Description
Bacillus subtilis species is a gram-positive, spore-forming, rod-shaped bacterium. The Bacillus subtilis DE111 strain was originally soil extracted from a site in Kennesaw, Georgia, United States. It is deposited in the Agricultural Research Culture Collection under the accession number NRRL B-67989. The ingredient is manufactured by aerobic fermentation using animal component-free fermentation media. It is then pasteurised, separated, and dried to the desired concentration of 100-200 billion CFU/g.
| Test | Method reference | Acceptance criteria |
|---|---|---|
| Characterisation | ||
| General properties | ||
| Appearance | Visual & Organoleptic evaluation | Light brown to brown powder |
| Water activity | USP <922> | <0.5 aw at 25oC |
| pH | USP <791> | 5.5-8.5 at 25oC |
| Macroscopy | Visual | Approximately 1.5 mm in diameter, circular, fuzzy, white to slightly yellow in colour with jagged edges colonies |
| Microscopy | USP <776> | 2 µm long, rod-shaped |
| Gram staining | USP <1113>
| Gram-positive |
| Water solubility | Ph. Eur. 04/2022:51100 | Very soluble |
Antimicrobial resistance and susceptibility
| Antimicrobial susceptibility test[1] | Susceptible to antibiotics: Vancomycin, Gentamicin, Kanamycin, Streptomycin, Erythromycin, Clindamycin, and Tetracycline
|
Whole genome screening using, at least 80% ID and 70% length threshold levels, ResFinder[2] and Resistance Gene Identifier[3] databases
| Absence of acquired resistance genes | |
| Absence of virulence factors | Whole genome screening for the potential virulent factors using (at least 80% ID and 70% length threshold levels) PathogenFinder[4] and ToxFinder[5] databases | Absent
|
| Identity | ||
| Polymerase Chain Reaction (PCR) | USP <1127> | Matches Bacillus subtilis (NRRL B-67989) |
| Assay | ||
Total Viable cell count
| In-house method[6] | 100-200 Billion CFU/g |
| Impurities and incidental constituents | ||
| Elemental impurities | ||
| Lead | USP <2232> | <1 ppm |
| Cadmium | USP <2232> | <1 ppm |
| Mercury | USP <2232> | <1 ppm |
| Arsenic | USP <2232> | <1 ppm |
| Microbial contaminants | ||
| Yeast and Molds | BP: SC I M. Microbial contamination | <300 CFU/g |
| Coliforms | BP: SC I M. Microbial contamination | <100 CFU/g |
| Enterobacteriaceae | BP: SC I M. Microbial contamination | <100 CFU/g |
| E. coli | BP: SC I M. Microbial contamination | Negative |
| Salmonella | BP: SC I M. Microbial contamination | Negative |
| Staphylococcus | BP: SC I M. Microbial contamination | Negative |
Footnotes
- Cockerill, F. R., et al. (2012). Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Eleventh Edition. Clinical and Laboratory Standards Institute M02-A11, 32(1).
- Florensa A. F., et al. (2022). ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes. Microbial Genomics, 8(1):000748.
- Alcock B. P., et al. (2020). CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Research, 48(D1):D517–D525.
- Cosentino S., et al. (2013). PathogenFinder - distinguishing friend from foe using bacterial whole genome sequence data. PLoS one, 8(10):e77302.
- Centre for Genomic Epidemiology, ToxFinder 1.0.
- B. subtilis was grown in a soybean-casein digest agar and broth at 37 ± 2°C for 18-24 hours prior to diluting and plating approximately 25-250 colonies per plate. The cell count was conducted manually under a light microscope.