电渗析/超滤内在耦合过程分离蛋清中溶菌酶

doi:10.16085/j.issn.1000-6613.2022-1293

摘要/Abstract

摘要：

溶菌酶（LYZ）是一种化学性质稳定、无毒无害的具有抗菌作用的天然活性蛋白，高纯度、高活性溶菌酶在食品与医药领域具有重要价值。本文尝试采用新型电渗析／超滤内在耦合（EDUF）技术，同时利用特定pH条件下蛋清中卵白蛋白（OVA）与LYZ荷电性及分子量的差异，从蛋清中直接分离、提取LYZ。重点考察了超滤膜材料及EDUF原料室和回收室内料液流量对过程分离性能的影响。结果表明，在操作电压为5.0V，采用PVDF100Ⅰ超滤膜，原料室和回收室料液流量均为50mL/min时，LYZ的回收率可达21.05%，所回收LYZ的纯度可达100%，其最终迁移速率为0.21g/(m²·h)，单位产品能耗为2.30kW·h/g。该研究证明了EDUF作为一项独特的电驱动膜分离技术，具有选择性高的显著特点，在活性蛋白高效分离领域具有较好的应用前景。

关键词: 电渗析/超滤内在耦合, 溶菌酶, 蛋清, 分离, 纯化, 蛋白质

Abstract:

Lysozyme (LYZ) is a natural active protein with stable chemical properties, non-toxic and antibacterial function. Lysozyme with high purity and high activity has great value in the fields of food and medicine. In this paper, a novel electrodialysis with ultrafiltration membrane (EDUF) technology was used to directly separate LYZ from egg white based on the difference in charge and molecular weight between ovalbumin (OVA) and LYZ under specific pH conditions. The effects of ultrafiltration membrane material and the solution flow rates in feed and recovery compartments on the separation performance of EDUF were investigated. Results showed that the recovery rate and purity of LYZ could respectively reached 21.05% and 100% by using PVDF100Ⅰmembrane when the operating voltage was 5.0V and the solution flow rates in feed and recovery compartments were both 50mL/min. The final transport rate of LYZ was 0.21g/(m²·h) and energy consumption per unit product was 2.30kW·h/g under the tested conditions. This study demonstrates that EDUF, as an unique electro-driven membrane separation technology, has remarkable characteristics with its high selectivity. EDUF technology has good application prospects in the field of high-efficient separation of active proteins.

Key words: electrodialysis with ultrafiltration membrane (EDUF), lysozyme (LYZ), egg white, separation, purification, protein

中图分类号:

X792

孙鲁芹, 卢会霞, 王建友. 电渗析/超滤内在耦合过程分离蛋清中溶菌酶[J]. 化工进展, 2023, 42(5): 2262-2271.

SUN Luqin, LU Huixia, WANG Jianyou. Separation of lysozyme from egg white by electrodialysis with ultrafiltration membrane(EDUF) process[J]. Chemical Industry and Engineering Progress, 2023, 42(5): 2262-2271.

图/表 10

图1 用于LYZ分离的EDUF膜堆构型AEM—阴离子交换膜；CEM—阳离子交换膜；UFM—PES100或PVDF100Ⅰ超滤膜；OVA-—荷负电的OVA；LYZ+—荷正电的LYZ

表1 各隔室料液组分及流量

隔室	料液组分	流量/mL·min^-1
原料室	溶解于2g/L KCl中，OVA浓度为1g/L，LYZ浓度为0.1g/L的蛋白混合溶液	30~70
回收室	2g/L KCl	30~70
保护室	40g/L Na₂SO₄	200
电极室	20g/L Na₂SO₄	200

图2 超滤膜材料对膜堆电流、电阻、原料液、回收液、电导率及原料液、回收液pH的影响

图3 采用不同超滤膜时回收液冻干蛋白质样品的SDS-PAGE分析结果M—marker；泳道1—原料液样品；泳道2—PES100膜的回收液蛋白样品；泳道3—PVDF100Ⅰ膜的回收液蛋白样品

图4 PVDF100Ⅰ超滤膜最终回收液样品的液相色谱峰图

图5 超滤膜对LYZ回收率、迁移速率及运行能耗的影响

图6 料液流量对膜堆电流、电阻、原料液、回收液电导率及原料液、回收液pH的影响

图7 不同料液流量条件下回收液冻干蛋白样品的SDS-PAGE分析结果M—marker；泳道1—原料液和回收液流量同为30mL/min时，回收液蛋白样品；泳道2—原料液和回收液流量同为50mL/min时，回收液蛋白样品；泳道3—原料液和回收液流量同为70mL/min时，回收液蛋白样品

