Identification of Cross-Species Marker Peptides for the Detection of Mammalian and Poultry Meat in Vegan and Vegetarian Foods.

Authentication of vegan and vegetarian foods is important since these increasingly popular food items could be adulterated with cheap meat to increase profit margins. In this study, nine marker peptides for the detection of meat (several species) were identified applying liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). These marker peptides enable the crucial differentiation of beef versus milk and chicken meat versus egg, demonstrated by the investigation of 19 commercial vegetarian meat substitutes containing milk and egg. Extensive experimental testing proved the presence of the cross-species meat marker peptides in 19 food-relevant types of mammals and poultry as well as their absence in more than 136 plant-based ingredients for the production of vegan and vegetarian foods. An authentic vegan sausage matrix based on an actual retail product was produced and spiked with 5.0%, w/w meat to confirm the high signal intensities and the heat stability of the marker peptides.

Barbecue conditions affect contents of oxygenated and non-oxygenated polycyclic aromatic hydrocarbons in meat and non-meat patties.

The contents of eight oxygenated polycyclic aromatic hydrocarbons (OPAHs; anthracene-9,10-dione, benzo[a]anthracene-7,12-dione, 11H-benzo[b]fluorene-11-one, 6H-benzo[cd]pyren-6-one, 7H-benzo[de]anthracene-7-one, 9,10-dihydro-8H-benzo[a]pyren-7-one, fluoren-9-one, and naphthacene-5,12-dione) and six PAHs (anthracene, fluorene, and PAH4) were investigated in barbecued meat and non-meat patties. The patties were prepared with ten setups (six replicates, each) of barbecue conditions defined by grill type, grate height, heating medium, and barbecue time. The highest median contents were observed with a disposable grill (OPAHs: 46.3 µg/kg; PAHs: 40.7 µg/kg) and a charcoal grill (OPAHs: 29.6 µg/kg; PAHs: 23.3 µg/kg). Fluoren-9-one and anthracene-9,10-dione were the dominant compounds within OPAHs, but also the four toxicologically most relevant OPAHs were detected with a total up to 11.8 µg/kg. Pairs of OPAHs and corresponding PAHs did not show strong correlations, as individual OPAHs and PAHs were affected differently by the barbecue conditions. No suitable markers for OPAH prediction could be found. We recommend to include OPAHs in future PAH investigations.

Simultaneous Mass Spectrometric Detection of Proteins of Ten Oilseed Species in Meat Products.

Food fraud is a common issue in the modern food industry. The undeclared use of foreign proteins in meat products is a major concern in this context. Oilseeds are ideal for this purpose due to their high protein content and since huge amounts of oil meal are obtained as a by-product of oil production. Therefore, a UHPLC-MS/MS method was developed for the simultaneous detection of chia, coconut, flaxseed, hemp, peanut, pumpkin, rapeseed, sesame, soy, and sunflower proteins in meat products. Potential tryptic peptide markers were identified by high-resolution mass spectrometry. The final twenty peptide markers selected, which are specific for one of the ten species targeted, were each measured by multiple reaction monitoring. To the best of our knowledge, twelve new heat-stable marker peptides for chia, coconut, flaxseed, pumpkin, rapeseed, sesame and sunflower have not been reported previously. Emulsion-type sausages with 0.01, 0.25, 0.50, 0.75 and 1.00% protein addition by each oilseed species were produced for matrix calibration. No false-positive results were recorded. In the quantification of the ten oilseed species, 466 of 480 measuring data points of the recovery rate in unknown sausages (0.15 and 0.85% protein addition by each oilseed species) were in the accepted range of 80-120%.

A sensitive GC-HRMS method for the simultaneous determination of parent and oxygenated polycyclic aromatic hydrocarbons in barbecued meat and meat substitutes.

