EFFECT OF HEAT TREATMENTS ON THE ANTIMICROBIAL ACTIVITY OF BETA-LACTAMS AND TETRACYCLINES IN MILK BERRUGA I1., ZORRAQUINO M. A.2, BELTRAN M. C.2, ALTHAUS R. L:3, MOLINA M. P.2 1 Departamento de Ciencia y Tecnología Agroforestal, ETSIA, Universidad de Castilla-La Mancha, 02071 Albacete, Spain. 2Departamento de Ciencia Animal, Universidad Politécnica, Camino de Vera 14, 46071 Valencia, Spain.3 Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, R.P.L. Kreder 2805, 3080 Esperanza, Argentina ABSTRACT
The presence in milk of residues of antimicrobial substances may have serious toxicological and technical consequences. The aim of the study was to analyse the effect of different heat treatments (40 ºC-10 minutes, 60 ºC-30 minutes, 83 ºC-10 minutes, 120 ºC-20 minutes y 140 ºC-10 seconds) on milk samples fortified with 8 beta-lactam and 4 tetracycline antibiotics. The method utilised was a bioassay that enables the detection of different groups of antibiotics. The bioassay uses different microorganism, reagents, culture medium and conditions for each antimicrobial group. The results showed that heating at 40 ºC-10 m caused no inactivation in most of the antibiotics studied. Treatment at 83 ºC-10 m produced an antimicrobial activity loss of over 20% in cephalexin, cephuroxime and chlortetracycline. Pasteurisation (60 ºC-30 m) provoked a slight inactivation in beta-lactams (6-20%) and in tetracyclines (18-31%), whereas sterilisation (120 ºC-20 m) led to the highest inactivation (65-92%). Lastly, inactivation percentages from 140 ºC-10 s (UHT) were in the range of 7-21% for beta-lactams and 27-39% for tetracyclines. In conclusion, only sterilisation produced a high inactivation level of beta-lactams and tetracyclines in milk. The other treatments were no guarantee that these molecules would lose their antimicrobial activity. Keywords: antibiotics, milk, thermostability, pasteurisation, sterilisation
Residues of antimicrobial agents in milk may cause technological problems in the milk processing industry, in the preparation of cheese, yoghurt, and other dairy products (Mourot and Loussouron, 1981; Brady and Katz, 1988). Moreover, the presence of residuals in milk may have serious consequences for public health; antibiotic residues in milk may provoke antibiotic resistance or allergies in consumers (Schwarz and Sher, 1984; Demoly and Romano, 2005). Nowadays, quality controls are essential in milk to establish the price that the producer receives, as well as to assure the quality of the product. In control laboratories, it is common practice to carry out treatments by heating the sample for its homogenisation, or for inactivation of the natural antimicrobials in the case of analysis of inhibitors. To date, there have been no studies investigating how these treatments affect the presence of antibiotic residues in milk. On the other hand, milk arrives at the consumer after undergoing different heat treatments in the industry, such as pasteurisation, sterilisation, etc. The studies referring to the influence of these treatments on antimicrobial residues in the dairy industry are very few, and centred on a very limited number of antibiotics. Moreover, in the case of the beta-lactams, almost all of the works have focussed on penicillin G (Moats, 1999), with very few studies on other penicillins and cephalosporins (Jacquet and Auxepaules 1978). In many of these studies, it is indicated that very prolonged heat treatments are required for the complete inactivation of penicillin residues. As for the tetracyclines, different authors (Moats, 1999) have reported a greater thermal instability, with lower inactivation times than for penicillin, for oxytetracycline, tetracycline and chlortetracycline, with heat treatments at temperatures over 70ºC. In contrast, Jacquet and Auxepaules (1978) assessed the thermal stability after a low pasteurisation at 63ºC for 30 minutes of chlortetracycline and tetracycline in milk, with low inactivation percentages of 2.6% and 6.1%, respectively. Given the cited lack of information and the importance of the presence of antibiotic residues in milk for the industry and the consumer, the aim of the study was to analyse the effect of different heat treatments (40 ºC-10 minutes, 60 ºC-30 minutes, 83 ºC-10 minutes, 120 ºC-20 minutes and 140 ºC-10 seconds) on milk samples fortified with 8 beta-lactam (penicillin, amoxycillin, ampicillin, cloxacillin, cephalexin, cefalonium, cefoperazone, cephuroxime) and 4 tetracycline antibiotics (doxycycline, tetracycline, oxytetracycline and chlortetracycline).
