INHIBITORS IN MILK: PROBLEMSand METHODS OF DETERMINATION

Joana ŠalomskienėKTU Food Institute, Kaunas, Lithuaniamikrobjs@lmai.lt, 8-37-312380

Inhibitors (antimicrobials, antibacterial substances) in dairy industry:

substances that inhibit growth of microorganisms:

a) substances of microbial origin –antibiotics;

b) substances of chemical origin –veterinary preparates (i.e. sulfa drugs),materials for washing and disinfection (biocides) and others.

Problems related with consumption of milk containing residues of inhibitorsn 10 % of people are sensitive or allergic to beta-

lactams. Residues of penicillin can be a reason of allergic illnesses;

n In the organism of man strains of pathogens resistant to antibiotics can appear;

n It is technologically impossible to produce cultured milk products of good quality when milk contains more than 0,005 IU/ml penicillin (growth of lactic acid bacteria is inhibited).

European legislation on Antibiotic Residues

The levels of residues in milk should not exceed the Maximum Residue Limits (MRLs) established by EU Regulation 2377/90:

n � 0,004 µg penicillin, amoxicillin or ampicillin in 1 ml;

n � 0,1 µg oxytetracycline or sulfamethazine in 1 ml;

n � 0,04 µg erythromycin in 1 ml;

n � 1,5 µg neomycin in 1 ml;

n � 0,2 µg streptomycin in 1 ml;

n � 0,05 µg trimethoprim in 1 ml.

Inhibitors in raw cow milk in Lithuania (in percent, from the number of investigated samples)

-0,3+0,50,40,30,5October

-0,70,70,81,21,0July

1,2

(March)

0,50,50,40,40,5April

0,90,40,40,30,40,4January

200920082007200620052004

The groups of methods used for determining the inhibitors in milk:

n microbiological, based on the growth or inhibition of sensitive test-organism (usually used for screening the samples);

n enzymatic, based on the interaction of enzymes with antibiotics;

n immunoassays or receptor assays,based on the immunoenzymatic reaction;

n physical methods (HPLC)(used for confirming the presence of antimicrobials)

Principle of the reference microbiological method in EU

Raw milk is added to the tube with a special medium including spores of Geobacillus stearothermophilus (bacteria sensitive to inhibitors) and indicator. Tubes are incubated at 64 oC for 3 hours.

No antimicrobials in milk sample or the concentration is lower then MRLs, spores germinate, grow and metabolize sugar. The acid produced changes the color of indicator.

Antimicrobials present in the milk sample, then germination and growth of spores is inhibited, color does not change.

The aim of our research

n To determine the different groups of inhibitors in raw milk;

n To make a comparison of different tests, long microbiological and rapid enzymatic and immuno/receptor tests.

Materials and methods

The experiments were conducted in the Microbiological laboratory of the Food Instutute of Kaunas University of Technology.

Objects of investigation:

1.Samples(10000) of raw milk from different regions of Lithuania delivered to the State Laboratory “Pieno tyrimai”;

2.Methods for determining of inhibitors in milk.

Scheme of determining groups of inhibitors in raw cow milk

Raw cow milk, 10 000 samples,

„Positive“ samples determined by analogue* of the reference method

Identification of „positive“ samples by microbiological methods**

Materials and methods

*Analogue of the reference methodThe agar-diffusion test with Geobacillus stearothermophilus was used for screening milk samples (i.e. selection of “positive” samples). Preparation (medium with spores of Geobacillus stearothermophilus and indicator dispensed into multiplates with 96 wells) was prepared according the SL “Pieno tyrimai” company’s standard ĮST 3381629-01-2000.

Materials and methods

**The modified EEC four-plate method was used for the detection of antimicrobials of different groups (penicillins, tetracyclines, sulphonamides, aminoglycosides and macrolides) in the samples of milk.

The method involves diffusion into agar: paper disks soaked in milk were placed on the surface of solid media (pH 6,0; pH 7,2 and pH 8,0) with Bacillus subtilis spores, and on the surface of solid medium (pH 8,0) with Micrococcus luteus. Merck media were used. The cultures, media and test plates were prepared according to R. Bogaerts and F. Wolf’s description.

Materials and methods

The plates with spores of B. subtilis BGA were incubated at 30 oC for 18-24 h and those with M. luteus ATCC 9341, at 37 oC for 18-24 h.

The presence of antimicrobials was indicated by the complete inhibition of growth on the surface of the agar around disks from a sample on one or more plates, in an annular zone not less than 2 mm wide.

