Introduction

Antibiotics are antimicrobial compounds obtained from the secondary metabolism of microorganisms, which could destroy bacteria or inhibit their growth via affecting their structural or metabolic elements needed for survival. In contrast to primary metabolites, secondary metabolites are not essential for growth and reproduction and are produced by microor-ganisms for various reasons (Welsh et al., 2019).

Long-term consumption of antibiotics at concentrations less than the therapeutic dose (1-10 mg/kg feed) results in an increased growth rate. In such conditions, the yield of feed digestion and weight gain are higher likely due to changes in microbial flora (World Health Organization, 2017). Irregular use of antibiotics for prevention and treatment for an inappropriate time causes the transfer of antibiotics into milk resulting in allergy and pathogenic bacterial resistance which is an important concern (Tumini et al., 2019). Milk is exposed to various chemical contaminations including hormones, antiseptics, nitrites, nitrates, and nitrosamine, insecticides, fungal toxins, toxic metals, dioxins, and antibiotics. These substances enter milk through medicines, rations, and a milking environment (Meyer, 2000).

Antibiotics are used to control systemic and localized infections in dairy herds, among which mastitis is the main reason for consuming antibiotics. In comparison with the water-soluble antibiotics, fat-soluble antibiotics may remain in the udder for a longer time. Scientists found that the addition of antibiotics and anabolic compounds, such as artificial estrogen into feed could elevate the weight gain of livestock. Therefore, antibiotics and estrogens were commercially produced, and the Food and Drug Administration (FDA) permitted the consumption of these compounds (Kang et al., 2005).

Various studies have been conducted to determine the contamination of milk and its products with antibiotics in different countries. Antibiotic residue in milk is not a new concern. Studies conducted before 1960 showed that 6% of milk in the US market contained antibiotics residue. This rate decreased to 3.7% after 1960. However, 7%-15% of milk specimens in 1975 contained detectable levels of antibiotics. A study conducted on 1100 milk samples selected from a milk tank in 1963 in France demonstrated that 3.6% of the specimens had penicillin. In Kenya, studies carried out on pasteurized and non-pasteurized milk collected from local suppliers indicated that 16% of milk samples were contaminated with antibiotics (Kang et al., 2005).

 In Iran, many studies have been conducted on antibiotics in milk. An investigation in Qazvin revealed that 43% of the milk samples were contaminated with antibiotic residues. The cause of this high contamination rate was stated to be the irregular use of antibiotics in cattle farms. In addition, another study was conducted in 2014 in different parts of Iran on raw and pasteurized milk samples and it was observed that 30.76% of raw milk and 22.2% of pasteurized milk obtained from milk processing plants and retail shops contained antibiotics, respectively (Razzagh Mahmoudi et al., 2014).

Antibiotics residue in milk is undesirable because it may cause antibiotic resistance by spreading antibiotic-resistant strains. Furthermore, some people, especially infants whose diet is mainly milk might develop an allergic reaction to antibiotics (Priyanka1 et al., 2017). This is supported by the susceptibility of some people to an insignificant amount of penicillin in foods.

In addition, antibiotics in milk destroy the beneficial bacteria used in making cheese yoghurt, and other fermented pro-ducts.Therefore, they reduce the quality and quantity of products (Priyanka et al., 2017). Milk is an important part of the daily diet and is easily contaminated with antibiotic residues. Given the fact that milk contaminated with antibiotics cannot be used for producing dairy fermented products, it is mainly utilized for producing pasteurized and sterilized milk resulting in many health problems in the consumers. The aim of this study was to compare YCT, FPT, and Copan test to identify antibiotic residues in raw and pasteurized milk produced in Chaharmahal and Bakhtiari province, Iran.

Materials and Methods

Sampling

A total of 200 milk samples (146 raw milk samples and 54 pasteurized milk samples) were randomly collected from farms and dairy processing plants during Des 2018-June 2019.  Samples were transferred to the laboratory on ice.

YCT

 First, fresh healthy milk was skimmed by centrifuge, heated, and sterilized at 121°C for 10-15 min. Next, the samples were cooled to 42-44°C and 10-15 gr starter granules containing Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were added to 0.5 L of sterilized milk. The inoculated milk was incubated at 42°C for 3-4 h until acidity reached 100° dormice. Afterwards, it was immediately transferred to the refrigerator. The obtained culture is known as mother culture (the Samples were stored at 5°C).

The samples were homogenized and heated to 85°C for 30 min in a warm water bath (Memmert WNB Co., Germany). The temperature of samples was rapidly reduced to 42-43^oC and 1 mL of fresh well-stirred lactic inoculum (homogenized) was mixed with the milk sample. Next, 0.1 mL of red methyl reagent was added to the samples and were incubated at 42°C for 3 h, and color variations in the samples were checked. An antibiotic-free sample was used as negative control and a sample with antibiotics was applied as a positive control (penicillin and tetracycline, 0.5 ppm) (Khaled, 2004).