MATERIAL & METHODS
The method utilised was a bioassay (Nouws et al., 1999) that enables the detection of different groups of antibiotics. The bioassay uses different microorganisms, reactives, culture media and methodological conditions (pH, incubation time and temperature) for each group. In the case of the beta-lactams and tetracyclines, the characteristics of the plates used were as follows: Beta-lactamic plates: 23.5 g/L PCA of Difco lyophilised at pH 8.0 ± 0.1. Inoculation with B. stearothermophilus var. calidolactis C953, (Merck, Ref. 1.11499) with a final concentration of 107 spores/mL. Plates were incubated at 55 + 1ºC for 6 hours. Tetracycline plates: 25 g/L of Standard II Närh-agar lyophilised at pH 6.0 ± 0.1 with 0.5% of a 100 µg/kg solution of chloramphenicol. Inoculation with Bacillus cereus var. micoydes ATCC 11778 to obtain a concentration de 105 spores/mL. Incubation was carried out at 37 + 1ºC for 16-18 hours. The commercial UHT milk samples were fortified with three concentrations (C1, C2=2C1 and C3=4C1) of antibiotics, whose sources and concentrations are presented in Table 1. Table 1. Concentrations of antibiotics used to study the effect of thermal treatments on the activity of beta- lactams and tetracyclines.
The treatments of 40ºC-10 min., 60ºC-30 min. and 83ºC-10 min. were carried out in Eppendorf tubes protected from the light, in a thermostatic water bath. Heating at 120ºC-20 min was performed in an autoclave (Selecta Presoclave 75, Pl) using glass tubes with “sero-tab” caps. In the treatment at 140 ºC-10 sec, a bath was used with silicon oil and stainless steel tubes with an outer diameter of 1.6 mm and 0.5 mm thick (Pagliani et al., 1990). For analysis, NINC square bioassay plates (243 x 243 x 17 mm) were used, with 36 perforations made for the samples, both thermally treated and without treatment, with the different concentrations of antibiotic. Three plates were used per treatment (15 plates per antibiotic). The measurements of the diameters of the inhibition zones (including the14 mm dish) were taken in duplicate with a digital calibre of ± 0,01 mm precision. For statistical analysis of the results, the stepwise option from the Multiple Linear Regression model (SAS, 1998) was applied, by means of the model: Yij = β0 + β1 log Ci + βTj ETj+ εij Where: Yjj: diameter of the inhibition zone (mm), log Ci: decimal logarithm of the antibiotic concentration (µg/kg), β0: X-axis intercept, β1: Slope of the straight line, βTj: Correction coefficients of the control line x-axis due to the effect of treatment “i”, ETj: effect of thermal treatment in terms of dummy variables: Without treatment (Z1 = 0, Z2 = 0, Z3 = 0, Z4 = 0, Z5 = 0), Treatment 40ºC-10 minutes (Z1 = 1, Z2 = 0, Z3 = 0, Z4 = 0, Z5 = 0), Treatment 83ºC-30 minutes: (Z1 = 0, Z2 = 1, Z3 = 0, Z4 = 0, Z5 = 0), Treatment 60ºC-10 minutes (Z1 = 0, Z2 = 0, Z3 = 1, Z4 = 0, Z5 = 0), Treatment 120ºC-20 minutes (Z1 = 0, Z2 = 0, Z3 = 0, Z4 = 1, Z5 = 0), Treatment 140ºC-10 seconds (Z1 = 0, Z2 = 0, Z3 = 0, Z4 = 0, Z5 = 1), εij: residual error of model. Calculation of the antimicrobian activity loss was performed by means of the percentage of thermal inactivation (%IT) using the following expression: % IT = [1 – 10 (-β /β )
For those antibiotics that did not present inhibition zones when subjected to thermal treatment at 120º-20 seconds, the minimum inhibitory percentage was calculated as the relative percentage between the maximum concentration assayed and the minimum concentration estimated by the regression model that did not produce inhibition zone (14 mm), by means of the formula: % IT = [(Cmáx – C 14mm))/Cmáx] x 100
