The depletion times of enrofloxacin and its metabolite ciprofloxacin as well as sulfaquinoxaline and oxytetracycline were evaluated in broiler chickens that had been subjected to pharmacological treatment. The presence and residue levels of these drugs in muscle tissue were evaluated using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method that was validated in this work. The results showed the presence of all antimicrobial residues; however, the presence of residues at concentrations higher than the drugs’ maximum residue limit (MRL) of 100 μg kg-1 was found only during the treatment period for oxytetracycline and until two days after discontinuation of the medication for enrofloxacin, ciprofloxacin and sulfaquinoxaline. It was concluded that the residues of all antimicrobials were rapidly metabolized from the broiler muscles; after four days of withdrawal, the levels were lower than the limit of quantification (LOQ) of the method for the studied analytes.
A quantitative and confirmatory high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of bioactive amines in the albumen and yolk of commercial eggs was developed, optimized and validated by analyte extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine standards were used to evaluate the following performance parameters: limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, precision, recovery and ruggedness. The LoD of the method was defined from 0.2 to 0.3 mg kg(-1) for the yolk matrix and from 0.2 to 0.4 mg kg(-1) for the albumen matrix; the LoQ was from 0.7 to 1.0 mg kg(-1) for the yolk matrix and from 0.7 to 1.1 mg kg(-1) for the albumen matrix. The validated method exhibited excellent selectivity and separation of all amines with coefficients of determination higher than 0.99. The obtained recovery values were from 90.5% to 108.3%, and the relative standard deviation (RSD) was lower than 10% under repeatability conditions for the studied analytes. The performance parameters show the validated method to be adequate for the determination of bioactive amines in egg albumen and yolk.
In order to evaluate the efficiency of the pasteurization process in liquid whole eggs, an UV/visible spectrophotometric method was developed and validated for the assessment of alpha-amylase activity. Samples were collected from 30 lots of raw eggs (n = 30) and divided into three groups: one was reserved for analysis of the raw eggs, the second group was pasteurized at 61.1°C for 3.5 minutes (n = 30), and the third group was pasteurized at 64.4°C for 2.5 minutes (n = 30). In addition to assessing alpha-amylase activity, the microbiological quality of the samples was also evaluated by counting total and thermotolerant coliforms, mesophilic aerobic microorganisms, Staphylococcus spp., and Salmonella spp. The validated spectrophotometric method demonstrated linearity, with a coefficient of determination (R2) greater than 0.99, limits of detection (LOD) and quantification (LOQ) of 0.48 mg kg-1 and 1.16 mg kg-1, respectively, and acceptable precision and accuracy with relative standard deviation (RSD) values of less than 10% and recovery rates between 98.81% and 105.40%. The results for alpha-amylase activity in the raw egg samples showed high enzyme activity due to near-complete hydrolysis of the starch, while in the eggs pasteurized at 61.1°C, partial inactivation of the enzyme was observed. In the samples of whole eggs pasteurized at 64.4°C, starch hydrolysis did not occur due to enzyme inactivation. The results of the microbiological analyses showed a decrease (P < 0.0001) in the counts for all the studied microorganisms and in the frequency of Salmonella spp. in the pasteurized egg samples according to the two binomials under investigation, compared to the raw egg samples, which showed high rates of contamination (P < 0.0001). After pasteurization, only one sample (3.33%) was positive for Salmonella spp., indicating failure in the pasteurization process, which was confirmed by the alpha-amylase test. It was concluded that the validated methodology for testing alpha-amylase activity is adequate for assessing the efficiency of the pasteurization process, and that the time-temperature binomial used in this study is suitable to produce pasteurized eggs with high microbiological quality.
The occurrence of antimicrobial resistance in microorganisms isolated from eggshells, especially Enterococcus , might serve as a parameter to assess the selection of bacteria due to the use of drugs in the diet of laying hens. In order to evaluate the frequency and the antimicrobial resistance profile of Enterococcus spp. isolated from the eggshells, 225 Hy-line laying hens were submitted to a 25-d long trial. The treatments were the following: hens fed ration without antimicrobials (control) and groups that received oxytetracycline (10 mg kg −1 ), doxycycline (20 mg kg −1 ), lincomycin (50 mg kg −1 ), and enrofloxacin (10 mg kg −1 ) in the ration for 5 d. Six replications were analyzed per treatment, composed of a pool of 5 eggs each. They were collected before treatment and on days 3, 6, 15, and 25, totaling 150 samples. Eggshells were submitted to counts of Enterococcus spp., which were identified by proteomic analyses. Antimicrobial resistance was determined by the disk-diffusion test. It was observed that 97.3% (n = 146) of the samples were contaminated with Enterococcus spp. There were no differences ( P > 0.05) in the bacterial counts between treatments on the same day of evaluation. E. faecalis and E. faecium were the most frequent on the eggshells of all treatments. Multiresistance to the four classes of antimicrobials was also verified in the isolated bacteria. A total of 83% of the Enterococcus isolates showed resistance to neomycin, which was not administered to the hens, demonstrating an environmental problem. Thus, feeding laying hens with diets added with antimicrobials induces drug resistance in Enterococcus spp., isolated form the eggshells.
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