A new method for detecting the animal origin of collagen

自1987年BSE/TSE（传染性海绵状脑病）危机以来，欧洲每年都有更严格的立法，旨在根除这种疾病。目前禁止在动物饲料中存在反刍动物蛋白质，然而，由于反刍动物蛋白质不能容易地与其他动物蛋白质区分，目前在动物饲料中完全禁止动物蛋白质。基于DNA的监管方法通常被认为是欺诈的解决方案，但不像最初希望的那样适合作为鉴定动物来源的手段，因为：（i）试剂中动物DNA的污染（ii）在侵略性处理过程中DNA的破坏（例如凝胶化）（iii）通过添加外源DNA实现的“物种改变”欺诈。蛋白质是产品，因此通过对蛋白质片段进行测序进行直接物种鉴定是不受欺诈的。作为NERC资助的CASE学生奖学金（NER/S/J/2004/13017）的一部分，我们已经开发了基于使用蛋白质质谱法对蛋白质片段进行选择性指纹识别的工具来识别胶原蛋白（及其变性产物，明胶）。我们的方法在SAFEED PAP EU项目组织的盲法试验中成功检测了8/8的141 C加工动物蛋白样品。然而，该方法是劳动密集型的。它依赖于使用昂贵的，专业的“高端”质谱仪，目前缺乏检测多个物种存在的低水平骨骼所需的灵敏度。该提案寻求在更常用的仪器上实施该方法，并开发标准操作程序，该标准操作程序包括一系列内部肽标准品，产生一系列用于控制该方法的肽标准品，作为试剂盒出售。加工动物蛋白：在欧盟范围内，有1000万吨肉类（占胴体的23% - 48%）不直接供人类消费。从历史上看，其中四分之一用于提高动物饲料的蛋白质含量，占动物饲料蛋白质总量的6%以上（相比之下，鱼粉为3%）。TSE危机改变了这一点，现在这种材料被作为危险废物处理。由于担心同类相食导致TSE感染的风险，禁止在动物饲料中使用哺乳动物和禽类蛋白。通过显微镜鉴定骨碎片是目前唯一官方的欧盟鉴定加工动物蛋白污染的方法，只能区分鱼类和其他动物。由于缺乏更好的区分方法，导致2003年的“延长饲料禁令”（EC法规1234/2003），几乎所有加工动物蛋白（鱼类除外）都被排除在饲料之外，估计每年给欧洲工业造成3.5亿欧元的损失。在动物饲料中检测到骨碎片是自禁令实施以来第二常见的EC动物饲料警报通知。2005年在英国，这些物质主要在甜菜浆中发现，尽管这些物质可能来自自然来源（例如啮齿动物和捕食者的粪便），但必须销毁“受污染”的饲料。明胶：每年生产超过300，000吨明胶，主要来自猪和牛皮（74%）和骨头（24%），在制药和食品行业中无处不在，在某些情况下，后者以欺诈方式（例如增加肉类的蛋白质氮含量，并包含在“素食”产品中）。明胶的动物来源的确定也是必不可少的，在一个世界上，猪和牛产品的消费是不同的世界宗教禁止自称超过三分之一的地球人口。在我们手中，在鸡肉膨胀剂（用于增加鸡肉含水量的明胶）中检测到的所有肽都被证明是牛源性的，尽管认证实验室只检测到了鸡的DNA。

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.