contained 4,043,105 bp, 34 contigs, 4,003 proteins, 596078 N50, 3 L50, 334 subsystems, 4192 coding sequences, and 43.7% G + C. It showed 98.61% OrthoANIu and 91.8% dDDH with Bacillus subtilis ATCC 6051. Both GBDP trees (Fig. 7ab) indicated close similarity with B. subtilis ATCC 6051.

The genome elements of DC-11 constituted genes for rRNA (17), tRNA (83), non-coding (101), putative (429), hypothetical/uncharacterized (1162), gene ontology (GO) term (1206), and pathway (4006) elements (Fig. 8a). The further characterization of coding sequences (CDS) revealed the genes for metabolic functions (Fig. 8b) and protein Clusters of Orthologous Groups (COGs) (Fig. 8c). The details of CDS, tRNA, rRNA, and tmRNA were given in circular genome map (Fig. 9).

Nineteen antibiotic resistance genes that showed more than 60% identity matching using loose cut-off criteria were considered (Table S1), since no antibiotic resistance genes were determined using strict criteria. At around 40 insertion elements (Table S2), 1 CRISPR spacer gene was detected, however, there were no virulence factors and plasmids. AntiSMASH analysis showed the genes for bacillane, bacillibactin, bacilysin, subtilosin A, surfactin, plipastatin, and fengycin.

In silico assessment of genome features contributing to probiotic properties

The proteins involved in probiotic attributes of Bacillus subtilis DC-11 i.e. acid tolerance, bile salt tolerance, adhesion, and stress resistance are depicted in Table 3.

In food fermentations, the number of microorganisms plays an important role in converting food components and improving nutritional and organoleptic properties [38]. Lactic acid bacteria (LAB) and yeast are explored extensively for their benefits and probiotic potential [39]. Despite its widespread presence in food fermentations, Bacillus subtilis has received little attention for its potential health advantages [40]. In this study, we investigated the genetic and phenotypic safety of probiotic Bacillus subtilis strain DC-11 isolated from traditionally fermented Idli Batter.

The beneficial effects of probiotic bacteria depended on their ability to withstand harsh gastrointestinal conditions and ability to adhere to gut epithelial cells. In this study, strain DC-11 retained 88.98% viability at the end of 3 h incubation in gastric juice, and 98.60% viability at 6 h in intestinal juice, indicating the strain's ability to survive under gastrointestinal transit. The viability recorded was comparatively higher than B. subtilis strain F24 (gastric juice, pH 2.5, 3 h- 42%; intestinal juice, pH 6.8, 6 h- 63%) reported by Dabiré et al. [41]. The in silico genomic analysis revealed the presence of F₀F₁ ATP proton pumps, amino acid decarboxylase, acid stress-sensitive anti-sigma factor RsiO, and bile acid symporter, which could be one of the reasons for maintaining cytoplasmic pH, repair and necessary metabolic activities under acidic and alkaline conditions [42].

Adhesion is a key to inhibit colonization of pathogens, nutrient absorption, enhancement of gut transit time, and immunity [43]. In this study, the strain's autoaggregation, negative membrane potential, and adhesion to non-polar solvent xylene, mucin, and Caco-2 cells indicated hydrophobic cell surfaces. The presence of flagellin, which forms the filaments of bacterial flagella, sortases an extracellular trans-peptidases responsible for covalently attaching secreted proteins (pili) to the peptidoglycan cell wall [44] and membrane anchor proteins in DC-11 further validated strain’s inherent adhesion ability and colonization thereof. Moreover, these results were coordinated well with previous findings [45].

Despite regular inadvertent consumption of Bacillus strains in fermented foods, their safety is a primary concern to the regulatory authorities around the globe, as they cater to a serious risk of enterotoxin formation and the transfer of antibiotic resistance genes [7]. In this study, B. subtilis DC-11 was found negative for enterotoxin production, mucin degradation, and antibiotic resistance to the commonly used therapeutic antibiotics. Strains MIC cut-off levels for clinically relevant antibiotics were in accordance with EFSA. Besides the aforementioned safety qualities, the strain's low cytotoxicity toward Caco-2 and HepG-2 cells further indicated the safety of B. subtilis DC-11. Overall, the results are well coordinated with the strain's genetic safety, as no antibiotic resistance genes, virulence factors, and plasmids were detected in WGS. The strain’s gelatinase production, which is one of the genetic properties of B. subtilis, could not be considered a safety threat, as several gelatinase-positive B. subtilis strains have been endorsed as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration (FDA) [46]. The DC-11 resistance to antifungal drugs could be useful for its application with antifungals.

In antimicrobial evaluation, B. subtilis DC-11 showed the production of the antimicrobial compound sactipeptide subtilosin A. The broad absorption peak at 270–310 nm is attributed to conjugation compounds, aromatic rings of amino acids, and peptide bonds [47]. FTIR indicated the aliphatic link between C and H could be due to hydrophobic amino acids. C = O, N-O, C = C = N, N = C = O, and N-H for peptide bonds, amide groups, and S-H for Cysteine S–H bonds. Similarly, the proton NMR signals observed for aromatic amino acid (6.5–9.5 ppm), alkyl amines (3–5 ppm), and –CH₃ resonance and − C−CH₂ − C− (0.5–2 ppm), suggesting the polypeptide nature of AMC [48]. The results of carbon NMR showed carbon signals for aromatic, carboxylic acid, ester and amide compounds, and are in agreement with proton NMR. Furthermore, the mass and WGS analysis confirmed and validated the synthesis of sactipeptide subtilosin A from subtilosin biosynthetic gene. Subtilosin A was stable (computed from instability index), hydrophobic (computed from the aliphatic index and grand average of hydropathicity) in nature [27], and found active against Gram-positive bacteria including MRSA. Studies have suggested that subtilosin interacts with membrane-associated receptors to inhibit the growth of bacteria, similar to that seen with the lantibiotic, Nisin [49].