A sensitive GC-HRMS method was developed to analyze six polycyclic aromatic hydrocarbons (PAH; anthracene, benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, chrysene, and fluorene) and eight oxygenated PAHs (OPAH; anthracene-9,10-dione, benzo[a]anthracene-7,12-dione, 7H-benz[de]anthracene-7-one, 11H-benzo[b]fluorene-11-one, 6H-benzo[cd]pyren-6-one, 9,10-dihydro-8H-benzo[a]pyren-7-one, fluoren-9-one, and naphthacene-5,12-dione) in barbecued meat and meat substitutes. After optimization of the conditions of the sample preparation, consisting of accelerated solvent extraction (ASE) and solid-phase extraction (SPE), high recoveries (PAH 72-109%; OPAH 74-106%) were obtained. The linear regression of the matrix calibration resulted in high correlation coefficients (0.959-0.999). For the first time, reasonably low limits of detection (PAH 0.03-0.17 µg/kg; OPAH 0.04-0.43 µg/kg) were achieved, allowing the analysis of samples barbecued under practical relevant conditions. In charcoal grilled samples, the sum content of the seven detected OPAHs (5.7-62.4 µg/kg) was higher than the sum content of the six PAHs (1.4-36.7 µg/kg). However, 9,10-dihydro-8H-benzo[a]pyren-7-one was not detected in these samples.

A UHPLC-MS/MS Method for the Detection of Meat Substitution by Nine Legume Species in Emulsion-Type Sausages.

Meat substitution by legume proteins in various types of meat products is a common practice. A reliable detection and quantification of these additives is required to control food specifications, especially regarding food fraud. Consequently, a UHPLC-MS/MS method for the simultaneous detection of alfalfa (Medicago sativa), broad bean (Vicia faba), chickpea (Cicer arietinum), lentil (Lens culinaris), lupine (Lupinus albus and Lupinus angustifolius), pea (Pisum sativum), peanut (Arachis hypogaea), and soy (Glycine max) proteins in meat products was developed. After protein extraction and tryptic digestion, three marker peptides for each legume species were measured by multiple reaction monitoring (MRM) using an optimized extraction protocol. To the best of our knowledge, the marker peptides for alfalfa, broad bean, chickpea, and lentil have not been reported previously. Emulsion-type sausages with 0.1, 0.4, 0.7, 1.0, 1.3, 1.6, 1.9, 2.2, and 2.5% meat substitution by each legume species, representing the concentration range between inadvertently transferred cross-contaminations and the conscious use for meat substitution, were produced for matrix calibration. No false-positive results were recorded in blank samples. In the quantification of alfalfa, broad bean, chickpea, lentil, pea, peanut, and soy, 673 of 756 measuring data of the recovery rate in unknown sausages were in the accepted range of 80-120%.

A rapid UHPLC-MS/MS screening method for the detection of the addition of porcine blood plasma to emulsion-type pork sausages.

The adulteration of meat products by the undeclared addition of commercially available blood plasma powder is quite conceivable due to low costs, high protein contents (about 70%), and advantageous functional properties. This applies particularly to pork, which has the highest meat production rate in the European Union. Evidence of this type of food fraud has been rather difficult to identify due to the lack of appropriate analytical methods, especially when adding plasma to meat of the same animal species. Consequently, a rapid UHPLC-MS/MS method for the detection of porcine blood plasma in emulsion-type pork sausages was developed. After protein extraction and tryptic digestion in a quick and simple one-pot process, species-specific marker peptides for porcine blood cell proteins (four markers) and plasma proteins (12 markers) were measured by UHPLC-MS/MS. Emulsion-type pork sausages were produced from a variety of raw materials that differed in the age or sex of the slaughtered pigs. Sausages were spiked with 0.5, 1, 1.5, 2, 3, or 5% meat substitution by one of two plasma powders, or produced as corresponding blank samples, and subjected to different thermal treatments as full or semi-preserves. Four plasma peptides were identified for the overall sample that allowed detection down to 0.7% meat substitution from the sum of their peak areas, with 5% error probability for both false positives and negatives.

