Determination of the antimicrobial activity of amino acid zootechnical mix: Three different batches examined

Metex-Noovistago (MNG) would like to examine the antimicrobial activity of a new product using requirements from EFSA “Technical Guidance, Microbial Studies, Prepared by the Panel on Additives and Products or Substances used in Animal Feed”, EFSA-Q-2008-461, adopted on 21 October 2008. To this aim three different samples of the Amino acid zootechnical mix were tested for their antimicrobial activity using the two-fold dilution method in broth following the recommendation broth in the referred EFSA Technical Guidance. For the antimicrobial activity tests, five different microorganisms were used to determine the antimicrobial activity of the mixture. The minimum inhibitory activity was determined by the third party - Nutricontrol. The three batches of the amino acid zootechnical mix were shipped from WFBR location to the third party (Nutricontrol) and handled as follows to execute the experimental protocol determining their antimicrobial activity: Broth micro-or macro-dilution is one of the most basic antimicrobial susceptibility testing methods. The procedure involves preparing two-fold dilutions of the antimicrobial agent in a liquid growth medium dispensed in a tube. The test material was incubated during 30 minutes at 90°C. After cooling, a pH correction and filtration, the analysis in aqueous BPW extract was carried out. Products were diluted in duplicate per bacteria culture and subsequently incubated under aerobic conditions at 37°C. The test tubes were visually judged after 24 hours and after 48 hours of incubation. The highest dilution used was: 30 gram product + 300 ml aqueous solution (BPW was used), i.e. 9.1%. this was followed by two-fold dilutions. Based on the above described experiments the following results were observed: For the batches AA Mix#1 and AA Mix#2, after 24 hours of incubation at 37°C, there was no antimicrobial effect observed by any concentration tested, and no MIC value could be derived. For AA Mix#3 a MIC value of ≥ 9.1% was observed providing inhibition to the growth of B. subtilis ATCC 6633 in one of the duplicate tubes. This was not observed for the other cultures E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and E. faecalis ATCC 29212. After 48 hours of incubation at 37°C, no antimicrobial effect was detected and no MIC value for the samples of batch AA Mix#1, batch AA Mix#2 and batch AA Mix#3 could be derived.