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.     

Polymerase chain reaction assay for identification of chicken in meat and meat products

The aim of this study was to develop polymerase chain reaction (PCR) assay for specific detection of chicken meat using designed primer pair based on mitochondrial D-loop gene for amplification of 442 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The PCR result was further verified by restriction digestion with HaeIII and Sau3AI enzymes for specific cutting site in amplified DNA fragments. The specificity of assay was cross tested with DNA of cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail, where amplification was observed only in chicken without cross reactivity with red meat species. However positive reaction was also observed in quail and turkey. In this study, no adverse effects of cooking and autoclaving were found on amplification of chicken DNA fragments. Thus, the detection limits was found to be less than 1% in admixed meat and meat products. The developed assay was found specific and sensitive for rapid identification of admixed chicken meat and meat products processed under different manufacturing conditions.     

The detection of commercial duck species in food using a single probe-multiple species-specific primer real-time PCR assay

A real-time PCR assay for the simultaneous detection of Mallard and Muscovy duck is described. Species-specific primers were designed for Mallard or Muscovy duck using the mitochondrial cytochrome b gene sequence. These primer sets were multiplexed with a single duck probe to produce a simple, rapid and robust real-time PCR assay. This assay was shown to be specific for duck compared to a wide range of commercially important meat species and was used for the successful detection of duck meat in complex food matrices. This is the first report of an assay that will detect all species of commercially available duck in commercial products using real-time PCR.     

Effect of cooking methods on the formation of heterocyclic aromatic amines in chicken and duck breast

Heterocyclic aromatic amines (HAAs), potent mutagens/carcinogens, are pyrolysis formed during the cooking of meat and fish. In the present study, the effects of various cooking methods, pan-frying, deep-frying, charcoal grilling and roasting on the formation of HAAs in chicken breast and duck breast were studied. The various HAAs formed during cooking were isolated by solid-phase extraction and analyzed by high-performance liquid chromatography (HPLC). Results showed that chicken breast cooked by charcoal grilling contained the highest content of total HAAs, as high as 112 ng/g, followed by pan-fried duck breast (53.3 ng/g), charcoal grilled duck breast (32 ng/g), pan-fried chicken breast (27.4 ng/g), deep-fried chicken breast (21.3 ng/g), deep-fried duck breast (14 ng/g), roasted duck breast (7 ng/g) and roasted chicken breast (4 ng/g). For individual HAA, the most abundant HAA was 9H-pyrido-[4,3-b]indole (Norharman), which was detected in charcoal grilled chicken breast at content as high as 32.2 ng/g, followed by 1-methyl-9H-pyrido[4,3-b] indole (Harman) and 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine(PhIP) at 32 and 31.1 ng/g in charcoal grilled chicken breast, respectively. The content of PhIP in pan-fried duck and chicken breast were 22 and 18.3 ng/g, respectively. Generally, the type and content of HAAs in cooked poultry meat varies with cooking method and cooking conditions.     

Identification of genetic markers associated with residual feed intake and meat quality traits in the pig

Residual feed intake (RFI) has become increasingly important and is being considered as a more reasonable approach to evaluate feed efficiency in livestock. However, the cost and technical difficulties in measuring this trait restrict the extensive adoption of RFI selection, and this makes marker assisted selection (MAS) a feasible tool. In addition, the effects on meat quality caused by low RFI selection have yet to be clarified. In this study, 11 SNPs from eight candidate genes were evaluated in a Yorkshire pig experimental population (n = 169) consisting of a low RFI selection line and a randomly selected control line. Associations of these SNPs with RFI, growth rate, carcass composition, and meat quality measures including water holding capacity, pH at 2 d postmortem, meat color and sensory traits were analyzed. The SNPs FTO p.Ala198Ala and TCF7L2 c.646+514A > G showed significant (P < 0.05) and suggestively significant (P < 0.1) associations with RFI, respectively. The MC4R SNP p.Asp298Asn was associated with backfat but it was not with ADG and meat quality attributes. Both SNPs within HNF1A were associated with intramuscular lipid content and sensory juiciness. The SNPs ACC1 c^∗384C > T and TCF7L2 c.646+514A > G were significantly (P < 0.05) associated with ADG. The SNPs CTSZ p.Arg64Lys and TCF7L2 c.646+514A > G were associated with both visual scoring of meat color and the objective L-value measure of meat color. This study has identified potential genetic markers suitable for MAS in improving RFI, ADG, and meat color traits, but these associations need to be validated in other larger populations.     

