Farm to table: Residues of different pesticides in tomato and tomato juice - Food safety aspects.

During plant cultivation, the pesticides can get into the tissue of vegetables due to crop protection processes, and thus into the food chain. Therefore, they constitute a potential risk to the consumer's health. Depletion of pesticides [spirotetramat (Movento), azoxystrobin and difenoconazole (Amistar Top)] was monitored by testing tomatoes treated individually or simultaneously and tomato juices prepared from the treated tomatoes. The investigations aimed to reveal any kinetic interaction between the compounds tested and changes in their elimination, and thus to assess their compliance with the official Maximum Residue Limits (MRLs). The co-presence of pesticides prolonged the elimination of the individual compounds which reached significantly higher residue levels (P < 0.0001) in tomato, especially difenoconazole (45%) and azoxystrobin (50%) on day 8 after treatment that can cause food safety issues to the human consumers. However, the concentrations of pesticides applied alone or simultaneously were found to be below the corresponding MRL values after the withdrawal period in all investigated tomato and tomato juice samples. Accordingly, the investigated pesticides can be safely used simultaneously, their concentrations are in compliance with the legal regulations and thus their concomitant presence does not pose any risk to the consumers' health.

Possible inhibitory effect of microbial transglutaminase on the formation of biogenic amines during Trappist cheese ripening.

Trappist cheese (semi-hard, rennet-coagulated cheese with round eyes) was manufactured and matured for 4 weeks at 12 ± 1°C, 85% relative humidity (RH). The effect of microbial transglutaminase (MTGase) was followed by measuring the levels of free amino acids (FAAs) and biogenic amines (BAs) every 2 weeks during 4 weeks of cheese ripening. Results show that MTGase can decrease the cadaverine production by 30%, but only at the initial stage of ripening. Application of MTGase results in 49% less putrescine, 12% less tyramine production at the end of 4 weeks ripening time, and can decrease histamine levels by 8% after 2 weeks of ripening time in the examined semi-hard cheese type.

Transfer of certain beta-lactam antibiotics from cow's milk to fresh cheese and whey.

The aim of this study was to investigate the transfer of cephalexin, penicillin-G, and ampicillin & cloxacillin from cow's milk to cheese and whey. For this purpose, raw milk was artificially contaminated to different antibiotic levels and then heat-treated to prepare fresh cheese from it. Antibiotic levels of the milk, whey and cheese were measured with LC-MS/MS. The extent of heat degradation was not sufficient to remove the antibiotic residues from milk. Antibiotic concentrations in whey and fresh cheese were in good accordance with the concentration of the same compound in milk suggesting that contamination of the milk will result in contamination of the product. The investigated antibiotics were transferred less into the cheese curd (1.6-12.5% of the original amount), than into the whey (33.2-74.1%). For penicillin-G even 100% (complete removal) was experienced.

Possible food safety hazards of ready-to-eat raw fish containing product (sushi, sashimi).

It is undeniable that with the popularity of sushi and sashimi over the last decade the consumption of raw fish has extremely increased. Raw fish is very appreciated worldwide and has become a major component of human diet because of its fine taste and nutritional properties. Possible hazards concerning fish safety and quality are classified as biological and chemical hazards. They are contaminants that often accumulate in edible tissue of fish and transmit to humans via the food chain affecting the consumer's health. Although their concentration in fish and fishery products are found at non-alarming level of a daily basis period, they induce hazardous outcome on human health due to long and continuous consumption of raw fish. Regular sushi and sashimi eaters have to be aware of the contaminants found in the other components of their dish that often add up to acceptable residue limits found in fish. Hence, there is the urge for effective analytical methods to be developed as well as stricter regulations to be put in force between countries to monitor the safety and quality of fish for the interest of public health.

Serum hepcidin measurements in healthy dogs using liquid chromatography/tandem mass spectrometry.

