Screening Of D-arabitoi Producing Yeast Strain And Research Of Culture Conditions

As a kind of functional sugar polyol, D-arabitol has a lot of advantages in themetabolic of mammals such as the high sweet taste with low calorie value, the propertyof prevent caries, no effect to the blood glucose level after intake. D-arabitol can also beused as a transport media through the blood-brain-barrier. For these reasons, D-arabitolcan be widely used in food, medicine, and chemical industry. In recent years, the newuse of D-arabitol is developed continually. However, the disadvantages of the directextraction and chemical synthesis limit the large-scale production of D-arabitol, whichmakes the study on the production of D-arabitol by fermentation more important. Thispaper concentrates on the screening and identification of the D-arabitol producing strain,the identification of the metabolites, the optimization of culture medium and cultivationconditions, the batch fermentation performed in5-liter fementor. The main results are asfollows:Different methods of qualitative and quantitative detection of the polyols productswere studied. In the first screening, paper chromatography was used for the detection ofthe metabolites. Normal butanol, pyridine and water were selected to be the developingsolvent in a volume ratio of100:30:30. Meanwhile, the saturated silver nitrate dissolvedby acetone and sodium hydroxide dissolved by ethanol were selected to be the colorreagent. Under this condition, the polyols and the glucose could be well separated in thepaper chromatography with clear spot. In the second screening, high performance liquidchromatography （HPLC） was used for the accurate analysis. The analysis methodsprovided good instruction for the screening of strains producing D-arabitol fromglucose.By a screening method with high concentration of glucose in the media, strainL-84was isolated from the grape and the yield of D-arabitol achieved43.8g/L. Themain product of strain L-84was demonstrated to be D-arabitol by HPLC analysis. Alittle glycerin was produced as the by-product. The yeild ratio of D-arabitol and glycerinwas4:1. With the following identification of colony morphology, physiological andbiochemical characteristics and26S rDNA gene sequence, the strain L536wasidentified as Pichia guilliermondii, named Pichia guilliermondii L-84.The culture medium and the fermentation conditions of the strain L-84producingD-arabitol were optimized by single factor experiment and orthogonal experiment. The optimum culture conditions were as follows （g/L）: glucose250, yeast paste4.4,steepwater8.8,（NH₄）₂SO₄4.4, MgSO₄·7H₂O0.2, broth’s volume in shake flask25mL/250mL, inoculation amount8%（V/V）, initial pH4.0, temperature28℃,rotationspeed of rocking bed180rpm,culture time112h. under the optimum condition, the yieldof D-arabitol achieved60.6g/L,which was increased by38.4%; and the percentconversion of glucose achieved26.11%,which was increased by0.83%. The yeild ofglycerin reduced1.2g/L after the optimization of the conditions, and the yeild ratio ofD-arabitol and glycerin raised to6:1. According to the result of orthogonal experiment，the initial glucose concentration had the most important effect on the yield of D-arabitol,concentration of nitrogen source the second, additive amount of surfactant the third, andtemperature had the least effect.Based on the above optimum condition, the enlargement test of the processingtechnique was performed with strain L-84in5-liter fermentor. The yield of D-arabitolachieved57.2g/L at84.5h, and the substrate conversion rate was24.76%, thefermentation rate was0.677g/L·h. Because of the high dissolved oxygen in thefermentor, the yeild of glycerin reduced to4.6g/L, and the yeild ratio of D-arabitol andglycerin reached11:1. The results laid a foundation for further study.