Food authenticity testing using modern techniques

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November 2013

The recent horse meat scandal has highlighted the need to have the scientific means to check the components of our foods. As our population grows, increasing amounts of food are produced in the UK and also imported from overseas. The ingredients of an individual food product may be sourced from a single farm or from a wide range of producers and manufacturers worldwide. The authenticity and origin of these ingredients must be labelled so that this information can be included on the food label of the final product, thus creating a ‘paper trail’ which follows ingredients as they are exported around the globe. However, as highlighted by the horse meat scandal, there is room for error or intentional adulteration of ingredients during this process. In order to protect consumer interests and public health, in addition to combating the growing problems of food fraud and adulteration, scientific expertise and technologies are constantly being developed and advanced to test the authenticity of foods and feeds.

Such methods, using the latest developments in DNA fingerprinting techniques, chromatography and mass spectrometry, have been applied during recent high profile cases of food fraud and adulteration reported in the media. Examples are the inclusion of illegal Sudan dyes in foods and food ingredients, the addition of bovine material to chicken fillets, the counterfeiting of popular wines and species determination of products of animal origin.

Adulteration can occur for a variety of reasons, often linked to financial gain. Increases in profitability may be achieved by adulterating to improve the perceived quality of products, mimic an established brand, reduce manufacturing costs or for product extension purposes.

Analytical approaches to screen for these recent food fraud cases are discussed below, focussing on methods involving DNA analysis, mass spectrometry and spectroscopy. These techniques include targeted approaches when the analyte of interest is known and specifically screened for, non-targeted approaches, isotopic measurements and ‘omics’ technologies including metabolomics and proteomics.

What's in my food?

Targeted analysis involves screening for pre-defined components in a sample. Foods are analysed using techniques such as liquid chromatography- and gas chromatography-mass spectrometry (LC-MS and GC-MS) or nuclear magnetic resonance spectroscopy (NMR). The resulting data can then be compared to information stored in databases or from analysis of authentic standards in order to identify the contents of a food and screen for a given adulterant. Targeted approaches, for example, have been used to interrogate foods for the presence of the illegal and potentially carcinogenic Sudan dyes (URL http://www.scientistlive.com/content/14486) by electrospray ionisation (ESI)-LC-MS/MS. Since July 2003, all chilli powder imported into the UK must be certified free of Sudan I. In 2005, the dyes were found to have been included in a batch of chilli which was added to Worcester sauce as a flavour enhancer in processed meals, resulting in the recall of hundreds of products as a precautionary measure.

Other food additives can be traced by LC-MS/MS to check food labelling claims. Certain meat binding products or ‘glues’, used to bind minced meat or off-cuts and trimmings of high value meats together into ‘steak-like’ products are often derived from blood. These colourless, odourless binding agents can also be used to produce processed meat products of exact or extraordinary shapes. The blood plasma protein fibrin can be mixed with meat and, along with addition of the blood clotting enzyme thrombin, clots to bind the meat together. The blood tends to be of bovine or porcine origin. This particular food binding process therefore raises the issue of products from one animal being added as an undeclared ingredient in the manufacture of other meat and fish products. Further, since binding agents are permitted for use as a food ingredient, there is concern that there is an opportunity for unscrupulous producers to use these products to fraudulently increase the declared meat content of foods. Therefore, in terms of food labelling, to support ethical and religious sensitivities and for enforcement purposes, there is a need for a method to determine the species of blood-derived binding agents added to foods. LC-MS/MS methods were developed to determine the species origin of blood plasma of bovine and of porcine origin to meat products (Grundy et al., 2007, 2008). Peptides are released during the blood clotting process on which the binding agent technology is based. These fibrinopeptides differ in mass depending on species, and thus fibrinopeptides derived from bovine blood can be differentiated from those from porcine blood by determining their mass and the characteristic manner in which they fragment when energy is applied within a mass spectrometer. Example mass spectra are shown in Figure 1.

