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Open access

Ewelina Kowalczyk and Krzysztof Kwiatek

Abstract

Introduction: Pyrrolizidine alkaloids (PAs) are probably the most widespread toxins of natural origin. More than 6,000 plant species produce these toxic compounds. Bees can forage on flowers of plants producing PAs, which leads to contamination of honey with the toxic compounds. To determine the contamination of honey with PAs, a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

Material and Methods: PAs were extracted with 0.05 M sulphuric acid and purified with MCX cartridges. A solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine (8:1:1:0.1:0.1, v/v) was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC–MS analysis.

Results: The developed method was validated according to SANTE/11945/2015 requirements. The recovery was from 80.6% to 114.5%. The repeatability ranged from 2.3% to 14.6%, and the reproducibility was from 4.9% to 17.7%.

Conclusions: A new method for the determination of PAs in honey has been developed and validated. All evaluated parameters were in accordance with the SANTE/11945/2015 guidance document. Out of 50 analysed honey samples, 16 (32%) were positive for the content of at least one PA.

Open access

Ewelina Kowalczyk and Krzysztof Kwiatek

Abstract

Introduction: Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

Material and Methods: PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis.

Results: The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%.

Conclusions: A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.

Open access

Ewelina Patyra and Krzysztof Kwiatek

Abstract

Introduction: The article presents a rapid and simple analytical procedure for determination of four tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) in animal medicated feedingstuffs. Material and Methods: Two-gramme samples were extracted by a Na2EDTA-McIlvaine buffer (pH 4)/methanol mixtures (40/60, v/v). The determination was achieved by liquid chromatography using a Zorbax Eclipse XDB C18 analytical column with mass spectrometer detection (LC-MS). Results: Recoveries of the antibiotics from spiked feed samples ranged from 78.2% to 113.5%. The LOD and LOQ for tetracyclines in feeds ranged from 2.8 to 4.2 and from 4.3 to 5.7 mg/kg, respectively. Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of tetracyclines in medicated feedingstuffs.

Open access

Joanna Stadnik, Małgorzata Karwowska, Zbigniew Dolatowski, Małgorzata Świątkiewicz and Krzysztof Kwiatek

Effect of Genetically Modified Feeds on Physico-Chemical Properties of Pork

The objective of this study was to evaluate the effects of genetically modified (GM), insect-resistant Bt maize (MON810) and the meal made of glyphosate-tolerant soybean (Roundup Ready MON40-3-2) used as the dietary components for pigs on the physico-chemical properties of meat. Forty-eight fatteners derived from Polish Landrace x Polish Large White sows mated to a Duroc x Pietrain boar were used. All animals received isonitrogenous and isoenergetic diets containing or not containing the genetically modified components. The design of the experiment was as follows: group I (control) - non-modified soybean meal and maize; group II - GM soybean meal and non-modified maize; group III - non-modified soybean meal and GM maize; group IV - GM soybean meal and GM maize. The examination of the pH values of loin and neck muscles indicated no statistically significant differences between pigs fed diets containing non-transgenic or transgenic feeds. No statistical differences were observed for water holding capacity (WHC) within dietary treatments. The introduction of transgenic maize and soybean meal into pig diets did not significantly affect the a* colour parameter of loin as well as neck muscles. The use of transgenic maize or soybean meal did not cause significant changes in the L* colour value of loin. Results obtained for neck muscles were more differentiated, possibly due to the natural heterogeneity of this primal cut. Pigs which had consumed the transgenic diet exhibited slightly decreased lipid stability of loin, as indicated by thiobarbituric acid reactive substances (TBARS). The decrease was statistically significant only in the case of muscles from group II. The addition of feeds derived from genetically modified crops into pig diets did not significantly affect the stability of neck muscle lipids; however, TBARS values of these muscles were twice those of loin muscles.

Open access

Tomasz Grenda, Elżbieta Kukier, Zbigniew Sieradzki, Magdalena Goldsztejn and Krzysztof Kwiatek

Abstract

The aim of this study was to perform an in-house validation of multiplex PCR method for C. botulinum detection in food and feed samples. The study was carried out on food and feed matrixes artificially contaminated by spores of C. botulinum reference strains. The following characteristic parameters for qualitative detection were estimated: limit of detection expressed as LOD50 according to the Spearman-Kärber formula, specificity, sensitivity, and accuracy according to the PN-EN ISO 16140:2004. The validated method showed high specificity. Specific PCR products were revealed only for DNA obtained from samples contaminated with C. botulinum spores. PCR inhibition was observed, especially during examination of contaminated feed. The calculated LOD50 for feed was nearly 10 times higher than for food. The implemented method enables to obtain test results during 3 d without time- consuming process of isolation and proving the ability of strains to produce botulinum toxins.

