Heat-treated Meat Products Research Articles

«Нақты уақыт» режиміндегі полимеразды тізбекті реакция әдісімен тамақ өнімдерінің түрлік фальсификациясын анықтау бойынша хаттама әзірлеу

Adulteration of meat products is becoming a serious problem all over the world, including inappropriate labeling, prohibited additives, or replacement of expensive ingredients with cheaper components. The consequences of such manipulations can be dangerous for the health of a consumer. For example, it may cause food allergies and poisoning. In this article, there was developed a protocol for the qualitative determination of the DNA of the mitochondrial genome of chicken (Gallus gallus) by the polymerase chain reaction in real-time in raw and heat-treated meat products. The study was carried out to determine mixtures of meat and meat products from chicken, turkey, horse, lamb, pork, and beef. There were prepared 11 samples of 9 types of meat and two types of plants for the study of falsification. DNA isolation kits PrepMan Ultra (Thermo Fisher, USA) were used to obtain DNA with a high concentration between 1,900 ng/ml and 140 ng/ml. The selection of special primers and fluorescent probes was based on the alignment of the sequence of the chicken mitochondrial gene (Gallus gallus) from GenBank. All circuits are aligned using Primer Designer 3.0 software and synthesized by ThermoFisher. The authors have developed a protocol to determine the specific falsification of food products by a polymerase chain reaction in "real-time" mode. The PCR protocol was tested on a collection of DNA samples to determine specificity, and PCR with different DNA content was delivered to determine sensitivity. Chicken DNA was clearly traced in the DNA matrix in the range of 0.01-10%. Studies have shown that the developed protocol is a highly sensitive and specific express method for determining chicken DNA by a polymerase chain reaction in "real-time" mode, which allows them to be used in the diagnosis of animal DNA detection.

Development of two specific multiplex qPCRs to determine amounts of Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta and Staphylococcus in meat and heat-treated meat products

Culturing methods are conventionally applied to investigate the contamination of food with several microorganisms after heat processing. However, with these methods, it is not possible to evaluate whether heat-treated meat products, such as cooked sausages, contained parts of spoiled meat. Therefore, two specific multiplex qPCRs were developed in this study in order to determine the microbiological quality of the raw materials used for these products. The PCR targets focused on four bacterial groups often found on meat (family Enterobacteriaceae, genus Pseudomonas, genus Staphylococcus and species Brochothrix thermosphacta). Specificity as well as sensitivity of the developed multiplex qPCRs, validated by using 68 microbial species, were 100%. The applicability of both multiplex qPCRs compared to culturing methods was performed using 96 meat samples (fresh and naturally spoiled) and 12 inhouse-made “Lyoner” sausages containing variable ratios of spoiled meat (0%, 5%, 12% and 25%; n = 3 for each group). Both methods showed similar results by evaluating the ∆log10 cfu/g, the relative accuracy and the t-test analysis (p > 0.05). Comparing qPCR results of the different sausage groups, a significant difference between sausages containing fresh meat and sausages containing spoiled meat (12% and 25%) was found only for Pseudomonas and B. thermosphacta in both raw and cooked sausages. The statistical difference between 5% vs. 12% and 25% spoiled meat in cooked sausages, was also found only for these two bacterial groups. The developed multiplex qPCRs were further applied to 30 commercially available “Bologna-type” sausages. The results showed a total of 14 sausages considered to be suspicious for Food Fraud. While the role of Staphylococcus spp. in meat spoilage remains unclear, Pseudomonas, Enterobacteriaceae and B. thermosphacta could together be used as an indicator for “spoiled meat” used in sausages. The developed qPCR systems in this study allow the detection of four relevant bacterial groups in the heated Bologna-type sausages and provide information about the hygienic quality of raw materials used. This method could thus be helpful for screening food suspected of Food Fraud.

Assessment of Polycyclic Aromatic Hydrocarbons in Heat Treated Meat and Fish

This study was conducted to assess the concentrations of the polycyclic aromatic hydrocarbons (16PAH, PAH4 and PAH8) in the examined samples of six heat-treated Egyptian charcoal-grilled meatball (kofta), gas chicken Shawerma, charcoal and gas grilled Chicken, charcoal-grilled Fish and cold smoked Herring which are widely consumed as a family meal, take-away or as sandwiches in Egypt. The samples were collected from different restaurants, Hyper-Markets, and grilled Shops in Ismailia Governorate. Four samples from each of the six heat-treated products were analyzed by using HPLC analysis using a Shimadzu LC20 series (Kyoto, Japan) with a fluorescence detector (RF-10AxL) and a ZORBAX Eclipse PAH (2.1×150 mm, 3.5 lm, Agilent) as a separation column. The PAHs concentration levels in examined samples varied mainly according to the heat-treated method. Charcoal and gas grilling cooking process were released high concentrations of total PAH4 and PAH8 in heat-treated meat products and fish.