Among other high value products which can be vulnerable to adulteration is monofloral honey. Often unique biomarkers are present in the nectar of specific plants on which the bees forage. For example, kynuric acid has been shown to be a robust biomarker of chestnut honey (Donarski et al., 2010a). Manuka honey is a bioactive monofloral honey prepared from the nectar of the Manuka bush (Leptospermum scoparium) in New Zealand and Australia. This honey is reported to have potent biocidal activity with many perceived health benefits. Activity is often rated by the Unique Manuka Factor (UMF™) and honeys with a higher UMF™ rating command a high value at market. There is, therefore, a financial incentive to fraudulently sell adulterated product.

Various methods can be combined to determine the authenticity of Manuka honey, these include direct measurement of specific marker compounds such as methylglyoxal (MGO) and dihydroxyacetone (DHA). NMR technology is well suited to analysis of complex mixtures (McKenzie et al., 2011). An NMR method has been developed (Donarski et al., 2010a) to directly quantify the antimicrobial MGO in Manuka honey which can also be measured by liquid chromatography–infrared spectrometry (LC-IR) after derivatisation. Further, non-peroxide activity is usually measured using the phenol equivalence test, a microbiological assay which provides a direct measurement of the antimicrobial properties of the honey. It is well established that the amount of MGO in Manuka honey can be fraudulently raised by heating (Adams et al., 2009). It is therefore common practice to monitor the level of hydroxymethylfurfural (HMF) in Manuka honey, as this is a robust indicator of heating. Values of less than 40 mg/kg would be expected for unheated honey, with slightly higher values expected in tropical climates. HMF is typically measured using liquid chromatography with refractive index detection.

PCR Technologies: Screening for horse meat

During the horse meat scandal, Polymerase Chain Reaction (PCR) techniques were used to screen for horse DNA in processed food samples (Chisholm et al., 2005). DNA is extracted from the meat portion of products such as lasagne and tested using real-time PCR. This is a highly sensitive test which allows specific species such as horse or pork to be detected in a rapid and reliable manner, allowing results to be delivered to customers quickly, ensuring no delays before product release. Products that were found to contain levels of horse meat and deemed to be adulterated were then tested for the anti-inflammatory drug phenylbutazone which is banned in the treatment of animals entering the human food chain as in rare cases, it produces blood disorders in some individuals. PCR techniques are used to combat other types of food fraud including screening for adulteration of basmati rice which may be mixed with cheaper rice, verification of fish species (e.g. Hird et al., 2012), detection of undeclared genetically modified food (Macarthur et al., 2007), detection of cow’s milk in buffalo mozzarella (López-Calleja et al., 2004 ), confirming meat is from a speciality breed (e.g. Gloucester Old Spot pork) (Conyers et al., 2012), and to determine the floral content of honey (Laube et al., 2010).

Where has my food been produced? Can we determine manufacturing quality and origin by taking a food sample?

Analysis of stable isotopes in foods can reveal economically motivated adulteration such as addition of cheap sugar syrups to extend honey and maple syrup, watering down of wine, preparation of fruit juice described as ‘freshly squeezed’ from concentrate, verification that chicken has been ‘corn-fed’, determination of whether ethanol and vinegar and flavourings are natural or synthetic, and differentiation between organic and conventional farming methods (Kelly 2003, Kelly and Bateman, 2009). As shown in Figure 2, in more sophisticated applications of multi-element stable isotope analysis, the geographic origin (rearing location) of animals used in meat production can be determined (Heaton et al., 2007). This approach can be applied to any agricultural product where provenance adds value, such as Saffron spice (Maggi et al., 2011). The use of stable isotope analysis to verify the provenance of premium food products is underpinned by systematic global variations in the distribution of hydrogen and oxygen isotopes in precipitation and ground water. Variations in carbon and nitrogen isotopes relate to the mode of photosynthesis used by plants and the use of chemical fertilisers or animal manures during cultivation. All of these factors may be utilised to establish a unique stable isotope fingerprint for a given geographical location. Furthermore, the relationship between these parameters and environmental conditions such as temperature, precipitation and humidity during production can be used to produce maps that show the distribution of isotopes in agricultural products, so called isoscapes (isotope landscapes). Isoscapes have the added value that they can be used to predict the isotope composition of foods from geographical locations which have not been sampled, thus reducing the costs of establishing databases of authentic food reference samples.

