A new method for detecting the animal origin of collagen

Since the BSE/TSE (Transmissible Spongiform Encephalopathies) crisis in farming in 1987 there has been stricter legislation year on year aimed at eradicating the disease from Europe. There is currently a ban on the presence of ruminant protein in animal feed, however since ruminant protein cannot be readily distinguished from other animal proteins, there is at present a total ban on animal protein in animal feed. DNA-based policing methods, often thought to be the solution to fraud, are less suitable than originally hoped as a means of identifying animal origin because of:- (i) contamination from animal DNA in reagents (ii) destruction of DNA during aggressive processing (e.g. gelatinization) (iii) 'species-change' fraud achieved by adding foreign DNA. The protein is the product and therefore direct species identification by sequencing pieces of the protein is immune to fraud. As part of NERC funded CASE studentship (NER/S/J/2004/13017) we have developed tools to identify collagen (and its denatured product, gelatin) based upon the selective fingerprinting of protein fragments using protein mass spectrometry. Our method was successful at detecting 8/8 of the 141C processed animal protein samples in a blind trial organised by the SAFEED PAP EU project. However the method is labour intensive. It relies on the use of expensive, specialised 'high-end' mass spectrometers, and currently lacks the sensitivity required to detect low levels of bone where more than one species are present. The proposal seeks to implement the method on more commonly-available instrumentation and to develop a standard operating procedure which includes a series of internal peptide standards producing a series of peptide standards for controlling the method, to be sold as a kit. Processed animal protein: Within the EU, 10 million tonnes of meat are not destined for direct human consumption (23% - 48% of the carcass). Historically, one quarter of this was used to enhance the protein content of animal feed, accounting for more than 6% of total animal feed protein (in contrast to 3% from fishmeal). The TSE crisis changed this and now this material is being handled as hazardous waste. Concerns regarding the risk of TSE infection caused by cannibalism prohibit the use of mammalian and avian protein in animal feed. The identification of bone fragments by microscopy is currently the only official EU method for identification of contamination with processed animal proteins and can only discriminate fish from other animals. The lack of methods allowing any better discrimination led to the 2003 'extended feed ban' (EC Regulation 1234/2003) that excludes almost all processed animal protein (other than fish) from feeds, at an estimated cost to the European industry of ¤350M per year. Detection of bone fragments in animal feed represented the second most common EC animal feed alert notification since implementation of the ban. In 2005 in the UK these were mainly found in sugar beet pulp, and despite the fact that these probably derived from natural sources (e.g. rodents and predator scats) the 'contaminated' feed had to be destroyed. Gelatin: More than 300,000 tonnes of gelatin are produced annually, principally from pig and cattle skin (74%) and bone (24%) and it is ubiquitous in both the pharmaceutical and food industry, in some of the latter cases in fraudulent ways (e.g. to increase the protein nitrogen content of meat, and inclusion in 'vegetarian' products). Determination of the animal origin of gelatin is also essential in a world where the consumption of pig and cow products is variously prohibited in world religions professed by more than one third of the planet's population. In our hands all the peptides detected in a chicken plumping agent (gelatin used to increase the water content of chicken meat) were shown to be of bovine origin despite that fact that an authentication laboratory had detected only chicken DNA.