Open access

Elżbieta Kukier, Magdalena Goldsztejn, Tomasz Grenda, Krzysztof Kwiatek, Dariusz Wasyl and Andrzej Hoszowski

Abstract

The aim of the study was the assessment of microbiological quality of compound feed used in Poland in 2007-2010. The examinations were done at all veterinary diagnostic laboratories operating in the frame of official laboratory system. The occurrence of Salmonella sp. and counts of Enterobacteriaceae family, mesophilic aerobic bacteria, total microorganisms, and fungi were assessed. Assays were done following Polish, European, and international standards. Percentage of contamination of compound feed for poultry, pigs, and cattle by Salmonella sp. ranged from 0% to 3.5%. The highest contamination level by Enterobacteriaceae bacteria were detected in wet petfood. No more than 106 cfu/g of aerobic bacteria and no more than 105 cfu/g of fungi were detected in the feed. The results of the study revealed that the microbiological quality of compound feed used in Poland in 2007-2010 was better than the quality of the feed used in 2003-2006.

Open access

Anna Weiner, Ilona Paprocka, Agata Gołębiowska and Krzysztof Kwiatek

Abstract

The aim of the study was to gather and analyse available data concerning the presence of terrestrial animal constituents in feeds in Poland. A microscopic method of identification of the contaminants was used. Between 2012 and 2013, overall 16 139 samples of feeds were analysed, from which 282 (1.75%) contained elements from terrestrial animals. The percentage of feeds contaminated with such elements was lower in 2013. In 2012, among 8 499 samples analysed, 203 (2.39%) contained ingredients of animal origin, and in 2013, the elements were found in 79 (1.03%) out of 7640 samples. The percentage of feed samples positive for processed animal protein was relatively low and steadily decreasing. Furthermore, the microscopic method demonstrated to be a very sensitive technique for the detection of constituents of animal origin.

Open access

Tomasz Grenda, Elżbieta Kukier, Magdalena Goldsztejn and Krzysztof Kwiatek

Abstract

A PCR-based procedure for detection of C. botulinum C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and accuracy were determined according to PN - EN ISO 16140:2004. Additionally, the specificity of the validated methods was proved by sequence analysis of PCR products obtained from examination of samples connected with botulism cases in cattle and mallard ducks. Limit of detection was estimated according to the Spearman - Kärber formula and expressed as LOD50. The obtained results showed that a 100% specificity was achieved. The sequencing of PCR products revealed 99% identity with sequences of bont/C and bont/D genes deposited in the GenBank. The sensitivity value ranged from 63.3% for C. botulinum type C to 75% for type D. The accuracy value varied from 72% for type C to 81.3% for type D. LOD50 was estimated at the levels of 0.272 (0. D 188-0395) spore/g for type C and 0.17 (0.1-0.289) spore/g for type D. The described PCR-based procedure enabled detection of C. botulinum C and D at the stage of liquid culture. This makes examination of feed samples possible without isolation process. The presented procedure could support the diagnosis of botulism by faster and specific laboratory examination process.

Open access

Tomasz Grenda, Magdalena Grabczak, Krzysztof Kwiatek and Andrzej Bober

Abstract

Introduction: The aim of this study was to evaluate the prevalence of Clostridium botulinum and Clostridium perfringens in food samples purchased from Polish producers. Material and Methods: The analyses were performed on 260 food samples collected in Lublin and Subcarpathian regions: 56 of smoked meat, 21 of pork meat, 20 of dairy products, 26 of vegetable and fruit preserves, 40 of ready-to-eat meals, 27 of fish preserves, and 70 of honey collected directly from apiaries. Results: C. botulinum strains were isolated from 2.3% (6/260) of samples and the isolates were classified as toxin types A (4/260) and B (2/260). C. perfringens strains were isolated from 14% (37/260) of samples. All the isolates were classified as toxin type A, 28 of them were able also to produce α toxin and 9 - β2 toxin. Conclusion: On the basis of the obtained results it could be suggested that risk assessment, especially regarding the entire honey harvesting process, should be provided in order to ensure the microbiological safety of the products to be consumed by infants and people with a weakened immune system.

Open access

Tomasz Grenda, Elżbieta Kukier, Magdalena Goldsztejn, Krzysztof Kwiatek and Nina Kozieł

Abstract

As the test material mink feed with natural microflora was used. The analyses were conducted using Wrzosek and TPGY broth media, and Willis–Hobbs and Zeissler differential agar media. Wrzosek, Willis–Hobbs, and Zeissler media are described in Polish Standards approved by the National Standards Body in Poland and routinely used in detection of anaerobic bacteria in Poland. Detection and identification of C. botulinum was performed with a previously validated real-time PCR method based on ntnh gene detection, which is common in all C. botulinum toxotypes. The use of Wrzosek broth and Zeissler agar in routine analyses for detection and identification of C. botulinum was ineffective and limited. The obtained results showed the highest culturing process effectiveness in TPGY broth with 72 h incubation at 30°C and isolation on Willis–Hobbs agar. The real-time PCR method based on ntnh gene detection used in this study could be utilised as a supplementary tool to the mouse lethality assay.