MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Techniques for microbiological analyses

Lesson 30
MONITORING ANTIBIOTIC RESIDUES AND AFLATOXIN M1

30.1 Introduction

Antibiotics are extensively used in dairy cattle management for preventing and curing disease like mastitis, brucellosis etc. Based on chemical structure, the most commonly used antibiotics in dairy animals are β-lactam, tetracycline, amino glycoside, sulfonamide and macrolides. Among these, β-lactam antibiotic is one of the major groups of antibiotics used for treatment of dairy cattle. The substantial excretion of these residues in milk is attributed to indiscriminate use of antibiotics, lack of medication records, use of unapproved drugs, contaminated milking equipment, purchase of treated cows, failure to observe withdrawal period in lactation animals. These residues in milk are allergic, carcinogenic and cause development of antibiotic resistant pathogenic strains .The presence of antibiotics residues in milk supply can have adverse affects during processing of dairy foods in terms of starter failure, poor ripening of cheese and efficiency of dye reduction test. Maximum residual limit (MRL) has been recommended for a number of anti-microbial agents for their compliance in milk (Table 30.1 & 30.2).

Table 30.1 List of antibiotics used in dairy husbandry

Table 30.2 Maximum residual limit (MRL) recommended for milk

30.2 Detection Methods for Antibiotics and Drug Residues in Milk

Various rapid antibiotic detection methods have been commercialized in last two decades. Currently, seven types of detection methods are commonly used for detection of antibiotic residues in milk i.e. microbial growth inhibitor assay, microbial receptor assay, enzyme-colorimetric assay, receptor binding assay, spectrophotometer assay, chromatographic methods and immunoassay. These methods are qualitative, quantitative or semi-quantitative. However, they have one or more limitations in terms of precision, accuracy, sensitivity, cost and infrastructural requirements. However, microbial inhibitor & immuno-receptor based tests have gained most popularity for dairy application globally.

30.2.1 Reference method

The EU reference method for the determination of antibiotic residues in raw milk and in heat-treated milk is the International Dairy Federation (IDF, 1991) microbial inhibition test. The IDF microbial inhibitor test uses B.stearothermophilus var. calidolactis, ATCC 10149 as the test organism due to its relatively high sensitivity to inhibitory substances. The IDF test procedure has been chosen as representative of similar procedures which in principle use B. stearothermophilus as the test organism. However, it is quite complex and lengthy to carry out as it involves the continual growth of large quantities of B. stearothermophilus spores. As the test involves a colour change, which is dependent on the growth of B. stearothermophilus, if the organism fails to grow then a false negative result may occur. Therefore, due to the afore-mentioned technical difficulties in carrying out the reference method, microbial inhibitor test kit assays based on the IDF method are the routine methods used for the determination of antibiotic residues in milk.

30.2.2 Microbial inhibitor test

The 'traditional' tests known as 'microbial inhibitor' tests, involve incubating a susceptible organism in the presence of the milk sample. In the absence of an antibiotic, the organism grows and can be detected visually either by opacity of the agar growth medium or by a color change resulting from acid production. In the presence of an antibiotic, or any other inhibitor, the organism fails to grow and a zone of inhibition or lack of a color change is observed (Fig.30.1: antibiotic detection). Such tests are exceptionally sensitive to ß-lactam antibiotics. They are generally reliable and cost-effective but require incubation for several hours before the result can be visualized.

30.2.2.1 Delvotest SP

The Delvotest developed by Gist-brocades BV, The Netherlands is the best known microbial inhibitor test. Its first version was developed, in the 1970s as Delvotest P, to detect ß-lactams. The target organism, B. stearothermophilus, is encapsulated in an agar medium containing a pH indicator, a nutrient tablet and milk sample both being dispensed onto the agar surface. The 'ampoule version' is designed for individual tests or small-scale testing whilst a micro-tire plate version is designed for mass testing where 96 tests can be undertaken simultaneously. A negative result is indicated by a color change from purple to yellow, due to acid development during incubation at 64°C for 2½ hours.

The Delvotest P has been used throughout the world and has sensitivity to penicillin G of 0.005 IU/ml. A more recent development, the Delvotest SP, is capable of detecting a wider spectrum of substances, notably sulphonamides, but also has increased sensitivity to tylosin, erythromycin, neomycin, gentamicin, trimethoprim and other antimicrobials. The Delvotest SP appears identical to the Delvotest P, the only difference being the need to incubate the Delvotest SP for 2¾ hours. The Delvotest SP is sold throughout the world and, universally, has sensitivity to penicillin G of 0.003-0.004 IU/ml.

