Veterinary drug residues in milk are a growing concern among consumers, because of the risk they might pose for health, i.e. generating allergies, toxic reactions or drug resistance (Alanis, Reference Alanis2005; Demoly & Romano, Reference Demoly and Romano2005; Sanders et al. Reference Sanders, Bousquet-Melou, Chauvin and Toutain2011), and technological implications in the manufacture of dairy products (Packham et al. Reference Packham, Broome, Limsowtin and Roginski2001; Adetunji, Reference Adetunji2011). Therefore, Maximum Residue Limits (MRLs) of drugs in different foodstuffs of animal origin, including milk, have been defined by Regulation (EC) 470/2009 (European Union, 2009) and established by Commission Regulation (EU) 37/2010 (European Union, 2010).
Currently, there are numerous screening tests commercially available to detect antimicrobial residues in milk (International Dairy Federation (IDF), 2010). In control laboratories, microbial inhibitor tests are widely used thanks to their simplicity, low cost and wide range of detection. Microbial inhibitor tests are based on the inhibition of spore outgrowth of the microorganism-test, the most commonly applied being Geobacillus stearothermophilus var. calidolactis; a thermophilic bacterium highly sensitive to β-lactam antibiotics. Screening microbial tests are non-specific methods and may be affected by different substances capable of inhibiting microorganism-test growth, causing positive results in antibiotic-free milk samples, such us: natural inhibitors of milk (Andrew, Reference Andrew2001), and preservatives (Molina et al. Reference Molina, Althaus, Balasch, Torres, Peris and Fernandez2003), among others.
Detergents and disinfectants used in the cleaning of milking parlours and milk tanks are a possible source of residues in milk and have occasionally been associated with the positive results obtained in microbial tests (Fabre et al. Reference Fabre, Moretain, Ascher, Brouillet and Berthelot1995).
The hygienic production of milk implies the use of cleaning products to prevent the proliferation of microorganisms on surfaces that come into direct contact with milk, such as milking machines and milk storage tanks (Pontefract, Reference Pontefract1991). Following good cleaning practices, the residues of detergents in milk should be minimal (⩽2 ppm; Reybroeck, Reference Reybroeck1997), although owing to errors in the washing temperature, dosage, and inadequate post-rinse the concentration of these cleaning products can be higher, which may alter the organoleptic characteristics of milk (Dunsmore et al. Reference Dunsmore, Makin and Arkin1985; Merin et al. Reference Merin, Rosenthal, Bernstein and Popel1985) and interfere in the activity of starter cultures in the industry (Guirguis & Hickey, Reference Guirguis and Hickey1987; Petrova & Dimitrov, Reference Petrova and Dimitrov1993).
Moreover, only few studies in cow milk have evaluated the effect of detergents on the presence of positive results in microbial inhibitor tests, showing controversial results. Some authors (Zvirdauskiene & Salomskiene, Reference Zvirdauskiene and Salomskiene2007; Salomskiene et al. Reference Salomskiene, Macioniene, Zvirdauskiene and Jonkuviene2013) only found false-positive results at very high concentrations of alkaline detergents, equal or superior to the dose recommended by the manufacturers. However, Schiffmann et al. (Reference Schiffmann, Schütz and Wiesner1992) obtained non-compliant results at lower concentrations (0·01%), whereas Merin et al. (Reference Merin, Rosenthal, Bernstein and Popel1985) for these concentrations did not obtain any positive results, although they employed a limited number of cleaning products and microbial methods. Furthermore, these studies focus on positive outcomes; there is no information about the effect of detergents on the detection capability of microbial methods.
Therefore, the goal of this study was to analyse the effect of detergents used in the cleaning of milking equipment on the performance of microbial tests for screening antibiotics in goat's milk.
