Screening Of High-β-galactosidase-yield Strain For Lactulose Production And Optimization Of Fermentation Condition

Lactulose are composed of galactose and glucose unit linked by 1,4-glycosidic bond. Lactulose are known to be non-digestible oligosaccharide with low sweetness,low caloric values,low cariogenicity,and selectively promote the growth of beneficial bacteria in the colon.So it is a potential ingredient in food industry,health industry and leechdom.Lactulose is currently produced by chemical synthesis. However, this method has several drawbacks, such as a high level of lactulose degradation and a considerable amount of colored byproducts that are difficult to separate. The required separation and purification steps to remove these by-products are costly and lead to low lactulose yields. Bioproduction of lactulose by enzymes is therefore a desirable strategy to overcome the disadvantage of lactulose production by chemical synthesis.The primary of this research was to screen a high-β-galactosidase-yield strain suiting for lactulose production.Studies on the strain screening, optimization of fermentation medium,e- nzymatic properties and factors affecting lactulose formation were systematically carried out in this paper.The main results were as follows.A strain suiting for lactulose production was obtained using 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside(X-Gal) as indicator from Kluyveromyces lactis with UV, ultrasonic and ethylmethane sulfonate (EMS) as mutagen.It was code Kluyveromyces lactis K.lactis1-16- 7. The activities ofβ-galactosidase to nitrodiphenol-β-D-gala-ctoside (ONPG) and lactose were increased by 3.00 times and 2.75 times, respectively and the yield of lactulose reached 44.88g/L.Then the fermentation medium and condition were optimizated. The optimizated fermentation medium was yeast extract 10.7g/L,peptoneF403 9.4g/L,lactose 12.1g/L,MgSO4 5.6g/L,KH2PO4 4.7g/L.The optimizated condition was temperature40℃,pH6.0,liquid volume 40mL/250mL,rotation speed 160rpm,inoculum6%,fermentation time24h. After the optimizat- ion, the yield ofβ-galactosidase was 10.64U/mL.The optimal temperature of K.lactis1-16-7 was 40℃. The optimal pH was 7.0.Na+,Cu2+can inhibit the activities ofβ-galactosidase while Co2+,Mg2+,Fe2+,Mn2+ can promote the activities ofβ-galactosidase.The fermentation conditions of K.lactis1-16-7 in 2L fermenter were studied. The active- ities reached 33.47U/mL and increased 213.8%.The optimum substrate and enzyme concentrations were 40% (w/v) lactose and 20% (w/v) fructose at 3.0 units/mL, respectively. The optimal temperature was 50℃. The optimal pH was 7.0.Under the optimum conditions, theβ-galactosidase from K.lactis1-16-7 produced approximately 57.83g/L lactulose in 10 h of reaction time .