Mitochondrial respiratory activity in porcine longissimus muscle fibers of different pig genetics in relation to their meat quality

The pig genetics of Duroc, Pietrain (MHS homozygote negative, PiNN), Pietrain (MHS homozygote positive, PiPP) and a F2-Duroc-Pietrain cross-breed were analyzed. The animals had comparable (P > 0.05) carcass weights, but the PiPP pigs had higher carcass yield and lean meat values (P < 0.05). Considering the meat quality characteristics, the PiPP showed a faster pH drop and higher electrical conductivity, drip loss, shear force as well as lightness and redness values (P < 0.05). The PiPP animals had less slow-twitch-oxidative (STO) and more fast-twitch-glycolytic (FTG) muscle fibers, whereas the results of the Duroc animals were converse (P < 0.05). The STO and FTG fibers of the PiPP animals were larger than those of the other genetics (P < 0.05). The analysis of the mitochondrial respiratory activity (MRA) using permeabilized longissimus muscle fibers resulted in no differences between the pig genetics before and immediately after slaughter. During chilling the MRA decreased in all pigs but to a higher extent in the PiPP pigs (P < 0.05).     

Mitochondrial markers for the detection of four duck species and the specific identification of Muscovy duck in meat mixtures using the polymerase chain reaction

A polymerase chain reaction (PCR) assay for the qualitative detection of four duck species in meat mixtures, and a second PCR assay for the specific identification of Muscovy duck, have been developed based on oligonucleotide primers targeting the 12S rRNA mitochondrial gene. The specificity of both assays was tested against a wide range of animal species. The technique was applied to raw and sterilized muscular binary mixtures, with a detection limit that ranged from 0.1% to 1.0% (w/w). The short length (less than 100 bp) of the DNA fragments amplified with these primer pairs was found to be essential for the successful amplification in samples with highly degraded DNA, and consequently, it could be very useful in inspection programmes to enforce labelling regulation of heat and pressure-processed products, for which other methods cannot be applied.     

Contents of biologically active polyamines in chicken meat,liver, heart and skin after slaughter and their changes during meat storage and cooking

Dietary polyamines, putrescine, spermidine (SPD) and spermine (SPM), participate in an array of important physiological roles, including tumour growth. Thus, reliable information on polyamine content in foods has been needed. We therefore determined polyamine contents in chilled chicken meat and giblets (n = 20) and skin (n = 10) 24 h after slaughter. The polyamines were determined, after extraction with perchloric acid, as dansyl derivatives, using an HPLC method. Mean SPD values were 4.8, 10.2, 11.4, 48.7 and 12.1 mg kg⁻¹ and SPM values were 36.8, 38.0, 24.3, 133 and 82.7 mg kg⁻¹ in breast, thigh, skin, liver and heart, respectively. Significant statistical correlations between SPD and SPM contents were observed in breast, thigh, skin and liver, whereas correlations were insignificant in heart. An increase of SPD and SPM was apparent in breasts and thighs stored at −18 °C for 6 months; however, it was significant only for SPM in thighs. The losses of both SPD and SPM were statistically insignificant during storage of aerobically packaged breasts up to 9 days at +2 °C. A significant decrease of SPM to about 60% of the initial contents was observed in both vacuum-packaged and in modified atmosphere (20% CO₂ and 80% O₂)-stored breasts on day 21 at +2 °C. For both SPD and SPM, roasting, grilling and frying of fresh breasts caused losses of about 40–60% of the initial contents (higher than boiling and stewing). Similarly, losses of SPM, due to roasting of breasts frozen for 3 or 6 months, were higher than those caused by stewing. Putrescine was detected only sporadically and at levels close to the detection limit of 1.0 mg kg⁻¹ (fresh matter).     

肉制品中鸭源性成分的实时荧光PCR检测

目的：建立基于实时荧光PCR技术的鸭源性成分的快速检测方法。方法：以鸭线粒体DNA序列为目的基因,设计并筛选了鸭源性特异性引物及探针,进行荧光定量PCR扩增,建立鸭源性成分检测方法。通过特异性、灵敏性、及盲样检测试验,对该体系进行验证。结果：该方法能够快速有效的检测鸭源性成分,具有较强的特异性及灵敏性,灵敏度约为0．01％（质量分数）;通过市售盲样肉制品的检测,表明该体系可用于定性检测加工肉制品中的鸭源性成分。结论：该方法特异性强,灵敏度高,可用于对肉制品中鸭源性成分的掺假鉴别。     

利用线粒体DNA Cyt b基因PCR-RFLP分析方法鉴别羊肉和鸭肉

建立了一种利用线粒体DNA（mtDNA） Cyt b基因PCR-RFLP分析来鉴别羊肉和鸭肉的方法。采用一对通用引物扩增绵羊、山羊和鸭的mtDNACytb基因,并对扩增产物用DNA限制性内切酶Bsu36I和SpeI进行酶切,电泳分析酶切产物的变化。结果表明通用引物可扩增羊和鸭472bp的PCR产物,经两种内切酶酶切后,绵羊、山羊和鸭的PCR产物分别被切为大小不同的片段,其中绵羊和山羊被SpeI切为213bp和259bp,而鸭则被Bsu36I切为95bp和377bp。利用PCR-RFLP分析mtDNA Cyt b基因的方法操作简单,是一种快速鉴别羊肉和鸭肉的可靠方法。      

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.     