Antibiotic-free samples showed pink color after incubation because of the growth of starter bacteria leading to lactic acid production, while positive samples remained orange without acid production. Moreover, the presence or absence of lactic coagulation in samples was obvious from their consistency. The acidity was measured by an electric pH meter (Metrohm, Switzerland). The acidity of suspicious samples was about 10°D less than that of natural milk indicating the presence of antibiotics or other inhibitory compounds. The samples with pH>5.3 were considered positive samples (Tumini et al., 2019).

FPT

The FPT has been recently investigated and its sensitivity as a standard method for identification of antibiotic residue has been approved by the EU. Five classes of antibiotics, including beta-lactam, tetracycline, sulfonamide, aminoglycoside, and a macrolide could be identified by this test (Koenen-Dierick et al., 1995) (Table 1).

Table1. Identifiable medium and antibiotics by the four-plate test

To prepare 100 mL of medium, 0.345 gr of both casein and meat peptone, 0.5 g sodium chloride, and 1.3 g trimethoprim agar were weighed and dissolved in 100 mL distilled water. Next, 0.24 gr trisodium phosphate was added to the prepared medium. The pH values of the prepared medium were adjusted by a digital pH meter with acetic acid and sodium hydroxide as 6, 7.2, and 8. The media were sterilized at 121°C for 15 min in an autoclave (Zaim Mega F.I.M, China).

The absorbance of the two bacterial cultures Bacillus subtilis (Ptcc 1365) and Micrococcus luteus (PTCC 1169) was read at 580 nm by a spectrophotometer (Spectronic, Sweden) (Society for General Microbiology, 2006). Afterwards, 0.1 mL of each dilution of B. subtilis was added to 100 mL of the medium at pH 7, 7.2, and 8. In addition, 0.1 mL of the dilution of M. luteus was added to 100 mL of the medium with pH 8.

Blank disks with a thickness of 2 mm were coated with raw and pasteurized milk samples and were placed on the media along with positive and negative control samples. The plates with four pH values containing B. subtillis at 30°C and the plates containing M. luteus at 37°C were incubated for 24 h. Next, the inhibition zone was measured by Collis Vernier, and inhibition zones equal and larger than 2 mm in diameter were considered as a positive sample (Gaudin et al., 2004). The inhibition zone in the FPT is only observed when antibiotics residue exceeds the permitted level.

Copan Test

Copan test is used for detecting antibiotics residue in raw, heated, and dry milk samples. Copan kit (Chr. Hansen, Denmark) is used for the qualitative monitoring of antibiotics in milk. It has 96 microplates containing agar with the spores of B. stearothermophilus var. calidolactis, as well as glucose and Bromocresol purple (Forouzan et al., 2014).

About 100 μl of milk sample was poured on the agar and put in a water bath at 64±1°C for 3 h. In the absence of antibiotics, the spore grows, ferments glucose, and produces acid. Consequently, the color of the medium changes to yellow.

Comparison of Tests for Detecting Tetracycline and Penicillin

First, healthy antibiotic-free milk was obtained from a healthy cow. It was homogenized for 5 min, heated at 85°C for 30 min, and tested for antibiotics by Copan test. Afterwards, dilutions 0.1, 0.5, 1, 2, 5, 10, and 20 mg/L of crystalline penicillin, as well as 0.1, 0.5, 1, 10, 25, 50, and 100 mg/L of tetracycline were added to milk. Negative and positive samples were prepared from antibiotic-free and antibiotic-containing milk, respectively. Finally, the samples were evaluated by YCT, FPT, and Copan test.

Statistical Analysis

Data were analyzed by SigmaStat 4 software using the Cochran test. P-value

Results

The number of samples containing antibiotics residue is given in Table 1. Eleven (7.5%) and 13 (8.9%) raw milk samples were positive by YCT and Copan test, respectively. Moreover, regarding the samples of pasteurized milk, 5 (9.3%) and 6 (11%) samples were positive with YCT and Copan test, respectively. Three (2%) raw samples and 1 (1.8%) pasteurized milk sample were positive by FPT (Figure 1). In the plate containing B. subtillis at pH 6, zones of inhibition >2 mm in diameter were observed indicating the residues of penicillin and tetracycline (Table 2).

The Chi-square test showed no significant difference between YCT and Copan test (P>0.05). However, significant differences were found between FPT and YCT, as well as between FPT and Copan test (P<0.05).

Table 2. Occurrence of antibiotics residue in raw and pasteurized milk producing in chaharmahal and Bakhtiari province-Iran.

Figure 1. Antibiotic residues in raw and pasteurized milk determined with YCT and Copan tests.

According to our results, the limit of detection of FPT was 313 and 500 ppb for penicillin and tetracycline, respectively. The limit of detection for YCT was 60 ppb for penicillin and 100 ppb for tetracycline. However, for Copan test was 6 and 100 ppb for penicillin and tetracycline, respectively (Table 3).

Table 3. Detection the limit of yogurt, four-plate, and Copan tests forpenicillin and tetracycline in milk.

Discussion