Abstract
Background/Aim: The presence of tetracycline (TC) and its residues in raw milk and milk dairy products poses a threat to human health due to the induction of antibiotic resistance of bacteria that can be transmitted between animals, humans, and the environment. The aim of this study was to investigate the transfer of TC from raw milk to different dairy products: pasteurized milk, boiled milk, sour milk, skimmed milk, and cottage cheese. We analyzed samples of milk from different sources: household farmers, local farms, and milk factories. Materials and Methods: The analyses of TC in milk and dairy products were performed using colloidal gold immunochromatography assay (GICA) and enzyme-linked immunosorbent assay (ELISA). Results: The highest content of TC was found in the milk purchased from local household farmers; therefore, these samples were chosen for the study of TC transfer to dairy products. TC was also found in sour milk at levels comparable with those obtained in raw milk. The average TC content decreased following heat treatment of the milk, as follows: for pasteurized milk 22.07% and for boiled milk 29.35%. The highest concentrations were determined in cottage cheese in the range 200-620 μg/kg. Conclusion: TC residues are transferred from milk to dairy products in various amounts depending on the preparation conditions, and due to their chemical properties, they accumulate in concentrated derivatives, such as cheese. Therefore, TC can be identified even in cheeses prepared from milk with undetected antibiotic levels.
Among the agricultural-food industries of the European Union, milk dairy production is one of the biggest industries (1), producing raw milk and different processed products. Therefore, research in milk industry is an important current topic with many and various directions. The most recent scientific directions include: technological solutions for milk processing (1), food-monitoring technologies (2), sensors for antibiotic detection (3) or environmental monitoring (4, 5), evaluation of bacterial diversity (6), developing methods for the determination of antibiotics in milk (7, 8), food security (9), impact on the human health (10).
Antibiotics are used in veterinary medicine for the treatment of bacterial infections and infectious diseases caused by other microorganisms. There are many different classes of oral or injectable antibiotics for veterinary use, depending on the bacteria or organisms causing the infection. In addition to the use of antibiotics for medical purposes, they can be used to induce faster growth in animals and to prevent infections that could occur in animals in the case of large groups (11).
When antibiotics are administered to an animal, it takes time for the antibiotics to be completely degraded and eliminated from the animal’s body, leading to the contamination of food products with antibiotic residues. Antibiotic residues can cause health problems in people who consume contaminated food (12, 13). The bioaccumulation of antibiotic residues in humans, on long-term, can lead not only to bacterial resistance (14), but also to hypersensitivity reactions (15), gastrointestinal and liver issues (16), as well as cancer and mutagenesis (17). Moreover, antibiotics used in the treatment of animals end up in waste (manure), leading to soil pollution (18, 19), changing its physical-chemical composition and enzymology (20, 21), and modifying its productivity, quality and activity correlated with agriculture, on short/medium and long term.
AdHoc Expert Group on Antimicrobial Resistance Recommendations (AMEG) classified antibiotics according to the potential public health consequences of increased antimicrobial resistance caused by use in animals, as well as according to the need for their use in veterinary medicine: Category A — to be avoided; Category B — restricted; Category C — administered with caution; Category D — administered with prudence.
That is why the most used antibiotics are those from category D (22). Tetracycline (TC) belongs to group D and exhibits widespread action against a range of atypical pathogens, some protozoan parasites, and some Gram-positive and Gram-negative bacteria (23).
TC has a rigid four-ring skeleton and numerous groups, including alkyl, hydroxyl, and amine. The first modern TC introduced into therapy was tigecycline (Figure 1A), followed by omadacycline (Figure 1B), eravacycline (Figure 1C), and sarecycline (Figure 1D).
Chemical structures of tigecycline (A), omadacycline (B), eravacycline (C), and sarecycline (D).
Typically, TCs are classified into three generations; the first generation includes biosynthesis derived TCs, such as chlortetracycline, oxytetracycline, and demeclocycline. Doxycycline, lymecycline, meclocycline, and metacycline are second-generation TCs. TC was partially synthesized to produce minocycline and roliTC. These compounds also belong to the second generation TCs. Tigecycline, which is made entirely synthetically, is included in the third generation. Modern third generation TCs include derivatives of sarecycline with more or less similar chemical structures (24).