Groups of inhibitors in raw cow milk in Lithuania

86 %

19 %

55 %

34 %

24 %

0

10

20

30

40

50

60

70

80

90

100

penicillins macrolides sulphonamides aminoglycosides other inhibitors

Groups of inhibitors

Num

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of sa

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ith d

iffere

nt in

hib

itors

,

% fro

m "

posi

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Microbiological tests for determining inhibitors in raw milkTest Producer Principle Scope

Delvotest SPanalogue ofreference method

DSM, Netherlands

Microbiological method with spores of G. stearothermophilus

Detection ofPenicillin G > 0,004±0,001 IU/ml

LPT,analog ue ofreference method

VĮ Pieno tyrimai,Lithuania

Detection ofPenicillin G 0,004 ± 0,001 IU/ml

MaI-1, analogue ofreference method

KTU Food Institute, Lithuania

Detection ofPenicillin G 0,004 ± 0,001 IU/ml

Copan,analogue ofReferencemethod

Christian Hansen, Italy

Detection ofPenicillin G 0,002±0,001 µg/mlIU/ml

Valio T-101 Valio Ltd, Finland

Microbiological method with S. thermophilus

Detection ofPenicillin G 0,004+0,001 IU/ml

Rapid tests for determining inhibitors in raw milk

Penicillin G 0,002 -0,004 µg/ml

Immunoenzymatic methodDSM, NetherlandsDelvo-X-PRESS

Penicillin G 0,003±0,001

TV/ml

Immuno/receptor assay

Nitrogen corporation,Belgium

β-STAR

Penicillin G 0,005-0,006

TV/mlEnzymatic method

Nitrogen corporation,Belgium

Penzym S

Penicillin G 0,004 µg/ml

Immuno/receptor assay

Charm Sciences, USAROSA

Penicillin G 0,004 µg/ml

Immunoenzymatic method

IDEXX Laboratories, Inc, USASNAP

ScopePrincipleProducerTest

Sensitivity of the tests to detergents

Concentration of the detergent, %

TestTAAB-1

0,2TAAB-2

1,0F18 Airol

1,0Ipa 300

0,6

PesettiANTIBACT

1,0

PesettiANTIBACT

5,0

LPT + +- - + +

MaI-1 + + - - + +

Copan ± ± - - + +

Valio T 101

+ + - - + +

PenzymS - + - - - -

β-STAR - - - - - -

SNAP - ± - - ± ±

ROSA test - - - - - +

Comparison of the reference method with other methods

Delvotest and MaI-1 -100 % (according the data of our investigations made before)

Coincidence of the results (+/+ ar -/-),n = 65 (raw milk samples investigated):

MaI-1 and Copan - 93,8 %;MaI-1 and Valio - 55,4 %;MaI-1 and Penzym S - 90,8 %;MaI-1 and beta-Star - 84,6 %;MaI-1 and SNAP - 98,5 %;MaI-1 and ROSA - 87,7 %.

Comparison of Delvo-X-PRESS with reference method and Penzym methods

---+122

+++-121

++--108-120

++-+103-107

----90-102

++++66-89

+-+65

+--41-64

---35-40

+++1-34

Penzym SPenzymDelvo-X-Press

Delvotest-SP-NT

(reference method)

Method

Sample

Comparison of Delvo-X-PRESS with reference method and Penzym methods

Delvo-X-PRESS and Delvotest-SP-N - 94,3 % Penzym and Delvotest-SP-NT - 73,7 % Penzym S and Delvotest-SP-NT - 68,0 % Penzym and Penzym S - 100,0 %Penzym and Delvo-X-PRESS - 68,4 %Penzym S and Delvo-X-PRESS - 64,8 %

Coincidence of the results (+/+ ar -/-), n=122 (samples of raw milk investigated)

Conclusions

n Penicillins were found in 86 % of the “positive”samples,sulphonamides, aminoglycosides and macrolides in 55, 34 and 19 % resp. 24 % of antimicrobials were not identified;

n Microbiological methods detected 4 from 6 detergentsinvestigated when rapid tests detected 1-2 detergents;

n All analogues of reference method (Delvotest, LPT, MaI-1, Copan) were equivalent according their sensitivity;

n Delvo-X-PRESS method showed high coincidence with a reference method and could be proposed to include it into “The amendment of Instruction for evaluation of the primary characteristics of raw milk quality”.

Contribution of KTU Food Institute to the solution of the problem of inhibitors in milk (1)

• Three company’s standards were prepared:1. Geobacillus stearothermophilus spores

suspension for inhibitor tests (ĮST 1195612-99:2000);

2. LPT test (multiplate test for 96 samples,ĮST 3381629-01:2000), used in State Laboratory “Pieno tyrimai”;

3. MaI-1 test (for individual sample analysis,ĮST 1195612-102:2001);

• Main groups of inhibitors determined in raw cow milk in Lithuania;

Contribution of KTU Food Institute to the solution of the problem of inhibitors in milk (2)

• Evaluation of microbiological and rapid tests in 2004 and 2006 was made and the recomendations for dairy plants were prepared;

• The second renewed edition of the “Instructions for microbiological control in dairy plants” was prepared in 2004. It includes 10 descriptions of inhibitor tests;

• Amendment of “Instructions for evaluation of the primary characteristics of raw milk quality” was prepared in 2006.

Problems

1. Antimicrobials in „positive“ samples are not identified in Lithuania;

2. Residues of chloramphenicol in milkChloramphenicol prevention program is performed by veterinary

service (milk from selected farms is investigated):a) by ELISA method (sensitivity - 0,00005 mg chloramphenicol/g milk) –

in SL “Pieno tyrimai”;b) confirmation by chromatographic method (approx. 90 % samples

are confirmed);

3. Residues of tetracycline – rapid Charm test for detection of tetracycline is used.

Thank you for your attention!