INHIBITORS IN MILK: PROBLEMS and METHODS OF DETERMINATION. Joana Šalomskienė KTU Food Institute, Kaunas, Lithuania

Transcription

1 INHIBITORS IN MILK: PROBLEMS ad METHODS OF DETERMINATION Joaa Šalomskieė KTU Food Istitute, Kauas, Lithuaia

2 Ihibitors (atimicrobials, atibacterial substaces) i dairy idustry: substaces that ihibit growth of microorgaisms: a) substaces of microbial origi atibiotics; b) substaces of chemical origi veteriary preparates (i.e. sulfa drugs), materials for washig ad disifectio (biocides) ad others.

3 Problems related with cosumptio of milk cotaiig residues of ihibitors 10 % of people are sesitive or allergic to betalactams. Residues of peicilli ca be a reaso of allergic illesses; I the orgaism of ma strais of pathoges resistat to atibiotics ca appear; It is techologically impossible to produce cultured milk products of good quality whe milk cotais more tha 0,005 IU/ml peicilli (growth of lactic acid bacteria is ihibited).

4 Europea legislatio o Atibiotic Residues The levels of residues i milk should ot exceed the Maximum Residue Limits (MRLs) established by EU Regulatio 2377/90: 0,004 µg peicilli, amoxicilli or ampicilli i 1 ml; 0,1 µg oxytetracyclie or sulfamethazie i 1 ml; 0,04 µg erythromyci i 1 ml; 1,5 µg eomyci i 1 ml; 0,2 µg streptomyci i 1 ml; 0,05 µg trimethoprim i 1 ml.

5 Ihibitors i raw cow milk i Lithuaia (i percet, from the umber of ivestigated samples) Jauary 0,4 0,4 0,3 0,4 0,4 0,9 April 0,5 0,4 0,4 0,5 0,5 1,2 (March) July 1,0 1,2 0,8 0,7 0,7 October 0,5 0,3 0,4 0,5 0,3

6 The groups of methods used for determiig the ihibitors i milk: microbiological, based o the growth or ihibitio of sesitive testorgaism (usually used for screeig the samples); ezymatic, based o the iteractio of ezymes with atibiotics; immuoassays or receptor assays, based o the immuoezymatic reactio; physical methods (HPLC)(used for cofirmig the presece of atimicrobials)

7 Priciple of the referece microbiological method i EU Raw milk is added to the tube with a special medium icludig spores of Geobacillus stearothermophilus (bacteria sesitive to ihibitors) ad idicator. Tubes are icubated at 64 oc for 3 hours. No atimicrobials i milk sample or the cocetratio is lower the MRLs, spores germiate, grow ad metabolize sugar. The acid produced chages the color of idicator. Atimicrobials preset i the milk sample, the germiatio ad growth of spores is ihibited, color does ot chage.

8 The aim of our research To determie the differet groups of ihibitors i raw milk; To make a compariso of differet tests, log microbiological ad rapid ezymatic ad immuo/receptor tests.

9 Materials ad methods The experimets were coducted i the Microbiological laboratory of the Food Istutute of Kauas Uiversity of Techology. Objects of ivestigatio: 1.Samples(10000) of raw milk from differet regios of Lithuaia delivered to the State Laboratory Pieo tyrimai ; 2.Methods for determiig of ihibitors i milk.

10 Scheme of determiig groups of ihibitors i raw cow milk Raw cow milk, samples, Positive samples determied by aalogue* of the referece method Idetificatio of positive samples by microbiological methods**

11 Materials ad methods *Aalogue of the referece method The agardiffusio test with Geobacillus stearothermophilus was used for screeig milk samples (i.e. selectio of positive samples). Preparatio (medium with spores of Geobacillus stearothermophilus ad idicator dispesed ito multiplates with 96 wells) was prepared accordig the SL Pieo tyrimai compay s stadard ĮST

12 Materials ad methods **The modified EEC fourplate method was used for the detectio of atimicrobials of differet groups (peicillis, tetracyclies, sulphoamides, amioglycosides ad macrolides) i the samples of milk. The method ivolves diffusio ito agar: paper disks soaked i milk were placed o the surface of solid media (ph 6,0; ph 7,2 ad ph 8,0) with Bacillus subtilis spores, ad o the surface of solid medium (ph 8,0) with Micrococcus luteus. Merck media were used. The cultures, media ad test plates were prepared accordig to R. Bogaerts ad F. Wolf s descriptio.

13 Materials ad methods The plates with spores of B. subtilis BGA were icubated at 30 oc for 1824 h ad those with M. luteus ATCC 9341, at 37 oc for 1824 h. The presece of atimicrobials was idicated by the complete ihibitio of growth o the surface of the agar aroud disks from a sample o oe or more plates, i a aular zoe ot less tha 2 mm wide.

