News

Detecting the mislabelling of thawed meat as fresh meat has been an analytical challenge for many years.  The most established technique, based on measuring the HADH enzyme (see FAN method explainers) is not perfect.

This paper (purchase requires) reports the development of an assay based on a different biochemical marker enzyme for the detection of previously-frozen pork The authors proposed mitochondrial citrate synthase (CS) as a candidate biomarker enzyme, and developed an ELISA to measure CS and test their hypothesis.

For the development of sandwich ELISA, polyclonal antibodies (pAb) against CS were produced in two different laboratory animals (rabbits and guinea pigs). A sandwich ELISA was optimized by utilizing rabbit anti-CS pAb as capture and guinea pig anti-CS pAb as detection antibody,  The limit of detection (LOD) and limit of quantification (LOQ) of the sandwich ELISA were calculated to be 3.71 ng/ml and 11.24 ng/ml, respectively. The sandwich ELISA was having 100.78 ± 1.66 % of accuracy, and the assay was found to be having good repeatability as well as reproducibility. The assay showed a good storage stability up to 12 weeks of storage at refrigeration temperature (4 ± 1 °C).

The authors report that there was a statistically significant difference between results from fresh/chilled and frozen/thawed pork meat.

Photo by patrick le on Unsplash

This paper (purchase required) describes the development and validation of a highly sensitive lateral flow immunoassay (LFIA) test kit for detecting trace pork in meat products. A rapid bead-based sample extraction was developed (1–5 min) for the  biomarker (porcine IgG).  The authors report that total test time to result was 20 minutes. The reported detection limit was 0.001 % (w/w), which is 5–500 × more sensitive compared to current commercial lateral flow kits. The LFIA was validated with a range of meat and processed food products, confirming its high specificity to pork without cross-reactivity to other animal species or non-meat ingredients. Moreover, a long-term stability study confirmed that the LFIA maintained its performance at room temperature storage for 2 years.

This paper (open access) reports the development of a label-free electrochemical immunosensor for the detection of low quantities of porcine gelatin.  The sensor is based on a boron-doped diamond electrode modified with aryl diazonium salt. The diazonium electrografting enabled stable covalent immobilization of anti-porcine gelatin antibodies via protein A, preserving anti­body orientation and activity.

The optimised conditions were a 500× antibody concentration, 60 minute antibody incubation, and 15 minute gelatin incubation. Detection was performed using differential pulse voltammetry with [Fe(CN)₆]3-/4- as a redox probe, allowing label-free monitoring of anti­body-antigen interactions based on changes in current.

The authors report that the immunosensor demonstrated excellent analytical performance, with a detection limit of 142.15 pg mL-1. Specificity testing showed no cross-reactivity with bovine gelatin.

Although suitable validation would be required, the authors conclude that this immunosensor has potential to form the basis of a rapid, highly sensitive, and specific platform for porcine gelatin detection, offering great potential for food authentication and halal verification.

Mechanically Separated Meat (MSM – sometimes called Mechanically Recovered Meat, MRM) must be declared on-pack if used in meat products.  Testing for undeclared MSM can be a significant analytical challenge.  Traditional official control methods rely on microscopy, which requires experienced interpretation and can be highly subjective. 

This paper (open access) builds upon previous published work from the same researchers to develop targeted LC-MSMS methods that are suitable for official control applications..

In contrast to a comparable study on MSM from poultry, the authors report that the use of cartilage/intervertebral disc material was not useful for porcine MSM. They therefore report a new marker protein from porcine MSM, protegrin-4, which allows the detection of 5/3/1 mm MSM. The validity of the developed assay was ensured by the investigation of 182 blinded samples. After unblinding, all samples containing 5/3/1 mm MSM and all negative control samples were correctly classified. Additional new results related to the investigation of the species specification of chicken, turkey, and pork also are presented.

They concluded that LC-MS/MS-based detection of of undeclared MSM has been successfully extended from poultry to porcine MSM. The assay was successfully transferred to a triple quadrupole LC-MS system to facilitate routine use.

Photo by Branimir Petakov on Unsplash

Differentiating gelatin species is an analytical challenge because of a lack of intact DNA.  Most speciation methods therefore target the profile of proteins.  Proteins are difficult to analyse - they are too large to measure directly by techniques such as LC-MS, without  prior breaking down, and their folded structure is also an important diagnostic parameter.  This structure is disrupted by many of the sampling and extraction procedures used in analytical method. Analysis of mixed gelatins is particularly difficult.

This method (open access) used a new approach based on the interaction of ethanol with amino acids inside a protein. Ethanol can denature globular proteins by disrupting intraprotein hydrogen bonds due to hydrophobic interactions. However, when added to solutions having proteins with considerable number of α-helices, ethanol can stabilize the protein structure and prevent aggregation. The specific effects of ethanol on protein structure and function can vary depending on the protein's composition and environment.

Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to leverage ethanol's differential effects on gelatin's amide bands for quantifying pork gelatin contamination in bovine gelatin.

The authors report that the method showed a strong linear correlation between contamination levels and amide band transmission, with detection and quantification limits of 0.85 and 2.85 mg/100 mg (pork in bovine), respectively. It effectively identified pork gelatin in halal candy, with recovery rates from 50.05 % to 103.69 %.