Effect of pasteurization on the residues of tetracyclines in milk

Abstract

The main aim of this work was to determine the effect of high pasteurization of milk (85 degrees C/3 s) on the residues of tetracycline and oxytetracycline. The samples of raw cow's milk, purchased from a vending machine, were spiked with standard solutions of tetracycline and oxytetracycline. The content of the residues of tetracycline antibiotics was measured before and after heating. Pre-cleaned samples were extracted by a mixed-mode solid phase extraction technique and analysed using high performance liquid chromatography/diode array detection. Whereas the residues of tetracycline decreased only by 5.74% and were not significantly different (P > 0.05), the residues of oxytetracycline decreased by 15.3% and this distinction was highly significant (P <= 0.01). Based on the results of our study, the tetracycline antibiotics were proved to have differences in the thermostability of particular substances at pasteurisation temperatures.

Full text

Effect of pasteurization on the residues of tetracyclines in milk
Eva Kellnerová, Pavlína Navrátilová, Ivana Borkovcová
University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Veterinary Hygiene and Ecology,
Department of Milk Hygiene and Technology, Brno, Czech Republic
Received July 31, 2014
Accepted February 25, 2015
Abstract
The main aim of this work was to determine the effect of high pasteurization of milk (85 °C/3 s)
on the residues of tetracycline and oxytetracycline. The samples of raw cow’s milk, purchased
from a vending machine, were spiked with standard solutions of tetracycline and oxytetracycline.
The content of the residues of tetracycline antibiotics was measured before and after heating. Pre-
cleaned samples were extracted by a mixed-mode solid phase extraction technique and analysed
using high performance liquid chromatography/diode array detection. Whereas the residues of
tetracycline decreased only by 5.74% and were not signicantly different (P > 0.05), the residues
of oxytetracycline decreased by 15.3% and this distinction was highly signicant (P ≤ 0.01).
Based on the results of our study, the tetracycline antibiotics were proved to have differences in
the thermostability of particular substances at pasteurisation temperatures.
Antibiotics, heat treatment, thermostability
Tetracyclines are a group of highly important broad-spectrum antibiotics used in
veterinary medicine to treat food-producing animals (Botsoglou and Fletouris 2001;
W ang et al. 2012). They are used to treat gastrointestinal, respiratory, genitourinary and
skin bacterial infections as well as infectious diseases of the musculoskeletal system
and systemic infections, and also in the treatment of cholera and sepsis (S amanidou
et al. 2007). However, they have a range of side effects, including disturbances in
healthy intestinal microora, allergic reactions, liver and kidney malfunctions,
hypersensitiveness and intense-light related dermatitis. Moreover, nowadays it is
necessary to take into account the relatively high probability of acquired tetracycline
resistance (Michalova et al. 2004).
In order to protect consumers’ health, EU legislation lays down the maximum residue limit
(MRL) in food of animal origin for veterinary medical products approved for use in food-
producing animals. Legislation establishes the MRL for three tetracycline antibiotics most
commonly used in lactating dairy cows. The MRL for tetracycline (TTC), oxytetracycline
(OTC) and chlortetracycline (CTC) in cow’s milk is 100 µg·kg
-1
(Commission Regulation
37/2010). When heated or exposed to acidic or highly alkaline environments, tetracyclines
are subject to chemical transformation processes, such as isomerization and epimerization
(Wang et al. 2012). For this reason, when establishing MRLs it is necessary to take into
account both the basic compound (tetracycline) and its epimers (the 4-epimer products of
TTC, OTC and CTC) (Commission Regulation 37/2010; Spisso et al. 2010).
Information concerning thermal stability of drug residues in food is toxicologically
important. Most food of animal origin is not eaten raw, but requires heat treatment: boiling
or poaching, frying, roasting or stewing. These culinary processes may lead to protein
denaturation, increase in temperature, water and fat loss and changes in pH, which in turn
may result in changes in the residues’ concentration, chemical structure and chemical
reactions as well as to their loss of solubility. Many drugs are chemically unstable to
ACTA VET. BRNO 2014, 83: S21–S26; doi:10.2754/avb201483S10S21
Address for correspondence:
MVDr. Navrátilová Pavlína, Ph.D.
Department of Milk Hygiene and Technology
Faculty of Veterinary Hygiene and Ecology
University of Veterinary and Pharmaceutical Sciences Brno
Palackého tř. 1/3, 612 42 Brno
Phone +420 541 562 716
E-mail: navratilovap@vfu.cz
http://www.vfu.cz/acta-vet/actavet.htm

