# Biopurification of Oligosaccharides by Immobilized _Kluyveromyces Lactis_
## 1 Introduction  
Human milk oligosaccharides (HMOs) are active carbohydrates observed in human milk. With more than a hundred different structural isomers, HMOs play critical roles in maintaining and improving the intestinal microbiota of breast-fed infants [1]. Human milk uniquely contains such complex oligosaccharides in high concentrations up to 20 g/L. Bovine or porcine milk has oligosaccharides as well but only less than 1 g/L [2]. Since HMOs are hardly hydrolyzed in the human digestive system [3, 4], they can reach the small and large intestines while retaining their intact structures. In the human gut, HMOs serve as prebiotics for promoting beneficial bacteria and soluble decoy receptors for preventing pathogen attachment to mucosal surfaces. In addition to this, they serve as critical factors in modulating the responses of epithelial and immune cells [5].  
For the commercial use of HMOs, bovine milk oligosaccharides (BMOs) can be considered as an alternative to HMOs [6, 7]. Since whey is one of the largest byproducts of dairy products, massive amounts of BMOs can be produced from it [8, 9]. However, about 50 g/L of lactose has to be eliminated from bovine milk for practical application of BMOs. Hence, pilot-scale filtration or chromatographicmanners have been studied for lactose removal [10; 11; 12; 13; 14]. A biological method using \(\beta\)-galactosidase (EC 3.2.1.23) was also studied [15].  
2'-Fucosyllactose (2'-FL) is one of the simplest but most abundant oligosaccharides in human milk. It is currently produced at an industrial scale by the recombinant yeast or bacterial system [16; 17; 18; 19] via two distinct pathways, the salvage pathway and de novo pathway. GDP-L-fucose is synthesized through the engineered metabolic pathway and then transferred to a lactose backbone, which results in the production of 2'-FL. In this process, purification of 2'-FL from the mixture of lactose and 2'-FL is crucial for maximizing the prebiotic activity of 2'-FL.  
Galactooligosaccharides (GOSs) are also non-digestible oligosaccharides used as prebiotics [20]. They have been used for the health-promoting components of infant milk formulas to mimic the beneficial effects of HMOs [21]. Advantageous functions like anticarcinogenic effects and lowering serum cholesterol levels have been reported, as well [22; 23]. In the enzymatic synthesis of GOSs, however, 50% of the lactose remains in its unreacted form [24; 25]. This residual lactose makes it difficult to apply to people with lactose intolerance and decreases the valuable effects of GOSs [26].  
_Kluyveromyces lactis,_ the Crabtree-negative yeast, is widely used in the food industry because it is considered as \"generally recognized as safe\" (GRAS) [27]. It is extensively studied for the host system to produce recombinant proteins. The most successful utilization of _K. lactis_ is as the production host for \(\beta\)-galactosidase. Additionally, _K. lactis_ can assimilate lactose efficiently because it harbors the _LAC4_ (\(\beta\)-galactosidase) and _LAC12_ (lactose permease) genes.  
In this work, we developed a biopurification system of oligosaccharidesamples using immobilized _K. lactis_. From the mixture of lactose and oligosaccharides, _K. lactis_ selectively consumed the lactose with high efficiency. To reduce the burden in the process, lactose removal in a nutrient-rich, minimal salt media or distilled water was examined. The immobilized cells with high density were applied to maximize the consumption of lactose. Then, biopurification of HMOs and GOSs was examined to evaluate the selective removal of lactose without altering the distribution of oligosaccharides.