Lactoperoxidase (LP) and lactoferrin (LF) are two interesting proteins present in dairy whey due to their antioxidant, anti-inflammatory, antibacterial, antiviral and antifungal properties. LP and LF are traditionally purified using cation-exchange chromatography, but since the concentration of these proteins in sweet whey are very low (0.03–0.06 g/L for LP and around 0.08–0.20 g/L for LF), clarification, diafiltration, concentration and/or depletion of others whey proteins prior to the ion-exchange chromatography is needed to obtain good adsorption yields. After their elution from the matrix, the overall process results in variable yields. Due to their similar physico-chemical properties, a high cross-contamination is usually present, especially for the LP fraction, where part of the LF is co-eluted.

The development of novel supports with immobilized affinity and/or multimodal ligands aims to the direct adsorption of the target proteins even at low concentration and without the need of a pre-treatment of the source. Herein, a new, low-cost support material using chitosan mini-spheres with Orange R-HE triazine dye as immobilized ligand (CMS-Orange R-HE) was synthesized, physico-mechanically characterized and used for the purification of LP and LF by a one-step direct adsorption from dairy whey and a two-step elution process, with minor cross-contamination. CMS-Orange R-HE matrix showed a compression modulus of 47.6 ± 9.8 kPa, predominantly elastic deformation and an open-porous structure with mesopores around 5–20 nm and without ink-bottle pores, ideal for protein purification applications. The affinity between LP and LF for CMS-Orange R-HE matrix was characterized using the Langmuir isotherm model (K_d = 0.48 ± 0.07 mg/mL and 0.31 ± 0.09 mg/mL; q^max = 77.5 ± 4.0 mg/g and 56.0 ± 2.1 mg/g, respectively). Adsorption studies showed that LP interaction with CMS-Orange R-HE was partially influenced by the presence of ionic strength (p < 0.05), whereas LF adsorption was not. These differences of both electrostatic and hydrophobic interactions between LP and LF with the CMS-Orange R-HE matrix were used to develop a differential elution process using Response Surface Methodology (RSM). By one-step direct adsorption from dairy whey and two differential elution steps –by using the CMS-Orange R-HE matrix- LP and LF were obtained with good yields (≈70% for LP and ≈60% for LF), high purity and with minor cross-contamination between them. The strategy presented here, could be applied in other cases of cross-contamination between two proteins, being especially interesting for its application in multimodal chromatography.

乳过氧化物酶（LP）和乳铁蛋白（LF）是乳清乳清中存在的两种有趣的蛋白质，因为它们具有抗氧化，抗炎，抗菌，抗病毒和抗真菌的特性。LP和LF传统上是使用阳离子交换色谱法纯化的，但是由于这些蛋白质在甜乳清中的浓度非常低（LP为0.03-0.06 g / L，LF为约0.08-0.20 g / L），因此需要进行澄清，渗滤，为了获得良好的吸附率，需要在离子交换色谱之前浓缩和/或清除其他乳清蛋白。从基质中洗脱后，整个过程会产生可变的收率。由于它们的相似的理化性质，通常会出现高交叉污染，尤其是对于部分LF共洗脱的LP馏分。

具有固定的亲和力和/或多峰配体的新型支持物的开发旨在即使在低浓度且不需要预处理的情况下也直接吸附靶蛋白。本文合成了一种新的低成本载体材料，该材料使用壳聚糖微球和橙色R-HE三嗪染料作为固定配体（CMS-Orange R-HE），对其进行了物理机械表征，并用于纯化LP和LF。一步从乳清乳清直接吸附和两步洗脱过程，轻微的交叉污染。CMS-Orange R-HE基质的压缩模量为47.6±9.8 kPa，主要表现为弹性变形，其开孔结构具有5-20 nm左右的中孔，并且没有墨水瓶孔，非常适合蛋白质纯化应用。_d  = 0.48±0.07 mg / mL和0.31±0.09 mg / mL; ^最大q 分别为77.5±4.0 mg / g和56.0±2.1 mg / g）。吸附研究表明，LP与CMS-Orange R-HE的相互作用部分受到离子强度的影响（p <0.05），而LF吸附则不受此影响。LP和LF与CMS-Orange R-HE基质之间的静电和疏水相互作用的这些差异被用于使用响应表面方法（RSM）开发差分洗脱过程。通过一步法从乳清乳清直接吸附和两个不同的洗脱步骤–通过使用CMS-Orange R-HE基质，可获得高产率的LP和LF（LP≈70％，LF≈60％），高纯度并且它们之间的交叉污染较小。此处介绍的策略可以应用于其他两种蛋白质之间交叉污染的情况，