Safety of ethyl lauroyl arginate (E 243) as a food additive in the light of the new information provided and the proposed extension of use.

The present scientific opinion deals with the evaluation of the safety of the food additive ethyl lauroyl arginate (E 243) in the light of a new interpretation of the available toxicological data and with respect to the proposed changes to the currently authorised conditions of use. Ethyl lauroyl arginate (E 243) is an already authorised food additive in the EU for use in heat‐treated meat products only, with some exceptions. The safety of ethyl lauroyl arginate (E 243) as a food additive has been evaluated in 2007 by EFSA and an acceptable daily intake (ADI) of 0.5 mg/kg body weight (bw) was set. The present assessment is based on a new interpretation of the available data elaborated by the applicant and on exposure estimates calculated by the Panel for both the current and the proposed changes to the authorised uses of this food additive. The Panel considered the new information provided, including the re‐examination of some of the results from the toxicological studies included in the original application dossier submitted for the initial evaluation of ethyl lauroyl arginate (E 243) in 2007. The Panel concluded that it does not contain new scientific evidence. The concerns and uncertainties expressed in the previous scientific opinions of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Foods (AFC) and Panel on Food additives and Nutrient Sources added to Food (ANS) remain to be addressed and there is no justification for changing the current ADI. Based on the above, the Panel concluded that the current ADI of 0.5 mg/kg bw would be reached in toddlers and children at the 95th percentile already for exposure estimates calculated using the currently permitted maximum level (ML) for ethyl lauroyl arginate (E 243). At the proposed new uses and use levels, the ADI would be exceeded at mean level of consumption in all age groups.

ANALYSIS OF SALMONELLA SPP. DETECTIONS IN EUROPEAN UNION COUNTRIES ACCORDING TO RASFF DATABASE

Ofcial data of RASFF (Rapid Alert System for Food and Feed) database on detection of Salmonella spp. in animal and plant raw materials in 2010–2015 are demonstrated. Total of 2,651 notifcations of Salmonella spp. detection were reported by RASFF member countries. The majority of such notifcations are associated with contamination of poultry meat and poultry products, feed, fruits and vegetables. Salmonella can survive in food for a long time (up to 6 months). The cause of salmonellosis in humans mostly involves consumption of raw or insufciently heat-treated eggs, meat and milk products. Regulation (EU) No. 142/2011 prescribes a requirement, which the heat-treated and processed food products shall comply with, i.e. absence of Salmonella in 25 g of the product. Analysis of RASFF data for 2010–2015 demonstrated annual increase of Salmonella spp. detections. The most frequently occurring is Salmonella enterica. Over the above mentioned period there were notifcations of 154 Salmonella serovars, the overwhelming serovars include S. enterica – 32.4%; Salmonella spp. – 18.8%; S. enteritidis – 6.3%; S. typhimurium – 4.6%, S. Agona – 2.6%; S. Lexington – 1.4%. Salmonella spp. were detected in poultry meat (19.5%), in feed for dogs and cats (5.6%), in pork (3.7%), in fshmeal (5.1%). During the period from 2010 to 2014, the increase of Salmonella notifcations in plant feed was reported. Over 2010–2015, RASFF reported of 42 salmonellosis outbreaks that resulted in 1,586 diseased humans.