Test procedure

The growth of B. stearothermophilus spores at 64°C initiates an acidification process which causes the turning of a pH indicator from purple to yellow. The presence of antibacterial substances will cause delay or inhibition of the spores, depending on the concentration of the residues. In the presence of residues the spores will not multiply and the pH indicator will remain purple. Following steps are involved in procedure:

1. Add 1 nutrient tablet to each of the agar wells in the strip.
2. Inoculate 100 μl of milk into the agar well plus nutrient tablet.
3. Seal the wells for incubation
4. Incubate the strip of wells in a water bath at 64°C ± 0.5°C for 2.50 hours (at the time the negative control has been changed to yellow)
5. Examine the strip for colour change from purple to yellow. A yellow reading indicates that no inhibitory substances are present; a purple reading indicates that antibiotic residues are present and a yellow/purple reading indicates a doubtful result.

30.2.2.2 Copen test

The Copan test is also based on the IDF standard method for determination of antibiotic residues in milk. This method is very similar to Delvotest SP, however,in this test the nutrient tablet is already added to the agar medium .

30.2.2.3 Charm farm test

The Charm Farm test is a microbial inhibition test which uses a one step single service vial. It is a broad screening assay for five families of veterinary drugs, including beta-lactams, sulphonamides, tetracyclines, aminoglycosides and macrolides in raw, commingled, bovine milk. The results are stable upto 8 hours after assay completion and can be read by visual colour comparison or optionally with a pH meter. The Charm Auto-Farm Equipment is required to run this test. The test can be completed in approximately 3.5 hours. Up to 12 tests can be run simultaneously.

30.2.2.4 Charm AIM-96

The Charm AIM-96 Test is designed for high volume, broad spectrum screening of raw, pasteurised, and homogenised or skim milk. The results can be read by visual colour comparison or optionally with a microplate reader. The Charm AIM-96 detects beta-lactams, sulphonamides, tetracyclines, aminoglycosides and macrolides. 96 tests can be completed simultaneously in approximately 4 hours.

30.2.2.5 MDR test

An analytical process which involves sporulation & activation of dormant spores of B. stearothermophilus in newly developed medium & their germination/ outgrowth in presence of selective germinant mixture has been developed and is available commercially as microbial drug residue (MDR) test in India ( & ). The validated process in line with AOAC approved charm 6602 assay and can be used effectively for semi-quantitative detection of antibiotic residues in different types of milk with results within 2.30-3.0 hours at MRL/ or above levels as recommended by the codex.

In addition, there are several other microbial inhibitor tests, produced by several companies. These include the Brilliant Black Reduction Test, the Valio T101 test, the Copan microbial inhibitor test, the Lumac rapid antibiotic test and the Biosys bioluminescence method. Microbial inhibitor tests are cheap and easy to perform, however, there are some limitations in the sense that they need longer incubation period and are not specific for antibiotics. There are occasional reports of positive reactions associated with other inhibitors such as lactoferrin, lysozyme or sanitizers.

30.2.3 Immune-receptor test

The desire for a more rapid and reliable result has promoted the development of tests that employ the 'immune receptor' test principle, which is a variation of the well-established enzyme-linked immunosorbent assay (ELISA). Essentially, a specific target antibiotic group is captured by immobilized antibodies, or by a broader-spectrum receptor such as a bacterial cell. Most tests involve a competitive principle in which antibiotic in the sample competes with an internal antibiotic standard for the immune receptor. The antibody-antibiotic complex is then usually linked to an enzyme that catalyses a color or fluorescence reaction and a comparison of the intensity of the 'test' reaction with that of a 'control' determines whether the sample is positive or negative. Because of their competitive principle, a low intensity usually means 'positive' whilst a high intensity is regarded as 'negative'. Immune receptor tests can be made quantitative but are generally used to provide a 'pass/fail' result. They are generally more expensive than microbial inhibitor tests but only detect substances that react immunologic ally with the immobilized receptor and they provide a result in less than 10 min.