German Government Official Methods Board Points the Way Forward: Launch of a New Working Group for Mass Spectrometry for Protein Analysis to Detect Food Fraud and Food Allergens.

The detection of food fraud and undeclared food allergens is one of the major challenges for competent authorities. Because adulterations are continuously adapted to the methods used to uncover them, the accomplishment of this task has become increasingly difficult over time. In recent years, various new promising methods for the detection of multiple food adulterants and multiple food allergens have been developed. Some of them utilize LC-MS to identify specific marker peptides. However, these methods have yet to be validated and standardized. For this reason, the German officials have established a working group with the objective of validating methods through multilaboratory validation studies. The experts of the working group also aim for the first time to standardize validated methods and to develop general validation criteria. This manuscript will highlight the current work of the group. For this purpose, an overview is given on the principles and applications of the new mass spectrometric methods. Moreover, requirements and the present work of other institutions regarding method validation are described.

A sensitive HPLC-MS/MS screening method for the simultaneous detection of barley, maize, oats, rice, rye and wheat proteins in meat products.

The use of grain proteins in various types of meat products is common practice. A reliable detection of these food ingredients is required to control specifications and regarding food fraud and allergenic potential. Consequently, a sensitive HPLC-MS/MS method for the simultaneous detection of barley, maize, oats, rice, rye and wheat proteins in meat products was developed. After protein extraction and tryptic digestion, three to four selected marker peptides for each grain species were measured by HPLC-MS/MS. Emulsion-type sausages with grain-based protein concentrations in the range of 5-1000 mg/kg and blank values were produced. The limits of detection of the method were ≤5 or ≤10 mg grain protein/kg meat product for each grain species and no false-positive or -negative results were obtained. The detectability of the marker peptides only slightly decreased after storage and grilling of sausages, whereas the influence of the canning process was noticeably higher.

SPIONs functionalized with small peptides for binding of lipopolysaccharide, a pathophysiologically relevant microbial product.

Systemic inflammation such as sepsis represents an acute life-threatening condition, to which often no timely remedy can be found. A promising strategy may be to functionalize magnetic nanoparticles with specific peptides, derived from the binding motives of agglutinating salivary proteins, that allow immobilization of pathogens. In this work, superparamagnetic iron oxide nanoparticles with stable polycondensed aminoalkylsilane layer were developed, to which the heterobifunctional linkers N-succinimidyl 3-(2-pyridyldithio)-propanoate (SDPD) and N-succinimidyl bromoacetate (SBA) were bound. These linkers were further chemoselectively reacted with the thiol group of singularly present cysteines of selected peptides. The resulting functional nanoparticles underwent a detailed physicochemical characterization. The biocompatibility of the primarily coated aminoalkylsilane particles was also investigated. To test the pathogen-binding efficacy of the particles, the lipopolysaccharide-immobilization capacity of the peptide-coated particles was compared with free peptides. Here, one particle-bound peptide species succeeded in capturing 90% of the toxin, whereas the degree of immobilization of the toxin with a system that varied in the sequence of the peptide dropped to 35%. With these promising results, we hope to develop extracorporeal magnetic clearance systems for removing pathogens from the human body in order to accelerate diagnosis and alleviate acute disease conditions such as sepsis.

A sensitive HPLC-MS/MS method for the simultaneous detection of microbial transglutaminase, and bovine and porcine fibrinogen/thrombin in restructured meat.