Maturation and shelf life

Meats which are allowed to mature for longer periods of time before sale can carry a premium price at market. Beef, for example, can be labelled as being matured for 21 days as a marketing tool to promote sales. Metabolomic methods have been developed to verify labelling claims linked to beef ageing and also to the temperature used for meat storage during ageing. These methods can determine whether the meat has been stored at ideal (4°C), sub ideal (-20°C) and above ideal (20°C) temperatures and the number of days (0 to 28) the meat has been allowed to mature. Samples of meat were analysed by 1H NMR spectroscopy using a simple extraction strategy to generate a non-targeted profile. As shown in Figure 3, underlying trends were identified for several analytes including alanine, phenylalanine, leucine, nicotinic acid and nicotinamide that systematically varied with both temperature and age and could be used to verify labelling claims about maturity (Donarski et al., unpublished data). This approach could also be used to verify compliance with ‘Best Before’ dates and to establish ‘shelf-life’. Preliminary work has demonstrated this for eggs (Charlton et al., unpublished data).

Testing for pork and beef additives in chicken, confectionery and desserts

Proteomic methods use high resolution mass spectrometry to identify unique proteins or peptides to determine food components. Further, the technology can be used in a more forensic approach, for example, to identify the plant or animal species incorporated within a food.

Protein-based methods are often useful for analysing highly processed samples in which the DNA is denatured during the production procedure and thus, is unsuitable as a marker of authenticity. A proteomics method was developed to determine the species origin of gelatine and similar highly processed hydrolysed proteins made from skin and bone and added to foods. Most gelatine is prepared from collagen material found in pig, cow and fish carcasses and thus, for many consumers, there are religious and ethical sensitivities regarding the types of gelatine that they consume. Gelatines and related collagen proteins are incorporated into many foods, confectioneries, beverages and pharmaceuticals as thickeners, gelling agents, clarifying agents and to enhance ‘mouthfeel’. Gelatine is manufactured from skin and bone material under an aggressive process involving high temperature and pressure and treatment by acid or alkali. The proteins are irreversibly altered during these treatments. The analytical method employs a proprietary database built up over a number of years that contains gelatine peptide mass data from mass spectrometry experiments encompassing scores of species and phylogeny data which is used to match unique peptides specific to a species or to a tissue (skin or bone). The method can determine the species provenance of a wide range of gelatines including pig, cow, horse, fish or poultry. An example spectrum is shown in Figure 4. The method was recently employed to investigate the suspected addition of hydrolysed protein to samples of chicken fillets as a water binding agent (URL. http://www.food.gov.uk/news/newsarchive/2009/jun/chicken) The fillets, labelled as ‘chicken only’ or as ‘Halal-slaughtered’, were shown to be adulterated with hydrolysed proteins derived from cow material.

Avoiding another BSE crisis

In a variation of the above method, the species contained in animal feed (meat and bone meal) can be determined. This method may be useful in supporting the relaxation of the European Commission (EC) Extended Feed Ban. This legislation prohibits the inclusion of mammalian proteins in ruminant feed and was implemented as a result of the bovine spongiform encephalopathy (BSE) outbreak of 1987 in order to eradicate the disease in Europe. Scientific and epidemiologic evidence show that meat and bone meal is the most probable vector of the disease. The EC now considers that amendments of certain parts of the feed ban are possible without endangering the health or the policy of eradicating BSE, provided that scientific conditions are in place to screen feeds. There are methods which show potential to determine the species origin of processed animal proteins in feed including classical microscopy (van Raamsdonk et al., 2013), near infrared microscopy (NIR) (Fernandez et al., 2013), PCR (Fumiere et al., 2013), immunological approaches (Reaney and Bremer, 2013), or combinations of these methods (Bremer et al., 2013). The gelatine method described above could be used as a confirmatory method to determine the species provenance of skin and bone material within a highly processed feed sample to determine whether ruminant material is present.