Where: Cmáx: maximum concentration assayed for each antibiotic and C 14mm: minimum concentration estimated by the linear regression model that did not produce inhibition zone (14 mm). RESULTS Table 2 resumes the mathematical equations obtained by means of application of the multiple linear regression model for the study of different thermal treatments that were significant in milk samples fortified with beta- lactams and tetracyclines. As may be seen in the table, the regression coefficients obtained were high (> 0.92) and the treatments that significantly affected the measurements of the inhibition zones were different depending on the antibiotic. Thus, of the laboratory treatments, the homogenisation of the samples (40ºC-10 m: Z1) only affected the cephalexin, whereas the heating to 83ºC-10 m (Z2) to inactivate the natural inhibitors presented significant reductions in the measurements of the diameters in all the antibiotics studied, with the exception of cloxacillin. The industrial treatments (Z3, Z4 and Z5) affected all four. Regarding the industrial treatments, it must be noted that in some beta-lactam antibiotics (amoxicillin and ampicillin) and in the four tetracyclines, the treatment at 120ºC -20 m (Z4) presented an important effect on antimicrobial activity, although, as it was not possible to take any measurements of the inhibition zone, this was not included in the statistical analysis. Heating to 60ºC-30 m (Z3) presented significant effects on all the antibiotics and heating to 140ºC-10 s (Z5) diminished the diameter of all the beta-lactams with the exception of Penicillin G and, to a greater extent, the tetracyclines. Table 2. Results of the statistical analysis of the effect of thermal treatments on the antimicrobial activity of beta- lactams and tetracyclines in milk Antibiotics Beta-lactams
D = 11.85 + 15.00 Log[Penicillin] - 0.61 Z2 - 0.42 Z3 - 6.93 Z4
D = 10.38 + 12.85 Log[Amoxicillin] - 0.55 Z2 – 0.64 Z3 - 0.84 Z5
D = 10.39 + 12.84 Log[Ampicillin] - 0.73 Z2 - 0.54 Z3 - 0.50 Z5
D = - 4.85 + 14.52 Log[Cloxacillin] - 0.43 Z3 – 7.99 Z4 - 0.49 Z5
D = - 4.59 + 12.28 log[Cephalexin] - 1.02 Z1 - 1.71 Z2 - 1.01 Z3 - 0.61Z5 0.9429
D = 4.52 + 13.67 Log[Cephalonium] - 0.82 Z2 - 0.68 Z3 - 0.80 Z5
D = 2.50 + 9.29 Log[Cephoperazone] - 0.86 Z2 - 0.78 Z3 - 0.58 Z5
D = - 7.64 + 15.06 Log[Cephuroxime] - 2.84 Z2 – 0.87 Z3 - 1.54 Z5
D = 1.07 + 10.69Log[Chlortetracycline] - 1.15 Z2 - 1.72 Z3 -1.57 Z5
D = 2.45 + 11.92Log[Doxycycline] - 0.75 Z2 - 1.05 Z3 - 2.51 Z5
D = -6.34 + 12.24 log[Oxytetracycline] - 0.42 Z2 - 1.36 Z3 - 1.67 Z5
D = -3.38 + 11.6 Log[Tetracycline] - 0.62Z2 - 1.14 Z3 - 1.67 Z5
The thermal inactivation percentages calculated for the beta-lactams and tetracyclines according to the different treatments applied are presented in Table 3. Of the two treatments carried out in the control laboratories, the results show that heating at 40 ºC-10 m caused no inactivation in most of the beta-lactams, except for cephalexin (17%), and none in the tetracyclines. In fact, heating at 40ºC-10 m is a mild treatment routinely carried out in dairy laboratories to achieve homogenisation of milk samples before carrying out physical-chemical determinations, and logically should not affect the milk composition. In turn, the treatment at 83 ºC-10 m, although producing losses in almost all of the antibiotics, these are equal to or greater than 20% in penicillin, cephalexin, cefuroxime and chlortetracycline. This fact raises the issue that although this treatment pursues the objective of diminishing the frequency of “false positive” results in screening methods used for the detection if inhibitors in milk (Molina et al., 2003), it also gives rise to a decrease in the activity of certain molecules, and as a result this heating prior to analysis should be evaluated in greater depth. As for the treatments similar to those employed in the dairy industry, pasteurisation (60 ºC-30 m) causes a low rate of inactivation in beta-lactams (9-18%), somewhat higher in the tetracyclines (18-31%). In contrast, sterilisation (120 ºC-20 m) produced a marked loss of antimicrobial activity (65-92%), with the percentage of inactivation having to be estimated in many cases, as no inhibition zone of the microorganism used in the method was present. In turn, the inactivation percentages of the treatment at 140 ºC-10 s (UHT) are moderate, being situated between 7-21% for beta-lactams and 27-39% for tetracyclines.