TCs are extremely popular in developing countries because they are relatively inexpensive and possess broad-spectrum antimicrobial properties. They have applications in medicine, particularly in the management of infections of the gastrointestinal tract, respiratory system, skin, locomotive organs, or genito-urinary tract, as well as systemic infections and sepsis (25, 26). However, TC residues ingested through food, can affect the growth of the fetus and infants (since they pass through the placenta and into milk) and the development of bones and teeth (27).
Antibiotic residues in foods may alter the intestinal microflora, metabolic activity, and endogenous compound metabolism (28). To prevent adverse impacts on humans, the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) established guidelines for acceptable daily intake (ADI) and maximum residue levels (MRLs) in foods. An MRL of 100 μg/kg for TC, OxyTC (OTC) and ChlorTC (CTC) has been established (27). Even if the MRL for antibiotics in foods of animal origin products are not exceeded, they can still lead to health problems on long term (29, 30), because they could cause bacterial resistance or teratogenicity risk in case of first trimester pregnant woman (31).
Different methods, such as thin layer chromatography (TLC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) have been developed for the quantitative, accurate, and reliable measurements of TC in milk and animal tissues (7, 32). All these techniques require complex, expensive equipment and complex procedures involving the use of many reagents and chemicals. Ready-made test kits are also often used to detect a range of antibiotic residues easily, when other laboratory facilities are not available. In some cases, the test kits are initially used to detect antibiotic residues in the samples and then subject them to a more detailed examination method to determine the residues more precisely (29).
Colloidal gold immunochromatography assay (GICA), a novel convenient and rapid immunological assay with numerous applications in the field of animal medicine (33-37) was used to identify TC residues. Confirmation of the presence of TC residues was followed by quantitative analysis using the enzyme-linked immunosorbent assay (ELISA) technique (38).
This study aimed to investigate the presence of TC residues in raw milk purchased from different sources in northwestern Romania, thus evaluating their quality control policies. Because the TC residues have been shown to be transferred to different dairy products from previously contaminated milk (by the addition of specific antibiotic amounts) (39), the presence and transfer of TC were studied in dairy products from raw milk, which originates from different sources, even if the levels of TC in milk were below the detection limits. We also examined the sources of milk with the highest concentrations of antibiotics and determined how the concentration is influenced following the processing of milk into dairy products.
Materials and Methods
Study design. In order to determine the presence of TC residues in milk samples, BALLYA rapid immunological lateral flow TC test kits (BALLYA, Guangzhou, PR China) were utilized (40, 41). This test is a colloidal gold immunochromatography assay (GICA) that was developed specifically for the quick, easy, and on-site identification of TC and its residues. It utilizes test strips, being compliant with the European Union maximum residue levels (MRL) (42) and has a detection range between 30-50 μg/kg. This assay integrates the antigen-antibody immune reaction with colloidal gold labelling. Due to the numerous benefits that this assay offers, including its speed, ease of use, low cost, excellent stability and no need for specialized, high-priced chemicals and apparatus, it has widespread application in the fields of clinical diagnosis and food analysis. The TC rapid test kit is a qualitative assay that identifies the presence of TC antibiotics residues in cow’s milk. Before use, the components of the kit are stored at room temperature, 22°C. Double distilled water was added to milk samples in a ratio of 2:1 before they were processed. The coating conjugate powder was dissolved and shaken up and down 4-6 times. After the sample was produced, it was allowed to sit for two min at room temperature and then the test results were read and interpreted.
The positive TC raw milk samples were subjected to Enzyme-Linked Immunosorbent Assay (ELISA), to allow the quantitative determination of total active TC molecules and its metabolites furazolidone, furaltadone and nitrofuran compounds.
For quantitative analysis of TCs, Ridascreen® Tetracycline R3505 (R-Biopharm AG, Darmstadt, Germany) – a competitive enzyme immunoassay for the quantification of tetracyclines in food samples that have very high DL, suitable for milk, cheese, and other types of dairy products – was used (43). Enzyme-linked immunosorbent assay (ELISA) begins with a coating step, in which the first layer (consisting of a target antigen or antibody) is absorbed onto a 96-well polystyrene plate. This is followed by a blocking step in which all unbound sites are coated with a blocking agent. Following a series of washes, the plate is incubated with enzyme-conjugated antibody. Another series of washes removes all unbound antibody. A substrate is then added, producing a colorimetric signal. Finally, the plate is read using a STAT FAX 2100 Microplate Reader spectrophotometer (Awareness Technology Inc., Ramsey, NJ, USA).