DETECTING MEAT FRAUD IN FOOD SUPPLY CHAIN

In recent years, numerous reports have repeatedly highlighted a series of food safety scandals involving contaminated and forged meat and fish products, grains and fruit products, juices, cooking oils, and spices and herbs, distilled beverages and pet treats. Foods that have been found to be stained with chemicals, illicit drug residues, additives and dyes, pathogenic microorganisms and other pests. Some foods enter the market have expired well or are unhygienic. Economic gain is the goal of food fraud. Food fraud and the prevention of such fraud are very important processes in the food industry. Such frauds are economically motivated, rated as criminal behaviour, and the moment we understand criminal behaviour and decision making we will be able to calculate and exclude the risk of food fraud. By analysing financially motivated fraud that combine opportunities, motivations, and inadequate control measures, we can assess the likelihood of fraud in any food product or component. The modified ingredients are specially designed to avoid quality assurance and quality control systems for customers. Only people who manipulate the ingredients know what substances and how to manipulate them. In addition, fraudulent ingredients are often unconventional substances that do not meet the requirements of food safety management systems, and become known only after they are incorporated into the supply chain. International standards for food and regulations address the risk of fraud food adulteration. European Union (EU) Directives, Global Food Safety Initiative (GFSI) Codex Alimentarius has continued work on a food fraud, or how, food fraud fits into their benchmarking. The problem of detection and typing of meat in meat products in the world and lack of research on them in the Republic of North Macedonia was the main goal for this paper. Our task was identification of meat type by ELISA method and proof of counterfeiting of meat products. Analyses are made in the laboratories of the Institute of Food at the Faculty of veterinary medicine in Skopje A total of 350 samples of various heat-treated meat products subgroups of meat sausages in pieces were examined for detection, typifying and quantifying the type of meat used for production.

Quality Assurance of Some Heat Treated Meat Products

The aim of this work was to assure the quality of some heat treated meat products, through organoleptic, chemical and microbiological aspects of these products. These products were Luncheon, Frankfurter and Hot dog. A total of 75 samples of final products of Luncheon, Frankfurter and Hot dog (25 / each) were collected randomly from supermarkets in Cairo Province and transferred directly (in a dry icebox) to the laboratory under complete aseptic condition with a minimum of delay. All samples were subjected to organoleptic, chemical and microbiological examination to evaluate their quality status. The results of organoleptic examination revealed that the odour ranged from good to very good, medium to very good and fair to good in Luncheon, Frankfurter and Hot Dog respectively while colour and consistency was good to very good in the three products. The results of chemical analysis revealed that the chemical indicators pH, Total Volatile Nitrogen (T.VN.) and Thiobarbituric Acid (T.B.A.) were within the permissible limits according to EOS (2005). For the chemical examination the mean value of total mesophilic bacterial count was 5.73×102 ± 2.08×102cfu/g, 2.07×106 ± 3.04×105cfu/g, and 1.83×106 ± 2.76×105cfu/g. Also, it was revealed that 100% of the examined samples of total mesophilic count of luncheon were less than the permissible limits according to EOS, (2005) while, 100% of samples of frankfurter and hot dog exceeded the permissible limits when compared with the same standard. The mean value of coliforms count were 2.92 ± 0.31cfu/g, 5.12 ± 0.25 cfu/g, and 6.92 ± 0.27cfu/g for Luncheon, Frankfurter and Hot Dog respectively. The three products were free from Salmonella. Luncheon was free from E.coli and S. aureus while frankfurter and hot dog both of E.coli and S. aureus were isolated in the rate of 80 and 12% respectively. And obtained results indicated that the gram negative coliforms and gram-positive bacteria were present predominantly, Moreover, Moulds and Yeasts counts. Moulds countscfu/g ranged from 0 to 2.1×104 with a mean value of 5.2×102 ± 1.5×102, from 2.2×102 to 3.5×104 with a mean value of 2.9×103 ± 2.1×102 and ranged from 3.1×102 to 4.6×104 with a mean value of 5.3×103 ± 2.2×102 in Luncheon, Frankfurter and Hot Dog respectively. Yeasts counts cfu/g were negative in Luncheon while, ranged from 0to 1.1×104 with a mean value of 3.8×103 ± 2.3×102 and ranged from 0to 1.6×104 with a mean value of 4.5×103 ± 2.1×102 in Frankfurter and Hot Dog respectively. So, it is highly recommended to apply restricted hygiene practices along all steps of processing and storage.

The Influence of Cooking Methods and Some Marinades on Polycyclic Aromatic Hydrocarbon Formation in Beef Meat

ABSTRACTThis study aimed to determine the effect of various marinades and their concentrations, as well as various cooking procedures, on polycyclic aromatic hydrocarbon (PAH) formation in cooked beef. The PAH levels in meat were determined, based on the saponification of lipids by methanolic/potassium hydroxide solution, followed by liquid–liquid extraction and QuEChERS method. Pan frying caused lower levels of BaP (1.39 versus 1.62 µg/kg) and PAH4 (5.58 versus 5.73 µg/kg) in beef meat than barbecuing. For sage and thyme extracts prepared at 0.5 to 2.0 °Brix, a significant decrease in the PAH levels of barbecued meat samples was achieved compared to the controls. The levels of PAHs in the samples containing the commercial marinating material were found to be higher than those in the non-marinated control. The BaP and PAH4 levels found in the meat samples marinated with sage and thyme extracts, were below the EU maximum levels. However, BaP was the highest for Control 2 (2.26 µg/kg), and exceeded the EU maximum limit of 2 mg/kg for BaP in heat-treated meat and meat products. The results show a reducing effect of sage and thyme extracts that are normally used during marinating of meat, on the formation of PAH compounds. Consequently, the extracts of sage and thyme, could be used in the commercial marinating material to reduce the level of PAH compounds formed in meat during cooking.