Material and methods
Microbial inhibitor tests
The microbial inhibitor tests were: Brilliant Black Reduction Test MRL (BRT MRL) (AiM Analytik in MilchProduktions-und Vertriebs-GmbH, Munich, Germany), Delvotest MCS (DSM Food Specialties, Delft, the Netherlands) and Eclipse 100 (Zeu-Inmunotec, Zaragoza, Spain). The tests were used according to each manufacturer's instructions. A negative control (antimicrobial-free milk) and a positive control (antimicrobial-free milk spiked with 4 μg/kg of benzylpenicillin) were included in each test. Visual interpretation of the test results was carried out independently by three trained technicians and was evaluated as ‘negative’ (yellow) and ‘positive’ (blue or purple).
Goat's milk samples
Antimicrobial-free milk samples to be used as ‘negative milk’ were obtained according to the requirements established by the In IDF (ISO13969/IDF183:2003). Therefore, mixed milk of 10 Murciano-Granadina goats in mid-lactation (more than 90 d and below 150 d postpartum) from the experimental flock of Universitat Politècnica de València (Valencia, Spain) was used. Animals had a good health status and did not receive any veterinary drugs before or during the experimental period. Moreover, goats were fed diets formulated and produced in the experimental feed processing plant of Universitat Politècnica de València using first-class raw materials without added antibiotics.
All milk samples were analysed to check the physico-chemical and hygienic quality parameters using MilkoScan 6000 (Foss, Hillerød, Denmark) to determine gross composition (fat, protein and total solids); somatic cell count (SCC) was obtained employing Fossomatic 5000 (Foss, Hillerød, Denmark); bacterial count (BC) was determined using Bactoscan FC (Foss, Hillerød, Denmark) and the pH value was measured by a conventional pHmeter (Crison, Barcelona, Spain).
Spiked milk samples: detergents and antibiotics
The detergents used for the study of their presence on the microbial test response were commercial detergents of the acid and alkaline type, which were added to the antibiotic-free goat's milk at concentrations of: 0, 0·25, 0·5, 0·75, 1, 1·5, 2, 2·5 ml/l for acid and 0, 0·5, 1, 2, 4, 6, 8, 10 ml/l for alkaline (Table 1). Concentrations tested for acid detergents were lower than those selected for the alkaline detergents, as higher concentrations produce milk coagulation. Each concentration was tested twelve times by microbial methods (BRT MRL, Delvotest MCS, and Eclipse 100).
Table 1. Brand name, composition and recommended dose of acid and alkaline detergents
† DeLaval International A.B., (Tumba, Sweden)
‡ OXA Chemical Specialties, CYGYC S.A., (Barcelona, Spain)
§ Grupanor-Cercampo S.A., (Madrid, Spain)
¶ Manovac S.L., (Valencia, Spain)
†† GEA Farm Technologies Ibérica, S.L., (Barcelona, Spain)
To evaluate the effect of detergents on the detection capability of microbial screening methods for penicillins, the detection limits (DLs) for ampicillin, amoxicillin, cloxacillin and benzylpenicillin were calculated according to ISO13969/IDF183:2003 specifications. To do so, two detergents were chosen, an acid one (Circoaction SF, Westfalia Surge Ibérica SL, Spain) and a basic one (Circoaction AF, Westfalia Surge, Ibérica SL, Spain), which were then added to antibiotic-free goat's milk at the maximum detergent concentration, not showing interferences in the response of the microbial tests, nor significantly altering the pH of milk (0·5 ml/l). Furthermore, goat's milk samples without detergents were used as control.
The goat's milk samples, with or without detergents, were spiked with eight different antibiotic concentrations (Table 2), prepared following the recommendations of the IDF (2003). The antibiotics selected for this study were supplied by Sigma-Aldrich (Madrid, Spain): amoxicillin (31 586), ampicillin (A-9518), cloxacillin (C-9393), and benzylpenicillin (PENNA). All the antibiotic standard solutions were prepared daily, and twelve repetitions of milk were analysed within four hours after spiking.