The test package includes all the reagents that are necessary to conduct an enzyme immunoassay. The manufacturer’s instructions were followed for the samples’ preparation and for the analysis procedure. Standard TC solution was provided in the test kit. The obtained standard curve was similar with the curve presented in the kit; therefore, the following kit instructions were followed step by step. The samples were centrifuged at 3,000 rpm/865×g for 10 min, at 10°C for the milk, 15 min at 4°C for the cheese, and 10 min at 10°C for the sour milk (method provided by the ELISA Kit) to separate the upper cream layer. The milk, cheese, and sour milk samples were diluted: 1:1, 1:5, and 1:10, respectively, considering the TC concentration calculus. To perform TC analysis, 50 μl of each standard solution or sample was added in each well. Then, 50 μl antibody was added, followed by incubation at room temperature for 1 h. The wells were washed twice using the buffer solution provided in the kit. Afterwards, 100 μl enzyme conjugate solution was added in each well, followed by incubation at room temperature for 15 min. The wells were washed twice and then 100 μl of substrate was added to each well and incubated in the dark, at room temperature for 15 min. Finally, 100 μl of stop reagent was added to each well and the absorbance at 450 nm was measured using the STAT FAX 2100 Microplate Reader. All samples were measured twice, by two different people and for the evaluation of test results, RIDA SOFTWIN software (Art. No. Z9999, R-Biopharm) was used.
The Hanna Instruments edge specialized pH-meter, model HI 2002 (Hanna Instruments Ltd, Leighton Buzzard, Bedfordshire, UK) was utilized to determine the pH levels of both the milk samples and the dairy products. All the measurements have been made at room temperature, 22°C.
EBA200 Hettich (Hettich, Kirchlengern, Germany) were utilized to skimming the samples. The Millipore Milli-Q academic (Merck KGaA, Darmstadt, Germany) instrument was used to produce HPLC grade water.
Samples preparation. Thirty milk samples, originating from a different location, were analyzed: 10 were collected from individual farmers, 10 were collected from nearby farms, and 10 were collected from milk factories, along with some dairy products that were obtained using the milk samples (pasteurized milk, boiled milk, sour milk, skimmed milk, cottage cheese). The investigation focused on milk that originated from the region of Bihor County, in the northwest of Romania. Samples were collected from August to September 2022 because summertime is peak infection season for livestock, which means that antibiotic administration is performed more frequently during that time.
The milk was heated to 63°C for 30 min to allow its pasteurization. The boiled milk was obtained by heating the milk to the boiling point, in a water bath for 15 min. To acquire skim milk, raw milk had to be stored in a refrigerator at a temperature of 7°C for 12 h before the fat was removed from the milk. The obtained raw milk was allowed to sit at ambient temperature for 36 h, and it was then homogenized before being used to produce sour milk. Cottage cheese was made from sour milk by heating the milk to 48-50°C, allowing it to chill for 50 min, then filtering it and allowing it to drain for 12 h (Figure 2).
A schematic of the manufacture of milk dairy products.
Results
In the first part of the study, the presence of TC in raw milk was investigated using the GICA method and the BALLYA TC Test Kit. The raw milk came from a variety of sources, including household farmers (Figure 3A), nearby farms (Figure 3B), and a milk factory (Figure 3C). Each sample of milk was tested four times to avoid any detection errors, and the average value was used in Figure 3. A total amount of 120 analyses were done. The TC amounts were determined using the ELISA test kit. A total of ten samples of each variety of raw milk were analyzed. The quantity of TC in some of the samples was below the detection limit (DL) of the GICA method, which was set at 30 μg/kg. As can be seen in Figure 3 and Figure 4, two samples of milk from household farmers, four samples from nearby farms, and six samples from the milk factory had TC levels below DL (Figure 5). The maximum residue levels (MRL) for TC in foods is established at 100 μg/kg. The MRL was exceeded in four samples gathered from householder farmers and one sample gathered from local farm. If the detection limit of 50 μg/kg of the BALLYA test was exceeded in the analyzed samples, 2:1 dilutions were made with double-distilled water, and the determinations were repeated.
Detected amount of tetracycline residue (μg/kg)-average values for each of the 10 samples of raw milk from A). household farmers; B) a local farm; C) a milk factory, using enzyme-linked immunosorbent assay (ELISA) test kit.
The proportion of compliant and non-compliant samples of analyzed raw milk.