图8 不同料液流量条件下回收液样品的液相色谱峰图

图9 不同料液流量对LYZ回收率、迁移速率及运行能耗的影响

参考文献 21

1	张铁华. 蛋清活性蛋白高压脉冲电场提取及功能特性研究[D]. 长春: 吉林大学, 2008.
	ZHANG Tiehua. Study on extraction and functional property of major activity protein from egg white by pulsed electric field technology[D]. Changchun: Jilin University, 2008.
2	ABEYRATHNE E D N S, LEE H Y, AHN D U. Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents—A review[J]. Poultry Science, 2013, 92(12): 3292-3299.
3	SHENG Long, WANG Yibo, CHEN Jiahui, et al. Influence of high-intensity ultrasound on foaming and structural properties of egg white[J]. Food Research International, 2018, 108: 604-610.
4	OLIVER W T, WELLS J E, MAXWELL C V. Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge[J]. Journal of Animal Science, 2014, 92(11): 4927-4934.
5	明辉, 李浩, 徐婷. 鸡蛋清溶菌酶分离纯化研究进展[J]. 农产品加工, 2021(11): 81-85.
	MING Hui, LI Hao, XU Ting. Research progress on isolation and purification of lysozyme from egg white[J]. Farm Products Processing, 2021(11): 81-85.
6	WAN Yinhua, LU Junren, CUI Zhanfeng. Separation of lysozyme from chicken egg white using ultrafiltration[J]. Separation and Purification Technology, 2006, 48(2): 133-142.
7	JI Shengnan, Dong Uk AHN, ZHAO Yunlong, et al. An easy and rapid separation method for five major proteins from egg white: Successive extraction and MALDI-TOF-MS identification[J]. Food Chemistry, 2020, 315: 126207.
8	周钦育, 黄燕燕, 赵珊, 等.蛋清溶菌酶的提取及其酶学性质探究[J].中国食品学报, 2021, 21(4): 148-158.
	ZHOU Qinyu, HUANG Yanyan, ZHAO Shan, et al. Studies on extraction and enzymatic properties of egg white lysozyme[J]. Journal of Chinese Institute of Food Science and Technology, 2021, 21(4): 148-158.
9	余龙, 黄鹏, 李宗林, 等. 一种用于分离溶菌酶的亲和层析介质及其制备方法: CN101352673A[P]. 2009-01-28.
	YU Long, HUANG Peng, LI Zonglin, et al. An affinity chromatography medium for separating lysozyme and preparation method thereof: CN101352673A[P]. 2009-01-28.
10	吕晓萌, 鲍宗源, 郝晓妤, 等. 聚乙二醇2000/磷酸氢二钾双水相体系萃取溶菌酶[J]. 农产品加工, 2019(19): 38-40.
	Xiaomeng LYU, BAO Zongyuan, HAO Xiaoyu, et al. Extracting lysozyme in PEG 2000/K₂HPO₄ aqueous two-phase system[J]. Farm Products Processing, 2019(19): 38-40.
11	刘瑞, 张弘弛, 周凤, 等. 溶菌酶的反胶束提取条件优化[J]. 中国实验方剂学杂志, 2015, 21(20): 30-33.
	LIU Rui, ZHANG Hongchi, ZHOU Feng, et al. Optimization of reverse micelles extraction conditions of lysozyme[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2015, 21(20): 30-33.
12	HUANG Weiwei, ZHU Yuanhong, DONG Bingzhi, et al. Investigation of membrane fouling mechanism of intracellular organic matter during ultrafiltration[J]. Scientific Reports, 2021, 11: 1012.
13	HAO Songze, ZHAO Xuehui, ZHANG Hongwei, et al. Effects of a novel bimetallic catalytic biofilter-based pretreatment technique on the form of ultrafiltration membrane fouling[J]. Chinese Journal of Chemical Engineering, 2020, 28(10): 2513-2522.
14	GALIER S, Roux-de BALMANN H. Electrophoretic membrane contactors[J]. Chemical Engineering Research and Design, 2005, 83(3) : 268-275.
15	AIDER Mohammed, BRUNET Serge, BAZINET Laurent. Electroseparation of chitosan oligomers by electrodialysis with ultrafiltration membrane (EDUF) and impact on electrodialytic parameters[J]. Journal of Membrane Science, 2008, 309(1/2): 222-232.
16	FIRDAOUS Loubna, DHULSTER Pascal, AMIOT Jean, et al. Concentration and selective separation of bioactive peptides from an alfalfa white protein hydrolysate by electrodialysis with ultrafiltration membranes[J]. Journal of Membrane Science, 2009, 329(1/2): 60-67.
17	KETNAWA Sunantha, SUWAL Shyam, HUANG Jen-Yi, et al. Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates[J]. International Journal of Food Science and Technology, 2019, 54(4): 1062-1073.
18	HE Rong, GIRGIH Abraham T, ROZOY Elodie, et al. Selective separation and concentration of antihypertensive peptides from rapeseed protein hydrolysate by electrodialysis with ultrafiltration membranes[J]. Food Chemistry, 2016, 197: 1008-1014.
19	SUWAL Shyam, ROBLET Cyril, AMIOT Jean, et al. Presence of free amino acids in protein hydrolysate during electroseparation of peptides: Impact on system efficiency and membrane physicochemical properties[J]. Separation and Purification Technology, 2015, 147: 227-236.
20	DURAND Rachel, PELLERIN Geneviève, THIBODEAU Jacinthe, et al. Screening for metabolic syndrome application of a herring by-product hydrolysate after its separation by electrodialysis with ultrafiltration membrane and identification of novel anti-inflammatory peptides[J]. Separation and Purification Technology, 2020, 235: 116205.
21	NDIAYE N, POULIOT Y, SAUCIER L, et al. Electroseparation of bovine lactoferrin from model and whey solutions[J]. Separation and Purification Technology, 2010, 74(1): 93-99.

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