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Compositional guideline: Streptococcus salivarius K12

27 June 2018

Name of the ingredient

Streptococcus salivarius

Definition of the ingredient

Streptococcus salivarius is a gram positive bacterium found as part of the normal human oral microflora, predominantly colonising the tongue. Streptococcus salivarius K12 (isolated from the saliva of a healthy child) is a probiotic intended for use in oral cavity. The ingredient is prepared by fermentation of culture of Streptococcus salivarius K12. The cell concentrate is mixed with lyoprotectant, freeze-dried and milled into powder. The quality and safety of the probiotic Streptococcus salivarius was assessed for the strain K12 and the specific requirements listed below are strain specific.

Table 1. Ingredient specific requirements
Test Method reference Acceptance criteria
Description
Appearance Visual evaluation Free flowing off-white powder
Odour/taste Organoleptic evaluation Proteinaceous taste
Characteristics
Particle size Sieve analysis 90% <500µm
Water activity (aw) USP <1112>[1] < 0.25
Identification
Microscopic morphology USP <1113> Gram staining[2]

Gram reaction: positive

Cellular shape: cocci in chains or pairs

Macroscopic morphology Visual examination of growth on Mitis salivarius agar at 37°C in 5% CO2 in air after 24-48 hr

Colony size: 1-2 mm in diameter

Colony shape:

  • Form: round
  • Margin: entire
  • Elevation: convex

Colony colour: blue

Colony texture: mucoid

Visual examination of growth on Blood agar [Columbia Agar Base with 5% human blood] at 37°C in 5% CO2 in air after 24-48 hr

Colony size: <1 mm in diameter

Colony shape:

  • Form: round
  • Margin: entire
  • Elevation: convex

Colony Colour: white

Colony Texture: butyrous

Haemolysis: None

Biochemical profile

USP <1113> biochemical tests[2]

And/Or

Automated microbial identification test (e.g. API 20 Strep test system)

Negative for: catalase

Positive for: acetoin production, β-glucosidase, alkaline phosphatase, leucine aminopeptidase, D-lactose, D-trehalose, inulin, and D-raffinose.

Molecular identification of strain Barretto, Alvarez-Martin et al. 2012[3] Matches the sequence for S. salivarius K12
Deferred antagonism, P-typing producer Wescombe et al. 2006[4] Matches the profile for S. salivarius K12
Antibiotic susceptibility profile

Clinical and Laboratory Standards Institute (CLSI) methods[5]

Or

Automated antimicrobial susceptibility testing system (e.g. Vitek 2 AST)

Susceptible to: penicillin, amoxicillin, tetracycline and erythromycin
Streptococcal virulence genes Burton et al. 2006[6] Absence of representative streptococcal virulence genes: emm, scpA, speB, smez-2, and sagA
Assay
Streptococcus salivarius K12 enumeration Viable plate count on CAB K12 agar cultured at 37°C in 5% CO2 in air after 24-48 hr per Ishijama 2012[7], Burton et al. 2011[8] > 1 x 1011CFU/g

Incidental metals and non-metals

While ingredient manufacturers are encouraged to include limits for incidental metals and non-metals, it is the product into which those substances are formulated that contains the ingredient, alone or in combination with other ingredients, which must comply with the acceptance criteria set in the United States Pharmacopoeia - National Formulary (USP-NF) general chapter '<2232> Elemental Contaminants in Dietary Supplements'.

Microbiology

While substance manufacturers are encouraged to include limits for objectionable microorganisms, it is the product into which those substances are formulated that is subject to a legally binding set of criteria. The Therapeutic Goods Order No. 77 'Microbiological Standards for Medicines' mandates that any finished product that contains the ingredient, alone or in combination with other ingredients, must comply with the microbial acceptance criteria set by Clause 9 of the Order.

Key to abbreviations

BP = British Pharmacopoeia

Ph. Eur. = European Pharmacopoeia

USP = United States Pharmacopoeia

CFU = Colony forming units


Footnotes

  1. United States Pharmacopoeia - National Formulary (USP-NF) General Chapter <1112>: APPLICATION OF WATER ACTIVITY DETERMINATION TO NONSTERILE PHARMACEUTICAL PRODUCTS.
  2. United States Pharmacopoeia - National Formulary (USP-NF) General Chapter <1113>: MICROBIAL CHARACTERIZATION, IDENTIFICATION, AND STRAIN TYPING.
  3. Baretto, C. & Alvarez-Martin, P. (2012). Genome sequence of the lantibiotic bacteriocin producer Streprococcus salivarius strain K12. J Bacteriol, 194(21), pp. 5959-5960.
  4. Wescombe et al, AEM, (2006) p. 1459-1466. Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva
  5. CLSI. Performance Standards for Antimicrobial disk Susceptibility Tests. 13th ed. CLSI standard M02. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
  6. Burton, J. P., Wescombe, P. A., Moore, C. J., & Chilcott, C. N. (2006). Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol., 72(4), 3050-3053.
  7. Ishijima, S.A., Hayama, K., Burton, J.P., Reid, G., Okada, M., Matsushita, Y., Abea, S. (2012) Effect of Streptococcus salivarius K12 on the In Vitro Growth of Candida albicans and Its Protective Effect in an Oral Candidiasis Model. Appl Environ Microbiol. 78(7):2190 - 9
  8. Burton, J.P., Cowley, S., Simon, R. R., McKinney, J., Wescombe,P.A., Tagg J.R (2011) Evaluation of safety and human tolerance of the oral probiotic Streptococcus salivarius K12: A randomized, placebo-controlled, double-blind study. Food and Chemical Toxicology.