Development and application of criteria for classification of hydrolysed proteins in the framework of feed safety

In the view of a circular economy, there is an increasing need for (re-)using animal by-products that have a wide range of applications and sufficient safety. Hydrolysates of animal proteins (HPs) are frequently used as feed ingredients. Nevertheless, clear criteria for legal use and methods for monitoring feed applications are not available. Here, a range of methods have been used and evaluated for characterizing a set of 26 samples of hydrolysed proteins, ‘hydrolysed’ feather meals and processed animal proteins (PAPs), with verification based on an additional set of eight samples. Methods included determination of ash content, sediment (mineral fraction) content, protein content, species identity, solubility, protein solubility, size exclusion chromatography and polyacrylamide gel electrophoresis (SDS-PAGE). After a comparison of results obtained with water and SDS, water was chosen as the solvent for environmental and occupational reasons. Typical HP samples have a protein content higher than 60%, a solubility exceeding 50% and a virtual absence of a mineral fraction. The first discrimination between HPs and PAPs could be based on the absence or presence, respectively, of a mineral fraction. An approach for HP characterization is designed using a Hydrolysation Index (HI) based on the fraction of peptides smaller than 10 kDa, the solubility of the sample and the fraction of soluble proteins. A simplified version (HI_s), exclusively based on the fraction of peptides smaller than 10 kDa and the solubility of the sample, shows a trend among the samples highly comparable to HI. Values for HI and HI_s exceeding 60% would characterise HPs. Feather meals, which are heat treated instead of treatment by a chemical process of hydrolysation, range among the PAPs and should not be indicated as “hydrolysed.” The HI_s can be used as an easy parameter for classifying HPs and for legal enforcement.从循环经济视角出发，对具备广泛应用前景且安全性合规的动物副产品的（再）利用需求正日益增长。动物蛋白水解物 HPs (Hydrolysates of animal proteins) 常被用作饲料原料，但当前尚无明确的合法使用标准及饲料应用监测方案。本研究针对26份水解蛋白、"水解"羽毛粉及加工动物蛋白 PAPs (processed animal proteins) 样本，开展了多种表征方法的开发与评估，并额外选取8份样本完成验证工作。所用表征方法涵盖灰分含量、沉淀物（矿物组分）含量、蛋白质含量、物种鉴定、总溶解度、蛋白质溶解度测定，以及尺寸排阻色谱法与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 SDS-PAGE (polyacrylamide gel electrophoresis)。通过对比以水与十二烷基硫酸钠为溶剂的检测结果后，基于环境友好与职业健康的考量，最终选定水作为实验溶剂。典型的动物蛋白水解物样本蛋白质含量高于60%，溶解度超过50%，且几乎不含矿物组分。区分动物蛋白水解物与加工动物蛋白的首要依据可基于矿物组分的有无：前者不含矿物组分，后者含有矿物组分。本研究基于分子量小于10 kDa的肽段占比、样本溶解度及可溶性蛋白占比，构建了用于动物蛋白水解物表征的水解指数 HI (Hydrolysation Index)。仅基于分子量小于10 kDa的肽段占比与样本溶解度构建的简化版水解指数 HI_s，其在样本间的变化趋势与完整水解指数高度一致。当水解指数与简化版水解指数的数值均超过60%时，即可将样本判定为动物蛋白水解物。羽毛粉仅经过热处理而非化学水解工艺，归类于加工动物蛋白范畴，不得标注为"水解"产品。HI_s可作为便捷参数，用于动物蛋白水解物的分类及法律合规监管。