Pork Adulteration Research Articles

A label free electrochemical immunosensor for sensitive detection of porcine serum albumin as a marker for pork adulteration in raw meat

A label free electrochemical immunosensor for sensitive detection of porcine serum albumin (PSA) is reported in this work. The immunosensor was constructed by first electrochemically reducing 4-carboxyphenyl diazonium salt, which had been electrochemically generated in situ, to a stable 4-carboxyphenyl layer on carbon nanofiber-modified screen printed electrode. Antibodies were covalently attached onto the electrode using carbodiimide chemistry between the carboxylic groups of the 4-carboxyphenyl layer and amine groups of the antibody. Using the strong affinities of serum albumins towards anions, the increase in cathodic peak current in anionic redox probe after immunocomplex formation with antibodies was used for PSA detection. The reported immunosensor demonstrated a linear range from 0.5 to 500pg/mL for the measurement of PSA with detection limit of 0.5pg/mL in buffer solution. Cross-reactivity studies have shown excellent specificity with satisfactory recovery of PSA in fresh meat samples without the need of sample dilution.

Application of class-modelling techniques to infrared spectra for analysis of pork adulteration in beef jerkys.

The use of chemometrics to analyse infrared spectra to predict pork adulteration in the beef jerky (dendeng) was explored. In the first step, the analysis of pork in the beef jerky formulation was conducted by blending the beef jerky with pork at 5-80% levels. Then, they were powdered and classified into training set and test set. The second step, the spectra of the two sets was recorded by Fourier Transform Infrared (FTIR) spectroscopy using atenuated total reflection (ATR) cell on the basis of spectral data at frequency region 4000-700cm(-1). The spectra was categorised into four data sets, i.e. (a) spectra in the whole region as data set 1; (b) spectra in the fingerprint region (1500-600cm(-1)) as data set 2; (c) spectra in the whole region with treatment as data set 3; and (d) spectra in the fingerprint region with treatment as data set 4. The third step, the chemometric analysis were employed using three class-modelling techniques (i.e. LDA, SIMCA, and SVM) toward the data sets. Finally, the best result of the models towards the data sets on the adulteration analysis of the samples were selected and the best model was compared with the ELISA method. From the chemometric results, the LDA model on the data set 1 was found to be the best model, since it could classify and predict 100% accuracy of the sample tested. The LDA model was applied toward the real samples of the beef jerky marketed in Jember, and the results showed that the LDA model developed was in good agreement with the ELISA method.

Methods for detection of pork adulteration in veal product based on FT-NIR spectroscopy for laboratory, industrial and on-site analysis

Three different methods for near infrared (NIR) based multivariate analyses were developed to reveal deliberate adulteration or accidental contamination of a pure veal product with pork and pork fat. More precise, methods for laboratory use of high performance Fourier transform-NIR (FT-NIR) desktop devices, methods suitable for industrial purpose like in- and on-line application with a fibre optic probe and methods applying a handheld spectrometer ready for on-site analyses were established. The methods were developed for the detection of pork adulteration in the meat and fat part of veal sausages. Therefore sausages were self-made based on a commercial veal product. Adulterations up to 50% (in 10% steps) with pork and pork fat were analyzed, respectively. Principal component analyses (PCA) were developed for every setup with previous data pre-treatment steps including wavelength selection, scattering corrections and derivatives of the spectral data. PCA scores were used as input data for support vector machines (SVM) classification and validation. Advantages and disadvantages of the equipment were discussed and the limits of detection regarding the setups were determined. Measurements were also carried out directly through a polymer packaging of the samples and compared to measurements through quartz cuvettes. Meat and fat adulteration could be detected up to the lowest level of contamination (10%) applying the laboratory setup and the industrial fibre optics setup, regarding measurements through quartz and polymer packaging. Analyses with the on-site setup led to successful separation up to the lowest degree of contamination (10%, measurement through quartz cuvettes) regarding meat adulteration and up to 20% and 40% contamination regarding the fat adulteration performing measurements through quartz cuvettes and through polymer packaging, respectively.