BACKGROUND: Hepcidin is a key regulator of iron homeostasis. The measurement of this hormone is essential for the diagnosis of iron deficiency anemia and might be useful as a prognostic factor in many diseases. Serum hepcidin levels have been infrequently evaluated in dogs. OBJECTIVES: The aim of this study was to measure serum hepcidin in a population of healthy dogs using liquid chromatography/tandem mass spectrometry (LC-MS/MS), considered the gold standard assay for measuring hepcidin like in human medicine. METHODS: Blood samples from 86 healthy dogs of 25 different breeds were measured with the LC-MS/MS method. Synthetic canine hepcidin was used as the standard reagent. Reference values were calculated based on the results. RESULTS: The mean hepcidin concentration of the study population was 16.6 ± 7.7 ng/mL. There reference interval (RI) was defined as 5.3-36.4 ng/mL. No significant difference was found between male and female dogs, or between different age and body weight groups. Hepcidin concentrations did not correlate with red blood cell counts, hemoglobin concentrations, iron levels, iron-binding capacities, and C-reactive protein concentrations in this healthy population. A weak negative correlation was found between hepcidin and the mean corpuscular volume. CONCLUSION: LC-MS/MS proved to be a reliable and time-effective method for the detection of canine hepcidin. The RI was similar but narrower compared with that of human studies.

Heterocyclic Amine Formation in Grilled Chicken Depending on Body Parts and Treatment Conditions.

Heterocyclic amines (HCAs) carcinogenicity is known since the 1970's, but the exact way of their formation is still unclear. During these examinations different body parts (breast filet with and without skin, thigh filet without skin and full wing with skin) of chickens from the same Ross 308 strain were analyzed after grilling with the combination of 3-3 temperature and duration levels (150-180-210 °C and 2.5-5-10 min per side). Five different kinds of heterocyclic amines (HAR, NOR, MeIQx, 4,8-DiMeIQx and PhIP) were detected by HLPC-MS/MS. The results obtained from the present study confirm that, in general, the higher the temperature and longer the duration of the grilling the more HCAs will be generated. Grilling of chicken thigh without bones and skin resulted in lower amounts of HCAs generated in comparison to the grilling of chicken breast without skin. The presence of skin on the chicken breast increased the amounts of HCAs formed, especially if grilling was performed at high temperature for longer duration, especially at 210 °C for 10 min. In case of grilling the chicken wings, the amounts of HCAs formed were lower than observed in the breast.

Predictive Correlation between Apparent Sensory Properties and the Formation of Heterocyclic Amines in Chicken Breast as a Function of Grilling Temperature and Time.

In the present set of experiments, we studied the correlation between the heterocyclic amine (HCA) concentration and the color changes of the chicken breast with or without skin during grilling under open or closed conditions as a function of the applied temperature and time. The concentration of the HCAs formed during grilling was measured by a validated LC-MS/MS method, whereas the color changes were determined either instrumentally or by visual observation. In general, higher temperatures and longer heat treatment times resulted in a more substantial HCA formation, especially on the surface of the samples and in the skin, where the total levels reached 746 ng/g. Results of regression analysis demonstrate a strong correlation (r > 0.7) between the HCA content of the grilled chicken breast and the L* and a* values indicating the significance of brightness and the red parameter of the color scale, respectively. In the case of open grilling, the skinless breast samples showed correlation (r > 0.7) between the HCA content and the color analysis results in both the full sample and the crust, respectively. Breast samples with skin exhibited the same level of correlation when they were grilled closed. In the case of open grilling the breast with skin, and closed-grilling the skinless breast, the linear regression analysis yielded a weaker correlation (0.7 > r > 0.4 or less) between the HCA concentrations and the color. Our results demonstrate that there is a predictive correlation between the color changes perceptible for the consumers and the HCA formation during grilling of chicken breast as a function of time and temperature depending on the type of grilling and the presence of skin.

Heavy metals in seafood purchased from a fishery market in Hungary.

Heavy-metal content (arsenic, cadmium, lead and mercury) was investigated and evaluated in shellfish, oysters and squids. The samples were collected weekly for 20 weeks from a fishery product market in Hungary. The concentration of heavy metals was measured by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) analysis after sample preparation. The results were analysed statistically by one-way ANOVA method. The average concentration of arsenic (3.01 ± 1.46 mg kg-1) in shellfish was significantly higher (p < 0.001) compared to oysters (2.88 ± 1.12 mg kg-1) and squids (1.28 ± 0.52 mg kg-1). The level of mercury was below the limit of detection (0.5 mg kg-1) in each sample and there was no statistical significance in the concentrations of cadmium (p = 0.351) and lead (p = 0.412) in the species investigated. The provisional tolerable intake values were also calculated. Based on the obtained results of the heavy-metal content of the investigated samples, the seafood is considered to be safe for human consumption. However, prolonged ingestion of oysters and squids at these levels may contribute to the consumers' cadmium burden.

