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Description:
Akkermansia muciniphila is a gram-negative, strictly anaerobic, non-motile, non-spore-forming, oval-shaped bacterium that was isolated from human faeces. It has been deposited under Collection de l’Institut Pasteur (CIP) accession number CIP 107961 and the American Type Culture Collection (ATCC) catalogue number ATCC BAA-835. The ingredient is manufactured under anaerobic fermentation conditions followed by pasteurisation. The heat-inactivated bacteria are then freeze-dried and concentrated to 2.5 x 1010 - 2.5 x 1012 Total Fluorescence Unit (TFU)/g equating to 34 billion non-viable cells of pasteurised Akkermansia muciniphila.
Test | Method reference | Acceptance criteria |
|---|---|---|
Characterisation | ||
General properties | ||
Appearance | Visual evaluation | Off-white to beige homogeneous powder |
Water activity | ISO 21807 | ≤0.43 aw |
Loss on drying | USP <731> | ≤10.0% (w/w) |
Total protein | Ph Eur 2.5.33 | 25 - 35% (w/w) |
Total fat | Determined by extraction after hydrolysis/gravimetry[1] | 0 - 2% (w/w) |
Total ash | Ph Eur 2.4.16 | 17 - 21% (w/w) |
Total carbohydrate | By calculation[2] | 36 - 48% (w/w) |
Identity | ||
Sanger Sequencing (16S rRNA) | USP <1126> | 100% sequence identity to the NCBI sequence ID NR-042817 |
Assay | ||
Non-viable Akkermansia muciniphila cell count | Ph Eur 2.7.24[3] | 2.5 x 1010 - 2.5 x 1012 TFU/g (equating to 34 billion non-viable cells of pasteurised Akkermansia muciniphila) |
Viable Akkermansia muciniphila cell count
| Plated cell count method[4] | Not more than 10 CFU/g |
Impurities and incidental constituents | ||
Elemental impurities | ||
Arsenic | USP <2232> | Not more than 0.3 ppm |
Cadmium | USP <2232> | Not more than 1.0 ppm |
Mercury | USP <2232> | Not more than 0.1 ppm |
Lead | USP <2232> | Not more than 0.5 ppm |
Microbial contaminants | ||
Aerobic mesophilic total count | ISO 4833-1 | ≤500 CFU/g |
Sulphite reducing anaerobes | ISO 15213-1 | ≤100 CFU/g |
Coagulase-positive Staphylococci | ISO 6888-2 | ≤10 CFU/g |
Enterobacteriaceae | ISO 21528-2 | ≤10 CFU/g |
Bacillus cereus | ISO 7932 | ≤100 CFU/g |
Listeria | ISO 11290-1 | Absent in 25 g |
Salmonella | ISO 6579-1 | Absent in 25 g |
Escherichia coli | ISO 16649-1 | Absent in 1 g |
Yeasts and moulds | ISO 21527-2 | ≤10 CFU/g |
Footnotes
- The sample is weighed and then subjected to acid hydrolysis on filter. The filter is then placed in a cartridge and the cartridge is place in a pre-weighed beaker. The fat content is ether extracted and the extracted fat content is then weighed. The fat content is determined by the difference in mass before and after extraction.
- Total carbohydrate (%) = 100 – (Total protein (%) + Total fat (%) + Loss on Drying (%) + Total ash (%)).
- Inactivated bacteria were identified by staining with Propidium Iodide.
- Plating BHI-GlcNac-AB (L03W01). The viable count of Akkermansia muciniphila (CFU) is determined on agar plates consisting of BHI-based medium. The plates are incubated for 48 to 96 hours at 37°C in anaerobic boxes together with an anaerobic gas pack to ensure the anaerobic conditions, and an oxygen indicator to check for potential oxygen presence due to leakage.
Abbreviations
BHI = Brain Heart Infusion agar
CFU = Colony forming unit
ISO = International Organization for Standardization
ICP-MS = Inductively coupled plasma - Mass spectrometry
Ph Eur = European Pharmacopoeia
TFU = Total fluorescent unit
USP = United States Pharmacopoeia
Version | Description of change (for official use only) | Effective date |
|---|---|---|
V1.0 | Original |