Pork adulteration in commercial meatballs determined by chemometric analysis of NIR Spectra

The use of chemometrics to analyse near infrared (NIR) spectra to determine pork adulteration in beef meatball was developed. Since, the adulteration of pork in beef meatball is frequently occurring. This study was aimed to develop a fast and simple technique for the determination and quantification of pork adulteration in beef meatball using NIR spectra and chemometrics. Both partial least-squares (PLS) calibration and linear discriminant analysis (LDA) model were developed to determine pork adulteration in beef meatballs. The models using the first derivative spectra, accurately classified 100 % of the pork adulterated beef meatballs samples using training set and test set. The PLS and LDA models were subsequently used for the determination of pork adulteration in real beef meatball samples. The results showed that the PLS and LDA models developed were in good agreement with the immunochromatographic method. Therefore, the potential of NIR spectra and chemometrics as a rapid method for halal authentication and identification of pork adulteration in beef meatball have been successfully developed.

Hyperspectral imaging in tandem with multivariate analysis and image processing for non-invasive detection and visualization of pork adulteration in minced beef

Pork adulteration in minced beef was detected for the first time using a hyperspectral imaging (HIS) technique.

Multispectral image analysis approach to detect adulteration of beef and pork in raw meats

The aim of this study was to investigate the potential of multispectral imaging supported by multivariate data analysis for the detection of minced beef fraudulently substituted with pork and vice versa. Multispectral images in 18 different wavelengths of 220 meat samples in total from four independent experiments (55 samples per experiment) were acquired for this work. The appropriate amount of beef and pork-minced meat was mixed in order to achieve nine different proportions of adulteration and two categories of pure pork and beef. After an image processing step, data from the first three experiments were used for partial least squares-discriminant analysis (PLS-DA) and linear discriminant analysis (LDA) so as to discriminate among all adulteration classes, as well as among adulterated, pure beef and pure pork samples. Results showed very good discrimination between pure and adulterated samples, for PLS-DA and LDA, yielding 98.48% overall correct classification. Additionally, 98.48% and 96.97% of the samples were classified within a ±10% category of adulteration for LDA and PLS-DA respectively. Lastly, the models were further validated using the data of the fourth experiment for independent testing, where all pure and adulterated samples were classified correctly in the case of PLS-DA, while LDA was proved to be less accurate.

Feasibility of Detecting Pork Adulteration in Halal Meatballs Using near Infrared Spectroscopy (NIR)

Halal food is widely accepted, with demand increasing yearly. However, due to advancements in food technology, pork adulteration, particularly in ho- mogenous meat products, occurs frequently through attempts to gain monetary benefits. In this study, we used near infrared spectroscopy (NIR) to detect pork adulteration in meatballs. A long wavelength NIR (1000-2500 nm) was used to create a partial least square (PLS) regression model. The results showed that the model of beef meatballs had an R 2 -val of 0.88, a SECV of 3.45% and a bias of 0.14%, respectively. For chicken meatballs, the model had an R 2 -val of 0.83, a SECV of 4.18%, and a bias of 0.22%, respectively. Considering the strictness of Halal guidelines, the obtained prediction model had a high SECV (3.45% and 4.18% for beef and chicken meatballs) for the detection of pork adulteration in Halal foods. NIR acquisition of meatballs at 25°C and grilled products provided a good result, but frozen meatballs did not. However, considering several advan- tages of the technique - speed, reduction in required sample preparation and low cost - NIR proved to be a promising technique for screening pork adulteration in Halal meatballs.

Determination of pork adulteration in meatballs using enzyme linked immuno sorbent assay (ELISA) techniques case study: Small medium enterprises meatballs traders at Jatinangor education area, sumedang district, west java, indonesia

Adulteration of meatballs using pork begins to bloom in a row with the increase of beef price in the market. There is a risk that can occur around the education area of Jatinangor. Therefore the identification of meatballs adulteration with enzymes linked immuno sorbent assay (ELISA) approach need to be done in order to create certainty and security guarantee of the consumed products. Results showed that Small Medium Enterprises (SMEs) in Jatinangor who process meat (meatballs) was 21 meatball traders around Jatinangor. ELISA tests on 21 meatball samples taken from the SMEs merchant in Jatinangor showed no pork-adulterated meatballs on all of the samples tested.

Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop

We describe a highly specific PCR assay for the authentic identification of pork. Accurate detection of tissues derived from pig (Sus scrofa) was accomplished by using newly designed primers targeting porcine mitochondrial displacement (D-loop) region that yielded an unique amplicon of 712 base pairs (bp). Possibility of cross-amplification was precluded by testing as many as 24 animal species (mammals, birds, rodent and fish). Suitability of PCR assay was confirmed in raw (n=20), cooked (60, 80 and 100°C), autoclaved (121°C) and micro-oven processed pork. Sensitivity of detection of pork in other species meat using unique pig-specific PCR was established to be at 0.1%; limit of detection (LOD) of pig DNA was 10pg (pico grams). The technique can be used for the authentication of raw, processed and adulterated pork and products under the circumstances of food adulteration related disputes or forensic detection of origin of pig species.

Analysis of pork adulteration in minced mutton using electronic nose of metal oxide sensors

The aims were to detect the adulteration of mutton by applying traditional methods (pH and color evaluation) and the E-nose, to build a model for prediction of the content of pork in minced mutton. An E-nose of metal oxide sensors was used for the collection of volatiles presented in the samples. Feature extraction methods, Principle component analysis (PCA), loading analysis and Stepwise linear discriminant analysis (step-LDA) were employed to optimize the data matrix. The results were evaluated by discriminant analysis methods, finding that step-LDA was the most effective method. Then Canonical discriminant analysis (CDA) was used as pattern recognition techniques for the authentication of meat. Partial least square analysis (PLS), Multiple Linear Regression (MLR) and Back propagation neural network (BPNN) were used to build a predictive model for the pork content in minced mutton. The model built by BPNN could predict the adulteration more precisely than PLS and MLR do.

Biogenic amine content, histamine-forming bacteria, and adulteration of pork in tuna sausage products.

Twenty-five tuna sausage products were purchased from retail markets in Taiwan. The rates of occurrence of biogenic amines, histamine-forming bacteria, and adulteration by pork and poultry were determined. The average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g (<0.05 to 1.85 mg/100 g). Thirteen histamine-producing bacterial strains isolated from tested samples produced 12.1 to 1,261 ppm of histamine in Trypticase soy broth supplemented with 1.0% L-histidine. Among them, Raoultella ornithinolytica (one strain), Enterobacter aerogenes (one strain), and Staphylococcus pasteuri (two strains) were identified as prolific histamine formers. PCR assay revealed that the adulteration rates were 80% (20 of 25) and 4% (1 of 25) for pork and poultry, respectively, in tuna sausage. The fish species in the tuna sausage samples were identified as Thunnus albacares for 22 samples (88%), Thunnus alalunga for 1 sample (4%), and Thunnus thynnus for 1 sample (4%), whereas the remaining sample was identified as Makaira nigricans (blue marlin).

Nanobiosensor for the detection and quantification of pork adulteration in meatball formulation

A 27-nucleotide AluI fragment of swine cytochrome b (cytb) gene was integrated to 3-nm diameter citrate–tannate-coated gold nanoparticles to fabricate a species-specific nanobiosensor. The biosensor was applied to authenticate pork adulteration in meatball formulation, which is a favourite food in many Asian and European countries. The sensor was found to be sensitive enough to detect 1% pork in raw and cooked meatballs, prepared from the previously mixed pork and beef in specific ratios (% w/w). The hybridisation kinetics of the hybrid biosensor was studied with synthetic targets from moderate to extreme target concentrations and a hyperbolic relationship was found. However, linearity was observed with probe/target ratios 4:1 to 1:2. This part of the curve quantified target DNA in ready-to-eat mixed meatball preparations with more than 90% accuracy. The biosensor probe was hybridised with a target DNA that was several-fold shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed meat products or extensively degraded samples where PCR-based identification technique might not work due to the fragmentation of comparatively longer DNA. We believe that the assay can be used as an alternative to qPCR for determining shorter size DNA sequences in degraded samples to address a range of biological problems, such as food analysis, bio-diagnostics, environmental monitoring, genetic screening and forensic investigations.

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 109bp fragment of porcine cytochrome b gene. Specificity test with 10ng 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 (r2=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.

Development of swine-specific DNA markers for biosensor-based halal authentication

The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.

