基于乳酸克鲁维酵母的高纯度低聚半乳糖的扩试制备工艺开发

Development of a Litre-Scale Process for the Production of High-Purity Galactoligosaccharides by Kluyveromyces Lactis

以乳酸克鲁维酵母(Kluyveromyces lactis)为研究对象,开发了一套扩试制备高纯度GOS的工艺包,包括透性细胞p-cells催化反应、全细胞np-cells发酵纯化.在实验室反应阶段(25 mL),以单位酶活来定义加酶量,消除了以湿细胞浓度为准引起不同实验批次的结果偏差.PBS浓度从50降至5 mmol/L后,对GOS催化制备无影响,但大幅度降低了GOS产品盐含量.在扩试反应阶段(1 L),通过改进加料方式,将最佳反应时间缩短了4倍,消除了放大效应对反应速率的影响.对搅拌转速优化后可得最优条件(加酶量100 U/mL、PBS浓度5 mmol/L、转速400 r/min)下,GOS收率达到19.15%.在扩试纯化阶段(1 L),经优化可得最佳条件(总糖质量浓度100 g/L、全细胞添加量50 g/L)下纯化所得GOS粗液,经喷雾干燥,GOS纯度达到96.21%,未检测到杂糖,盐含量仅为3.79%,达到低聚糖国标要求.

A litre-scale process for the production of high purity GOS by Kluyveromyces lactis(K.lactis)was developed,including catalytic reaction using permeabilized K.lactis cells and purification using nonpermeabilized K.lactis cells.In the laboratory reaction stage(25 mL),the specific activity(U/mg)was defined to eliminate the deviation caused by wet cell concentration in different batches.It was found that the concentration of PBS decreased from 50 to 5 mmol/L,which had no effect on the catalytic production of GOS,but greatly reduced the salt content of GOS product.At the expansion reaction stage(1 L),a new feeding method of substrate was adopted,which shortened the optimum reaction time by 4-fold and eliminated the effect of amplification.The yield of GOS reached 19.15%under the optimum reaction conditions(enzyme dosage 100 U/mL,salt concentration 5 mmol/L,stirring speed 400 r/min)after optimization of stirring speed.At the purification stage,the purity of GOS reached 96.21%under the optimum conditions(carbohydrate concentration 100 g/L,non-permeabilized K.lactis cells concentration 50 g/L)after spray drying.The GOS product is no sugar detected and has the salt content at only 3.79%,which meets the national standard of oligosaccharides.