Detection of Pork in Processed Meat by an Enzyme-linked Immunosorbent Assay Using Antiswine Antisera

An antiserum to autoclaved porcine muscle extract was raised in sheep and tested for the detection of pork in mixtures of pork in mutton (P/ S) or beef (P/B) heated at 70, 100, and 120°C for 30 min using a competitive enzyme-linked immunosorbent assay (ELISA) method. The results indicated that the antiserum detected low percentages of pork (1%) mixed in beef and in mutton (0.5%) even when the meat mixtures were heated at 70, 100 or 120°C for 30 min, which correspond to heat treatments of commercially processed meat products. Regression analysis showed a high positive correlation (r > 0.99) between absorbance (OD) values and different percentages of experimental or commercially processed P/B meat mixtures. It was concluded that the combination of anti-porcine sera raised in sheep and the ELISA method can be used for the detection of low percentages of pork in processed meats.     

Analysis of Pork Adulteration in Commercial Burgers Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by TaqMan Probe Real-Time Polymerase Chain Reaction

A TaqMan probe real-time polymerase chain reaction assay was developed for the determination of pork adulteration in commercial burgers. The assay combined porcine-specific primers and TaqMan probe for the selective amplification and detection of a 109-bp fragment of swine cytochrome b (cytb) gene. Specificity test with 10 ng DNA of 11 different meat-providing animal and fish species yielded a quantification cycle (Cq) of 15.5 ± 0.20 for the pork and negative results for the others in a 40-cycle reaction with a change of analysts and sources. Analysis of beef burger formulations with spiked pork showed the assay can determine 100–0.01% contaminated pork with a PCR efficiency (E) of 93.8% and a correlation coefficient (R ²) of 0.991. A plot of actual value against real-time PCR-predicted value also yielded a good linear regression, R ² 0.998, and small root mean square error of calibration, RMSEC 0.42. A strong correlation was found between the partial least square (PLS)-predicted values and real-time PCR-determined values. The accuracy of the method was ≥90% in all determinations of the standard set. Residual analysis also revealed a high precision in all determinations. Finally, a random analysis of 10 ng DNA of commercial burgers from pork, beef, chicken, mutton, and chevon yielded a Cq of 15.56 ± 0.22 to 16.24 ± 0.35 from pork burgers, and negative results from the others, showing the suitability of the assay to determine pork in commercial burgers with a high accuracy and precision.     

Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10⁸ to 10⁶ CFU) and low (10⁵ to 10⁰ CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 °C. Nucleic acid was extracted using the TRIzol^® method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 °C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10² CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10⁶ CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. Practical Application: The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocylcer but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.     

Oxidative Effects of Meat Grinder Wear on Lipids and Myoglobin in Commercial Fresh Pork Sausage

Pork ground before and after the replacement of worn meat grinder plates and knives was formulated as commercial fresh pork sausage containing antioxidants and processed into 454-g chubs. The sausage was frozen at ^?15°C for 4, 8, 12, and 16 wk and monitored for oxidation after 1 day, 2 wk and 3 wk of postfrozen refrigeration at 1°C. Estimated average grinder metal in sausage was 136 ppb. Meat temperature rise during grinding was higher (P<0.05) with the worn hardware (2.3°C vs 1.6°C). Hunter “a” values were greater (P<0.05) with the sharp than with the worn equipment. 2-Thiobarbituric acid (TBA) numbers did not differ with equipment state of wear. Significant correlations (P<0.05) indicated in inverse relationship between the oxidation of lipids and myoglobin.     

Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed Products

A monoclonal antibody‐based sandwich enzyme‐linked immunosorbent assay (ELISA) was developed for the sensitive detection of porcine skeletal muscle in raw and heat‐processed meat and feed products. Heat treatment of meat samples up to 132 °C for 2 h did not affect the assay performance. The assay uses a pair of monoclonal antibodies (MAbs 8F10 and 5H9) specific to skeletal muscle troponin I (TnI). MAb 8F10, reacting to mammalian TnI, is the capture antibody and the biotin‐conjugated MAb 5H9, specific to porcine TnI, the detection antibody. The sandwich ELISA is able to detect 0.05% (w/w) of laboratory‐adulterated pork in chicken, 0.1% (w/w) pork in beef mixtures, 0.05% (w/w) pork meal in soy‐based feed, and 1% commercial meat and bone meal (MBM), containing an unknown amount of pork, in soy‐based feed. This new assay provides a rapid and reliable means to detect the contamination of meat and feed products with trace amounts of porcine muscle tissue to ensure product quality and safety.     