Conclusion

A wide variety of scientific techniques have been developed to protect consumers and to screen for adulteration in the food and feed chain. The analytical testing services underpinned by scientific expertise, detailed knowledge of current and emerging regulations, and by internationally recognised quality standards are required to protect against food fraud. A range of sophisticated tools and continuing research effort should continue to be applied to address emerging food quality issues and to ensure brand and consumer protection.

References

Adams, C.J., Manley-Harris, M. and Molan, P.C. (2009) The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey Carbohydrate Research 344 1050–1053

Bremer, M. et al. (2013). Combination methods for PAP detection and species determination of animal particles. In Detection, identification and quantification of processed animal proteins in feedingstuffs. pp. 113-124. Edited by Jorgensen, JS and Baeten, V. Presses Universitaires de Namur.

Chisholm, J., Conyers, C., Booth, C., Lawley, W. and Hird, H. (2005). The detection of horse and donkey using real-time PCR Meat Science; 70 (4): 727-732.

Colyer, A., Macarthur, R., Lloyd, J., and Hird H.J. (2008). Comparison of calibration methods for the quantification of Basmati and non-Basmati rice using microsatellite analysis Food Additives and Contaminants Part A-Chemistry Analysis Control Exposure & Risk Assessment 25(10),1189-1194.

Conyers, C.M., Allnutt, T.R., Hird, H.J. Kaye, J. and Chisholm J. (2012). Development of a Microsatellite-Based Method for the Differentiation of European Wild Boar (Sus scrofa scrofa) from Domestic Pig Breeds (Sus scrofa domestica) in Food Journal of Agricultural and Food Chemistry; 60(13):3341-3347.

Donarski, J.A., Jones, S.A., Harrison, M., Driffield, M. and Charlton, A.J. (2010a) Identification of botanical biomarkers found in Corsican honey. Food Chemistry 118; 987–994

Donarski, J.A., Roberts, D.P.T. and Charlton, A.J. (2010b). Quantitative NMR spectroscopy for the rapid measurement of methylglyoxal in manuka honey Anal. Methods; 2: 1479-1483.

Fernandez Pierna, J.A., Bæten, V., Boix, A., von Holst, C., Perez Marin, D, Garrido Varo, A. (2013) Near Infrared Microscopy (NIRM). In Detection, identification and quantification of processed animal proteins in feedingstuffs. pp. 113-124. Edited by Jorgensen, JS and Baeten, V. Presses Universitaires de Namur.

Fumiere, O., Close, R., Karanam, M. and Berben, G. (2013) DNA based approaches for the species-specific identification of animal material. In Detection, identification and quantification of processed animal proteins in feedingstuffs. pp. 113-124. Edited by Jorgensen, JS and Baeten, V. Presses Universitaires de Namur.

Grundy, H.H., Reece, P., Sykes, M.D., Clough, J.A., Audsley, N., Stones, R. (2007). Screening method for the addition of bovine blood-based binding agents to food using liquid chromatography triple quadrupole mass spectrometry. Rapid Commun. Mass Spectrom.; 21(18):2919-25.

Grundy, H.H., Reece, P., Sykes, M.D., Clough, J.A., Audsley, N., Stones, R. (2008). Method to screen for the addition of porcine blood-based binding products to foods using liquid chromatography/triple quadrupole mass spectrometry. Rapid Commun. Mass Spectrom; 22(12):2006-8.

Heaton, K., Kelly, S.D., Hoogewerff, J. and Woolfe, M. (2007). Verifying the geographical origin of beef: The application of Multi-element Isotope and Trace Element Analysis. Food Chemistry; 107: 506–515.

Hird, H., Chisholm, J., Kaye, J., Colyer, A., Hold, G., Conyers, C., Irazu Nunez, J. & Macarthur, R. (2012). Development of Real-Time PCR assays for the detection of Atlantic Cod (Gadus morhua), Atlantic Salmon (Salmo salar) and European Plaice (Pleuronectes platessa) in Complex Food Samples. European Food Research and Technology; 234:127-136.