Effects of Nitrite and Erythorbate on Clostridium perfringens Growth during Extended Cooling of Cured Ham

To control the growth of Clostridium perfringens in cured meat products, the meat and poultry industries commonly follow stabilization parameters outlined in Appendix B, "Compliance Guidelines for Cooling Heat-Treated Meat and Poultry Products (Stabilization)" ( U.S. Department of Agriculture, Food Safety and Inspection Service [USDA-FSIS], 1999 ) to achieve cooling (54.4 to 4.4°C) within 15 h after cooking. In this study, extended cooling times and their impact on C. perfringens growth were examined. Phase 1 experiments consisted of cured ham with 200 mg/kg ingoing sodium nitrite and 547 mg/kg sodium erythorbate following five bilinear cooling profiles: a control (following Appendix B guidelines: stage A cooling [54.4 to 26.7°C] for 5 h, stage B cooling [26.7 to 4.4°C] for 10 h), extended stage A cooling for 7.5 or 10 h, and extended stage B cooling for 12.5 or 15 h. A positive growth control with 0 mg/kg nitrite added (uncured) was also included. No growth was observed in any treatment samples except the uncured control (4.31-log increase within 5 h; stage A). Phase 2 and 3 experiments were designed to investigate the effects of various nitrite and erythorbate concentrations and followed a 10-h stage A and 15-h stage B bilinear cooling profile. Phase 2 examined the effects of nitrite concentrations of 0, 50, 75, 100, 150, and 200 mg/kg at a constant concentration of erythorbate (547 mg/kg). Results revealed changes in C. perfringens populations for each treatment of 6.75, 3.59, 2.43, -0.38, -0.48, and -0.50 log CFU/g, respectively. Phase 3 examined the effects of various nitrite and erythorbate concentrations at 100 mg/kg nitrite with 0 mg/kg erythorbate, 100 with 250, 100 with 375, 100 with 547, 150 with 250, and 200 with 250, respectively. The changes in C. perfringens populations for each treatment were 4.99, 2.87, 2.50, 1.47, 0.89, and -0.60 log CFU/g, respectively. Variability in C. perfringens growth for the 100 mg/kg nitrite with 547 mg/kg erythorbate treatment was observed between phases 2 and 3 and may have been due to variations in treatment pH and NaCl concentrations. This study revealed the importance of nitrite and erythorbate for preventing growth of C. perfringens during a much longer (25 h) cooling period than currently specified in the USDA-FSIS Appendix B.

Simultaneous determination of 9 heterocyclic aromatic amines in pork products by liquid chromatography coupled with triple quadrupole tandem mass spectrometry

Heterocyclic aromatic amines (HAAs) are potent mutagens that formed at high temperature in cooked, protein-rich food. Owing to their frequent intake, an accurate method is essential to access human health risk of HAAs exposure through detecting these compounds in various heat-treated meat products. In this study, a liquid chromatography-electrospray tandem mass spectrometry (LC--ESI-MS/MS) method was developed to perform the determination of 9 mutagenic heterocyclic amines (HAAs) in meat samples with multiple reaction monitoring (MRM) mode. Ultrasound assisted extraction and diatomaceous earth was employed to extract HAAs from food samples, and the analytes were purified and enriched using tandem solid phase extraction, with propyl sulfonic acid coupled to a C18 cartridge. Two parameters, extraction time and eluent, were carefully optimized to improve the extraction and purification efficiency. The LC separation was carried out using a Zorbax SB-C18 (3.5 μm particle size, 2.1 × 150 mm i.d.) column and optimized some parameters, such as pH, concentration and volume. Under the optimal experimental conditions, recoveries ranged from 52.97% to 97.11% with good quality parameters: limit of detection values between 0.02 and 0.24 ng mL-1, linearity (R2>0.998), and run-to-run and day-to-day precisions lower than 9.81% achieved. To evaluate the performance of the method in high throughput analysis of complex meat samples, the LC-MS/MS method was applied to the analysis of HAAs in three food samples, and the results demonstrated that the method can be used for the trace determination of HAAs in pork samples.