some extent and thus prone to degradation during storage and thermal or technological
processing (Botsoglou and Fletouris 2001).
One of the basic methods of thermal processing of raw milk is pasteurization. Compared
to other food of animal origin, milk is subjected to only a very short heat treatment. The
results of many studies show that pasteurization does not lead to full inactivation and
degradation of drug residues (Botsoglou and Fletouris 2001).
Even members of the same class of antibiotics with the same structure may exhibit vast
differences in thermal stability, depending on different matrix types and conditions of the
treatment. When heated, tetracyclines split and degrade into metabolites, which indicate
lack of thermal stability among this group of drugs. The degree of instability may vary
with temperature. Many other questions are still to be answered. Thermal processing may
not only reduce the concentration of drug residues in food, but it can also change their
pharmacological and toxic effects (Hsieh et al. 2011). Heat treatment may give rise to
new chemical compounds with higher levels of toxicity than that of the parent compound
(Botsoglou and Fletouris 2001).
The presence of veterinary drug residues in milk is important from the hygienic point
of view as an important quality marker, and also from the technological point of view.
Presence of drug residues poses a risk especially for dairy product manufacture, for it may
interfere with dairy cultures.
The main objective of this study was to determine the effect of the most basic heat
treatment used in dairy industry – pasteurization – on the residues of tetracycline (TTC)
and oxytetracycline (OTC) in milk.
Materials and Methods
Milk samples
Raw cow’s milk was obtained from a vending machine during July and August 2013. All milk samples came
from the Agros Vyškov-Dědice, a.s. farm.
Sample preparation
A preliminary heating of the sample to 40 °C was carried out prior to pasteurization. Then the sample was
cooled to ca 20 °C and subjected to a centrifugation process at 2,490 × g for 10 min in order to remove fatty
constituents. Subsequently, an antibiotic solution was added to create milk samples with concentrations close to
1.5 × MRL (c = 150 µg·l
-1
). The concentration of tetracyclines in the samples was measured before and after heat
treatment (high pasteurization at 85 °C for 3 s).
Pre-analytical processing of the milk samples consisted in precipitation of protein components, which was done
by mixing the samples with McIlvaine buffer with ethylenediaminetetraacetic acid (EDTA) and centrifugation
at 2490 × g for 5 min. The supernatant obtained was adjusted to pH 10 by sodium hydroxide (NaOH) solution
(c = 1 mol·l
-1
). Sample purication was done using a solid phase extraction (SPE) vacuum system and Mixed-
Mode Oasis MAX columns (3 cc, 60 mg, Waters, USA). Subsequently, the sample was eluted by a 45:55 mixture
of acetonitrile and oxalic acid (c = 7.5 × 10
-2
mol·l
-1
). Before analysis the solution was diluted with distilled water
to the volume of 1.5 ml.
Chromatographic conditions
Measurements were carried out using a high performance liquid chromatograph (HPLC) Alliance 2695 with
photo diode array detector (PDA) 2996 (Waters, USA). Separation was performed on a Nova-Pak C8 column,
4 µm, 3.9 × 150 mm, UV detection at 355 nm. Non-linear gradient elution was used. Mobile phase A consisted of
oxalic acid (c = 1.2 × 10
-2
mol·l
-1
) and mobile phase B consisted of a mixture of acetonitrile and methanol (1:1)
with a ow rate of 0.8 ml/min. Column temperature was 35 °C, sample injection 30 µl.
Validation indicators of HPLC method
Method calibration was carried out by means of matrix samples analysis. Standards of OTC, TTC, and CTC
were added to the samples of raw cow’s milk to create concentrations ranging from 0.02 to 1.0 mg·l
-1
, and they
were analysed using a standard procedure. The linearity of determination was established from the values of the
calibration curve and was veried on the basis of the values of the correlation coefcient R (CTC R = 0.994, TTC
R = 0.997, OTC R = 0.988). Recovery and repeatability of the OTC, TTC, and CTC determination was established
by means of parallel analysis of the milk samples with the addition of standards of known concentration at levels
of 0.5 and 0.1 mg·l
-1
for each analyte (Table 1).
S22

