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Name of the ingredient
Bacillus coagulans (ABN)
Definition of the ingredient
The Bacillus coagulans species is a lactic-acid producing, gram-positive, spore-forming, rod-shaped bacterium that is aerobic to microaerophilic. The microbial culture is isolated from a food source (green malt). The Microbial Type Culture Collection (MTCC) designated strain accession number is MTCC 5856. The ingredient is prepared by fermentation of culture of MTCC strain 5856 to 200 - 300 billion CFU/g. The quality and safety of the microorganism B. coagulans was assessed for the MTCC strain 5856 and the specific requirements listed below are strain specific.
| Test | Method reference | Acceptance criteria |
|---|---|---|
| Description | ||
| Appearance | Visual & Organoleptic evaluation | Pale brown-to-brown coloured powder |
| Characteristics | ||
| Solubility | USP - General notices - 5.30 | Very slightly soluble in water, insoluble in methanol |
| Loss on drying | USP (731) | Not more than 10.0% w/w (dried at 105˚C) |
| Sieve test (Passes through) - 60 mesh | USP (786) | Not less than 95% w/w |
| Identification | ||
| Microscopy | In-house method[1]
USP (776) |
Gram positive, slightly bent rods having terminal spores, which stains Malachite green. |
| Lactic acid producing capacity | In-house method[2] | Not less than 20 ml of 0.05N NaOH is consumed. |
| Phenotypic identification | ||
| Purity |
Macroscopic & Microscopic examination In-house method [3] USP (776) |
White colonies with smooth margins, slightly bent rods having terminal spores, which stains Malachite green. To comply as per Bacillus coagulans Hammer 1915, Type Strain ATCC 7050. |
| Gram staining |
In-house method [4] USP (776) |
Gram positive, slightly bent rods having terminal spores, which stains Malachite green. |
| 16S ribosomal RNA gene sequencing | Polymerase chain reaction [5] | Homology: Not less than 99.0% with Bacillus coagulans ATCC 7050. |
| Antimicrobial susceptibility test | Clinical Laboratory Standards Institute (CLSI, 2012), M07-A9 & EFSA (2012) guidelines for MIC [6] |
Sensitive to clindamycin, kanamycin, ampicillin, streptomycin, vancomycin, erythromycin, gentamycin, tetracycline, and chloramphenicol. Should comply as per EFSA (2012) guidelines for MIC. |
| Cell viability |
MTT assay Supriya D. Mahajan et al. [7] |
Not less than 70% cell viability reduction with respect to Positive control (Bacillus cereus ATCC 14579). |
| Cytotoxity assay |
Verocell based cytotoxity assay Cecil From et al. [8] |
Microscopy: No swelling, rounding and dissemination of Vero cells with respect to Positive control (Bacillus cereus ATCC 14579). |
| Antimicrobial production for the strain |
Well diffusion assay method Cintas et al. [9] |
Zone of inhibition: Not less than 8 mm. |
| Toxigenicity | PCR based Detection of Bacillus cereus-like enterotoxin genes[10] | 200 to 300 Billion CFU/g |
| Assay | ||
| Viable spore count | In-house method [11] | 200 to 300 Billion CFU/g |
| Test | Method reference | Acceptance criteria |
|---|---|---|
| Incidental metals and non-metals | ||
| Lead | USP (2232) | Not more than 2.0 ppm (µg/g)* |
| Arsenic | USP (2232) | Not more than 1.5 ppm (µg/g) |
| Cadmium | USP (2232) | Not more than 0.5 ppm (µg/g) |
| Mercury | USP (2232) | Not more than 0.1 ppm (µg/g) |
| *The lead content in the specification is given as 2 ppm as an upper limit for the ingredient. In the finished product, the lead content must comply with the acceptance criteria set out in the United States Pharmacopoeia – National Formulary (USP-NF) general chapter ''(2232) Elemental Contaminants in Dietary Supplements'. | ||
| Microbiology | ||
| Other aerobic microorganisms | In-house method [12] | Not more than 0.3 Million cfu/g |
| Total yeast and mould count | USP (2021) | Not more than 100 cfu/g |
| Escherichia coli | USP (2022) | Negative/10 g |
| Salmonella | USP (2022) | Negative/10 g |
| Staphylococcus aureus | USP (2022) | Negative/10 g |