14 Groups of ihibitors i raw cow milk i Lithuaia Number of samples with differet ihibitors, % from "positive samples" % % % % % peicillis macrolides sulphoamides Groups of ihibitors amioglycosides other ihibitors

15 Microbiological tests for determiig ihibitors i raw milk Test Delvotest SP aalogue of referece method Producer DSM, Netherlads Priciple Microbiological method with spores of G. stearothermophilus Scope Detectio of Peicilli G > 0,004±0,001 IU/ml LPT, aalog ue of referece method MaI1, aalogue of referece method Copa, aalogue of Referece method VĮ Pieo tyrimai, Lithuaia Detectio of Peicilli G 0,004 ± 0,001 IU/ml KTU Food Istitute, Lithuaia Detectio of Peicilli G 0,004 ± 0,001 IU/ml Valio T101 Valio Ltd, Filad Christia Hase, Italy Detectio of Peicilli G 0,002±0,001 µg/ml IU/ml Microbiological method with S. thermophilus Detectio of Peicilli G 0,0040,001 IU/ml

16 Rapid tests for determiig ihibitors i raw milk Test Producer Priciple Scope SNAP IDEXX Laboratories, Ic, USA Immuoezymatic method Peicilli G 0,004 µg/ml ROSA Charm Scieces, USA Immuo/receptor assay Peicilli G 0,004 µg/ml Ezymatic method Peicilli G 0,0050,006 TV/ml Pezym S Nitroge corporatio, Belgium βstar Nitroge corporatio, Belgium DelvoXPRESS DSM, Netherlads Immuo/receptor assay Immuoezymatic method Peicilli G 0,003±0,001 TV/ml Peicilli G 0,002 0,004 µg/ml

17 Sesitivity of the tests to detergets Cocetratio of the deterget, % Test F18 Airol 1,0 Ipa 300 0,6 Pesetti ANTIBACT 1,0 Pesetti ANTIBACT 5,0 ± ± Valio T 101 PezymS βstar SNAP ± ± ± ROSA test TAAB1 0,2 TAAB2 1,0 LPT MaI1 Copa

18 Compariso of the referece method with other methods Delvotest ad MaI1 100 % (accordig the data of our ivestigatios made before) Coicidece of the results (/ ar /), = 65 (raw milk samples ivestigated): MaI1 ad Copa MaI1 ad Valio MaI1 ad Pezym S MaI1 ad betastar MaI1 ad SNAP MaI1 ad ROSA 93,8 %; 55,4 %; 90,8 %; 84,6 %; 98,5 %; 87,7 %.

19 Compariso of DelvoXPRESS with referece method ad Pezym methods Method DelvotestSPNT (referece method) DelvoXPress Sample Pezym Pezym S

20 Compariso of DelvoXPRESS with referece method ad Pezym methods Coicidece of the results (/ ar /), =122 (samples of raw milk ivestigated) DelvoXPRESS ad DelvotestSPN 94,3 % Pezym ad DelvotestSPNT 73,7 % Pezym S ad DelvotestSPNT 68,0 % Pezym ad Pezym S 100,0 % Pezym ad DelvoXPRESS 68,4 % Pezym S ad DelvoXPRESS 64,8 %

21 Coclusios Peicillis were foud i 86 % of the positive samples, sulphoamides, amioglycosides ad macrolides i 55, 34 ad 19 % resp. 24 % of atimicrobials were ot idetified; Microbiological methods detected 4 from 6 detergets ivestigated whe rapid tests detected 12 detergets; All aalogues of referece method (Delvotest, LPT, MaI1, Copa) were equivalet accordig their sesitivity; DelvoXPRESS method showed high coicidece with a referece method ad could be proposed to iclude it ito The amedmet of Istructio for evaluatio of the primary characteristics of raw milk quality.

22 Cotributio of KTU Food Istitute to the solutio of the problem of ihibitors i milk (1) Three compay s stadards were prepared: 1. Geobacillus stearothermophilus spores suspesio for ihibitor tests (ĮST :2000); 2. LPT test (multiplate test for 96 samples, ĮST :2000), used i State Laboratory Pieo tyrimai ; 3. MaI1 test (for idividual sample aalysis, ĮST :2001); Mai groups of ihibitors determied i raw cow milk i Lithuaia;

23 Cotributio of KTU Food Istitute to the solutio of the problem of ihibitors i milk (2) Evaluatio of microbiological ad rapid tests i 2004 ad 2006 was made ad the recomedatios for dairy plats were prepared; The secod reewed editio of the Istructios for microbiological cotrol i dairy plats was prepared i It icludes 10 descriptios of ihibitor tests; Amedmet of Istructios for evaluatio of the primary characteristics of raw milk quality was prepared i 2006.

24 Problems 1. Atimicrobials i positive samples are ot idetified i Lithuaia; 2. Residues of chlorampheicol i milk Chlorampheicol prevetio program is performed by veteriary service (milk from selected farms is ivestigated): a) by ELISA method (sesitivity 0,00005 mg chlorampheicol/g milk) i SL Pieo tyrimai ; b) cofirmatio by chromatographic method (approx. 90 % samples are cofirmed); 3. Residues of tetracyclie rapid Charm test for detectio of tetracyclie is used.

25 Thak you for your attetio!