Comprehensive Screening of Salinomycin in Feed and Its Residues in Poultry Tissues Using Microbial Inhibition Tests Coupled to Enzyme-Linked Immunosorbent Assay (ELISA).

Salinomycin is a coccidiostat approved as a feed additive for the prevention of coccidiosis in poultry. Official control of its residues is set by the Commission Delegated Regulation (EU) 2022/1644. The aim of our study was to assess the suitability of three microbial inhibition tests (MITs), Premi®Test, Explorer 2.0, and the Screening Test for Antibiotic Residues (STAR) linked to the enzyme-linked immunosorbent assay (ELISA), for the screening of salinomycin residues in the tissues of broiler chickens (breast and thigh muscle, heart, liver, gizzard, kidneys, lungs, spleen, skin, and fat) fed commercially produced feed containing 70 mg.kg-1 of salinomycin in the complete feed. The first residue screening (Sampling A) was performed on the last day of administration of the salinomycin-medicated feed (day 30), and the second screening (Sampling B) was performed on the day of slaughter (day 37) after the expiry of the withdrawal period with the feeding of non-medicated feed. Based on the quantitative confirmation of salinomycin residues in the examined chicken tissues by the ELISA method (Sampling A from 0.025 to 0.241 mg.kg-1; Sampling B from 0.003 to 0.076 mg.kg-1), all the MITs with the preference of the bacterial strain Bacillus stearothermophilus var. calidolactis ATCC 10149 demonstrated the ability to detect the residues of salinomycin in the examined tissues of broiler chickens at the level of the maximum residue limits set from 0.015 to 0.150 mg.kg-1 by Commission Implementing Regulation (EU) 2017/1914 and confirmed the relevance of their sensitivity to the coccidiostat salinomycin.

Influence of proline and hydroxyproline as antimicrobial and anticancer peptide components on the silver(I) ion activity: structural and biological evaluation with a new theoretical and experimental SAR approach.

Silver(I) complexes with proline and hydroxyproline were synthesized and structurally characterized and crystal structure analysis shows that the formulas of the compounds are {[Ag2(Pro)2(NO3)]NO3}n (AgPro) (Pro = L-proline) and {[Ag2(Hyp)2(NO3)]NO3}n (AgHyp) (Hyp = trans-4-hydroxy-L-proline). Both complexes crystallize in the monoclinic lattice with space group P21 with a carboxylate bidentate-bridging coordination mode of the organic ligands Pro and Hyp (with NH2+ and COO- groups in zwitterionic form). Both complexes have a distorted seesaw (C2v) geometry around one silver(I) ion with τ4 values of 58% (AgPro) and 51% (AgHyp). Moreover, the results of spectral and thermal analyses correlate with the structural ones. 1H and 13C NMR spectra confirm the complexes species' presence in the DMSO biological testing medium and their stability in the time range of the bioassays. In addition, molar conductivity measurements indicate complexes' behaviour like 1 : 1 electrolytes. Both complexes showed higher or the same antibacterial activity against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus as AgNO3 (MIC = 0.063 mM) and higher than silver(I) sulfadiazine (AgSD) (MIC > 0.5 mM) against Pseudomonas aeruginosa. In addition, complex AgPro exerted a strong cytotoxic effect against the tested MDA-MB-231 and Jurkat cancer cell lines (IC50 values equal to 3.7 and 3.0 µM, respectively) compared with AgNO3 (IC50 = 6.1 (5.7) µM) and even significantly higher selectivity than cisplatin (cisPt) against MDA-MB-231 cancer cell lines (SI = 3.05 (AgPro); 1.16 (cisPt), SI - selectivity index). The binding constants and the number of binding sites (n) of AgPro and AgHyp complexes with bovine serum albumin (BSA) were determined at four different temperatures, and the zeta potential of BSA in the presence of silver(I) complexes was also measured. The in ovo method shows the safety of the topical and intravenous application of AgPro and AgHyp. Moreover, the complexes' bioavailability was verified by lipophilicity evaluation from the experimental and theoretical points of view.

Humic Substances as a Versatile Intermediary.

