大孔阳离子交换树脂提取ε-聚赖氨酸工艺优化与放大研究

发布时间：2018-12-08 09:12

【摘要】：ε-聚赖氨酸(ε-poly-L-lysine,简称ε-PL)是一种绿色、安全的生物防腐剂。2014年,我国正式批准ε-PL及其盐酸盐为新的食品添加剂品种。经过多年的菌种选育和发酵策略优化,发酵产量已达到工业生产要求,然而,国内关于ε-PL分离提取方面的研究尚不完善,严重制约了ε-PL产业化。因此,研发能够用于工业生产的ε-PL分离提取技术对促进ε-PL产业化具有十分重要的现实意义。在前期研究工作的基础上,本研究对离子交换的动态条件进行系统优化,建立了以离子交换技术为核心的提取工艺,并对工艺的稳定性进行了考察,最后成功的将该提取工艺应用到试生产。取得的研究成果如下:(1)基于对钠型和铵型Amberlite IRC-50树脂动态条件的优化和比较研究,确定钠型IRC-50树脂更适合工业化应用。钠型树脂最适动态条件为:上样pH为7.0,上样速度为5 BV·h-1,洗脱剂为0.25 mol·L-1 NaOH,洗脱速度为2 BV·h-1。在最适操作条件下,钠型IRC-50树脂解吸率达到95%以上,工作交换容量为303.25 mg·g-1,ε-PL纯度从初始的24.60%提升至75.86%,蛋白和色素去除率均在60%以上。(2)针对离子交换洗脱液中存在的杂质,探索了利用离子交换手段去除该杂质的可能。通过分析杂质的来源和性质,初步判断该杂质来源于发酵,带电性质与ε-PL近似,分子量在900-2000 Da之间。采用钠型IRC-50树脂梯度洗脱、负吸附结合正吸附和分部收集、强酸树脂等方法,只能部分去除杂质。由此可知,杂质等电点和ε-PL十分接近,依靠离子交换方法难以实现其有效去除。(3)研究了钠型IRC-50树脂在不同放大规模下,对ε-PL分离提取的影响。在十个批次的提取工艺中树脂工作交换容量没有明显下降、解吸率均在95%以上、洗脱液纯度在70%以上,工艺相对稳定。对离子交换单元进行8倍和27倍实验室规模的放大,放大后离子交换分离性能良好。在离子交换柱总容为5.5 m3的试生产提取工艺中,离子交换杂质去除情况与实验室的结果一致,ε-PL损失率仅为2.62%。试生产所提取样品外观为接近白色的粉末,液相检测纯度为99.21%,对提取样品的理化指标进行测定和分析,结果均符合国家标准要求。质谱分析结果表明提取样品为L-赖氨酸聚合物,聚合度在27-34。
[Abstract]:蔚-poly-L-lysine, (蔚-PL) is a green and safe biological preservative. In 2014, 蔚-PL and its hydrochloric acid were approved as a new food additive in China. After years of strain selection and fermentation strategy optimization, the fermentation yield has reached the requirements of industrial production. However, the domestic research on the isolation and extraction of 蔚-PL is not perfect, which seriously restricts the industrialization of 蔚-PL. Therefore, the development of 蔚-PL separation and extraction technology which can be used in industrial production is of great practical significance to promote the industrialization of 蔚-PL. Based on the previous research work, the dynamic conditions of ion exchange were systematically optimized, and the extraction process with ion exchange technology as the core was established, and the stability of the process was investigated. Finally, the extraction process was successfully applied to trial production. The results obtained are as follows: (1) based on the optimization and comparison of dynamic conditions of sodium and ammonium Amberlite IRC-50 resins, it is determined that sodium IRC-50 resin is more suitable for industrial application. The optimum dynamic conditions of sodium resin were as follows: the sample pH was 7.0, the sample rate was 5 BV h-1, and the eluent was 0.25 mol L-1 NaOH,. The elution rate was 2 BV h-1. Under the optimum operating conditions, the desorption rate of sodium IRC-50 resin was over 95%, the working exchange capacity was 303.25 mg g-1, and the purity of 蔚-PL was raised from 24.60% to 75.86%. The removal rate of both protein and pigment is over 60%. (2) the possibility of removing the impurity in the ion exchange eluate by ion exchange is explored. By analyzing the source and properties of impurity, it was preliminarily determined that the impurity originated from fermentation. The charged property was similar to 蔚-PL, and its molecular weight was between 900-2000 Da. Sodium IRC-50 resin gradient elution, negative adsorption combined with positive adsorption and partial collection, strong acid resin and other methods, can only partially remove impurities. It can be seen that impurity isoelectric point is very close to 蔚-PL, and it is difficult to achieve its effective removal by ion exchange method. (3) the influence of sodium IRC-50 resin on the separation and extraction of 蔚-PL was studied in different scale. In the extraction process of 10 batches, the working exchange capacity of the resin was not obviously decreased, the desorption rate was above 95%, the purity of the eluent was over 70%, and the process was relatively stable. The ion exchange unit was amplified by 8 and 27 times of laboratory scale, and the ion exchange separation performance was good after amplification. The results of ion exchange impurity removal are in agreement with the results of the laboratory, and the loss rate of 蔚-PL is only 2.62% in the trial process of ion exchange column with a total capacity of 5.5 m ~ 3. The appearance of the extracted samples was close to white powder, the purity of liquid phase detection was 99.21. The physicochemical indexes of the extracted samples were determined and analyzed, and the results met the requirements of the national standard. The result of mass spectrometry showed that the extracted sample was L- lysine polymer with polymerization degree of 27-34.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS202.3;TQ028.33

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