Evaluation of sensitivity of modified star protocol microbiological method for beta-lactame antibiotics detection in raw cow milk

Antibiotic residues when present in animal tissues, through food chain, can enter human body, causing allergic reactions or facilitating the development of resistant bacterial strains. In order to determine the presence of antibiotics in animal tissues, it is appropriate to use convenient, reliable and sensitive methods. Microbiological methods applied for the detection of antibiotic residues in primary products of animal origin are based on the sensitivity of specific bacterial strains to a particular group of antibiotics. Regulatives on the amount of pesticides, metals and metalloids and other toxic substances, chemotherapeutics, anabolics and other substances which can be found in food ("Off. Gazette", No. 5/92, 11/92 - corr. and 32/02), state that milk and milk products can be used in commercial purposes only if not contain antibiotics in quantities that can be detected by reference methods. The applied method is modified STAR (Screening test for detection of antibiotics) protocol, regulated by the CRL (Community Reference Laboratory) Fougeres, France, in which the initial validation of the method had been carried out. In accordance with the demands of Regulative Commission EC No657/2002, the sensitivity of modified STAR protocol for beta lactam antibiotics group was examined , that is, there was carried out a contracted validation of the method, which initial validation had been performed at CRL. In a couple of series of experiments, 20 blank samples of raw cow milk originating from animals not treated by antibiotics, had been examined. By the beginning of the experiment samples were stored in a freezer at -20?C. Samples of raw cow milk enriched by working solutions of seven beta-lactam antibiotics, in order to obtain concentrations at the level of 0.5, 1 and 1.5 MRL (Maximmum Residue Limit) for each given antibiotic (Commission Regulation EC No. 37/2010). For detection of beta-lactam antibiotics, there was used Kundrat agar test with previously inoculated G.stearothermophilus ATCC 10149 strain. Aliquots of 30 _l of working solution at 0.5, 1 and 1.5 MRL concentration level, for each antibiotic, were inflicted on two paper disks placed on inoculated Kundrat agar surface. Petri plates with Kundrat agar previously inoculated with G.stearothermophilus , on which the samples were deposited, were incubated for 12-15h at 55oC. The obtained width of microorganisms growth inhibition zone, that is supposed to be at least 2.0 mm, measured from the disc edge, demonstrated the capability to detect all the tested 7 antibiotics from the beta lactam group at a level below the MRLs. Consequently, this proves that use of this method it is possible to meet the demands of Regulative Commission EC No. 37/2010.