Sample Preparation & Applications in food and feedstuff. 样品制备 - 食品和饲料应用

Purpose and Scope 应用目的和领域 Aim 目的 Hydrolysis of the peptide bonds joining the amino acids in the proteins to liberate them in solution prior to chromatography 分析前水解蛋白质中连 接氨基酸的肽键 Type of samples 样品种类 Pure protein sources (natural or synthetic) 纯蛋白（天然或合成） Cereals and feedstuff 谷物和饲料 Plants and fungi 植物或真菌 Hydrolysis procedure 水解步骤 Inorganic acid hydrolysis 无机酸水解 Organic acid hydrolysis 有机酸水解 Alkaline hydrolysis 碱水解 Factors to consider 考虑因素 Does the sample need to be oxidised? 样品需要氧化水解吗？ Does tryptophan need to be quantified? 需要定量色氨酸吗？ Does the sample contain carbohydrates? 样品包含糖类吗？

Factors to consider 需要考虑的因素 Hydrolysis time 水解时间 Complete liberation of the amino acids depends on the nature of the linkages in each particular protein 氨基酸彻底解离与特定氨基酸的联接有关 Ex: peptide bonds involving the amino acids Leucine, Isoleucine or Valine can require long hydrolysis times to cleave 如：在亮氨酸、异亮氨酸或缬氨酸间的肽键需要长时间水解才能解离 True values of the amino acids should be determined by performing hydrolysis with different hydrolysis times 氨基酸的真实结果应该通过不同水解时间确定 Alternative hydrolysis methods 选择水解方法 Needed when amino acids destroyed by acid hydrolysis need to be quantified. 通过酸水解定量的氨基酸需考虑这个问题 No single hydrolysis method can totally recover all the protein amino acids 没有任何一个水解方法得到的结果能够定量所有的蛋白氨基酸

Oxidation procedure 氧化步骤 Preparation of the oxidation mixture 氧化试剂的制备 Oxidation of the sample at 0°C in ice bath for 16 hrs 样品在冰浴中 0 度氧化 16 小时 Decomposition of excess reagent using Sodium Metabisulphite 使用焦亚硫酸钠分解过量试剂 Reflux hydrolysis with 6N HCl at 110°C for 24 hours 用 6 摩尔 / 升盐酸回流水解 24 小 时 Adjust the pH to 2.2 with 7.5 N NaOH 用 7.5 摩尔、升的 NaOH 调节 PH 到 2.2 Dilution and filtration of the sample through a 0.22 μm membrane 稀释样品后通过 0.22 微米膜过 滤 Analysis 分析

Alternative hydrolysis methods for Tryptophan 色氨酸的水解方法 1-5 mg protein hydrolysed with 0.5 ml 5N NaOH (or BaOH) containing 25 mg of starch (antioxidant) 每 1-5mg 蛋白 用 0.5mlNaOH( 或 BaOH) 水解，其中还包含 25mg 淀粉（抗氧化） Gives rise to artefacts and is not suitable for most amino acids 对大多数氨基酸不适合 Alkaline hydrolysis (recovery of tryptophan 100%) 碱水解（色氨酸回收率 100% ） mg of protein hydrolysed with 1ml 6N HCl containing 2% thioglycolic acid mg 蛋白用 1ml ，包含 2% 巯基乙酸的 6 摩尔 / 升盐酸水解 Thioglycolic acid (recovery of tryptophan 85 %) 巯基乙酸（色氨酸回收率 85% ） 2-3 mg protein hydrolysed with 1 ml 3N p-toluensulphonic acid containing 0.2 tryptamine 2-3mg 蛋白用包含 0.2% 色胺的 1ml3 摩尔 / 升对甲苯磺酸 p-toluenesulphonic acid (recovery 94 % in absence of carbohydrate 72 % at 30 % carbohydrate) 对 - 甲苯磺酸（不含糖类回收率 94% ， 30% 糖类回收率 72% ） mg protein hydrolysed with 1 ml 3N Mercaptoethanesulphonic acid 0.5-2mg 蛋白用 1ml3 摩尔 / 升的巯基乙磺酸水解 Mercaptoethanesulphonic acid (recovery 95 %) 巯基乙磺酸（回收率 95% ）

Protocol 协议 Oxidation with Performic acid at 0ºC for 16h 过甲酸 0 度 16 小时水解 Sample containing 10mg Nitrogen 含氮量 10mg 样品 Hydrolysis in 6N HCl at 110ºC for 24h 。 6 摩尔 / 升盐 酸 110 度水解 24 小 时 Extraction of free AA with loading buffer 用上样液提取游 离氨基酸 Deproteinisation 除蛋白 Neutralisation: NaOH 用 NaOH 中和 Hydrolysis in 6N HCl at 110ºC for 24h 6 摩尔 / 升盐酸 110 度水解 24 小时 Neutralisation: NaOH 用 NaOH 中和 Combined results give global protein AA content 蛋白中结合氨基酸含量 Analysis 分析 Free amino acid content 游离氨基酸含量

Polyamine analysis 多胺分析  Biogenic amines are mostly detrimental and can generally be considered as markers of protein degradation.  生物胺多是有害的，且通常是蛋白降解物  in large doses histamine can cause food poisoning and become toxic.  大量的组胺可导致食物中毒或称为有毒物质  According to the 91/493/EEC directive the average level of histamine in fish should not exceed 100 mg/kg.  按照 91/493/EEC 指令，鱼肉中组胺平均水平不超过 100mg/kg 。  Requires a specific column and buffer to run on the Biochrom AAA  在 BiochtomAAA 上需要特定分离柱和缓冲液分析

Analysis of Protein Hydrolysates on Lithium Systems 在锂系统中分析蛋白水解样品 Arg elutes in 60 min (total analysis time 93 min) 净分析时间 60 分钟（全分析时间 93 分钟） Standard 标准品 Hydrolysate 水解样