Table 3. Percentage of thermal inactivation (%) of antibiotics in milk
NS: Effect not significant compared with samples without thermal treatment; *: minimum inactivation percentage calculated on the basis of estimated concentration that did not produce an inhibition zone (14 mm) Other authors cited in the bibliographic references of Moats (1999) indicate that milk samples with Penicillin reach low percentages of inactivation (8%) for a treatment at 62ºC-30 (Shahani et al., 1956), value similar to that shown in Table 3. In the case of ampicillin and cephalexin in milk samples treated at 63ºC-30 (Jacquet and Auxepaules, 1978), the percentages found were lower, with values of 1.7% and 6.1%, respectively. The inactivation percentages of Penicillin were also higher in other works, between 20-60%, when the milk samples were heated to temperatures over 100ºC (Shahani et al., 1956; Pilet et al., 1969; Konecny, 1978). By way of synthesis, it may be established that the beta-lactams antibiotics, in general, are stable when heated at low temperatures, such as those used in the laboratories at 40ºC-10 min and 83ºC-10 min, although the latter may affect some cephalosporins (cephalexin and cefuroxime) to a greater extent, as does low temperature pasteurisation (60ºC-30 min), or even at high temperatures but with shorter times (140ºC-10 sec). In contrast, all the beta-lactams underwent a high degree of antimicrobial loss when the milk samples were subjected to high temperatures for prolonged periods, such as in conventional sterilisation (120ºC-20 min). As for the tetracyclines, Moats (1999) presented studies (Shahani, 1957, 1958) where similar reductions in the activity of chlortetracycline (16.6% and oxytetracycline (23.6%) were obtained with pasteurisation treatments (62ºC-30 m). Likewise, in the bibliographic references cited, it is indicated that high temperatures over 100ºC produce inactivation percentages of between 75-100% for oxytetracycline (Sanz et al., 2002) and tetracycline (Shahani , 1957, 1958; Pilet et al., 1969). From all of the above, it may be inferred that tetracyclines are stable in mild treatments such as those used in quality control laboratories, with average stability in low pasteurisation and UHT, and unstable in conventional sterilisation. CONCLUSIONS Of the two treatments applied in the control laboratories, the results show that heating at 40 ºC-10 m caused no inactivation in most of the beta-lactams, except in cephalexin (17%) and in none of the tetracycline. However, the treatment at 83 ºC-10 m produced losses of activity equal to or greater than 20% in penicillin, cephalexin, cephuroxime and chlortetracycline. As for the treatments similar to those of the dairy industry, pasteurisation (60 ºC-30 m) produced low inactivation in beta-lactams (6-20%) and tetracyclines (18-31%). In contrast, sterilisation (120 ºC-20 m) caused a high loss of antimicrobial activity (65-92%). In turn, the inactivation percentages of treatment at 140 ºC-10 s (UHT) are moderate, between 7-21% for beta-lactams and 27-39% for tetracyclines. In conclusion, only sterilisation produces a high degree of inactivation in beta-lactams and tetracyclines in milk. The other thermal treatments do not guarantee the loss of activity of the residues of these antibiotics and their possible consequences for the transformation processes and the consumer. ACKNOWLEDGEMENTS This work forms part of Project AGL2003-03663, funded by the Interministerial Science and Technology Commission (Madrid, Spain) and the European Regional Development Fund ERDF/FEDER. The authors also wish to thank the Polytechnic University of Valencia for funding the internship of R. L. Althaus at the Animal Science Department. English translation by N. Macowan. BIBLIOGRAPHY Brady M. S., Katz S. E., 1987. Simplified Plate Diffusion System for Microbial Assays of Antibiotics. J. Assoc. Off. Anal. Chem., 70: 641-646. Demoly P., Romano A., 2005. Update on Beta-lactam allergy diagnosis. Curr Allergy Asthma Rep., 1:9-14. Jacquet J., Auxepaules M., 1978. Le problème de la pollution du lait par les antibiotiques. État actuel de la question. Bull. Acad. Vét. de France, 51: 73-79. Konecny S., 1978. Effect of temperature and time on biological activity reduction of some kinds of antibiotics in milk. Vetemarstvi, 28: 409-410. Molina, M.P., Althaus, R.L., Balasch, S., Torres, A., Peris, C., Fernández, N., 2003, Evaluation of screening test for detection of antimicrobial residues in ewe milk. J. Dairy Sc., 86: 1947-1952. Moats W. A., 1999. 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XIX Reunión de especialistas en control de mamitis y calidad de leche (G-Temcal). Palencia. SAS Institute Inc. (1998). SAS Users guide: statistics version 6.12. Cary, NC. Schwartz H. J., Sher T. H., 1984. Anaphylaxis to penicillin in a frozen dinner. Ann. Allergy, 52: 342-343. Shahani K. M., Gould I. A., Weiser H. H., Slatter W. L., 1956. Stability of small concentrations of penicillin in milk as affected by heat treatment and storage. J. Dairy Sci., 39: 971-977. Shahani K. M., 1957. The effect of heat and storage on the stability of Aureomycin in milk, buffer, and water. J. Dairy Sci., 40: 289-296. Shahani K. M., 1958. Factors affecting Terramycin activity in milk, broth, buffer, and water. J. Dairy Sci., 41: 382-391.
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DR. GILDO BERTONCINI nato ad Aulla (MS) il 21.02.1958 STUDI COMPIUTI Titolo di Studio Laurea in Medicina e Chirurgia presso l'Università degli Studi di Milano con la valutazione di 110 e lode. Specializzazione in Cardiologia presso l'Università degli Studi di Pisa ESPERIENZE PROFESSIONALI - Servizio di guardia medica territoriale ASL n° 1 Massa Carrara. - Docente presso la S