A sensitive HPLC-MS/MS method for the simultaneous detection of microbial transglutaminase (TG) from Streptomyces mobaraensis, and bovine and porcine fibrinogen/thrombin in restructured meat was developed using tryptic marker peptides of TG (five markers), and bovine and porcine fibrinogen (six markers each). Meat binding experiments with beef and pork were performed using a technical TG mixture (Activa, Ajinomoto), and bovine and porcine plasmapowder FG (PPFG; Sonac B.V.). The method developed allows the simultaneous detection of the use of these cold-set binders in raw and heated samples. The peak areas of the fibrinogen marker peptides were increased by a factor of about 100, compared to blank values originating from the occurrence of residual blood in meat, using a concentration of 0.6% bovine and porcine PPFG. A differentiation between the use of blood plasma powder and PPFG using the ratios of fibrinogen to serotransferrin peptide peak areas seems to be possible.

Production of dry-cured formed ham with different concentrations of microbial transglutaminase: Mass spectrometric analysis and sensory evaluation.

Dry-cured formed hams were produced with different concentrations of microbial transglutaminase (TG; 0.05, 0.1, 0.2, 0.5, and 0.8% Activa PB) and glucono-delta-lactone as control. A sensory evaluation was performed during a 43-day storage to determine cohesion, cavities, and local separation of dry-cured formed ham. Rising TG concentrations resulted in a slight increase in the evaluation of all sensory parameters, whereas amounts of TG higher than 0.5% led to an only very minor improvement. Dry-cured formed ham samples were analyzed by a sensitive HPLC-MS/MS method for the detection of TG using five tryptic marker peptides. Even very small amounts of Activa PB (0.05%) were detectable unambiguously. A decrease of TG detectability during the storage time of dry-cured formed ham was not observed. Using four marker peptides, no false-positive or -negative results were obtained. The amounts of two marker peptides were calculated using isotope-labeled peptides. They showed high correlations to the amount of Activa PB (R2>0.995).

A sensitive high performance liquid chromatography-tandem mass spectrometry method for the detection of microbial transglutaminase in different types of restructured meat.

A sensitive HPLC-MS/MS-method for the detection of microbial transglutaminase (TG) from Streptomyces mobaraensis in different types of restructured meat (pork, beef, chicken, and turkey) was developed using six tryptic marker peptides (8-11 amino acids). Meat binding experiments were performed with two technical TG mixtures with and without caseinate. After optimization of the conditions of extraction and tryptic digestion, restructured meat and blank values (total samples: 62) were analyzed in a raw and heated state. By investigation of samples pre-treated with oil marinade, emulsion marinade, seasoning salt as well as breadcrumbs, only very little effects of the type of pre-treatment on the detectability of TG were found. Using four marker peptides, no false-positive or false-negative results were obtained. The limit of detection (LOD) was about a factor of 10 below the recommended amount of transglutaminase for raw as well as heated restructured meat.

Polycyclic aromatic hydrocarbons (PAH) and phenolic substances in meat products smoked with different types of wood and smoking spices.

The contents of polycyclic aromatic hydrocarbons (15+1 EU PAH) and phenolic substances (guaiacol, 4-methylguaiacol, syringol, eugenol, and trans-isoeugenol) were investigated in smouldering-smoked Frankfurters and mini-salamis. For the 51 smoking experiments wood chips of oak, poplar, hickory, spruce, fir, alder, beech, and beech with an apple-smoking spice mix, cherry-smoking spice mix, and a mix of juniper berries and bay leaves were tested. The use of poplar and hickory led to a decrease in the PAH contents in the range of 35-55% compared to the commonly used beech wood. Higher PAH contents by using softwood were not observed. The use of the rapidly growing poplar seems to be a reasonable approach for reducing the PAH contents in smoked meat products. Furthermore, the sum contents of the five phenolic substances in sausages smoked with poplar were higher, or only slightly lower, when compared to the use of beech.

Contents of polycyclic aromatic hydrocarbons (PAH) and phenolic substances in Frankfurter-type sausages depending on smoking conditions using glow smoke.

