Determination of the antimicrobial activity of the amino acid L-isoleucine

Metex Noovistago (MNG) would like to examine the antimicrobial activity of one of their amino acids, Lisoleucine, using the 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 L-isoleucine were tested for their antimicrobial activity using the two-fold dilution method in broth, following the recommendation in the referred EFSA Technical Guidance. For the antimicrobial activity tests, five different microorganisms were used to determine the antimicrobial activity of the amino acid. After incubation the minimum inhibitory activity was determined. The determination of the antimicrobial activity was carried out by a third party - Nutricontrol. The three batches of the amino acid L-isoleucine were shipped from WFBR location to the third party (Nutricontrol). The batch numbers were 210912, 210923 and 210924. Broth micro-or macro-dilution is one of the most basic antimicrobial susceptibility testing methods. The procedure is described below to execute the experimental protocol determining their minimum inhibitory activity. The procedure involves preparing two-fold dilutions of the antimicrobial agent in a liquid growth medium dispensed in a tube. The test material (diluted with aqueous solution 1/10) was incubated for 30 minutes at 90°C. After cooling, a pH correction and filtration, the analysis in the aqueous BPW (buffered peptone water) solution 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 concentration tested was 9.1% (start dilution was 30 gram product + 300 ml aqueous solution). Based on the above described experiments, the following results were observed: • After 24 hours of incubation at 37°C, no inhibition of micro-organisms was observed. • After 48 hours of incubation at 37°C, no inhibition of micro-organisms was observed. Conclusion: 9.1% or lower concentrations of the tested amino acid L-isoleucine did not show any antimicrobial activity in these experiments, which were designed according to the EFSA guideline ‘Technical Guidance, Microbial Studies, Prepared by the Panel on Additives and Products or Substances used in Animal Feed’.