Determination of pig sex in meat and meat products using multiplex real time-PCR

For specific production lines, European retail companies demand exclusively female pork meat. To control the quality of their suppliers the identification and a quantitative detection of the animal sex origin of the meat is therefore of importance for meat processors. To enable a fast and reliable detection of male pig meat, a real time-PCR-system was designed in the present study. This was based on the genes AMEL-X and AMEL-Y. The real time-PCR assay allowed the detection of male pig meat at a concentration of 1% yielding a detection probability of 100% while the detection probability investigating meat samples containing 0.1% male pig meat was 44.4%. The analytic sensitivity of this system was assessed to be <5 pg DNA per PCR reaction. The assessment of the accuracy of the real time-PCR assay to correctly identify sex individuals was investigated with 62 pigs including males (n=29) and females (n=33) belonging to different breeds/lines. With the newly designed test all analysed animals were correctly sexed. No amplification was obtained with cow, goat, sheep, turkey and chicken genomic DNA. The presented assay can be used for sex diagnosis, for the detection of male pig meat and for meat quality control.     

Cold atmospheric gas plasma disinfection of chicken meat and chicken skin contaminated with Listeria innocua

Gas plasmas generated at atmospheric pressure and ambient temperatures offer a possible decontamination method for poultry products. The efficacy of cold atmospheric gas plasmas for decontaminating chicken skin and muscle inoculated with Listeria innocua was examined. Optimization of operating conditions for maximal bacterial inactivation was first achieved using membrane filters on which L. innocua had been deposited. Higher values of AC voltage, excitation frequency and the presence of oxygen in the carrier gas resulted in the greatest inactivation efficiency, and this was confirmed with further studies on chicken muscle and skin. Under optimal conditions, a 10 s treatment gave > 3 log reductions of L. innocua on membrane filters, an 8 min treatment gave 1 log reduction on skin, and a 4 min treatment gave > 3 log reductions on muscle. These results show that the efficacy of gas plasma treatment is greatly affected by surface topography. Scanning electron microscopy (SEM) images of chicken muscle and skin revealed surface features wherein bacteria could effectively be protected from the chemical species generated within the gas plasma. The developments in gas plasma technology necessary for its commercial application to foods are discussed.     

Manipulation of the fatty acid composition of pig meat lipids by dietary means

Pigmeat products have been associated with an unhealthy image due to the relative proportions of polyunsaturated and saturated fatty acids. The aim of this experiment was to improve the fatty acid profile of the carcass fat by feeding various dietary sources of fat. Groups of 10 female Large While × Landrace pigs were fed one of four experimental diets. Five in each group were slaughtered at 70 kg live weight and the remaining five at 100 kg live weight. The diets were offered ad libitum and daily intake was recorded. The diets were based on barley, soya bean meal and fishmeal. Diet 1 contained 50 g tallow kg^?1, a relatively saturated fat, and diets 2, 3 and 4 contained 50 g soya oil kg^?1, an unsaturated fat. Diet 3 also contained 7.5 g GLA oil kg^?1, which is rich in gamma linolenic acid, with the aim of increasing the production of arachidonic acid in the body fat. Diet 4 was supplemented with 9.5 g EPAnoil kg^?1, which is rich in eicosapentaenoic acid and docosahexaenoic acid. There were no significant differences between dietary treatments in performance (daily liveweight gain or the efficiency of food conversion to liveweight gain) of the pigs slaughtered at 70 kg live weight, but small differences were observed at 100 kg live weight, where pigs on treatments 2, 3 and 4 performed slightly better than those on treatment 1. At both slaughter weights the lipid content of the m semitendinosus was higher than that of the m longissimus dorsi (approximately 24 and 13 g kg^?1, respectively). The pattern of fatty acids in the dietary fat was reflected to varying degrees in the carcass fat. Diet 1 resulted in the highest levels of palmitic, palmitoleic, stearic and oleic acids whereas diets 2, 3 and 4 gave high levels of linoleic and linolenic acids. The extra gamma linolenic acid in diet 3 did not result in a consistently significant increase in the production of arachidonic acid. The supplement of EPAnoil gave significant increases in the levels of eicosapentaenoic and docosahexaenoic acids in the body lipids. The polyunsaturated to saturated fatty acid ratio of the body lipid was increased with diets 2, 3 and 4 to about 1.0. In spite of high levels of linoleic acid, there were no adverse effects during the processing of the carcasses and the taste panel evaluation did not reveal any treatment differences.     