Statistical evaluation
The obtained results were processed and evaluated using a paired t-test in STAT Plus (Unistat software ver.
5.1; Unistat Ltd. 1998). This program was also used to establish the basic statistical parameters. Differences were
considered signicant at P ≤ 0.05, highly signicant at P ≤ 0.01.
Results
The effect of heat treatment (high pasteurization at 85 °C for 3 s) on OTC and TTC
residues in milk is shown in Table 2. Table 2 also reports the results of statistical analysis
of measured values. The acquired results conrm the differences in thermostability between
the tetracycline analytes. The mean concentration of TTC was 169 ± 15.84 µg·l
-1
in raw milk
and 159.84 ± 9.23 µg·l
-1
in pasteurized milk. The pasteurization caused a mean decrease
in TTC residues by 5.74%. Paired t-test evaluation of both groups (non-heated and heated
milk) did not reveal any signicant differences (P > 0.05). In the case of OTC samples,
however, the differences in mean values of raw and pasteurized milk were found to be highly
signicantly different (P ≤ 0.01); pasteurization decreased the OTC concentration by 15.3%.
The mean concentration of OTC in milk samples dropped from 163.28 ± 13.94 µg·l
-1
to
138.29 ± 11.35 µg·l
-1
during heat treatment. These results indicated that TTC was more stable
than OTC. Results suggested that heating under pasteurization conditions could cause only
a partial reduction of OTC and TTC residues.
S23
Parameter Oxytetracycline Tetracycline Chlortetracycline
Recovery (%) 93.30 91.50 88.10
RSD (%) 5.10 11.70 9.90
n 12 12 12
Repeatability
RSD (%) 9.26 12.18 11.70
n 15 15 15
Table 1. High performance liquid chromatography method parameters.
RSD = relative standard deviation, n = number of measurements
Oxytetracycline [µg·l
-1
] Tetracycline [µ·gl
-1
]
Raw milk Pasteurized Raw milk Pasteurized
milk milk
n 12 12 12 12
163.28 138.29 169.58 159.84
SD 13.94 11.35 15.84 9.23
Min 135.90 116.10 148.30 143.30
Max 177.10 152.00 201.60 180.00
Paired t-test Highly signicant difference Non-signicant differences
(p = 0.01) (p > 0.05)
Table 2. Statistical evaluation of oxytetracycline and tetracycline residues content in raw and pasteurized milk
(85 °C/3 s).
- arithmetic mean, n - number of measurements, SD - standard deviation, Min - minimum concentration,
Max - maximum concentration
x
x

Discussion
Most foods of animal origin are not eaten raw, but require heat treatment before
consumption. Heat treatment involving varying combinations of time and temperature
depending on the intended use of the starting product – while at the same time ensuring
health quality – is an essential part of industrial treatment of raw milk. Along with ultra
heat treatment (UHT) and sterilization, pasteurization is one of the basic and most common
heat treatment methods. In accordance with current legislation, pasteurization involves
either short time heat treatment at very high temperatures (at least 72 °C for 15 s) or long
time treatment at low temperatures (at least 63 °C for 30 min) and/or the use of any other
combination of time and temperature that has an equivalent effect. Pasteurized milk must
show a negative reaction to alkaline phosphatase test (Commission Regulation 1662/2006).
The effect of heat treatment on residues of antimicrobial substances has been studied
by a number of researchers (Botsoglou and Fletouris 2001; Hassani et al. 2008;
Loksuwan 2002; Hsieh et al. 2011). However, these studies often use matrices other than
milk (e.g. meat, aqueous solution, buffer solution) and they also use different temperatures
and different methods of thermostability evaluation. Very few studies have dealt with the
effects of pasteurization on the concentration of tetracycline residues in milk.
Examples include Loksuwan (2002), who studied the effects of low-temperature long-time
(LTLT) pasteurization (63 °C/30 min) on OTC, CTC, and TTC residues in raw milk. The OTC
residues were in samples with concentration of 100 µg
·l
-1
inactivated to such an extent that
they could not be detected; at concentrations of 200 µg·l
-1
and 300 µg·l
-1
the starting OTC
concentrations were found to have dropped by 86.7% and 79.36%, respectively. In case of
TTC residues, the decrease was distinctively smaller: it amounted to 54.75% in samples with
TTC concentration of 100 µg·l
-1
and to 22.97% and 37.45% in concentrations of 200 µg·l
-1
and
300 µg·l
-1
, respectively. In contrast, degradation of CTC was very limited: the drug residue
concentration decreased by only 9.57% (100 µg·l
-1
), 4.88% (200 µg·l
-1
), and 3.71% (300 µg·l
-1
).
The temperature and time used in our study were different than in the study of Loksuwan
(2002). If we compare the decrease in TTC and OTC concentrations in the two studies,
it is clear that the heat treatment we chose (85 °C/3 s) was less effective in eliminating
tetracycline residues. The temperature we used (85 °C) was higher; however, it appears
that the degradation of residues is affected by the heating time as well, which explains the
signicant difference in the values obtained in the two studies. Both studies show that OTC
in milk is more labile when subjected to heat treatment than TTC. Overall, our data conrm
the published results which show that pasteurization temperatures lead to the decrease
in OTC and TTC residues in milk, but they do not cause complete degradation of the
substances.
Pasteurization is not the only technological procedure employed in the dairy industry.
Other ways of thermal processing of milk include ultra heat treatment (UHT) and
sterilisation, which aim to devitalise the vegetative bacterial forms and spores in raw milk,
including enzyme inactivation, in order to extend the shelf life of milk. Both UHT and
sterilisation use temperatures above 100 °C. Ultra heat treatment requires temperatures of
at least 135 °C for a period of up at least 1 second (usually 3–5 s), which is followed by
aseptic lling (Deeth and Datta 2003; Commission Regulation 1662/2006). Sterilization,
on the other hand, involves continuous sterilization with aseptic lling and hermetic
sealing in nal containers, using an effective heat treatment duration; higher temperatures
are combined with shorter times (Hinrichs and Atamer 2003).
Hsieh et al. (2011) studied the effects of the above heat treatment on tetracycline
thermostability, using double-distilled water as a matrix. They used two different heating
temperatures (100 °C, 121 °C) with the same time of exposure (15 min). Their ndings
show that higher temperatures (121 °C/15 min) cause tetracycline degradation of up
S24

