Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in meat products sold in Navarra,Spain

Patterns of resistance in β-lactamase-producing Enterobacteriaceae family were investigated in isolates from 141 meat products (beef, poultry and pork) purchased in Spain. The strains that grow in ChromID ESBL agar plates were confirmed using the paired disk diffusion method. Resistance to amoxicillin/clavulanic acid, ceftazidime, ceftriaxone, aztreonam, cefpodoxime, gentamicin, doxycycline, cotrimoxazol, norfloxacin, piperacillin/tazobactam, fosfomycin and cefoxitin were tested following CLSI recommendations. Minimum inhibitory concentrations were determined by the MicroScan® NM37 panel and β-lactamase genes were detected using multiplex PCR and sequencing. Results show poultry as the meat product having the highest prevalence (84%), with Escherichia coli being the predominant bacteria (71.3%). Predominant β-lactamase types were CTX-M (37.8%), followed by CTX-M + TEM combination (20.7%), TEM (17%), SHV (12.2%), TEM + SHV combination (10.9%) and OXA (1.2%). 93.9% of the strains were resistant to one or more β-lactam antibiotics. Results indicate a widespread distribution of ESBL-producing Enterobacteriaceae in meat products, with a high rate of β-lactam resistance and a low rate of AmpC cephalosporinase-producing strains.     

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C _q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r ² = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.     

The devolopment of duplex real-time PCR based on SYBR Green florescence for rapid ıdentification of ruminant and poultry origins in foodstuff

SDRT-PCR (SYBR duplex real-time polymerase chain reaction) was designed for an assay that can combine the advantages of real-time PCR and multiplex PCR to identify animal genes more quickly. SDRT-PCR based on melting temperature (tm) discrimination by using SYBR Green fluorescence dye was developed for the analysis of ruminant and poultry contained in foodstuffs. Primers specific to ruminant and poultry were designed for species-specific gene-amplification. Appropriate mixtures of poultry and ruminant meat DNAs were used to develop the assay. Gene products of ruminant and poultry were represented in two melting peaks generated simultaneously at temperatures of 83.2 and 86.5 °C, respectively. Duplexing results obtained with one of the multiplex polymerase mixes correlated extremely well with the singleplex reference. The objective of the study was to design a rapid, specific and accurate duplex real-time PCR assay by using SYBR Green fluorescence dye that is cheaper than double labelled probes to detect a group of mixed meats simultaneously.     

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10³ CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.     

Identification of hare meat by a species-specific marker of mitochondrial origin

Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat.     

Identification of chicken,duck, pigeon and pig meat by species-specific markers of mitochondrial origin

In the present study, PCR based method for meat species identification of chicken, duck, pigeon and pig was achieved by developing species-specific markers. Using mitochondrial sequences species-specific primers were designed and the sizes of them were 256 bp, 292 bp, 401 bp and 835 bp for chicken, duck, pigeon and pig, respectively. The species-specific PCR products were sequenced to confirm the specificity of the product amplified. These markers were subsequently tested for cross amplification by checking them with beef, mutton, chevon, pork, rabbit, chicken, duck, turkey and pigeon meat. DNA markers developed in this study can help identify the species of fresh, cooked and autoclaved meat of chicken, duck and pigeon and fresh and cooked meat of pig. The process of identification is simple, economical and quick as compared to other methods such as RAPD, PCR-RFLP and sequencing method of species identification.     

Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods

The method performance characteristics of commercially available PCR kits for animal species identification were established. Comminuted meat products containing different levels of pork were prepared from authentic beef, chicken, and turkey. These meat products were analysed in the raw state and after cooking for 20 min at 200 °C. For both raw and cooked meats, the PCR kit could correctly identify the animal species and could reliably detect the addition of pork at a level below 0.1%. A survey of 42 Turkish processed meat products such as soudjouk, salami, sausage, meatball, cured spiced beef and doner kebap was conducted. Thirty-six samples were negative for the presence of pork (< 0.1%) and four were found to be correctly labelled as containing pork. However, one sausage sample was labelled as containing 5% beef, but beef DNA was not detected and a meatball sample labelled as 100% beef was found to contain chicken. Another turkey meatball sample was predominantly chicken.     

