Study On Lactase Production By Kluyveromyces Fragilis

β-galactosidase(β-D-galactose-galactosidase enzyme,β-D-galactoside galatohydrolase, EC3.2.1.23)is usually called lactase.This enzyme can hydrolyze lactose into galactose and glucose,and it can also transfer galactoside.With the economic development of China,milk has become one of our major foods,however,lactose is rich in milk,and many of our adults and children are lack of lactase or have a lower level of lactase activity.Lots of people suffer from lactose intolerance,which restricts the consumption of milk.Therefore,the use of lactase for producing low-lactose milk and dairy products can be one of the effective ways to solve the problem of lactose intolerance and it has an important theoretical and practical value.A factory producing scale lactase from K.fragilis(LSF-0612)was studied,including the optimization of seeds and fermentation medium composition,the optimization of fermentation culture,the analysis of the lactase properties and the lactose enzymatic hydrolysis test.K.fragilis LSF-0612 seeds medium was established through the single factor tests and orthogonal tests.The optimum seeds culture medium compositions for cultivating Kluyveromyces fragilis LSF-0612 were as follows:4%glucose,4%yeast extract powder and 0.5mmol/ml MnCl₂.and the cultivating time 24 h.The wet cell weight can be achieved 1.79g/100ml under these conditions.K.fragilis LSF-0612 fermentation medium and fermentation conditions were studied through the single factor tests and orthogonal tests.The optimum liquid fermentation medium compositions were as follows:4%lactose,3%yeast extract powder and 1mmol/ml MnCl₂;The optimum fermentation conditions are as follows:10%of inoculum,temperature 28℃,initial pH 7,the oscillation incubators rotation speed 180 r/min,incubation time 36h.Under these conditions,the K.fragilis LSF-0612 lactase activity was up.to 1100U/ml,which was same as 4140 U/g.dry cellThe analysis of K.fragilis LSF-0612 lactase properties showed that:the enzyme best reaction temperature was 40℃,and the thermal stability of this lactase was equal,as reserves more than 90%activity in 40℃for 2 hours preservation and after 4℃preservation for 2 month its activity stated getting downwards;and it has the optimal pH 7.0 and pH stability in the range 6.5-7.5.The lactose hydrolysis test operated under optimal conditions showed that,K.fragilis LSF-0612 lactase hydrolysis rate of lactose could eventually reach up to 60%,and hydrolysis time can be shortened when increasing the amount of lactase.Hydrolysis the rate can reach up to 60%after five hours when adding 2‰amount of lactase.The best temperature of lactose hydrolysis was 40℃,the optimum pH 7,which was close to the natural pH of milk,and it is suitable to apply to the hydrolysis of lactose in the milk.