SUMMARY
      The presence of antimicrobial residues in milk constitutes a public health risk as they may provoke allergies, resistance to antibiotics and intestinal alterations, among other things. It is, moreover, one of the dairy industry’s main concerns, as they may have a negative effect on the manufacturing process of some dairy products, such as yoghurt and cheese. The amount of information available on the heat stability of the residues in milk is very limited, particularly so when referring to the refrigeration and freezing temperatures used throughout the production stages, transport and analysis, as well as the heat treatments used in the control laboratories and dairy industries.
      Therefore, the aim of this study is to carry out a two-fold analysis. On the one hand, to evaluate the influence of refrigerated and frozen storage conditions on the presence of antimicrobial substances belonging to groups of betalactams, tetracyclines and sulphonamides and, on the other, to analyse the effect that heating milk to different temperatures has on the stability of these substances.
      The influence that refrigeration (4 ± 2 ºC) and freezing (-20 ± 2 ºC) has on antimicrobial presence in milk has been studied by means of analysing samples of 20 substances that have been spiked at Maximum Residue Limits (MRL) equivalent concentrations. These substances belong to the betalactam (penicillins and cephalosporins), tetracycline and sulphonamide antimicrobial groups and their corresponding extracts. HPLC-MS/MS equipment was used to carry out the chromatographic analysis of the milk samples and extracts after 0, 1, 2, 3, 7, 10, 15 and 30 days of storage. How much refrigeration and freezing time and the type of matrix (milk and extracts) influenced the loss in concentration of the antimicrobial substances was determined by applying the multiple linear regression model and estimating the degradation percentages of each substance.
      From the obtained results, it can be concluded that refrigeration and freezing do affect the betalactam and tetracycline antibiotics present in both the extracts and the milk samples, but have no effect on the stability of sulphonamides. It may also be seen that the stability of each substance depends on temperature, storage time and the interaction with the type of matrix (milk and extracts). Although the estimated losses in the milk samples refrigerated for 7 days ranged between 14 and 90% for every substance, the most unstable molecules were the betalactam antibiotics (with the exception of cephalexin). Nearly all the extracts experienced smaller losses. When frozen, most of the antimicrobial samples were more stable than the extracts. After 7 days of frozen storage, the degradation percentages ranged between 6.7 and 68.4%, and once again, the betalactams were the antibiotics that degraded the most, except for cephalexin and cefquinome.
      To analyse the effect of heating on antimicrobial stability in milk, an experiment was performed consisting of 5 heat treatments at 60, 70, 80, 90 and 100 ºC for different periods of time on samples spiked with 22 antimicrobial substances. In this study, the antimicrobial substances belonging to the betalactam and tetracycline antibiotic groups were analysed by preparing milk solutions that have been spiked at a concentration of 5000 µg/Kg and by analysing the samples treated for different periods of time at each of the temperatures, using two pieces of equipment: HPLC-UV for penicillins and HPLC-PDA for cephalosporins and tetracyclines. In the case of the sulphonamides, the milk samples were spiked at a concentration equivalent to 200 µgKg and were analysed using HPLC-MS/MS. At the different temperatures and after different treatment times, each substance loses concentration and these losses were analysed by applying the first order kinetic model and the Arrhenius equation. From these analyses, the degradation percentages of each antimicrobial substance were estimated for the heat treatments that are widely used both in the control laboratories and the dairy industry.
      From the results obtained in this study, it can be concluded that it is suitable to apply the first order kinetic model to study heat degradation of antimicrobial substances in milk. It may also be deduced, from the calculation of the activation energy and the half-lives, that cephalosporins are the most unstable molecules, whereas tetracyclines and sulphonamides are much more heat resistant. As regards the estimation of degradation percentage as influenced by different treatments, it may be seen that those used in laboratories (40 ºC-10 min and 83 ºC-10 min ) produce hardly any effects, except for the treatment at 83 ºC for 10 min on some cephalosporins. Concerning the industrial treatments (63 ºC-30 min, 72 ºC-15 sec, 120 ºC-20 min y 140 ºC-10 sec), it seems that most of them have no effect upon antimicrobial degradation, with two exceptions; the treatment at 63 ºC for 30 minutes has a degrading effect on cephalosporins, reaching percentages ranging between 14% (cefuroxime) and 36.2% (cefapirin) and the milk sterilization (120 ºC-20 min) which leads to significant losses (37.4-100%) in all the betalactam, tetracycline and sulphonamide antibiotics, except for sulfadimethoxine (6.5%) and sulfathiazole (9.9%).
      It can be concluded from this that milk sample storage conditions, both refrigeration and freezing, can affect the stability of betalactam and tetracycline antibiotics. This is why it is advisable to keep the milk samples for the shortest possible time prior to analysis (both at 4 ºC and -20 ºC) and also to study the possibility of storing extracts of those molecules that are mores stable then the milk samples. In this way, the aim is to reduce the possible variability in the results of the analysis carried out on the same milk sample over a period of time and which may be behind the contradictory results obtained during the different control stages.
      Regarding the heat treatments, neither those performed in the laboratories nor the ones used in the dairy industry, produce hardly any degradation effects on antimicrobial substances, except for milk sterilization (120 ºC-20 min). This means that, as most of the heat treatments do not act as a barrier preventing antimicrobial substances from reaching the consumer, it is necessary to apply the preventative and control methods correctly throughout the whole milk production chain to avoid any potential risk caused by the presence of these substances.
      The results of this work establish a starting point for future studies and it would be of interest to analyse the stability of other antimicrobial groups that are being used more and more in milk. It is also relevant to study the molecular changes that can be brought about in the antibiotics by the heat treatments and the possible toxicological and/or technological effects of their metabolites. All of this contributes towards improving one of the basic principles of food safety: consumer protection.
Summary

Summary