Biogenic amine formation in Nham,a Thai fermented sausage,and the reduction by commercial starter culture, Lactobacillus plantarum BCC 9546

Biogenic amines are of concern for sausage due to their toxicological effects on nervous, blood pressure, gastric and intestinal systems. In this study, the influence of raw pork meat quality and starter culture inoculation on biogenic amines accumulation in Nham, a Thai traditional fermented pork, were studied. Before Nham processing, pork meat was stored at 30 °C for 6 h, and at 4 and −20 °C for 2 days. Formation of biogenic amines (cadaverine, putrescine, histamine and tyramine) was significantly higher in Nham processed from stored meat. Accumulation of these biogenic amines in Nham reduced significantly by the addition of Lactobacillus plantarum BCC 9546, a commercial Nham starter culture. The results highlight the importance of using fresh meat products and the inclusion of an appropriate starter culture to minimise the formation of biogenic amines during the process of Nham fermentation.     

Evaluation of the effects of selected plant-derived nutraceuticals on the quality and shelf-life stability of raw and cooked pork sausages

The effect of lutein (200 μg/g meat), sesamol (250 μg/g meat), ellagic acid (300 μg/g meat) and olive leaf extract (200 μg/g meat) on total viable counts (TVC), pH, water holding capacity (WHC), cooking loss, lipid oxidation (thiobarbituric acid-reactive substances, TBARs), colour stability, texture and sensory evaluation of fresh and cooked pork sausages stored in aerobic or modified atmosphere packs (MAP) was investigated. Addition of sesamol, ellagic acid and olive leaf extract reduced (P < 0.001) lipid oxidation in all packaged raw and cooked pork sausages. Antioxidant potency followed the order: sesamol 250 > ellagic acid 300 > olive leaf extract 200 > lutein 200 for both raw and cooked pork sausages. Addition of sesamol increased (P < 0.001) WHC on days 2 and 12 of MAP storage. Meat addition of lutein, sesamol, ellagic acid and olive leaf extract had no detrimental effect on pH, cooking losses, TVCs, tenderness, juiciness, texture or product flavour. Lutein, sesamol, ellagic acid and olive leaf extract were effective as natural functional ingredients in suppressing lipid oxidation and have the potential to be incorporated into functional raw and cooked pork sausages.     

Heterocyclic amines and azaarenes in pan-fried meat and its gravy fried without additives and in the presence of onion and garlic

The effect of onion and garlic on the formation of heterocyclic amines (HAs, aminoazaarenes) and azaarenes (aza-PAHs) was evaluated by comparing the concentrations of several compounds in meat and gravy samples, obtained from three pork dishes prepared in the presence and absence of these spices. The concentrations of individual HAs (8-MeIQx, MeIQ, 4,8-DiMeIQx, PhIP) were from 0.5 ng g⁻¹ to 10.5 ng g⁻¹ of meat and of azaarenes (benzo[a]acridine, benzo[c]acridine, dibenzo[a,c]acridine, dibenzo[a,j]acridine and dibenzo[a,h]acridine) – from 0.06 ng g⁻¹ to 0.99 ng g⁻¹. The addition of onion (30 g/100 g of meat) in the dishes investigated, caused a decrease in heterocyclic amines concentration (considering total contents in meat and gravy) in the range of 31–49% and of azaarenes by 21–48%. Garlic (15 g/100 g of meat) lowered the concentration of HAs by 26–36% and azaarenes by 33–40%; the changes in concentrations caused by these spices were different for particular compounds. Components of onion and garlic intensify the extraction of heterocyclic amines and azaarenes from meat in gravy.     

Roselle (Hibiscus sabdariffa L.) and soybean oil effects on quality characteristics of pork patties studied by response surface methodology

Response surface methodology was used to investigate the effect and interactions of processing variables such as roselle extract (0.1–1.3%), soybean oil (5–20%) on physicochemical, textural and sensory properties of cooked pork patties. It was found that reduction in thickness, pH, L* and b* values decreased; however, water-holding capacity, reduction in diameter and a* values increased, respectively, as the amount of roselle increased. Soybean oil addition increased water-holding capacity, reduction in thickness, b* values of the patties. The hardness depended on the roselle and soybean oil added, as its linear effect was negative at p < 0.01. The preference of color, tenderness, juiciness, and overall quality depend on the addition of roselle and soybean oil. The maximum overall quality score (5.42) was observed when 12.5 g of soybean oil and 0.7 g of roselle extract was added. The results of this optimization study would be useful for meat industry that tends to increase the product yield for patties using the optimum levels of ingredients by RSM.     