Hird, H.J., Powell J., Johnson, M.L. and Oehlschlager, S.L. (2003). Determination of percentage of Roundup Ready soya in soya flour using real-time polymerase chain reaction: Interlaboratory study Journal of AOAC International; 86(1):66-71.

Kelly, S.D. (2003). Using stable isotope ratio mass spectrometry in food authentication and traceability. Food Authenticity and Traceability, Michele Lees(Ed). Woodhead Publishing, Cambridge, UK, ISBN 0-8493-1763-0, pp 156-183.

Kelly, S.D. and Bateman, A.S. (2009). Comparison of mineral concentrations in commercially grown organic and conventional crops - tomatoes (Lycopersicon esculentum) and lettuces (Lactuca sativa)” Food Chemistry: 119: 738–745.

Langrell, S.R.H., Allnutt, T.R., Laval, V., Bertheau, Y., Pla, M., Papazova, N. and Lee, D. (2011). Validation of a Real-Time PCR On-Site Quantification Method for MON810 Maize. Food Analytical Methods DOI: 10.1007/s12161-010-9167-z (2011).

Laube, I., Hird, H., Brodmann, P., Ullmann, S., Schöne-Michling, M., Chisholm, J. and Broll, H. (2010). Development of primer and probe sets for the detection of plant species in honey Food Chemistry; 118 (4): 979-986.

López-Calleja, I. González Alonso, I., Fajardo, V., Rodríguez, M.A., Hernández, P.E., García, T., Martín, R. (2004). PCR detection of cows’ milk in water buffalo milk and mozzarella cheese. International Dairy Journal 15(11): 1122–1129

Macarthur R;Murray AWA;Allnutt TR;Deppe C;Hird HJ;Kerins GM;Blackburn J;Brown J;Stones R;Hugo S; (2007) Model for tuning GMO detection in seed and grain Nature Biotechnology 25(2),169-170.

Maggi, L., Carmona, M., Kelly, S.D., Marigheto, N. and Alonso, G.L.(2011). Geographical origin differentiation of saffron spice (Crocus sativus L. stigmas) – preliminary investigation using chemical and multi-element (H, C, N) stable isotope analysis. Food Chemistry; 128: 543–548.

McKenzie, J.S, Donarski, J.A., Wilson, J.C. and Charlton, A.J. (2011). Analysis of complex mixtures using high-resolution nuclear magnetic resonance spectroscopy and chemometrics. Progress in Nuclear Magnetic Resonance Spectroscopy; 59(4): 336-359.

Reany, S. and Bremer, M. (2013) Immunological approaches for processed animal protein detection in animal feeds. In Detection, identification and quantification of processed animal proteins in feedingstuffs. pp. 113-124. Edited by Jorgensen, JS and Baeten, V. Presses Universitaires de Namur.

Reece, P., Chassaigne, H., Collins, M., Buckley, M., Bremer, M. and Grundy, H. (2013) Proteomic analysis of meat and bone meal and animal feed. In Detection, identification and quantification of processed animal proteins in feedingstuffs. pp. 113-124. Edited by Jorgensen, JS and Baeten, V. Presses Universitaires de Namur.

van Raamsdonk, L.W.D., Pinotti, L., Veys, P., Vancutsem, J, Pridotkas, G. and Jorgensen, J.S. (2013). Markers for microscopic detection. In Detection, identification and quantification of processed animal proteins in feedingstuffs. pp. 113-124. Edited by Jorgensen, JS and Baeten, V. Presses Universitaires de Namur.

Web references

Sudan dyes in Worcester sauce URL http://www.scientistlive.com/content/14486 Accessed 12.06.2013. Bovine material found in chicken fillets and chicken injection mixtures URL: http://www.food.gov.uk/news/newsarchive/2009/jun/chicken . Accessed 12.06.2013.

 

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