30.2.3.1 Commercially available immuno-receptor test

The commercially available immune receptor tests employ several variations of capture mechanism and color reaction but most possess the common features of an immunological reaction coupled with a change in color (or fluorescence). There are, however, two exceptions. The Penzym test (UCB Byproducts, Belgium) employs the inhibition of an enzyme reaction (DD-carboxypeptidases activity), instead of an immune reaction, to detect the presence of a ß-lactam and it visualizes this by a color change. The test produces pink color when a sample contains no antibiotics while a yellow color is interpreted as positive. Conversely, the Charm II assay (Charm Sciences Inc., USA) employs an immune reaction to bind the antibiotic to a microbial receptor but detects this complex using a low-level ³H or ¹⁴C radio-label, instead of an enzyme reaction.

The Charm II assay (Charm Sciences Inc., USA) is not a single test but a family of separate tests for specific groups of antibiotics, notably ß-lactams, sulphonamides, tetracycline, novobiocin, amino glycosides and macrolides, as well as various other substances such as chloramphenicol. The Charm II assay is an immune receptor test but is suitable for large laboratories only, requiring a range of laboratory equipment, including a centrifuge and sample mixers to prepare samples as well as a scintillation counter to detect the radio-label. Calibration curves need to be prepared for each group of antibiotics and a 'negative control' sample must be tested each day. The charm β-lactam test uses bacteria with specific receptor sites that bind all β-lactam drugs. The bacteria are added to a milk sample along with an exempt amount of [¹⁴C] labeled penicillin G. Any β-lactam already in milk competes for the binding sites with the labeled penicillin G. The amount of [¹⁴C]-penicillin G that binds to the receptor sites is measured compared to a previously determined control point or to a standard curve. The greater the amount of [¹⁴C]-penicillin G measured, the lower the β-lactam concentration in the sample ( & Fig. 30.3 B : Procedure of Charm II Assay).

30.2.4 Novel iodometric test

The developed test is working on principle of spore germination and induction of β-lactamase enzyme in presence of inducer i.e. specific β-lactam group in milk. In case when specific group i.e. β-lactam is absent, the induction of β-lactamase enzyme during germination as well as production of penicilloic acid as a result of enzymatic action will be minimal resulting in no color change of starch & iodine mixture (Animation 5). However, in presence of inducer there will be reduction of starch iodine mixture as a result of significant induction of marker enzyme and production penicilloic acid (Fig. 30.4). The change in color of starch iodine mixture from blue to colourless will indicate the presence of β-lactam group in milk when incubated at 35 ± 2^oC for 15-20 minutes.

Fig. 30.4 Detection of β-Lactam group in milk based on induction principle

30.2.4.1 Novel features

1. Real time test (Result within 15-20min)
2. Cost effective
3. Semi-quantitative detection at Codex MRL
4. No cross reactivity with non β–Lactam Groups
5. Validated with AOAC approved charm 6602 assay
6. Stability of test kit Up to 7-8 months under refrigeration storage
7. Wide spectrum of application with raw, pasteurized and dried milks.

30.3 Test Methods for Aflatoxin M1 in Milk

Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds. They have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans throughout the world. Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. Chemically they are defined as di-furano-cyclo-pentano-cumarines or di-furano-pentano-lido-cumarines, i.e. aflatoxins contain a dihydrofuran or a tetrahydrofuran ring, to which a substituted cumarin system is condensed. Out of about 20 known aflatoxins, the moulds Aspergillus flavus and A. parasiticus produce exclusively aflatoxin B1, B2, G1 and G2, and all the other aflatoxins are derivates of these four aflatoxins.

Aflatoxin M1 was the first metabolite of Aflatoxin B1, which could unequivocally be detected in the milk. Out of this reason this first derivative was called Aflatoxin M1 (milk). When cows are fed contaminated feed, aflatoxin B1 is converted by hydroxylation to aflatoxin M1, which is subsequently secreted in the milk of lactating cows. Aflatoxin M1 is quite stable towards the normal milk processing methods such as pasteurization and if present in raw milk, it may persist into final products for human consumption. Most controlling government agencies worldwide have regulations regarding the amount of aflatoxins allowable in human and animal foodstuffs. Many countries have declared limits for the presence of aflatoxin M1 in milk and milk products. In the codex the limit for the presence of M1 in milk is set at 0.5 µg/ L or 0.5 parts per billion (ppb).

