The Green Production Of High-purity Galacto-oligosaccharides By Kluyveromyces Lactis

In this thesis,a recyclable strategy was developed to produce the high-purity GOS by K.lactis,in which the K.lactis not only serves as the catalyst for the synthesis of GOS but also the requirement for the purification of GOS.On one hand,the permeabilized cells of K.lactis(p-cells)were prepared by the treatment of ethanol in order to overcome the mass-transfer barrier of K.lactis.Due to the liposoluble nature of ethanol,the permeability of K.lactis can be increased sufficiently to release the products of GOS,so the p-cells was used as a recyclable biocatalyst for the synthesis of GOS from lactose.The undesired lactose,glucose and galactose can enter the u-cells and be completely consumed through the cultivation of K.lactis without adding any other nutrients.It is not suitable for industrial production of high purity GOS because it requires complex and costly operation.A strategy which used lactose as raw materials and p-cells as the catalyst for the production of high purity GOS was developed.The production of high-purity GOS by free ?-galactosidase and K.lactis cells was also investigated.The conclusions can be summarized as follows.The production of high-purity GOS by free ?-galactosidase and K.lactis cells: The production of high-purity GOS by ?-galactosidase and yeast fermentation was investigated.In order to improve the GOS yield,the optimum conditions for GOS synthesis were optimized as 37?,pH 8.0,30U/g(Enzyme/lactose)?-galactosidase,400g/L lactose and reacted for 2 h.The maximum GOS yield(22.78%)was obtained.Furthermore,the by-products and lactose in raw GOS solution can be removed by the selective fermentation via kluyveromyces lactis cells.The optimal conditions were 100g/L total sugar concentration,75g/L cells and treated for 15 h.Finally,high-purity GOS(91.02%)was achieved.Synthesis of GOS by permeabilized cells of K.lactis: The p-cells exhibited superiority in stability,which is more practical for the industrial applications.The highest yield(35%)of GOS was obtained in 1.5 h under 18.9 g/L p-cells,400 g/L lactose,40 oC and pH 8.0.The maximum GOS concentration of 130.1 g/L with 35.0% of YGOS and 46.6% of SGOS were achieved,and the conversion of lactose was 71.5%.The purification of GOS by the untreated cells of K.lactis: The u-cells were preferred due to their intrinsic mass-transfer barrier toward GOS,which led to the selective consumption of carbohydrate during the cultivation.By this way,the undesired glucose,galactose and lactose in raw GOS solution can be completely removed.Finally,high-purity GOS(>95%)can be achieved when the initial carbohydrate concentration was 100 g/L and the ratio of u-cells to carbohydrate was 0.15.The batch production of the high-purity GOS: The yield of GOS remained 35.0±2.5% for at least 14 batches which reflected the high stability of p-cells.The purity of GOS decreased slightly in the batch purification of GOS through recovering the untreated cells of K.lactis after 9 batches.Another bonus of this strategy is the spontaneously generated ethanol which is just the solvent required to prepare p-cells.Thus,ethanol was collected after every round of distillation and directly used to treat the fresh K.lactis or the recycled u-cells.The activities of p-cells prepared from different cells and ethanol were at the same level,which indicated the high stability of the microorganism and the feasibility of this recyclable strategy.