Assessing the quality of mechanically and manually recovered chicken meat

The biochemical composition and histological characteristics of meat recovered mechanically by the auger/sieve (a/s) and the hollow drum/belt (hd/b) principles from two different chicken carcass parts were compared with meat recovered manually. The quality of meat recovered mechanically by the hollow drum/belt principle was equal to or higher than the quality of manually recovered meat. The degradation of muscle structure was highest in the meat recovered by the a/s principle and lowest in the manually recovered meat. For the biochemical measurements with the exception of collagen, determinations of a single sample were sufficient to achieve a repeatability of 0.9, whereas for the histological measurements at least 8 samples were necessary. It is suggested that a quality-grading scale based on biochemical composition and level of degradation of muscle fibre structure should be established for all types of minced meat regardless of whether the meat is obtained by mechanical or manual procedures and that legislation concerning the use of MRM should be based on such a scale.     

Effect of laccase and transglutaminase on the textural and water-binding properties of cooked chicken breast meat gels

The effects of laccase and transglutaminase (TG) on the firmness and weight loss of cooked chicken meat homogenate gels were investigated at laboratory scale. The salt, trisodium pyrophosphate and meat contents were also used as variables. Laccase decreased firmness and increased weight loss of phosphate-free, low-meat (65%) and low-salt (1%) gels, although it modified myosin and troponin T and reacted with isolated myofibrils. By applying both low-salt (1%) and low-phosphate (0.17%) amounts, gel firmness decreased and weight loss increased (p<0.05) greatly. A high dosage of TG significantly improved (p<0.05) the strength of phosphate-free, low-meat and low-salt homogenate gels compared to the corresponding no-enzyme controls. TG improved gel firmness of the low-meat homogenate to the level of the homogenate containing 75% meat. Weight loss was increased significantly (p<0.05) in all cases when the high-TG dosage was used. Enzymes were not capable of improving either texture or water-holding capacity in the low-salt–low-phosphate system. The firmness and cooking loss of the chicken meat products containing different amounts of meat, salt and TG were investigated at pilot scale. Under the conditions and dosages used, TG was capable of improving (p<0.05) firmness of the products without a significant reduction in water-holding capacity.     

H NMR and multivariate data analysis of the relationship between the age and quality of duck meat

To contribute to a better understanding of the factors affecting meat quality, we investigated the influence of age on the chemical composition of duck meat. Aging probably affects the quality of meat through changes in metabolism. Therefore, we studied the metabolic composition of duck meat using ¹H nuclear magnetic resonance (NMR) spectroscopy. Comprehensive multivariate data analysis showed significant differences between extracts from ducks that had been aged for four different time periods. Although lactate and anserine increased with age, fumarate, betaine, taurine, inosine and alkyl-substituted free amino acids decreased. These results contribute to a better understanding of changes in duck meat metabolism as meat ages, which could be used to help assess the quality of duck meat as a food.     

Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR

The identification of pork DNA in meat extracts is very important for Halal authentication and Muslim consumers demand protection from falsely labelled meat products. A pig‐specific SYBR green I real‐time PCR assay has been developed to address this issue. Using specific primers for pig mitochondrial DNA, successful amplification has been obtained by DNA extracted from control meat samples. With SYBR green I real‐time PCR, the specificity of the amplification was showed by Tm value. Detection limit of the real‐time PCR was down to 0.1 ng of porcine DNA. An appropriate linearity was obtained by construction of a standard curve based on Ct value and different concentrations of porcine DNA. By conventional PCR, no amplification was shown by porcine DNA less than 0.1 ng. The established method was conducted on commercially available meat extracts for detection and quantification of porcine DNA. The results showed the SYBR green I real‐time PCR could be considered a robust method for Halal authentication of meat extracts.     

Comparing the histochemical characteristics and meat quality traits of different pig breeds

The purpose of this study was to compare the muscle histochemical characteristics and meat quality traits between Berkshire, Landrace, Yorkshire, and crossbred pigs. A total of 594 pigs were evaluated. A clear difference between histochemical properties was observed from the results for fiber type composition. In Berkshire pigs, the area percentage of type I fibers was higher (P < 0.001) and that of type IIb fibers was lower (P < 0.05) than those of other breeds. The muscle pH_45min and pH_24h were significantly higher in Berkshire pigs. Drip loss and color parameters were significantly different between the breeds (P < 0.001). The Berkshire pigs, which showed the highest muscle pH and lowest drip loss and L^* values, contained a significantly higher percentage of type I fibers than the other breeds. By comparing the fiber type compositions of the different breeds, the results imply that the longissimus dorsi muscle of Berkshire pigs is more oxidative than that of other breeds. A high pH value in Berkshire pigs is due to a high percentage of type I fibers and a low percentage of type IIb fibers. Based on these results, we conclude that muscle fiber composition can explain in parts the variation of meat quality across and within breeds.