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This study (purchase required) used, high-performance liquid chromatography, combined with chemometric analysis, to classify buffalo vs cows ricotta based on the profile of water soluble peptides.  The authors then identified specific peptides that could be used as species markers . Both mid-infrared spectroscopy and electrophoresis were also investigated as peptide measurement methods by were found to give insufficient discrimination, with IR overly affected by storage time of the extracts.

The authors created  11 experimental cheese formulations by increasing the proportions of cow whey mixed with buffalo whey. Water-soluble peptides were analysed using mid-infrared spectroscopy, high-performance liquid chromatography and electrophoresis. The data obtained from mid-infrared spectroscopy and high-performance liquid chromatography were statistically processed using principal component analysis, analysis of covariance and multiple linear regression..

High-performance liquid chromatography identified 14 peptide peaks, with three recognized as specific markers for cow whey in adulterated samples. PCA explained 77% of the variance, distinguishing pure and adulterated ricotta. Multiple linear regression modelling of high-performance liquid chromatography data predicted cow whey concentration with a correlation of R = 0.87. High-performance liquid chromatography with chemometrics was effective for detecting buffalo ricotta adulteration.

When applied to 14 commercial samples, the model suggested that nine contained adulteration ranging from 10% to 100% cow whey.

Photo by Conor Brown on Unsplash

Spain has a legal limit of 3% for undeclared vegetable proteins in meat patties.  The aim of this open-access study was to evaluate the feasibility of point-based near infrared spectroscopy (NIRS) and hyperspectral imaging (HSI) to verify compliance.

The model was trained on patties prepared in-house.  They were all prepared from the same cut of beef, so the robustness of the model has not been verified.  A total of 240 patties were fabricated, of which 60 contained pea (PP), 60 contained soybean (SP), and 60 chickpea protein (CP) at levels from 1 up to 6 % (w/w). 60 pure beef patties were included.

The authors report that they could clearly discriminate the type of protein added, using either partial least squares-discriminant analysis (PLS-DA) or linear discriminant analysis (LDA), with >90 % of the samples in the test set correctly classified. Based on protein inclusion, LDA discriminated 100 % of the PP, SP and CP samples with both NIR and HSI. PLS-DA classified 100 % of the PP and CP burgers using the NIR instrument. To manage double classification tasks, a hierarchical model classifier (HMC) was proposed for both NIR and HSI spectra, achieving classification rates of at least 83% by combining LDA and PLS-DA models at the nodes.

The authors conclude that NIR spectroscopy is suitable for detecting low levels (1 %) of vegetable protein flours added to beef burgers.

This article (open access) discusses some of the current challenges in testing animal feed for compliance with European legislation using microscopy (one of the official methods mandated by the legislation).

Although the article is pre-publication and not peer-reviewed, it is generously illustrated with colour photographs of microscope slides - such as that shown here - which could be a valuable training aide to Official Control analysts who are relatively new to microscopy.  The author discusses key areas where expert interpretation is needed, describing the examples of bovine spray-dried plasma protein (legal, within restrictions, in the US but banned in the EU in ruminant feed), differentiating milk powder from blood powder, and differentiating hydrolysed proteins from vegetable vs animal sources.

In this study (open access) the researchers proposes using a MALDI-ToF and LC-Q-ToF dual approach, following trypsin digestion, as a method to verify fish species.  Trypsin digestion breaks the proteins down into peptides, and they used peptide fingerprints to identify peptides that were unique markers for specific species. The advantage of their approach over DNA methods, and in comparison to MALDI-ToF-MS analysis of undigested proteins, is that it can be applied to complex and heat-processed samples.

The study aimed to differentiate six fish species—carp, mackerel, pike, pollock, salmon and trout. Matrix-assisted laser desorption/ionization–time ff flight mass spectrometry (MALDI-TOF MS) was employed to identify characteristic species-specific m/z values to differentiate raw and cooked fish meat. Additionally, liquid chromatography–electrospray ionization–quadrupole–time tf flight (LC-ESI-Q-TOF) was used to determine specific amino acid sequences in carp and salmon, selected as model species.

Two or more distinct species-specific m/z markers were identified for all six fish species, enabling their differentiation in both raw and processed form. A slightly larger list of distinct markers were found for cooked, compared to raw, fish.  In carp and salmon, hundreds of peptide sequences were detected, leading to the identification of a panel of peptide markers that determine both the fish species and the type of meat processing. The results confirm that mass spectrometry-based proteomic approaches can serve as effective tools for the authentication of fish meat.