Humic substances are organic ubiquitous components arising in the process of chemical and microbiological oxidation, generally called humification, the second largest process of the carbon cycle. The beneficial properties of these various substances can be observed in many fields of life and health, whether it is the impact on the human organism, as prophylactic as well as the therapeutic effects; animal physiology and welfare, which is widely used in livestock farming; or the impact of humic substances on the environment and ecosystem in the context of renewal, fertilization and detoxification. Since animal health, human health and environmental health are interconnected and mutually influencing, this work brings insight into the excellence of the use of humic substances as a versatile mediator contributing to the promotion of One Health.

The Effect of the Supplementation of Humic Substances and Fermented Products in the Feed on the Content of Salinomycin Residues in Poultry Tissues.

The presence of antimicrobial residues in products of animal origin is a constant problem for consumer health. The aim of this study was to observe the effect of the addition of humic substances (H), fermented products (F) and a mixture of both (FH) to feed supplemented with the coccidiostat salinomycin, compared with a control group (C), on the content of salinomycin residues in the edible tissues of broiler chickens using two microbial inhibition screening methods, Explorer 2.0 test and the Screening Test for Antibiotic Residues (STAR), and a confirmatory competitive enzyme immunoassay analysis (Salinomycin ELISA Kit). The results of the microbial inhibition tests showed a gradual decline in the positive results in the tissue samples from the last day of salinomycin administration (30th day) tothe last day of fattening (37th day, day of slaughter) in group C and no positive results in the tissue samples from experimental groups H, F and FH slaughtered on the last day of fattening. Using the Salinomycin ELISA Kit, salinomycin was detected in the chicken muscle tissues of all the control and experimental groups. However, no sample from any group contained salinomycin at a concentration exceeding the maximum residue limits set by European law. The high level of significance (p < 0.001) confirmed the positive influence of the administration of humic substances and fermented products on the content of salinomycin residues in chicken tissues.

Detection of Gluten in Gluten-Free Foods of Plant Origin.

The work deals with the issue of standardization and more accurate methodology for the isolation of gluten DNA in gluten-free products of plant origin, which is more demanding due to the more complex structure of plant cells. Three isolation methods were compared, of which the combination of glass and zirconium beads, Proteinase K and a commercially produced isolation kit was confirmed to be the most effective procedure. The given isolation procedure was more effective in one-component gluten-free foods, where the concentration of the obtained DNA ranged from 80.4 ± 0.7 to 99.0 ± 0.0 ng/µL. The subsequent PCR reaction revealed the presence of gluten not only in guaranteed gluten-free products (40%), but also in naturally gluten-free foods (50%). These were mainly gluten-free sponge cakes, gluten-free biscuits "Cranberries", cocoa powder, coffee "3in1", and instant coffee.

Determination of lasalocid residues in the tissues of broiler chickens by liquid chromatography-tandem mass spectrometry.

Lasalocid is a polyether ionophoric coccidiostat used for the prevention of coccidiosis in poultry at a prescribed concentration and during a certain time interval. Due to a public health concern about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the levels of lasalocid residues in the edible tissues of broiler chickens (breast muscle, thigh muscle, heart, liver, gizzard, kidneys and skin/fat) fed commercially produced feed containing 100 mg kg⁻¹) of lasalocid in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) with triple quadrupole. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.47 and 1.44 µg kg⁻¹, respectively. The average recovery based on the matrix-fortified calibrations for chicken tissues ranged between 79% and 98%. Lasalocid was found to accumulate in the liver, followed by the heart, skin/fat, kidneys, thigh muscle and gizzard. The lowest concentrations of lasalocid residues were found in the breast muscle. On day 5 of the WP, residue concentrations of lasalocid did not decline below the LOQ of the method, but were far below the maximum residue level (MRL) established for lasalocid in poultry from 20 to 100 µg kg⁻¹ by European Commission Regulation (EU) No. 37/2010. The results confirmed that the WP established for lasalocid is sufficient to ensure the decline of its residues in the tissues of broiler chickens to the safe residue level.

Post-mortem changes in the concentration of lactic acid, phosphates and pH in the muscles of wild rabbits (Oryctolagus cuniculus) according to the perimortal situation.