Heat-Resistance of Escherichia Coli O157:H7 in Meat and Poultry as Affected by Product Composition

The effects of fat level and low fat formulation on survival of Escherichia coli O157:H7 isolate 204P heated in ground beef [7%, 10% and 20% fat], pork sausage [7%, 10%, and 30% fat], chicken (3% and 11% fat), and turkey (3% and 11% fat) were determined by D- and z-values. D-values for E. coli 0157:H7 in lowest fat products were lower than in traditional beef and pork products (P < 0.05). Overall, higher fat levels in all products resulted in higher D-values. D₆₀ values (min) ranged from 0.45–0.47 in beef, 0.37–0.55 in pork sausage, 0.38–0.55 in chicken and 0.55–0.58 in turkey. D₅₅ and D₅₀ values were respectively longer. Z-values ranged from 4.4–4.8°C. Product composition affected lethality of heat to E. coli O157:H7.     

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.     

Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction

A study on development of Fourier-transform infrared spectrophotometric method combined with principle component analysis as well as real-time polymerase chain reaction for determination of pork–beef mixture in meatballs has been performed. A lipid component extracted from pork and beef in meatballs is analyzed using Fourier-transform infrared spectroscopy, while DNA extracted from meatball was analyzed using real-time polymerase chain reaction. The correlation between actual and predicted concentration of lard using Fourier-transform infrared spectroscopy was performed by aid of partial least squares, while grouping of lard and beef fat components in meatball was carried out by Fourier-transform infrared spectra coupled with principle component analysis. The results showed that Fourier-transform infrared spectra at wavenumbers of 1000–1200 cm^?1 coupled with partial least square and principle component analysis are successfully used for quantification and classification of pork in beef meatballs. The relationship between actual value and predicted value of lard (lipid fraction obtained from meatballs containing pork) with Fourier-transform infrared spectrophotometric method revealed good correlation, with coefficient determination (R²) value of 0.997 and standard error of calibration of 0.04%. Principle component analysis is able to classify samples containing pork and beef meatballs. Fourier-transform infrared spectroscopy using normal spectra is fast technique for identification and quantification of lard extracted from pork in meatball. In addition, real-time polymerase chain reaction using Leptin Primer–AJ 865080 can be used for amplification of pork DNA specifically in meatballs containing pork.     

Functional and Sensory Properties of Meat Emulsions Produced by using Enzymatically Modified Gelatin

Mettwurst and Bologna sausage were produced from pork using enzymatically modified gelatin (EMG-8), which had been produced with covalent attachment of L-leucine n-octyl ester. EMG-8 improved spreadability, smoothness and softness of the Mettwurst. With the addition of EMG-8, the texture of the Bologna sausage was kept soft over a 1 to 7 day period of storage at 4°C or 2 to 4 weeks of storage at -20°C. Microscopic observation showed that fat globules in the Bologna sausage produced with EMG-8 were more homogeneously distributed than those in a control Bologna sausage.     

Compositional Factors that Influence Lipid Peroxidation in Beef Juice and Standard Sausages

In order to identify how different additives influenced lipid peroxidation formation, a sausage only using beef juice as pigment source and a standard beef–pork meat sausage were studied. The effects of different additives, including fish oil, myoglobin, nitrite, clove extract, and calcium sources on oxidation and sensory properties were examined. Both sausage systems were stored in 3 different manners prior to testing: (1) frozen immediately at ?80 °C; (2) chilled stored for 2.5 weeks followed by fluorescent light illumination at 4 °C for another 2 wk; (3) frozen at ?20 °C for 5 mo. The frozen group 3 showed the highest peroxide formation and thiobarbituric acid reactive substances (TBARS) for both sausage systems. Unpolar peroxides dominated in both systems. The clove extract could offset the peroxide formation from myoglobin/beef juice and/or fish oil, but the addition of clove flavor was recognized by the sensory panelists. Calcium addition reduced lipid peroxide formation. Added nitrite and fish oil seemed to interact to stimulate nitroso‐myoglobin formation. Nitrite was identified to interact with clove addition and thereby, relatively speaking, increased TBARS. The 2 sausage systems generally ranked the additives similarly as pro– and antioxidants.     

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration by digital PCR

The alarming problem of meat adulteration emphasises the demand for accessible analytical approaches for food regulatory agencies to detect and, specially, to measure altered meat fractions. This study proposes a novel cross-species triplex droplet digital polymerase chain reaction (ddPCR) assay to simultaneously identify and quantify the ratios of pork/beef meat fractions from a total DNA content, including processed and autoclaved meat, without requiring a standard, achieving high sensitivity with a limit of quantification estimated at 0.1% (w/w) and a limit of detection down to 0.01% (w/w). A single copy nuclear gene, β-actin, was employed as a target, accompanied with myostatin gene as a cross-species target to quantify the meat background. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and time.     

