A new method for detecting the animal origin of collagen

​Key Findings

​We are able to use peptide mass fingerprinting to identify the animal origin of collagen in highly processed samples of animal tissue​

Final Report

Collagen is one of the main signature materials in animals, occurring in bone, horn, ivory and skin. We developed a method that recognised the type of animal from its collagen when it occurred in minute traces – as on archaeological sites. The research team realised that this could have major impact on the detection of animal parts used fraudulently in human and animal food – for example horse in beef dinners. The results of deploying this research in the food and livestock industry have been revolutionary and have enhanced the lives and respected the beliefs of people the world over.

The project sought to develop a commercial application of peptide mass fingerprinting. Working with colleagues at the Veterinary Laboratories Agency, the Food Standards Agency and Fera we tested methods for the detecton of meat and bone meal samples​.​

Zooarchaeology by Mass Spectrometry (ZooMS) is a procedure developed at York as the consequence of a long struggle to identify the animal origin of worked bone and minuscule fragments from archaeological sites. Collagen, a majority constituent in skin, cartilage and bone was thought to decay into its component parts (peptides), so making it hard to identify (1). But at York Matthew Collins (Archaeology) and Jane Thomas-Oates (Chemistry) overturned this model of degradation, showing that collagen molecules in bone and (chrome tanned) leather were actually extremely stable, due to their being tightly compressed (the link-lock hypothesis) (3). While other proteins are decayed and lost from bone, intact collagen survives and is effectively purified, persisting for millions of years. This opened up the potential to use collagen as a fingerprint for identification not only in ancient bone (5), but also in processed tissues (leather). The collagen is identified by mass spectrometry, since the mass of particular molecules, is diagnostic of particular animal species. The method is economical and has few of the problems of DNA analysis: for example, the reassembling of long chain molecules by PCR and the rigorous measures required to avoid contamination.

The research is ​also ​proving invaluable in archaeology, since archaeologists prepare collagen every day (from bone, dentine, ivory and antler), either to date samples (using 14C) or to conduct stable isotope analysis (mainly 13C and 15N to infer diet). Now they have the tools to identify animals used in the making of artefacts: for example, e walrus ivory combs or whalebone boxes or the parchment of cattle or sheep used to write illuminated manuscripts.

The identification of minute traces on site will be of ever-increasing value as pressure grows to conserve archaeological resources rather than dig them. It will strengthen the new generation of techniques of ‘nano-excavation’ used in the field to map invisible activities in houses and fields and so enhance the historical yield (Carver Making Archaeology Happen 2011, ch 2).

The chief public impact has been in the application of the new research to detecting contaminants in food. In the two decades to 2007, the price of food for consumers fell, and the share of British household income spent on food dropped to 10%. Today 5% of our total food bill is £74bn is spent on ready meals containing meat, and these economic drivers have increased the risk of fraud. The ability to recover and identify the animal origin and state of degradation of collagen has been used to uncover food fraud, and ZooMS now provides an internationally approved method for identification of gelatin, a key product of the food industry (processed bone and skin), and has prepared a commercial database of collagen sequences hosted at the University of York.

This initiative led to us being approached (anonymously) by an investigative team from the BBC to test chicken breasts supplied to Asian restaurants. We were able to detect pig and cattle gelatin which derived from a so-called ‘plumping agent’, used to re-hydrate air-freighted products (‘What’s really in our food?’ broadcast BBC1 14th July 2009). The addition of other animal protein to meat stated to be from a single source is illegal. This resulted in an investigation with the Food Standards Agency (FSA, 2009-2010) into the illegal use of pig and cow-derived gelatin. As a consequence of our work it was evident that a complex process had been developed to dupe DNA testing, enabling low cost (pork and cattle) gelatin to be sold as ‘chicken protein’. Two factories (in Germany and Spain) producing the plumping agents were raided by local inspectors. The process compromised the food security of 2.8 million Muslims and 0.5 million Hindus in the UK. As a direct consequence of our work, Euro Foods, the largest supplier of poultry to the Asian market in the UK, announced that in order to “eradicate any future question marks and/or confusion over non chicken protein detection levels in our product range, Euro Foods Group has decided to switch to a vegetable derived protein instead, a move which was completed by 29th June 2009” (b). Due to the sensitive nature of issue of contaminating foodstuffs with pork and cow protein (condemned on air by religious leaders) we do not know the full impact of this change.