In this study changes in the concentrations of lactate, phosphates, and pH values of water extracts of muscles of transported and hunted rabbits during ripening were determined. Concentrations of lactate were higher in the muscles of hunted rabbits. The highest differences were obtained 24h after kill/hunt. Concentrations of lactate in the muscles of hunted rabbits were decreasing, while in the muscles of transported rabbits we observed it to increase in the 7th day and then decrease in the 14th day. Higher concentrations of phosphates were found in the muscles of transported wild rabbits. During the ripening process concentrations of phosphates were decreasing in muscles of both groups. Muscles of hunted rabbits had lower pH values during the whole ripening process. Our research showed that concentrations of lactate, phosphates and pH value post-mortem depended on the perimortal situations.

Liquid chromatography tandem mass spectrometry determination of maduramycin residues in the tissues of broiler chickens.

Maduramycin is a polyether ionophoric coccidiostat used to prevent coccidiosis in poultry at a prescribed concentration over a certain time interval. Due to public health concerns about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the level of maduramycin residues in the tissues of broiler chickens fed commercially produced feed containing 5 mg kg(-1) of maduramycin in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography (LC) coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.3 and 0.8 microg kg(-1), respectively. The average recovery based on matrix-fortified calibrations for chicken tissues was 90%. Maduramycin was found to be rapidly distributed in all tissues. The highest concentrations of maduramycin residues were found in the heart followed by the skin, liver, gizzard, kidneys and, finally, muscle (thigh and breast). On day 5 of the WP, residue concentrations of maduramycin did not decline below the LOQ of the method. Our results emphasize the need to establish a maximum residue limit (MRL) for maduramycin to control its residue levels in edible tissues from chickens before slaughter.

Evaluation of three different microbial inhibition tests for the detection of sulphamethazine residues in the edible tissues of rabbit.

The aim of the present study was to evaluate three microbial inhibition tests (MIT) based on inhibition of growth of the test organisms: (a) four plate test (FPT) containing Bacillus subtilis BGA, (b) screening test for antibiotic residues (STAR) containing Bacillus stearothermophilus var. calidolactis_ATCC 10149 and (c) the Premi(R)Test containing Bacillus stearothermophilus var. calidolactis. The tests were used to determine sulphamethazine (SMZ) residues in edible tissues of rabbit after oral administration up to day 15 of the withdrawal period (WP). A solvent extraction procedure was used to enhance the capability of the tests to detect SMZ residues at or below the maximum residue limit (MRL). Para-aminobenzoic acid (PABA) was employed to previously identify SMZ residues in the first stage of the residue screening. The presence of SMZ residues in the samples was confirmed and quantified by a validated HPLC method. The Premi(R)Test detected SMZ residues in the muscle and heart tissue up to day 9 of the WP, and in the liver, lungs and kidneys up to day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits up to day 8 of the WP. The kidneys were positive up to day 5 of the WP, the liver until day 4 of the WP and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. By using the solvent extraction procedure, SMZ residues were detected in the muscle extract up to day 10 of the WP and the muscle was positive until day 6 of the WP. No detection sensitivity was observed using the FPT. After solvent extraction, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscle was positive until day 3 of the WP. No positive results were detected after the addition of PABA into/onto the agar medium. PABA at a concentration of 10 microg ml(-1) completely reversed the inhibitory activity of SMZ and enabled reliable identification of SMZ in the examined samples. Using HPLC, SMZ was detected in the muscle samples until day 10 of WP (0.02 mg kg(-1)) and in the liver until day 12 of the WP (0.09 mg kg(-1)). The results obtained by the HPLC method and the limit of detection (LOD) of screening tests for SMZ (FPT 0.4 microg ml(-1), STAR 0.2 microg ml(-1), Premi(R) Test 0.05 microg ml(-1)) allowed us to state that the most suitable screening tests for the detection of SMZ residues in the edible tissues of rabbits at level corresponding to the MRL of 0.1 mg kg(-1), established for sulphonamides, are the Premi(R)Test and STAR in conjunction with the solvent-extraction procedure.