Table 2. Antibiotic concentrations used for the detection limit calculation of microbial inhibitor tests in goat's milk
Statistical analysis
To evaluate the effects of the acid (D AC) or alkaline (D AK) detergent on the response of the microbial inhibitor tests, the logistic regression model was used:
$$L_{ij} = {\rm Logit}[P_{ij} ] = {\rm \beta} _0 + {\rm \beta} _1 [{\rm atb}]_i + {\rm \beta} _2 D_{{\rm AC}} + {\rm \beta} _3 D_{{\rm AK}} + {\rm \varepsilon} _{ij} $$where: L ij=Logit model; [P ij]=probability for the response category (positive or negative); β0=intercept; β1, β2, β3=parameters estimated for the model; [atb]i=effect of antibiotic concentration (n=8); D AC=effect of the acid detergent; D AK=effect of the alkaline detergent on the dummy variable (without detergent: D AC=0, D AK=0; acid detergent: D AC=1, D AK=0; alkaline detergent: D AC=0, D AK=1), εij=residual error of model.
The detection limits (DLs) were calculated as an antibiotic concentration producing 95% positive results (ISO13969/IDF183:2003).
Statistical analysis was performed using Statgraphics Centurion XVI 5.1 (Statpoint Technologies, Inc, Warrenton, VA).
Results and discussion
Effect of detergents in goat's milk on false-positive results of microbial screening tests
The goat's milk samples presented an adequate physico-chemical quality (fat: 4·36±0·12%, protein: 3·61±0·07%, dry matter: 14·24±0·28%, and pH value: 6·66±0·02) and hygienic-sanitary parameters (SCC: 889±79×103 cell/ml, and BC: 331±51×103 cfu/ml) to be used as antimicrobial-free milk.
The presence of the acid detergent in goat's milk did not affect the response of the microbial inhibitor tests employed, the results were always negative. Moreover, the acid detergent addition decreased the pH of milk samples, reaching pH between 5·72 and 5·98 for the highest concentration (2·5 ml/l) of the five detergents, being lower compared with the average of pH cited for goat's milk (6·5–6·8; Park et al. Reference Park, Juárez, Ramos and Haenlein2007). These very low pH values can simulate the effect of acid generation, which is produced as a consequence of the metabolism of the microorganism, favouring the change in colour of the indicator present in the medium test.
The alkaline detergent addition in goat's milk produced positive results at concentrations ⩾2 ml/l (Table 3). Besides, at these concentrations, the pH of the milk samples was high, reaching pH of 9·82–10·80 for the highest concentrations tested, which could also inhibit the growth of the microorganism and thus, prevent the colour change of the test indicator system.
Table 3. Effect of the alkaline detergent concentrations in goat's milk on the positive results of microbial screening tests
These results agree with Zvirdauskiene & Salomskiene (Reference Zvirdauskiene and Salomskiene2007) and Salomskiene et al. (Reference Salomskiene, Macioniene, Zvirdauskiene and Jonkuviene2013) who studied the effects of various commercial detergents on the microbial test response in cow milk, and found positive results at alkaline detergent concentrations equivalent to the dose recommended by the manufactures and above. Although, it should be noted that these high concentrations are very unlikely to be found in practice, even with poor cleaning routines. Also, these authors did not find interferences due to the presence of acid detergents in milk. At lower alkaline detergent concentrations (⩽1000 mg/l), Merin et al. (Reference Merin, Rosenthal, Bernstein and Popel1985) and Salomskiene et al. (Reference Salomskiene, Macioniene, Zvirdauskiene and Jonkuviene2013) did not find any positive results for the Delvotest microbial test; similar results to those obtained for most alkaline detergents tested in goat's milk (Table 3). However, Schiffmann et al. (Reference Schiffmann, Schütz and Wiesner1992) observed doubtful and positive results at very low concentrations (0·01 mg/ml) using one acid detergent (Calgonit S) and a basic one (Calgonit D) in different versions of the Brilliant Black Reduction Test (BRT).