Effects of encapsulated starter cultures on microbial and physicochemical properties of traditionally produced and heat treated sausages (sucuks)

Starter cultures (Lactobacillus plantarum and Staphylococcus xylosus) were encapsulated by emulsion method using alginate-starch mixture to increase the live cell number when they used in the heat treated (at ∼70 °C for 20 min) and in fermented sucuks. Effects of encapsulated bacteria on microbial and physicochemical properties of heat treated sucuk were investigated. Freeze dried micro particles were characterized in terms of survival rate surface morphology and release profile. The highest survival rate was observed for L. plantarum and S. xylosus at 2% and 0.5% concentrations, respectively. Release test was completed for 14 days with the release rates of 70.21% and 70.34% for S. xylosus and L. plantarum, respectively. The starter cultures were protected by encapsulation against the harsh conditions during fermentation and heat treatment with lower reduction rates suggesting that the microencapsulation of starter cultures is a robust option for the use in the production of heat treated meat products.

Microbial Evaluation of Heat Treated Meat Products

Microbial Evaluation of Heat Treated Meat Products

Producion of low salt frankfurter with microbial transglutaminase

Several scientific papers suggest that microbial transglutaminase (mTG) is capable of reducing the salt content of cured and/or heat-treated meat products (ham, frankfurters, meat ball). These scientific results are not widely known in Hungary, and as a result of this, only little experience was gathered in our meat industry. According to this lack of knowledge, our aim was to lower the curing salt to a still microbiological safe level using mTG by frankfurters, one of the most well-known heat treated meat products in Hungary. The observed technofunctional properties suggest to use mTG enzyme preparation at 0.5% concentration. This enzyme dosage can reduce the average 1.8% salt content to 1.6% and it also may contribute to extended shelf life of popular frankfurters. Our sensorial analysis revealed that the panellists have not found a loss in quality between 1.4% and 1.6% salt.

Identification of pork in meat products using real-time loop-mediated isothermal amplification

In this study, a one-step, real-time, loop-mediated isothermal amplification (RealAmp) assay was developed, for the highly specific detection of pork DNA. For the assay, the mtDNA of cytochrome b (cytb) gene was amplified at 63 °C using SYBR Green I for 45 min with a Real-Time Polymerase Chain Reaction (PCR) System that measured the fluorescent signal at one-minute intervals. As little as 1 pg of template DNA could be detected, without any cross-reactivity with non-target species. Meat mixtures, heat-treated at 100 °C for 15 min, prepared by mixing pork meat with beef at different ratios (0.01%–10%) were tested, and the RealAmp assays allowed the detection of as little as 0.01% pork in the meat mixtures. Thus, this work showed that RealAmp could be used for specific identification and sensitive quantification of meat species, even for heat-treated meat products.

Analysis of the baseline survey on the prevalence of Listeria monocytogenes in certain ready‐to‐eat foods in the EU, 2010–2011 Part B: analysis of factors related to prevalence and exploring compliance

A European Union-wide baseline survey on Listeria monocytogenes was carried out in 2010 and 2011. Packaged (not frozen) hot or cold smoked or gravad fish, soft or semi-soft cheeses (excluding fresh cheeses) and packaged heat-treated meat products were sampled in 26 European Union Member States and in one country not belonging to the European Union. Multiple-factor analysis (Generalized Estimating Equations) was used to investigate the statistical association between several factors on which information was gathered during the baseline survey, and two outcomes: prevalence of Listeria monocytogenes and proportion of samples with counts exceeding 100 cfu/g, in the surveyed fish and meat products (no analysis is presented for cheese samples, owing to the small number of contaminated samples). Sparseness issues led to instability of the effect estimates for some of the factors. For fish samples, factors that exhibited a stable association with at least one of the two outcomes were ‘subtype of the fish product’ (factor related to the type of processing), ‘Number of antimicrobial preservatives and/or acidity regulators’ and ′Possible slicing′. For meat products, the corresponding factors were ′Type of the meat product′, ′Animal species of the origin of the meat product′, ′Possible slicing′ and ′Remaining shelf-life′ (days between sampling and ′Use by date′). Furthermore, a statistical model was developed that allowed the use of estimates of the proportion of samples with an L. monocytogenes count > 100 cfu/g obtained from a single-unit sample survey of a population of RTE foods, in order to estimate the probability that if a five-unit sample had been taken from the same population, no individual unit, out of n = 5 units constituting the sample, would have exceeded the level of 100 cfu/g. The model was applied using data from the baseline survey for fish, cheese and meat products, at the end of shelf-life.