| Pseudomonas aeruginosa | USP (62) | Negative/10 g |
| Bile tolerant Gram negative bacteria | USP (2021) | Not more than 100 cfu/g |
| Coliforms | BAM 2001, 8th Edition, Chapter 4 | Less than 10 cfu/g |
Footnotes
| [1] | (SSL/STP-MB/030): Sample solution is prepared in sterile saline water and one loop full mounted on a slide. Stain the slide with primary stain 'Crystal violet' and iodine and later rinsed with ethanol. Stain the spores on the slide with malachite green/Schaeffer and Fulton spore stain S029. Check under microscope 100x immersion oil. |
|---|---|
| [2] | (SLL/STP-L-015): Sample is suitably diluted and after incubation, the incubated media is titrated with 0.05 N NaOH with bromothymol blue- neutral- red indicator till it starts turning green. |
| [3] | (SSL/STP-MB/030): Sample solution is prepared in sterile saline water and one loop full mounted on a slide. Stain the slide with primary stain 'Crystal violet' and iodine and later rinsed with ethanol. Stain the spores on the slide with malachite green/Schaeffer and Fulton spore stain S029. Check under microscope 100x immersion oil. [Reference: Bergey's manual of systematic bacteriology, Vol. 2. pp. 1122; Please also refer: Appendix 3.5.1.1 - Phenotyping study]. |
| [4] | (SSL/STP-MB/030): Sample solution is prepared in sterile saline water and one loop full mounted on a slide. Stain the slide with primary stain 'Crystal violet' and iodine and later rinsed with ethanol. Stain the spores on the slide with malachite green/Schaeffer and Fulton spore stain S029. Check under microscope 100x immersion oil. |
| [5] | Ed. Kary Mullis, Francois Ferre, Richard A. Gibbs, Springer Science + Business Media, LLC 1994. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis, Jethro S. Johnson, et al., Nature Communications, Vol. 10, Article number: 5029 (2019). |
| [6] | Standards Institute (CLSI), M07-A9: By standard broth micro dilution method, (https://www.researchgate.net/file.PostFileLoader.html?id=564ceedf5e9d97daf08b45a2&assetKey=AS%3A297254750572544%401447882463055) and EFSA guidelines 2012. (https://www.efsa.europa.eu/sites/default/files/consultation/120323.pdf) and also refer: Appendix 3.6.3 – Safety and Efficacy (3.6.3.1) |
| [7] | Supriya D. Mahajan et al. Methods in Enzymology, Nanomedicine: Chap.3, sec.6.3, Pg.51 Ed. Nejat Duzgunes, Elsevier First Edition 2012. ISO Report. ISO 10993–5: biological evaluation of medical devices - Part 5: tests for in vitro cytotoxicity. Geneva: International Standard Organization; 2009. Please also refer: Appendix 3.6.3 – Safety and Efficacy (3.6.3.3) |
| [8] | Toxin-Producing Ability among Bacillus spp. Outside the Bacillus cereus Group, Appl. Environ. Microbiol 71(3), 2005, Pp.1179. Please also refer: Appendix 3.6.3 - Safety and Efficacy (3.6.3.4) |
| [9] | Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Appl Environ Microbiol 61:2643-2648. Please also refer: Annexure-4 Pg.7-8, Table No.1. |
| [10] | Ed. Kary Mullis, Francois Ferre, Richard A. GibbsSpringer Science + Business Media, LLC 1994. Please also refer: Appendix 3.6.3 Safety & Efficacy. 3.6.3.2: PCR based detection of Bacillus cereus-like enterotoxin genes. Pages 9 to 21 for complete details. |
| [11] | (SLL/STP-B-014): Take 1 mL of aseptically prepared saline test solution poured on petri plates with GYEA (Glucose, Yeast extract, Agar) media and incubated at 37°C for 72 hours. Count the number of colonies per plate. |
| [12] | (SLL/STP-O-009): Prepare Soyabean Casein Digest Agar (Tryptic Soy Agar) media. Pour diluted sample on to the plate and incubate at 30-35°C for 17-20 hours. Count the number of colonies forming units (cfu). |
Key to abbreviations
BAM = Bacteriological Analytical Manual
USP = United States Pharmacopeia
OECD = Organisation for Economic Co-operation and Development
ISO = International organisation for standardisation
CFU = Colony forming units
μg/g = Microgram/ gram
ppm = Parts per million
Ph Eur = European pharmacopoeia