Tetracycline average values in the analyzed raw milk samples.
As demonstrated in Figure 4 it should not be surprising that the milk production environment has a direct correlation with the percentage of compliant and non-compliant samples of analyzed raw milk. The percentage of noncompliant samples was 40% for household producers, 10% for local farm, and 0% for milk factory.
Milk samples with TC levels under the DL were discovered in proportions of 20% for household farmers, 40% for local farms, and 60% for milk factories. Traces of TC were discovered in raw milk in the following concentrations: 30-121 μg/kg for milk produced by household producers; 30-103 μg/kg for milk produced by local farms; and 30-97 μg/kg for milk produced by milk factories. Figure 5 illustrates the typical levels of TC found in samples derived from a variety of different kinds of raw milk.
There are some additional significant points that should be taken into consideration. The samples of milk from the factory are the final marketed product, and it is common knowledge that the actual raw milk from cows comes from different sources/producers, and the raw milk is subjected to a process of standardization (27). Figure 6 and Figure 7 show the decrease of TC amount in the controlled environment of milk production.
Comparative amounts of tetracycline in different dairy products and raw milk for the ten milk samples from household farmers.
Tetracycline average values for each milk dairy product from household farmers.
Similar average values of TC amount were found in the samples from milk factory and local farm. These values were very close to the test’s lower detection limit of 30 μg/kg. Considering the samples’ average TC values, we find that they were very close to complying with the requirements, in all the environments.
Because the milk purchased from local household farmers contained the greatest concentration of TC, these milk samples were used for the study of TC transfer to dairy products. Using an ELISA Biopharm Ridascreen tetracycline test kit, the levels of TC in the dairy products obtained from raw milk produced by household farmers were determined, and the findings are depicted in Figure 6.
Cottage cheese and sour milk are the milk dairy products that were considered for these subsequent investigations. Because the TC amount in samples 1 and 2 was below the DL, these samples were considered to contain 30 μg/kg. Figure 7 presents the TC values that are typical for each of the 10 examples of dairy products.
The pH values of samples of milk and dairy products from local farmers were within the allowed limits; the average values obtained were: 6.7 for raw milk, 6.7 for skimmed milk, 4.83 for sour milk and 5.00 for cottage cheese.
The following observations can be made after comparing the typical concentrations of TC found in raw milk and various dairy products. The ranges of TC content of the samples were: 30-98 μg/kg for pasteurized milk, 30-88 μg/kg for boiled milk, 30-119 μg/kg for sour milk, 30-109 μg/kg for skimmed milk and 98-620 μg/kg for cottage cheese. The average amount of TC found in dairy products was compared to the average amount of TC found in raw milk from householder producers, which was 83.8 μg/kg.
After the heat treatment of the milk, changes in the average TC content were detected; specifically, a decrease of 22.07% for pasteurized milk and a decrease of 29.35% for boiled milk. The TC content of the pasteurized milk was the lowest. When compared to raw milk, the amount of TC residue found in sour milk showed a slight reduction of 1.55%. TC was found to be 11.69% lower after skimming. This reduction in comparison to that of sour milk may be explained by the fact that during the process of milk skimming, a small portion of the TC residues passes into the fat fraction (cream).
The highest TC amounts were identified in cottage cheese in the range of 109-620 μg/kg, with an increase in the average value of 437.7%. Samples 1 and 2 of cottage cheese provided conclusive evidence that the samples of raw milk also contained TC, although at a concentration below the detection level.
Discussion
The treatment and prevention of illnesses or acceleration of animal development are two common uses of antibiotics in the animal production sector. Antibiotic residues in milk represent a major public health concern. This research indicates that more than 40% of samples from household farmers exceeded the allowed TC amount (100 μg/kg), as has also been reported in other regions of the world including Italy (8), Poland (6), Serbia (44), or Iran (31). Inadequacies in the vaccination strategy may lead to the introduction of infectious diseases into herds of cattle, necessitating the administration of antibiotics. The inappropriate use of antibiotics in both human and veterinary medicine has contributed to the contamination of the environment, the disruption of the delicate microbial equilibrium in ecosystems, and the proliferation and selection of organisms that are resistant to antibiotics (45, 46).
It was found that the content of TC in milk decreased following heat treatment of milk by an average of 22.07% for the milk that was pasteurized at 63°C and 29.35% for the milk that was boiled at 100°C, these reductions being caused by the TC’s thermal instability (43).