Effect of Acid and Neutral Pyrophosphates on the Natural Bacterial Flora of a Cooked Meat System

The microbiological effects of selected pyrophosphates were studied in cooked, vacuum-packaged pork sausage stored at 7°C for 21 days, or at 20–22°C for 24 and 48 hr. Neutral trisodium pyrophosphate (PYRO-3) and sodium acid pyrophosphate (SAPP) were tested at 0 and 0.4% meat weight, separately or in combinations. Both phosphates had an effect after 21 days refrigerated storage (7°C), with lower mesophilic counts than controls with no phosphate. Both phosphates also resulted in significantly lower counts of mesophilic and facultative anaerobic microorganisms in sausage after 48 hr elevated temperature (20–22°C) holding. The organisms affected were mainly streptococci.     

微滴式数字聚合酶链式反应对香肠制品中鸡、猪、牛源性成分的定量分析

为实现肉制品中动物源性成分的定量检测,基于微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction,ddPCR）,以鸡的转化生长因子β-3基因、猪的朊蛋白基因和牛的生长激素基因为靶基因,建立香肠制品中鸡、猪、牛源性成分的定量检测方法。结果表明：所建立ddPCR方法特异性强,能特异性检测相应的鸡、猪、牛源性成分;根据肉粉质量（mg）与DNA质量浓度（ng/μL）、DNA质量浓度（ng/μL）与基因拷贝数浓度（copies/μL）之间的线性关系,得到鸡肉粉、猪肉粉和牛肉粉质量（x1）与基因拷贝数浓度（ y 2） 之间的关系式为： 鸡：x1=（n*y2）/273.946 - n/886.557 + 0.216; 猪：x1=（n*y2）/64.950 + n/60.644 + 0.215; 牛：x1=（n*y2）/62.839 + n/12.646 + 1.218（n为稀释倍数）;基于建立的ddPCR方法对64 份市售不同种类香肠样品进行检测,在1 份原料标识仅为“猪肉”的香肠中检出鸡源性成分含量为18.68%。本研究建立的ddPCR方法能够实现香肠中鸡、猪、牛源性成分的准确定量检测,并可以此判断“蓄意掺假”或“无意带入”。     

Identification of Meat from Game and Domestic Species

Isoelectric focusing combined with specific enzyme staining for creatine kinase was used to characterize banding patterns in meat from pronghorn, mule deer, white-tailed deer, sheep, moose, pork, bison, elk, caribou, red deer, beef and goat. Processed and cooked pork was differentiated from all species and beef and elk were separated from pronghorn. It was not possible to differentiate beef from elk, or prong-horn from sheep. The inability to separate some game from some domestic species and the lability of the staining proteins after heating above 67°C limits the application of this technique.     

Palatability and Appearance Traits of Beef/Pork Meat Patties

Eight beef/pork ground meat blends were made from mature (cows or sows) and youthful (steers or barrow) beef and/or pork lean. Blends were stored at — 27°C for either 14 or 150 days. Storage time decreased overall desirability scores of blends made with 20% pork fat and 80% youthful beef lean. No differences were found for flavor or overall desirability scores within the 14-day storage treatment. The consumer panel did not detect differences among treatments for source of fat or species. Results indicated beef/pork patties containing 40–80% mature lean and a minimum of 10% beef fat were equal to all-beef controls (100% beef patty) for visual and palatability traits.     

Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction

A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.     

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.     

Ready-to-use single tube quadruplex PCR for differential identification of mutton,chicken, pork and beef in processed meat samples

ABSTRACT

A reliable and sensitive identification method is required to tackle food adulteration mainly in meat production. We developed a dry reagent based ready-to-use single tube quadruplex PCR assay for accurate identification of chicken, mutton, beef and pork. The assay was found to be specific and reproducible. Thermo-stability studies of lyophilized PCR master mix were conducted at different temperature and time intervals, which revealed significant stability for 75 days at 4°C and for 60 days at 25°C. The developed assay was shown to be sensitive down to 16 pg DNA per reaction and the detection limit was found to be 0.01% (w/w) of each species. Furthermore, this method has been applied to the analysis of 68 commercial meat products and the results indicated that nine samples contained non-declared meat components. This dry reagent-based quadruplex PCR assay can be utilized to monitor various processed food products and also to maintain quality control in food industries mainly in the resource-limited settings.     

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg^?1 for sesame and 1.4 mg kg^?1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.     

Sodium Lactate Effects on Shelf-Life, Sensory, and Physical Characteristics of Fresh Pork Sausage

The objective of our study was to evaluate the effects of various levels (0%, 1%, 2%, 3%) of sodium lactate on selected physical, sensory, and microbiological characteristics of fresh pork sausage stored at 4°C for 28 days. Samples containing 0% and 1% SL reached 10⁸ CFU/g after 10 days refrigerated storage. Addition of 2% or 3% SL to fresh pork sausage delayed microbial deterioration, pH decline, and development of sour- and off-flavors 7 days at 4°C. Samples containing 2% SL did not reach total plate counts of 10⁸ CFU/g until 24 days storage. SL appeared to protect red color, and to “enhance” pork-and salty-flavors in sausage. TBA, L-, a-, and b-values were unaffected by SL level.