30.3.1 Source of aflatoxins in milk

Aflatoxin M1 contamination in milk results primarily from the conversion of aflatoxin B1 that is metabolized by enzymes found primarily in the liver. After aflatoxin M1 is formed, it is excreted in the urine and milk of the cow. The action level for afla-toxin B1 is 20 parts per billion (ppb) for feed fed to lactating dairy cows. As both aflatoxins B1 and M1 may cause cancer in humans, the action level of 0.5 ppb of aflatoxin M1 in milk is strictly enforced by the United States Food and Drug Administration (FDA).Aflatoxin B1 in feed is a mycotoxins produced by Aspergillus that grow on grain, especially corn, cotton seed and sometimes peanuts. Feed does not contain aflatoxin M1 as it is found only in milk.

30.3.2 Rapid screening methods

Rapid screening methods such as microbial inhibition assay, enzyme-linked immunosorbent assay, immunoaffinity, and lateral flow tests are used by industry and state laboratories for screening milk samples. Positive samples may require further analysis by validated methods such as the officially approved high-performance liquid chromatography (HPLC) for aflatoxin M1 in milk. With any methodology, there are concerns about the sensitivity, precision, and reproducibility of the method and the subsequent rate of false-positive, false-violative (positive test result with non-actionable levels in the sample), and false-negative results. Rapid screening methods need to provide detection at the action level but not be overly sensitive as to cause the loss of milk due to false violatives. The commercially available methods used in detection of Aflatoxin M1 in milk are given in Table. 30.2.

Table 30.3 Commercially available methods for aflatoxin M1

30.3.2.1 Spore based assay for aflatoxin M1

The bacterial spores have unique ability to sense environmental changes in response to specific “germinant” and transform rapidly into growing vegetative cells. This characteristic can be effectively used as biosensor for tracking microbial and non–microbial contaminants. A test based on the specific spore germination and its inhibition in presence of specific analyte, i.e., aflatoxin M1 has been developed (Kumar, 2012). In case where analyte is absent in milk, specific indicator enzyme (s) are produced by active bio-sensing molecules which will act specifically on chromogenic/or fluorogenic substrate resulting in colored reaction/or fluorescence as end product which is measured semi-quantitatively by either visually/or using optical system at specific excitation/emission spectra ( & )*
*(Patent Reg # 3064/DEL/2010)

30.3.2.2 Enzyme linked immune sorbent assay (ELISA)

ELISA is a widely used biochemical technique for the detection of an antigen in a sample. The sandwich ELISA utilizes two antigen specific antibodies, a capture antibody bound to a solid phase and an enzyme linked detection antibody. Direct enzyme conjugation of the detection antibody ensures an easy-to-use and sensitive assay with minimal background signal. A competitive assay in which there is a competitive binding of an antigen-specific, biotin-linked antibody to sample antigen or to antigen bound to the microtiter well. Bound antibody is detected with enzyme-linked streptavidin.

30.3.2.3 Lateral flow assay

The Charm Safe Level Aflatoxin M1 Quantitative (SLAFMQ) test is a colloidal gold lateral flow immunoassay. Aflatoxin M1 in a milk sample competes with the antibody gold beads for binding to 2 test lines. Remaining unbound binder forms on the control line. The test and control lines are compared with a reflectance reader, and a ppt concentration is determined with an algorithm. A negative interpretation with a reading of [less than or equal to] 400 ppt and a positive interpretation with a reading >400 ppt was designed to detect 500 ppt, the U.S. and Codex violative level at 90% positive with 95% confidence.

30.3.2.4 Charm ІІ test for aflatoxin (competitive assay)

The test procedure is as follow:

• Add 300µL.solution AF to test tube
• Fill tube3/4 full with milk sample or standard
• Centrifuge the sample at 3400 rpm for 5 minutes
• Cool to 4ºC±2 ºC
• Add white tablet to empty test tube
• Add 300±100µL.water
• Mix 10 seconds to break up tablet (take additional time if required to be sure tablet is broken up)
• Add 5.0±0.25mL centrifuged sample or standard from below fat layer(new tips for each sample, milk temp. 4ºC±2 ºC)
• Immediately add purple tablet
• Immediately mix by swirling milk up and down 15 times for 15seconds
• Incubate at 35±2 ºC for 3 minutes
• Centrifuge at 3400 rpm for 5 minutes.
• Immediately remove from centrifuge and pour off milk
• Remove fat ring wipe dry with swabs. Do not disturb pellet
• Add 300±100µL.water
• Mix thoroughly to break up pellet
• To one tube at a time, add 3.0± 0.5 mL scintillation fluid. Cap, invert (or shake) until mixture has uniform cloudy appearance.
• Immediately count in analyzer for 60seconds. Read CPM (count per minute) on (³H) channel