In 2011 we signed an agreement with FERA giving them commercial access to our collagen databases for use in the future forensic work for a fee of 5% of income generation, this has raised approximately £3,000 in fees to date.

In 2013 FERA commissioned us to develop a procedure to identify horse gelatin, and have applied our method to test stock cubes. Writing about the impact of the team’s relationship with FERA, Paul Brereton, Head of Food and Health Research at FERA, wrote “FERA’s close collaboration with Professors Thomas-Oates and Collins have led to a number of technologies that we routinely exploit to deliver ongoing project work and to underpin project proposals, papers and publicity material. In particular, we have undertaken a number of studies to determine peptide sequences that can be used for the species origin determination in collaboration with you and your colleagues. These projects have led to Fera offering an international service for the species identification of gelatin and MBM in food and feed, respectively".

​In addition the research has had a number of unexpected consequences:

Illegal cremations

In 2010 on behalf of Gloucester County Council we used ZooMS analysis of cremated remains to provide evidence in the successful prosecution of John Cooper Livestock Services (11 Feb 2011). Owners paid for the cremation of pet horses and donkeys but were returned cremated cattle bone ash, part of a fraud perpetrated by the accused, who had instead rendered the animals.

County Archives

Our history until the 15th C mainly was mainly written on parchment, our laws still are.

County archives are faced with the task of conserving 100s of millions of membranes. Five city and county archives now post their PVC eraser waste (used to remove dirt prior to conservation) to York. We can update in real time on shared records, our ZooMS results (1000 analyses to date) which provide the animal origin and state of deterioration of the parchment to guide conservation treatment and interpretation.

Dissemination

● The Jorvik Centre introduced a Science focused gallery (400,000 visitors per year), for which we wrote much of the text and continue to train the staff. This exhibition included a section on our collagen degradation work (used to identify boiled bones).

● The same method featured in a National Geographic Documentary Lost Cannibals of Europe (broadcast 25/1/11) which provided supporting evidence for earlier claims of cannibalism at Herxheim (Antiquity, 2009, 83, 322).

● Workshop for English Heritage Science Advisors (07/10/2011) on the latest developments in Biomolecular archaeology.

● We have developed a free web based tool www.thermal-age.eu (based upon or rate analysis for DNA, collagen and amino acid degradation) to enable museum curators to assess whether collections should be released for destructive analysis.

The RCUK has included a ZooMS module Protein structure and analysis by mass spectroscopy: investigating ancient antler combs produced by CP-educational as a Cutting Edge Science Vignette for teachers, and post-16 students.

1 Collins MJ, Riley MS, Child AM, Turner-Walker G. 1995. A Basic Mathematical Simulation of the Chemical Degradation of Ancient Collagen. J Arch Sci 22:175–183. The most widely reported model of collagen diagenesis (81 citations) correcting the mathematics of a previous Russian model by Rudakova & Zaikov

2 Allentoft, M. E., Collins, M., Harker, D., Haile, J., Oskam, C. L., Hale, M. L., ... & Bunce, M. (2012). The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils. Proceedings of the Royal Society B: Biological Sciences, 279 (1748), 4724-4733 6 citations, the first accurate measurement of the rate of DNA degradation in bone, decay conforming to a random chain scission model. Widely reported in the media as the final nail in the coffin of Jurassic Park.

3 Covington, A. D., Song, L., Suparno, O., Collins, M. J., & Koon, H. E. C. (2008). Link-Lock: the mechanism of stabilising collagen by chemical reactions. J. Soc. Leather Technol. Chem, 92, 1-7. “[The Link lock hypothesis] has made it possible to take quantum steps forward in developments in tanning technology” (Tanning Chemistry: The Science of Leather. p 464). 9 citations.

4 Collins, M. J., Penkman, K. E., Rohland, N., Shapiro, B., Dobberstein, R. C., Ritz-Timme, S., & Hofreiter, M. (2009). Is amino acid racemization a useful tool for screening for ancient DNA in bone?. Proceedings of the Royal Society B: Biological Sciences, 276(1669), 2971-2977 20 citations.

5 Buckley, M., Collins, M., Thomas-Oates, J., & Wilson, J. C. (2009). Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid communications in mass spectrometry, 23(23), 3843-3854. 37 citations.We are able to use peptide mass fingerprinting to identify the animal origin of collagen in highly processed samples of animal tissue​.