Predictive Model for Growth of Clostridium perfringens during Cooling of Cooked Beef Supplemented with NaCl, Sodium Nitrite and Sodium Pyrophosphate

This paper presents a model for predicting relative growth of Clostridium perfringens in ground beef products at different percentages of salt (0 to 3%), sodium pyrophosphate (0 to 0.3%), and nitrite (0 and 200 ppm). The results of the experiments indicates that salt was the primary variable affecting the amount of growth, and that growth in general, was significantly affected by the presence of nitrite. The inclusion of SPP did not significantly improve the model’s fit with observed results. The primary growth model, derived from growth experiments at constants temperatures, is based on a common form of Baranyi’s growth curves and the secondary model is based on cardinal temperatures, relating maximum specific growth rates as a function of temperature. When product is heated and then cooled, the model predicts an initial decline in the number of cells. The model’s predictions compared well with results obtained from dynamic temperatures experiments, for salt concentrations up to 1.5% and nitrite, to 200 ppm, when samples were initially heated and then cooled. The model can be used by processors to evaluate the risk of C. perfringens spore germination and outgrowth during cooling (stabilization) deviations or in custom cooling schedules in case the processors cannot follow the USDA FSIS Compliance Guidelines (Appendix A) for Cooling of Heat-Treated Meat and Poultry Products (Stabilization).

Prevalence and level of Listeria monocytogenes in ready-to-eat foods in Sweden 2010

An increasing trend in human listeriosis cases over the past five years (2005–2009) in Sweden encouraged the authorities to examine the prevalence and levels of Listeria monocytogenes in ready-to-eat (RTE) foods in 2010. The combined results of two surveys are presented: the Swedish part of an EU-wide survey and a national survey. A total of 1590 samples covering three categories of RTE food able to support growth of L. monocytogenes: (i) soft and semi-soft cheeses (mould- and smear-ripened); (ii) heat-treated meat products; and (iii) smoked and gravad fish, were collected at retail outlets and analysed at the end of shelf life. L. monocytogenes was detected in 0.4% of 525 cheese samples, 1.2% of 507 meat-product samples and 12% of 558 fish samples. In the latter category, L. monocytogenes was found in 14% of both gravad and cold-smoked fish samples and in approximately 2% of hot-smoked fish samples. The percentage of cold-smoked or gravad fish testing positive for L. monocytogenes was significantly lower in samples processed in Sweden (8%) than in samples processed in other countries (45%). Levels of L. monocytogenes exceeding 100cfu/g were found in one (0.2%) of the cheese samples and in three (0.5%) of the fish samples. The high prevalence of contaminated cold-smoked and gravad fish samples suggests that these products constitute the main problem. This has induced the development of a national strategy plan with the aim to halve the prevalence of L. monocytogenes in cold-smoked and gravad fish at retail in Sweden by the end of year 2015.

Detection of Chicken and Turkey Meat in Meat Mixtures by Using Real‐Time PCR Assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.

GC/MS detection of central nervous tissue as specified BSE risk material in meat products and meat and bone meals: thermal stability of markers in comparison with immunochemistry and RT-PCR

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNA(GFAP) in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120-140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products.

Outbreak of trichinellosis in eastern Slovakia

Abstract Trichinellosis is a zoonosis caused by ingestion of undercooked raw meat from animals that harbour infectious larvae. In most of the Slovak regions there is ongoing life cycle of circulating trichinellosis in wild carnivores and wild boar population. The outbreak of trichinellosis occured in Rožňava district east Slovakia during spring in 2008. Ten members of farmer’s family and their relatives got ill while processing meat from home-made pig-slaughter for meals and meat products intended for wedding dinner. During the meat processing all of them tasted raw meat. Moreover, another 45 persons were exposed to this infection by eating heat-treated meat products. The most common predominant clinical signs were: myalgias, fever, fatigue, exanthema and periorbital oedema. On the 40th day after infection there were intermediate to high titres of trichinella IgG antibodies detected (10 patients), high levels of eosinophilia (10 patients) with maximum of 6.76 × 109/l (55 %) and profound changes in selected laboratory parameters: decreased levels of total proteins, increased levels of alpha 1-globulin and C reactive protein. Presence of IgG antibodies as well as aforementioned laboratory parameters was important markers of trichinellosis in our study, whereas other laboratory changes (leukocytosis, high levels of activity lactate dehydrogenase and creatine kinase) were detected only in few hospitalized patients.