Noncovalent Interaction of Tilmicosin with Bovine Serum Albumin.

Tilmicosin is a widely used antibiotic in veterinary applications. Its antimicrobial activity is ranged from Gram-positive and some Gram-negative bacteria towards activities against Mycoplasma and Chlamydia. Adsorption affinity of tilmicosin antibiotics towards bovine serum albumin was investigated by both spectroscopic (UV-vis, Photoluminescence) and calorimetric methods. The interaction was determined on the basis of quenching of albumin by tilmicosin. Results confirm noncovalent binding of tilmicosin on bovine serum albumin with 1:1 stoichiometry associated with pK = 4.5, highlighting possible removal of tilmicosin molecules from the albumin surface through exchange reactions by known competitor molecules. Calorimetric measurements have confirmed the weak interaction between tilmicosin and albumin and reflect enhanced denaturation of the albumin in the presence of tilmicosin antibiotic. This process is associated with the decreased activation energy of conformational transition of the albumin. It opens a new, very quick reaction pathway without any significant effect on the product by noncovalent binding the tilmicosin molecules to the protein molecules. Results highlight the medical importance of these investigations by considerable docking of the selected antibiotic molecules on serum albumins. Although the binding may cause toxic effects in living bodies, the strength of the binding is weak enough to find competitor molecules for effective removals from their surface.

LC-MS study of the heat degradation of veterinary antibiotics in raw milk after boiling.

The aim of our study was to examine the degradation of veterinary antibiotics in milk during boiling. Raw cow milk samples were fortified with the target compounds and boiled for various short time-intervals prevailing in household practice. Antibiotic concentrations were determined by LC-MS/MS measurements. Degradation rate constants, half-lives and degradation percentages were calculated. Cefoperazone and cloxacillin proved to be the less and the most heat-stable substance, with 78.3% and 9.6% degradation in 300 s respectively. Aminoglicosides exhibited intermediate (33.8-43.6%), tetracycline (30.4%) and trimethoprim (22.6%) intermediate to high heat stability. The results demonstrate that antibiotic residues possibly present in raw milk exhibit high heat stability when treated for few seconds at around 100 °C. Keeping the milk at this temperature for at least two minutes would allow varying decrease in the amount of some compounds, but does not totally eliminate the potential risks to the consumer's health.

Rapid in-house detection method of Campylobacter spp. from food by redox potential monitoring combined with real-time PCR.

The rapid detection of Campylobacter spp. is of utmost importance for the reduction of infections in humans by contaminated food products. The standard culturing method (ISO 10272-1:2006) involves a high time and labour demand. In this paper, we present a method that reduces the detection time of Campylobacter spp. to or below one third as compared to the ISO method, at a reduced cost per test. We used redox potential change of enrichment cultures (Bolton broth with Bolton selective supplement) for reliably selecting Campylobacter-contaminated raw milk and broiler meat samples. Identification of Campylobacter spp. in the contaminated samples was done by real-time PCR method. Culturing time to conclusive redox monitoring varied between 6 and 24 h for positive samples, depending on the contamination rate, in contrast to 136 h with the standard culturing process. However, now the Campylobacter-negative majority of food samples will not need to be tested by real-time PCR because redox potential monitoring can identify them in the selective enrichment phase. This method could be potentially used as a faster alternative to the current standard ISO 10272-1:2006, for nonregulatory monitoring purposes.

Food safety aspects of primary environmental contaminants in the edible tissues of roe deer (Capreolus capreolus).