Physicochemical and sensory properties of healthier frankfurters as affected by walnut and fat content

This study evaluates the physicochemical and sensory properties of healthier frankfurters with 25% added walnut (WF) versus low-fat frankfurters (6% pork fat) (LF) and traditional frankfurters (18% pork fat) (NF). Results reveal that cooking losses were unaffected (p ? 0.05) by the formulation of frankfurters. The addition of walnut led to higher (p < 0.05) redness and yellowness values, while colour parameters did not differ significantly between LF and NF sausages. Frankfurters with added walnut (WF) presented higher (p < 0.05) hardness and chewiness values than LF and NF frankfurters. Differences in composition were also accompanied by changes in the microstructure of the gel/emulsions. Frankfurters with added walnut presented a flavour significantly different from meat and scored lower (p < 0.05) on texture preferences. However, all frankfurters scored the same for overall acceptability.     

Determination of tetracyclines in multi-specie animal tissues by pressurized liquid extraction and liquid chromatography–tandem mass spectrometry

A specific, sensitive and robust pressurized liquid extraction (PLE) and liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determining tetracycline, chlortetracycline, oxytetracycline and doxycycline in bovine, swine, poultry and lamb muscle tissues is presented. PLE was performed using an ASE^® 200 from Dionex and water as extractant, followed by solid-phase extraction (SPE) using an Oasis HLB cartridge. The method was validated for beef, chicken, pork and lamb meat in compliance with the requirements set by Commission Decision, 2002/657/EC [Commission Decision 2002/657/EC (2002). Implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. Official Journal of European Communities, L239, 66–98. (Available at: <http://europe.eu.int>)]. The average recoveries of the different meat samples, spiked with the four tetracyclines at three levels (1, 100 and 200 μg kg⁻¹ of each tetracycline), were always higher than 89% with intraday and interday precision lower than 15% and 17%, respectively. A good linearity was established for the four tetracyclines in the range from 5 to 10,000 μg kg⁻¹ with r > 0.995. The limits of quantification (LOQs) were between 0.5 and 1 μg kg⁻¹, which are well below the tolerance levels set by the European Union. The decision limit (CCα) and the decision capability (CCβ) were in the range 101–116 and 112–130 μg kg⁻¹, respectively. Compared with previous methods, sample preparation time required for the analysis and LOQs, are reduced. The method demonstrated its successful application for the analysis of 100 meat samples. Two samples of beef and one sample of chicken out of 25 of each type tested positive while none of 25 samples of either, lamb or pork, tested positive.     

Physicochemical properties and tenderness of meat samples using proteolytic extract from Calotropis procera latex

This study was conducted in order to tenderise muscle foods (pork, beef and chicken) by using crude enzyme extract from Calotropis procera latex. Chunks of knuckle muscle from pork and beef as well as of breast muscle from chicken were marinated with distiled water (control) and 0.05%, 0.1%, 0.2%, 0.3% and 0.5% (w/w) of crude enzyme extract powder for 60 min at 4 °C. The marinated samples were then subjected to various physical and chemical property determinations. A decrease in moisture content was observed when the crude enzyme extract was added. Firmness and toughness of the muscle samples significantly decreased with the increased addition of crude enzyme extract (p < 0.05). The water holding capacity and cooking yield of the treated samples showed no significant difference throughout the crude enzyme extract addition (p > 0.05). Crude enzyme extract had no effect on the pH of the pork sample, but it slightly increased the pH in the beef and chicken. An increase in protein solubility and TCA-soluble peptides content was observed in all of the treated samples. The electrophoresis pattern of the muscle treated samples also revealed extensive proteolysis occurring in each muscle type. From the results, it is determined that latex from Calotropis procera can be used as an alternative source of proteolytic enzymes for the effective tenderising of meat.     

