Since there is no internationally recognized standard available for the test, EFSA guidance is followed.
The inhibitory activity of culture supernatants is assessed against reference strains susceptible to a range of antimicrobials, or against other reference strains.
(e.g. Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633)
We recommend that the supernatants are sampled from authentic production conditions to better reflect the metabolism of the microorganism at the time of harvesting. The sample can be filtered to remove bacterial cells from the supernatant.
Agar well diffusion and agar disc diffusion methods (FAO, 2006) are commonly used for the assessment of antimicrobial activity; Biosafe Ltd. uses the agar disc diffusion method. Positive controls (antimicrobial) and negative controls (solvents, growth medium) are needed to verify the validity of the results.
If the test microorganism produces antimicrobial(s), the substance(s) should be identified. Furthermore, the absence of carry-over into the final product should be demonstrated using a sensitive method.