to 99%. At 100 °C the degradation was less extensive, amounting to as little as 54.4%.
The results of this study clearly show that the degree of TTC and CTC degradation is
temperature-dependent. In OTC samples, however, both temperatures led to the same
degree of degradation. In addition to these ndings, tetracyclines have been found to have
different degradation proles at 100 °C. The results of this study demonstrate, similarly as
ours, that even members of the same group of tetracyclines with the same structure may
exhibit vast differences in thermostability. For this reason, the thermostability of drugs
cannot be predicted from their membership in a particular group of medicaments.
Hassani et al. (2008) set out to determine the thermostability of OTC and TTC in
McIlvaine buffer with varying pH value (pH 7.0, 5.5 and 4.0) and in the same McIlvaine
buffer of pH 7 containing sodium chloride (NaCl) at high and ultra high temperatures
ranging from 110–140 °C. The results of the study showed that sterilization (118 °C/30 min
and 121 °C/20 min) reduced the concentration of TTC and OTC to very small, negligible
amounts (less than 0.01%). The UHT, on the other hand, reduced OTC concentrations
by more than 40% and TTC concentrations by roughly 30%. At 135 °C/15 s the UHT
inactivated OTC residues by 44% and TTC residues by 24%. It follows that while the
sterilization process degraded tetracyclines in milk by more than 98%, UHT milk still
retained about 50 to 90% of the initial concentrations of tetracyclines. Differences in pH
levels and water activity (a
w
) had little effect on tetracycline thermostability.
Comparing the results of some of the studies with our own is difcult due to differences
in matrix, result verication methods and applied thermal conditions. The degradation and
inactivation of antibiotic residues during heat treatment can be inuenced by a number
of factors: matrix, temperature, chemical structure of the antibiotic and its stability (the
formation of active and inactive metabolites from the original substance). Other factors can
also impact the results, e.g. the binding capacity of antibiotics to other milk components
and the used methods of antibiotic residue measurement (biological or physicochemical
methods). The basic method of heat treatment of milk used in the dairy industry –
pasteurization – does not ensure a complete degradation of tetracycline antibiotics. From
the technological point of view, the decreasing of TTC and OTC in milk during high-
temperature pasteurization (85 °C for 3 s) determined in this study is not signicant.
Our study conrms that the thermostability of individual drugs varies signicantly
within the group of tetracycline antibiotics. Compared to TTC, OTC displays lower
thermostability during high-temperature pasteurization (85 °C/3 s). The content of OTC
residues dropped by 15.3% when subjected to this type of heat treatment. The content of
TTC residues, on the other hand, dropped by only 5.7%.
Acknowledgements
This work was created with the nancial support of NAZV “KUS” QJ1230044 project.
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