The contents of polycyclic aromatic hydrocarbons (PAH) and phenolic substances in Frankfurter-type sausages were investigated depending on hot smoking conditions (glow smoke). For the 24 smoking experiments (performed in duplicates) three different smoke densities and ventilator velocities as well as wood chips with five different moisture contents were tested. During the smoking process, concentrations of O(2), CO(2) and CO, humidity and temperature in the smoking chamber as well as smoke generation temperature were determined. The chemical analysis included the contents of the 15+1 EU priority PAH and the phenolic substances guaiacol, 4-methylguaiacol, syringol, eugenol and trans-isoeugenol. The smoking conditions had a significant influence on smoke generation temperature, organoleptic properties and the formation of PAH and phenolic substances. The PAH contents increased with smoke density and ventilator velocity. No correlation between the contents of PAH and phenols was observed.

Fast-GC/HRMS to quantify the EU priority PAH.

For the analysis of the 15 polycyclic aromatic hydrocarbons (PAH) classified as priority from the Scientific Committee on Food (SCF) and benzo[c]fluorene (BcL) assessed to be relevant by the Joint FAO/WHO Experts Committee on Food Additives (JECFA) in food, a sensitive analytical method is necessary. In the past, many methods were established for only the analysis of the 16 priority PAH recommended by the US Environmental Protection Agency (16 EPA-PAH) but not for the 15 SCF-PAH and BcL. Therefore, at the Max-Rubner-Institut, Federal Research Institute of Nutrition and Food in Kulmbach, Germany, an analytical method with a runtime of 72 min was developed. To shorten this runtime without a loss of separation, a Fast-GC/high resolution MS (HRMS) method with a runtime of only 25 min using a TR-50ms column (10 m x 0.1 mm x 0.1 microm) was created. The repeatability (n = 3) of spiked PAH concentrations in test-matrix tea ranged from 0.1 to 11%. The analytical parameters LOD (0.01-0.02 microg/kg) and LOQ (0.03-0.06 microg/kg) also in the test-matrix tea were determined. A good linearity for all PAH (R(2) >0.99) and averaged recoveries from 75 to 117% in the concentration range of 1-20 microg/kg were achieved.

Polycyclic aromatic hydrocarbons (PAHs) in different types of smoked meat products from Serbia.

The contents of the16 EU priority PAHs in six different meat products from Serbia (beef ham, pork ham, bacon without skin, bacon with skin, cajna sausage and sremska sausage) were examined during the process of smoking. All these meat products from meat industry Zlatiborac, Mackat, Serbia presented in this study, have not previously been analysed concerning to their contents of PAH compounds. Determination and quantification of PAHs in meat products were performed by a Fast GC/HRMS method. The maximum level for benzo[a]pyrene (BaP) of 5µg/kg in smoked meat products was not exceeded in any samples. BaP comprises in general 4.6% of the total sum of the 16 EU priority PAHs and 15.2% of the total sum of the 12 IARC PAH compounds. The suitability of BaP as a marker both for 16 EU priority PAHs and 12 IARC probably and possibly carcinogenic PAHs was checked by applying correlation analysis.

Dioxin and polychlorinated biphenyl analysis: automation and improvement of clean-up established by example of spices.

To analyze polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/PCDFs) and polychlorinated biphenyls (PCBs) in spices by gas chromatography-mass spectrometry, a new clean-up method had to be developed owing to the high content of essential oils in the samples. A solid-phase extraction (SPE) column with activated silica endowed with sulfuric acid and sodium hydroxide was used. Under these conditions, clean-up was achieved using at least 5-7 g of pepper and even higher amounts of other spices. The automatized clean-up comprised three additional chromatographic steps after accelerated solvent extraction (ASE) followed by gel permeation: chromatography on a florisil SPE column, extract cleaning with the above-mentioned silica SPE column and chromatography with an activated charcoal column. On the basis of this automatized clean-up, a method that is more effective, rapid, simplified and economical than the available methods for PCDD/PCDF and PCB analysis is proposed. In model studies, the average recoveries for PCDDs/PCDFs ranged between 82.6% and 105.6% and for the PCBs between 71.3% and 113.3%.