The muscle, liver, kidney and fat samples of 20 roe deer of both sexes originating from a hunting area in central Hungary were investigated for the presence of heavy metals such as As, Cd, Hg and Pb, and their contents were evaluated for possible health risk to consumers. Both As and Hg were found at a level below the limit of detection (< 0.5 mg/kg wet weight) in all samples. The median of the measured Cd concentrations was significantly higher in both the kidney and the liver (p = 0.0011) of bucks than of does. In bucks, Cd levels exceeded the respective maximum limits laid down in the European legislation in four kidney and three muscle samples, whereas in does, the measured concentrations were below the respective limits in all samples. The detected amounts of Pb exceeded the maximum limits in the kidney of one buck and eight does, in the liver of two bucks and six does, in the muscle of six bucks and nine does, whereas in all fat tissues of both bucks and does. The concentration of Pb (p = 0.02) was significantly greater in the kidney of does compared to roebucks. Based on data obtained from the present study, the consumption of organs and tissues of the investigated roe deer could be objectionable from food-toxicological point of view and may pose risk to the high consumers of wild game due to their cadmium and lead contents.

Toxic heavy metals in the muscle of roe deer (Capreolus capreolus)--food toxicological significance.

The study was performed on 20 (10 males, 10 females) roe deer (Capreolus capreolus) to investigate the concentration of cadmium, lead, mercury, and arsenic in the muscle tissue. They reside in forest and meadow, about 50 km distance from industrial activities and traffic. Samples were taken from the musculus biceps femoris of each deer without external contamination after shooting during the regular hunting season on a hunting area close to Eger in Hungary. The determination of heavy metal contents was carried out by inductively coupled plasma optical emission spectrometry (ICP-OES). The statistical analysis was performed by statistical package for the social sciences (SPSS) version 11.0. The measured residue concentration of cadmium was below the limit of detection in the roe deer meat indicating no health risk for the consumers. The average lead concentration (0.48 ± 0.21 mg/kg wet weight) exceeded the regulated maximum limit, but its calculated weekly intake was below the provisional tolerable weekly intake (PTWI). The residue level of mercury is not regulated and the average mercury content of roe deer meat (0.87 ± 0.40 mg/kg wet weight) was about half of PTWI, but the consumption of meat with the highest detected concentrations results in higher PTWI than recommended. The measured concentration of arsenic (0.27 ± 0.20 mg/kg wet weight) in the roe deer meat may not pose any health risk for the human consumers according to the PTWI set by the World Health Organization.

Simultaneous determination of praziquantel, pyrantel embonate, febantel and its active metabolites, oxfendazole and fenbendazole, in dog plasma by liquid chromatography/mass spectrometry.

A liquid chromatography-electrospray-mass spectrometry method (LC/MS) has been developed and validated for determination of praziquantel (PZQ), pyrantel (PYR), febantel (FBT), and the active metabolites fenbendazole (FEN) and oxfendazole (OXF), in dog plasma, using mebendazole as internal standard (IS). The method consists of solid-phase extractions on Strata-X polymeric cartridges. Chromatographic separation was carried out on a Phenomenex Gemini C6 -Phenyl column using binary gradient elution containing methanol and 50 mm ammonium-formate (pH 3). The method was linear (r(2) ≥ 0.990) over concentration ranges of 3-250 ng/mL for PYR andFEB, 5-250 ng/mL for OXF and FEN, and 24-1000 ng/mL for PZQ. The mean precisions were 1.3-10.6% (within-run) and 2.5-9.1% (between-run), and mean accuracies were 90.7-109.4% (within-run) and 91.6-108.2% (between-run). The relative standard deviations (RSD) were <9.1%. The mean recoveries of five targeted compounds from dog plasma ranged from 77 to 94%.The new LC/MS method described herein was fully validated and successfully applied to the bioequivalence studies of different anthelmintic formulations such as tablets containing PZQ, PYR embonate and FBT in dogs after oral administration.

In vitro study on the effect of doxycycline on the microbial activity of soil determined by redox-potential measuring system.

The potential effect of doxycycline on the microbial activity was investigated in three types of soil. Soil samples were spiked with doxycycline, incubated at 25°C and tested at 0, 2, 4 and 6 days after treatment. The microbiological activity of the soil was characterized by the viable count determined by plate pouring and by the time necessary to reach a defined rate of the redox-potential decrease termed as time to detection (TTD).The viable count of the samples was not changed during the storage. The TTD values, however exhibited a significant increase in the 0.2-1.6 mg/kg doxycycline concentration range compared to the untreated samples indicating concentration-dependent inhibitory effect on microbial activity. The potency of the effect was different in the 3 soil types. To describe the combined effect of the doxycycline concentration and time on the biological activity of one type of soil a mathematical model was constructed and applied.The change of microbial metabolic rate could be measured also without (detectable) change of microbial count when the traditional microbiological methods are not applicable. The applied new redox potential measurement-based method is a simple and useful procedure for the examination of microbial activity of soil and its potential inhibition by antibiotics.

Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR.

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detect Listeria monocytogenes in artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. For Listeria species the measuring time was maximum 34 h. The absence of L. monocytogenes could reliably be proven by the redox potential measurement method, but Listeria innocua and Bacillus subtilis could not be differentiated from L. monocytogenes on the basis of the redox curves. The presence of L. monocytogenes had to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g of L. monocytogenes in a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection of L. monocytogenes in food.

Ecotoxicological assessment of doxycycline in soil.

Doxycycline has been used in continually increasing quantities for mass treatment of food animals because of its greater bioavailability relative to older tetracyclines. The study presented in this paper was undertaken to investigate the degradation rate of the tetracycline derivative in manure-amended soil. In the present experiment, following composting, the doxycycline-contaminated manure was applied to agricultural land, and a field study was performed to investigate the degradation rate of doxycycline in soil. By the end of the 20-week sampling period, about 20 %, 33 % and 18 % of the initial doxycycline concentrations could be measured in soil samples taken at three different soil depths. The calculated half-life of doxycycline in the soil was 66.5, 76.3 and 59.4 days at depths of 0 cm, 25 cm and 50 cm, respectively. The potential effect of doxycycline on soil microbial activity was demonstrated by the nitrogen transformation test performed in compliance with the Organisation for Economic Co-operation and Development (OECD) Guideline No. 216. On day 28, the following nitrate concentrations of the control soil sample were found in the soil samples treated with different amounts of doxycycline: 76.9 %, 53.0 %, 65.6 %, 59.7 % and 77.1 %.

Degradation of doxycycline in aged pig manure.

Two types of studies were carried out to characterise the degradation rate of doxycycline in manure. In the laboratory experiment, pig manure containing doxycycline was stored in biochemical oxygen demand (BOD) bottles in a thermoregulated room at a temperature of 20 ± 3.5 °C to maintain the humid and anaerobic incubation conditions, and the relevant temperature. In the field study, the traditional manure composting process used by small farms and household plots was demonstrated, in which the manure heap containing doxycycline was exposed to the regular environmental conditions. In the laboratory experiment more than 30% and in the field study about 10% of the initial doxycycline amount could be detected in manure samples after 16 and 12 weeks of manure ageing period, respectively. Based on the results obtained from the studies performed under laboratory and field conditions, the half-life of doxycycline in manure was calculated to be 52.5 and 25.7 days, respectively.

Biochemical background of toxic interaction between tiamulin and monensin.

Tiamulin, a diterpene antibiotic, is used for treatment of pulmonary and gastrointestinal infections in swine and poultry. Combined administration of tiamulin and ionophores (e.g. monensin) to farm animals may lead to intoxication manifested in severe clinical symptoms. Tiamulin metabolite complex with cytochrome P450 has been suggested to be the basis of drug-interactions. However, the formation of metabolic intermediate complex is questionable. The effect of tiamulin-treatment on cytochrome P450 activities was investigated in rats. Ethylmorphine and aminopyrine N-demethylation activities as well as monensin metabolism (O-demethylation) increased in liver microsomes of tiamulin-treated (200 mg/kg) animals. CYP3A1 induction caused by tiamulin was confirmed by the results of Western blot analysis. To test metabolic intermediate complex formation as a result of tiamulin treatment, cytochrome P450 activities were also determined in the presence of potassium ferricyanide. The findings together with those of in vitro complex formation suggested that formation of metabolic intermediate complexes of tiamulin with cytochrome P450 could be excluded. On the other hand, the results of inhibition studies showed significant decrease of ethylmorphine or aminopyrine as well as monensin demethylation in the presence of tiamulin. Our results proved that tiamulin has dual effect on cytochromes P450. It is able to induce and directly inhibit CYP3A enzymes, which are predominantly responsible for monensin O-demethylation. The direct effect of tiamulin as an inhibitor might play a more important role in toxicity than its putative effect as a chemical inducer of CYP3A enzymes.