Development Of A Litre-scale Process For The Production Of High-purity GOS Using Lactose And Whey Powder As Substrate

Galacto-oligosaccharide(GOS)is an important functional oligosaccharide which has been marketed internationally.It has been found that GOS can improve the digestive function of infants,promote the absorption of calcium,strengthen the metabolism of liposomes,and enhance the physical fitness of infants and has broad application prospects.Whey powder is a natural by-product which generated from the production of milk.The process for preparing high-purity GOS using whey powder as substrate can not only reduce the process cost,but also bring social benefits.The core work of this paper is to expand the high-purity GOS preparation process including synthesis and purification stages.The conclusions can be summarized as follows:Development of a litre-scale process for high-purity GOS production from lactose:During the litre-scale production of GOS,lactose was used as substrate and K.lactis p-cells as biocatalyst.The optimum reaction conditions were 100 U/m L of enzyme concentration,400 g/L of lactose concentration,5 mmol/L of PBS concentration,400 rpm of stirring speed,lactose and K.lactis p-cells were dissolved separately.In 1 L reaction system,the highest GOS yield was 19.15%after 2 h.K.lactis p-cells still had 10.90%GOS yield after 6 times of reuse and had good recycling effect.In the GOS purification stage,based on the principle of selective permeation of yeast cell membrane,K.lactis np-cells were used to ferment and consume the residual lactose and by-product glucose and galactose.The optimum conditions for the purification of lactose-derived GOS crude products were 50 g/L of K.lactis np-cells and 100 g/L of total sugar.The purity of GOS reached 95.98%after 34 hours of purification.The K.lactis np-cell after purification also had high catalytic activity after permeabilized treatment,and the yield of GOS decreased from 16.28%to 11.74%after six cycles of GOS synthesis from lactose.The purified GOS powder was obtained by spray drying.The optimum conditions were as follows:the feeding speed was 485 m L/h and the inlet temperature was 200?.The purity of lactose-derived products is as high as 95.98%,which is suitable for infant food additives.Development of a low cost process for GOS production based on catalytic mechanism analysis:Homology modeling and molecular simulation of the interaction between?-galactosidase and?-galactose showed that the catalytic sites of the enzyme were Asp201,His391,His540,Glu461,they bind to?-galactose by hydrogen bonding;In addition,residues such as Asn102,Trp999,Asn460,Tyr503 bind to?-galactose via van der waals force;Mg²⁺binds to?-galactose through the"electron-metal"interaction within the enzyme.Therefore,adding Mg²⁺to the medium during the fermentation stage increased transglycoside activity of?-galactosidase nearly twice,which is beneficial to GOS synthesis and can reduce the cost of biocatalyst preparation.During the litre-scale production of GOS from whey powder,the highest GOS yield was 16.25%after 45 minutes under the optimized catalytic conditions.The yield was slightly lower than that of lactose,but the reaction time was shortened by more than one time.And the GOS yield decreased to 10.94%after 6 times of reuse.The optimum conditions were the same as lactose process in the stage of litre-scale purification.The purity of GOS product from whey powder decreased from 63.28%to 47.36%after six cycles of GOS purification(30 hours each cycle).This is due to the 11%impurities in whey powder.The K.lactis np-cell after purification also had high catalytic activity after permeabilized treatment,and the yield of GOS decreased from 15.91%to 11.64%after six cycles of GOS synthesis from whey powder.Finally,two kinds of GOS products were obtained.The purity of whey powder derived products is 63.28%,which meets the GOS food safety national standard(GOS purity is more than 57%)and can be used in common food additives.