Nanobioprobe for the Determination of Pork Adulteration in Burger Formulations

We report the development of a swine‐specific hybrid nanobioprobe through a covalent integration of a fluorophore‐labeled 27‐nucleotide AluI‐fragment of swine cytochrome b gene to a 3 nm gold nanoparticle for the determination of pork adulteration in processed meat products. We tested the probe to estimate adulterated pork in ready‐to‐eat pork‐spiked beef burgers. The probe quantitatively detected 1–100% spiked pork in burger formulations with ≥90% accuracy. A plot of observed fluorescence against the known concentration of AluI‐digested pork DNA targets generated a concave curve, demonstrating a power relationship (y = 2.956x0.509) with a regression coefficient (R2) of 0.986. No cross‐species detection was found in a standard set of pork, beef, chicken, mutton, and chevon burgers. The method is suitable for the determination of very short‐length nucleic acid targets which cannot be estimated by conventional and real‐time PCR but are essential for the determination of microRNA in biodiagnostics and degraded DNA in forensic testing and food analysis.

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 (R2) of 0.991. A plot of actual value against real-time PCR-predicted value also yielded a good linear regression, R2 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.

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.

Gold Nanoparticle Sensor for the Visual Detection of Pork Adulteration in Meatball Formulation

We visually identify pork adulteration in beef and chicken meatball preparations using 20 nm gold nanoparticles (GNPs) as colorimetric sensors. Meatball is a popular food in certain Asian and European countries. Verification of pork adulteration in meatball is necessary to meet the Halal and Kosher food standards. Twenty nm GNPs change color from pinkish‐red to gray‐purple, and their absorption peak at 525 nm is red‐shifted by 30–50 nm in 3 mM phosphate buffer saline (PBS). Adsorption of single‐stranded DNA protects the particles against salt‐induced aggregation. Mixing and annealing of a 25‐nucleotide (nt) single‐stranded (ss) DNA probe with denatured DNA of different meatballs differentiated well between perfectly matched and mismatch hybridization at a critical annealing temperature. The probes become available in nonpork DNA containing vials due to mismatches and interact with GNPs to protect them from salt‐induced aggregation. Whereas, all the pork containing vials, either in pure and mixed forms, consumed the probes totally by perfect hybridization and turned into grey, indicating aggregation. This is clearly reflected by a well‐defined red‐shift of the absorption peak and significantly increased absorbance in 550–800 nm regimes. This label‐free low‐cost assay should find applications in food analysis, genetic screening, and homology studies.

Nanobiosensor for Detection and Quantification of DNA Sequences in Degraded Mixed Meats

A novel class of nanobiosensor was developed by integrating a 27-nucleotideAluIfragment of swine cytochrome b (cytb) gene to a 3-nm diameter citrate-tannate coated gold nanoparticle (GNP). The biosensor detected 0.5% and 1% pork in raw and 2.5-h autoclaved pork-beef binary admixtures in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic andAluIdigested real pork targets from moderate to extreme target concentrations and a sigmoidal relationship was found. Using the kinetic curve, a convenient method for quantifying and counting target DNA copy number was developed. The accuracy of the method was over 90% and 80% for raw and autoclaved pork-beef binary admixtures in the range of 5–100% pork adulteration. The biosensor probe identified a target DNA sequence that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed or degraded samples where PCR amplification was not possible due to template crisis. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences to address a wide ranging biological problem in food industry, diagnostic laboratories and forensic medicine.

Analysis of pork adulteration in beef meatball using Fourier transform infrared (FTIR) spectroscopy

Meatball is one of the favorite foods in Indonesia. The adulteration of pork in beef meatball is frequently occurring. This study was aimed to develop a fast and non destructive technique for the detection and quantification of pork in beef meatball using Fourier transform infrared (FTIR) spectroscopy and partial least square (PLS) calibration. The spectral bands associated with pork fat (PF), beef fat (BF), and their mixtures in meatball formulation were scanned, interpreted, and identified by relating them to those spectroscopically representative to pure PF and BF. For quantitative analysis, PLS regression was used to develop a calibration model at the selected fingerprint regions of 1200–1000 cm −1. The equation obtained for the relationship between actual PF value and FTIR predicted values in PLS calibration model was y = 0.999x + 0.004, with coefficient of determination (R 2) and root mean square error of calibration are 0.999 and 0.442, respectively. The PLS calibration model was subsequently used for the prediction of independent samples using laboratory made meatball samples containing the mixtures of BF and PF. Using 4 principal components, root mean square error of prediction is 0.742. The results showed that FTIR spectroscopy can be used for the detection and quantification of pork in beef meatball formulation for Halal verification purposes.