In conclusion, only the presence of alkaline detergents in goat's milk at concentrations ⩾2 ml/l, can produce positive results in microbial inhibitor tests. However, these amounts are not reached if the rinsing of the milking equipment is carried out in an effective manner after cleaning (Reybroeck, Reference Reybroeck1997).
Effect of detergents in goat's milk on the penicillin detection capability of microbial screening tests
Table 4 shows the equations resulting from statistical analysis used to predict the positive results for the penicillins and the detection limits (DL) of the microbial inhibitor tests. The goodness-of-fit test shows that the experimental values are similar to those estimated by the logistic model, suggesting a suitable adjustment of this model. The DLs calculated for microbial tests in detergent-free milk were lower than those indicated by Sierra et al. (Reference Sierra, Sánchez, Contreras, Luengo, Corrales, de la Fe, Guirao, Morales and Gonzalo2009) in goat's milk, which in most cases were closer to MRLs than those calculated in the present study. These differences could be related to modifications carried out by manufacturers to improve the sensitivity of these screening tests.
Table 4. Effect of the detergents in goat's milk on the penicillin detection capability of microbial inhibitor tests
L=ln (Probability (+)/1− Probability (+)); [atb]: antibiotic concentration; D AC=effect of the acid detergent; D AK=effect of the alkaline detergent on the dummy variable (detergent-free: D AC=0, D AK=0; acid detergent: D AC=1, D AK=0; alkaline detergent: D AC=0, D AK=1); DLDF: detergent-free detection limit, DLAC: acid detergent detection limit, DLAK: alkaline detergent detection limit; MRL: Maximum residue limits; —: no significant differences P>0·05
The presence of acid detergents in goat's milk did not affect or slightly increased the sensitivity of the Delvotest MCS and Eclipse 100, showing lower DLs than those calculated for detergent-free milk (Table 4). However, in the case of BRT MRL, the acid detergent decreased the sensitivity to detect most penicillins in goat's milk (Table 4). For the alkaline detergent, the DLs calculated for penicillins were below or equal to those obtained for detergent-free milk (Table 4), except for ampicillin in the Eclipse 100, which was slightly higher (4·3 v. 4 μg/l).
In spite of the statistical significant effect of the presence of detergents in goat's milk on the detection capability of the microbial tests, the DLs calculated were generally below MRLs established for each antimicrobial substance (Regulation EC 37/2010). Therefore, the presence of acid or basic detergent at concentrations equivalents to 0·05% does apparently not have any influence on the detection of raw milk containing penicillins above the safety levels when microbial screening tests are applied in the goat's milk quality control programmes.
However, it is not known if a higher concentration of detergents in milk could have a serious effect on the detection capability of the methods employed to detect antibiotics in milk. No reference concerning the effect of detergents on the sensitivity of inhibitor tests for the penicillins or other antimicrobial agents was found; therefore the comparison of the results with other authors is not possible.
Conclusions
The response of microbial screening tests in goat's milk can be affected by the presence of alkaline detergents at high concentrations equal or greater than 2 ml/l. However, acid detergents did not produce any interference. Small amounts of acid and alkaline detergents in goats’ milk (⩽0·05%) do not influence the sensitivity of the BRT MRL, Delvotest MCS and Eclipse 100 methods to detect penicillins. To avoid alterations in the milk quality and interferences in microbial screening tests employed in control programmes, the implementation of proper cleaning procedures to minimise the presence of detergent residues in milk is crucial.
This work forms part of the Project AGL 2009-11524 financed by the Ministry of Science and Innovation (Madrid, Spain) and the Generalitat Valenciana ACOMOP/2012/164 (Valencia, Spain). The authors are grateful to AiM Analytik in MilchProduktions-und Vertriebs-GmbH (Munich, Germany), DSM Food Specialties (Delft, Netherlands), ZEU-Inmunotec (Zaragoza, Spain) for their technological support.