The authors conclude that it is possible to use two complementary mass spectrometry techniques for reliable and rapid authentication of fish species. By focusing on peptide-level markers and leveraging accessible tools, they believe that the approach offers a cost-effective and innovative alternative for fish meat authentication.

This study (purchase required) assessed the nutrient composition and labelling accuracy of twenty-nine commercially available insect-based pet foods: twenty-four dog foods and five cat foods.  All were labelled as complete and balanced. Twenty were labelled as hypoallergenic. The products were analysed for proximate composition, essential amino acids, and mineral content (calcium, phosphorus, potassium, magnesium, copper, iron, zinc, selenium, mercury, and molybdenum) according to AOAC guidelines. The ‘hypoallergenic’ products were assessed for animal DNA using next-generation sequencing.

The results were compared with label declarations, considering nutritional and legal tolerances, as well as recommendations from FEDIAF and NRC for the intended species and life stages (g/1000 kcal ME). Heavy metals were compared to maximum tolerable limits from the FDA.

The analysis revealed that 22 products (76%) did not comply with declared nutritional values and tolerances for at least one nutrient, with nine products (31%) showing discrepancies in two or more; key issues were in crude fibre and metabolizable energy. Three products (10%) met FEDIAF’s recommendations, and seventeen (59%) met NRC’s recommendations. Only one (3%) adhered to both label and FEDIAF’s recommendations. Most nutritional inadequacies were seen in selenium, calcium, phosphorus, Ca/P ratios, and taurine, potentially posing health risks to pets.

Fifteen out of twenty (75%) hypoallergenic-labelled products complied with the labelled species.

Despite the potential benefits of insect-based pet foods, this study underscores the need for further research and stricter quality control to ensure safety and efficacy, ultimately improving pet nutrition and consumers’ trust.

Photo by Hulki Okan Tabak on Unsplash

There is a growing market for the use of insect protein in feed.  The cricket species Gryllus assimilis. is approved in the EU for feeding farmed animals whilst the closely-related G. locorojo is only permitted for pets. The two are difficult to distinguish analytically in a highly processed product.

This paper (purchase required) reports a method developed on the basis of the cytochrome oxidase I gene, (COI), which was sequenced with thoroughly characterised G. locorojo and G. assimilis samples. The method is highly sensitive, detecting 0.8 pg G. locorojo-DNA or 0.1% G. locorojo incurred in feed, respectively. Authentic G. assimilis specimens were used to ensure that the G. locorojo method (Gloco-PCR) discriminates this closely related sister taxon, with a comfortable Ct-difference of 10-15. For cross analysis of true G. assimilis, similar primers with another probe were employed (Gassim-PCR) and the annealing temperature was increased from 60 °C to 62 °C.

Under these conditions, authentic G. assimilis crickets were detectable with Ct-values around 20, while G. locorojo samples showed a low detection at cycles around Ct 35. An investigation of ten ‘G. assimilis’ samples collected from Germany and four other European countries revealed that all of them were of the G. locorojo type.

The authors conclude that this small preliminary survey proves the usefulness of the method and supports the assumption that many G. assimilis crickets marketed in the EU indeed belong to the species G. locorojo. Consequently, European legislation, currently based on a white list of allowed insect species, is critically questioned.

Photo by Ivan Ivanovič on Unsplash

Meat species identification has always been a challenge in highly processed foods, such as gelatines and stocks.

One approach is to measure proteins and protein patterns using mass spectrometry (MS).  A previous research project, under the UK Department of Environment, Food and Rural Affairs (Defra) Food Authenticity Programme, developed and in-house validated a method using proteomics.

That work has now been built upon by another 3 Defra projects to streamline the method to look for specific markers, in a format that can be used routinely by testing laboratories, and to fully validate the routine method including by interlaboratory trial.

All four research reports are now signposted on FAN’s Research pages.  Scroll through the table to find the appropriate report reference number:

• FA0166 – the original 2019 project – “Development, optimisation and validation of a non-targeted proteomics method for meat species identification”
• FA0165 – “Liquid chromatography targeted mass spectrometry method to determine the animal origin of gelatine - transfer to a high throughput, low cost platform with single lab evaluation”
• FA0177 – “Gelatine species determination, completion of method validation and determination of a quantitative method”
• FA0187 – “Interlaboratory trial of a mass spectrometry method for meat species determination”

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 %.