A new method for detecting the animal origin of collagen

自从1987年农业中的疯牛病/TSE（传染性海绵状脑病）危机以来，每年都有更严格的立法，旨在从欧洲根除这种疾病。目前禁止在动物饲料中添加反刍动物蛋白，但由于反刍动物蛋白与其他动物蛋白难以区分，因此目前全面禁止在动物饲料中添加动物蛋白。基于DNA的警务方法，通常被认为是欺诈的解决方案，并不像最初希望的那样适合作为识别动物来源的手段，因为：(i)试剂中动物DNA的污染；（ii）在积极加工（例如糊化）过程中DNA的破坏；（iii）通过添加外来DNA实现的“物种改变”欺诈。蛋白质是产物，因此通过测序蛋白质片段直接进行物种鉴定是不存在欺诈的。作为NERC资助的CASE奖学金（NER/S/J/2004/13017）的一部分，我们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）。在SAFEED PAP欧盟项目组织的盲法试验中，我们的方法成功检测了8/8的141C加工动物蛋白样品。然而，这种方法是劳动密集型的。它依赖于使用昂贵的、专门的“高端”质谱仪，目前缺乏检测存在不止一种物种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，并制定一个标准操作程序，其中包括一系列内部肽标准，生产一系列用于控制该方法的肽标准，将作为工具包出售。加工动物蛋白：在欧盟内部，1000万吨肉类不是直接供人类消费的（23% - 48%的胴体）。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%）。东京证券交易所的危机改变了这一点，现在这些材料被当作危险废物处理。由于考虑到同类相食导致的传染性脑炎感染风险，禁止在动物饲料中使用哺乳动物和禽类蛋白质。通过显微镜鉴定骨碎片是目前欧盟鉴定加工动物蛋白污染的唯一官方方法，并且只能将鱼类与其他动物区分开来。由于缺乏更好的区分方法，导致了2003年的“延长饲料禁令”（EC法规1234/2003），该禁令将几乎所有加工动物蛋白（鱼类除外）排除在饲料之外，据估计，欧洲工业每年的成本为3.5亿欧元。自禁令实施以来，在动物饲料中检测到骨头碎片是第二大常见的欧共体动物饲料警报通知。2005年，在英国，这些主要是在甜菜浆中发现的，尽管这些可能来自自然来源（例如啮齿动物和捕食者的粪便），“受污染”的饲料必须被销毁。明胶：每年生产超过30万吨明胶，主要来自猪和牛的皮（74%）和骨头（24%），它在制药和食品工业中无处不在，在后者的一些情况下，以欺诈的方式（例如，增加肉类的蛋白质氮含量，并将其加入“素食”产品中）。确定明胶的动物来源也至关重要，因为世界上超过三分之一的人口信奉的世界宗教禁止食用猪和牛产品。在我们手中，所有在鸡肉丰满剂（用于增加鸡肉含水量的明胶）中检测到的肽都被证明是牛源的，尽管认证实验室只检测到鸡的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.