Increasing omega-3 levels through dietary co-extruded flaxseed supplementation negatively affects pork palatability

To clarify the impact of feeding co-extruded flaxseed on carcass quality and pork palatability, 96 pigs (48 barrows and 48 gilts) were fed three different levels of flaxseed (0%, 5% and 10% of dietary intake) for 76 days. Carcass quality and meat quality characteristics of pure loin muscle and ground pork (20% fat) were evaluated. Fat hardness and belly firmness decreased (P < 0.001) with increasing co-extruded flaxseed. Pigs fed co-extruded flaxseed levels had higher lean yield (P = 0.045) and total lean content (P = 0.034). Loin from barrows had higher fat content compared to gilts (P < 0.001). Co-extruded flaxseed supplementation increased (P < 0.001) omega-3 content in loin and ground pork. Pork flavour intensity and off-flavour intensity scores lowered (P < 0.001) with increasing levels of co-extruded flaxseed, being more accentuated (P = 0.023) in reheated pork chops from barrows. Diet affected all texture and flavour sensory characteristics (P < 0.05) as tissue levels of omega-3 fatty acids increased, likely as a result of increased lipid oxidation.     

The suitability of plasma powder for cold-set binding of pork and restructured dry ham

To determine the ability of cold-set binder plasma powder (PP) for manufacturing restructured deboned dry ham, the effect of meat pre-treatment and PP preparation on the binding rate (k) and maximum binding force (BF_max) of pork model systems and deboned ham were evaluated. In pork model systems, the highest values for k (about 0.4 Ncm^− 2 h^− 1) and BF_max (about 2.5 Ncm^− 2) were obtained when powder or rehydrated plasma [in water or in NaCl aqueous solution at 0.5%] was applied onto the meat surface without additional pre-treatment or prior immersion in saline aqueous solution. Similar meat pre-treatment and PP preparation were used to restructure fresh deboned leg resulting in stable meat binding performances during salting and drying. An important increase in the binding force (BF_max > 10 Ncm^− 2) occurred over the drying period (after 4 weeks). Scanning electron microscopy showed different morphologies of the binding area, mainly depending on whether powder or rehydrated plasma was used.     

A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods.     

Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat

The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants.     

Oxidation in HiOx-packaged pork Longissimus muscle predisposes myofibrillar and sarcoplasmic proteins to N-nitrosamine formation in nitrite-curing solution

The effect of meat protein in situ oxidation on the formation of N-nitrosodiethylamine (NDEA) was investigated. Fresh minced pork was untreated (Con) or treated with 700 mg/kg α-tocopherol (Toc) or 300 mg/kg tea polyphenols (PPE), packaged in a HiOx atmosphere (78.8% O₂, 18.8% CO₂, 2.4% N₂), then stored at 2 ± 1 °C for up to 10 days. Crude myofibrillar (MP) or sarcoplasmic (SP) protein (20 mg/mL) extracted from stored meat was reacted with 43 μM sodium nitrite at 80 °C for 1 h. Lipid oxidation was totally inhibited in PPE pork but increased in Con and Toc samples after 10 days. There was significant protein oxidation (losses of sulfhydryls, formation of protein carbonyls) in both MP and SP in all samples during storage. However, the Con group suffered more extensive protein oxidation than Toc and PPE and produced more NDEA (P < 0.05), indicating that protein oxidation promoted nitrosation.     

The antioxidative properties of mustard leaf (Brassica juncea) kimchi extracts on refrigerated raw ground pork meat against lipid oxidation

The efficacy of varying concentration of mustard leaf kimchi ethanolic extracts (MK) in retarding oxidative rancidity was tested with raw ground pork. Freshly ground pork meat was assigned to one of the following five treatments: control (no antioxidants); AC-0.02 (0.02% ascorbic acid); MK-0.05, 0.1, and 0.2 (0.05%, 0.1% and 0.2% MK, respectively). The pH of the samples decreased and the TBARS values and free fatty acids (%) increased considerably (P < 0.05) during storage. The total bacterial count was lower in MK-0.1 and MK-0.2 than the control during storage. The internal L∗ value and a∗ value decreased (P < 0.05) with the addition of MK. The internal b∗ value of MK treatments were higher (P < 0.05) than that for the control and increased incrementally with MK concentration. The TBARS values and free fatty acids (%) of MK-0.02 was lowest among the treatments. The peroxide value of the control increased until 7 days and reached the maximum value at a certain storage time and decreased thereafter. In the other treatments it increased. All treatments had lower concentration of conjugated dienes (P < 0.05) compared to the control sample, after the first day. Mustard leaf kimchi ethanolic extracts exhibited a protective effect against lipid oxidation in raw ground pork.