The modest reduction in the amount of TC found in sour milk in comparison to raw milk can be attributed to the change in pH, from 6.7 (raw milk) to 4.83 (sour milk), as well as the TC instability in acidic medium (47). Following the skimming procedure, the transfer of TC residues from raw milk to cream was 11.16%.
The increased levels of TC metabolites found in cottage cheese, which reached up to 620 μg/kg, are consistent with the expected rise given that TC accumulates in this product. It is common knowledge that to produce one kilogram of cheese, four to five litters of milk are required. Considering this, the high concentration of TC found in cheese could also be explained by the fact that TC-based drugs contain many hydroxyl groups, enol hydroxyl groups, and carbonyl groups, which can create insoluble chelates with a wide variety of metal ions, such as calcium or magnesium.
The presence of traces of TC in raw milk highlight the requirement for a more comprehensive supervision of milk production in the household factories and small factories in Romania. The obtained results demonstrate that the TC residues not only transfer to dairy products (in various amounts depending on the preparation conditions) but, due to their chemical properties, accumulate in the concentrated derivatives such as cheese, therefore TC content could be measured in prepared cheese even using milk with undetectable levels of antibiotic.
The originality of the study consists in the fact that, at least in Romania, there is no public scientific information regarding the presence of antibiotics in milk/milk products. An appropriate withdrawal interval following any medication treatment must be respected before milk collection. Because of these factors, regulatory authorities ought to implement stringent controls over the administration of these medicines and the vaccination of livestock. Although in the antibiotic administration leaflet there is a recommendation that milk should not be consumed during the treatment or a few days after stopping the antibiotic treatment, this is difficult to monitor, at least at the level of individual farmers. This raises the issue of health education, and at the same time finding other treatment solutions, more friendly to the environment, to replace the use of antibiotics.
The limitations of the study include the relatively small number of samples and the short time interval during which the samples were collected. The study will be continued over a longer period and obviously using a higher number of samples to increase the degree of relevance of the experimental determinations. However, the advantages of this study lie in the fact that it brings to the attention of the public and specialists the degree of contamination with antibiotics, which is always higher and increasing. Therefore, an environmentally friendlier approach regarding the consumption and discharge of antibiotics is required, as well as appropriate policies to stop the contamination of the environment with such substances.
Conclusion
Considering the obtained results, milk obtained from milk factories or local farms is more reliable; underlining that it is safer to consume milk from a controlled environment. However, consuming cheese or other concentrated milk products from less monitored sources, prepared by householding farmers, could be questionable. If more milk and other dairy products are consumed, there is a greater probability that the maximum residue levels of TC will be exceeded somewhere along the food chain. Our research revealed that the majority of the raw milk samples examined complied with the standards, except for the samples collected from the local farmers. The overall average TC concentrations, for all the studied milk samples, which range from 52.6-83.8 μg/kg, was within the allowed level, which is defined as being lower than 100 μg/kg. However, the findings acquired for cottage cheese, which had an average TC value of 366.8 μg/kg, unequivocally point to an accumulation, and consequently, a possible risk. This is because milk and other dairy products contain trace amounts of antibiotics.
Monitoring the amount of antibiotics that are present in food is one of the methods that can be used to anticipate potential dangers to human health and, as a result, prevent these dangers from occurring.
Footnotes
Authors’ Contributions
Conceptualization, Alexandrina Fodor; Data curation, Alexandrina Fodor and Gabriela Badea; Investigation, Alexandrina Fodor, Anda Ioana Petrehele, Alina Groze; Methodology, Gabriela Elena Badea and Delia Mirela Tit; Software, Alexandrina Fodor and Delia Mirela Tit; Supervision, Delia Mirela Tit and Simona Gabriela Bungau; Validation, Alexandrina Fodor; Visualization, Gabriela Elena Badea and Delia Carmen Nistor Cseppento; Writing – original draft, Alexandrina Fodor, Gabriela Elena Badea and Simona Gabriela Bungau; Writing – review & editing, Delia Carmen Nistor Cseppento, Delia Mirela Tit, Gabriela Elena Badea and Simona Gabriela Bungau. All Authors have read and agreed to the published version of the manuscript.
Funding
University of Oradea supports the APC through an internal project.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received May 1, 2023.
- Revision received May 18, 2023.
- Accepted May 25, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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