Co-aggregation has been shown to improve probiotic bacteria colonization efficiency, which may be important for managing microbiota communities [50]. In this study, B. subtilis DC-11 showed the highest co-aggregations with E. coli, compared to P. mirabilis, and C. albicans, indicating its ability to lower colonization of opportunistic pathogens like E. coil, P. mirabilis, and yeast. The results are well coordinated with B. subtilis P223 co-aggregation [51], however, the co-aggregation percentages were mostly dependent on strains and the time of the assay [50]. Besides this, B. subtilis DC-11 exhibited biofilm formation, which is one of the traits for efficient colonization of the gut.

According to the food safety and regulatory standards, WGS is crucial to establish the safety of candidate probiotics [9, 10]. In this study, strain DC-11 was identified as Bacillus subtilis, based on close similarity with Bacillus subtilis ATCC 6051 in GBDP and OrthoANIu and dDDH analysis. 31% of genes were observed to contribute amino acid/derivatives and carbohydrate metabolism, 11% for protein metabolism, 9% for cofactors and vitamins, 7% for dormancy and sporulation, and rest for other essential activities. The insertion elements identified (less than 50% scores) were not found in the vicinity of regions of concern such as antibiotic resistance genes. Besides this, no plasmid and virulence factor genes suggest stability and safe use of the strain. The presence of a CRISPR spacer in strain DC-11 ensured the immunity of the strain to combat future infections of mobile DNA elements. In AntiSMASH and BAGEL analysis, B. subtilis DC-11 strain showed the presence of different genes for secondary metabolites and antimicrobial compounds production. However, the in vitro production of subtilosin A confirmed the expression of the sactipeptide biosynthesis gene. The number of stress response genes mapped in the genome could be useful for strain's survival under harsh environmental conditions including human and animal gastrointestinal tract.

In conclusion, Bacillus subtilis strain DC-11 isolated from traditionally fermented Idli Batter showed promising survival under gastrointestinal conditions, adhesion, biofilm formation, co-aggregation potential, and production of antimicrobial sactipeptide subtilosin A. In phenotypic safety assessment, B. subtilis DC-11 was found negative for enterotoxin production, mucin degradation, and antibiotic resistance to the commonly used therapeutic antibiotics. The whole genome sequence analysis showed close similarity with B. subtilis ATCC 6051, absence of transmissible antibiotic resistance genes, virulence factor genes, and plasmids, which further confirmed the strain’s genotypic safety. Moreover, the presence of genes for proteins involved in probiotic attributes ensured the probiotic potential of the strain. Overall, B. subtilis DC-11 isolated from traditionally fermented Idli Batter, could be a safe probiotic candidate for further applications.

Funding

The authors did not receive support from any organization for the submitted work.

Competing Interests

Ahire JJ was employed by Dr. Reddy’s Laboratories Limited. Dr. Reddy’s Laboratories had no direct and indirect role in the design/analysis/writing/publication of this research article. Other authors have no conflict of interest to declare.

Data Availability Statement

All data is included in the text, however, the raw data of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Author Contributions

Conceptualization: Chaudhari DN, Ahire JJ; Methodology: Chaudhari DN, Ahire JJ; Formal analysis and investigation: Chaudhari DN, Ahire JJ; Writing - original draft preparation: Chaudhari DN, Ahire JJ; Review – Chaudhari DN, Ahire JJ, Kulthe AA, Ghodke S. Resources: Chaudhari DN, Ahire JJ, Kulthe AA, Ghodke S; Supervision: Ahire JJ.

Ethics approval

This study does not contain any work related with participation of humans and/or animals.

Informed consent

Not applicable

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Table 1 to 3 are available in the Supplementary Files section.

Competing interest reported. Ahire JJ was employed by Dr. Reddy’s Laboratories Limited. Dr. Reddy’s Laboratories had no direct and indirect role in the design/analysis/writing/publication of this research article. Other authors have no conflict of interest to declare.

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Genomic and phenotypic safety assessment of probiotic Bacillus subtilis DC-11 isolated from traditionally fermented Idli Batter

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Abstract

Figures

Introduction

Materials and Methods

Isolation of bacterial strain

Probiotic characterization

Gastric and intestinal juice tolerance

Adhesion potential

Autoaggregation

Adhesion to xylene

Adhesion to mucin

Adhesion to Caco-2 cells

Zeta potential

Phenotypic safety

Detection of hemolytic and non-hemolytic enterotoxins

Gelatinase activity

Mucin degradation

Cytotoxicity

Antibiotic sensitivity

Antimicrobial activity

Screening for antimicrobial activity

Production and isolation of antimicrobial compounds (AMC)

Purification of AMC

Characterization of AMC

Co-aggregation with pathogens

Biofilm formation

Complete genome sequence analysis

DNA extraction

Library preparation and sequencing

Genome BLAST Distance Phylogeny (GBDP) analysis

Genome assembly, annotation, and safety

In silico assessment of genome features contributing to probiotic properties (dup: abstract ?)

Strain deposition and WGS accession number

Statistical analysis

Results

Isolation of bacterial strain

Probiotic characterization

Gastric and intestinal juice tolerance

Adhesion potential

Phenotypic safety

Antimicrobial activity, production, purification, and characterization of AMC

Co-aggregation

Biofilm formation

Complete genome sequence analysis

In silico assessment of genome features contributing to probiotic properties

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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