Study On Construction Of Deacidfication Wine Yeast By Protoplast Fusion

The research for construction of deacidification wine yeast was conducted by protoplast fusion with Saccharomyces ellisoideus 1450 and Oenococcus oeni SD-2a selected by College of Enology, Northwest Sci-Tech University of Agriculture & Forestry, and the results showed as below:1. As to cells suspension of O.oeni SD-2a,when OD600 value got up to 0.6,the cells were on the middle exponential growth phase, and the cells density was about 3×109cfu/ml.Using SMM buffer as osmotic stable solution,the cells were treated with 1mg/mL lysozyme at 37℃ for 30min,the formation rate and regeneration rate of protoplasts were up to 98.2% and 15.9% respectively.2. As to cells suspension of S.ellisoideus 1450 ,when the cells density got up to 2～3×107cfu/ml,the cells were on the middle exponential growth phage. Using PBS buffer as osmotic stable solution, the cells were pretreated with 0.3%β-mercaptoethanol- 0.1%EDTANa2 at 37℃ for 10～15min,then treated with 2% snail enzyme at 37℃ for 40min, the formation rate and regeneration rate of protoplasts were up to 96.9% and 16.1% respectively.3. The results of xylose fermentation test by O.oeni SD-2a showed that, with the same other culture conditions, the growth rate of O.oeni SD-2a incubated in the medium used xylose as carbon resource was far lower than the growth rate in the medium used glucose as carbon resource, which behold that it did not have or had lost xylose fermentation ability.4. The cells of S.ellisoideus 1450 on the middle exponential growth phage, whose density was adjusted to 2～3×108cfu/ml, were spread over YPD-medium plate. 7.5mg/L amphtericin B could not restrained the cells growth significantly; on the plate which contained 500u/ml nastin, no colony showed within 3 days ,then the nastin-resistant colonies showed; on the plate which contained 50ug/ml actidione, only several actidione-resistant colonies showed 5 days later,yet no colony showed on the plate which contained 100ug/ml actidione after 7days.5. The fusion conditions of PEG4000 30%,Cacl2 50mM,fusion time 30min,fusion temperature 30℃,were adopted. Using 100ug/mL actidione＋20ug/mL ampenicillin as fusants selecting pressure and the yeasts cells treated with the same procedure as contrast,117fusants were gained, whose colony properties were similar to those of S.ellisoideus 1450.One fusant F-20 which could degrade L-malic acid strongly was selected through target-fusant selection experiment.6. Single cell of fusant F-20 was separated and incubated. From 20 cells, one single colone strain named as F-20-7 with strong activity was selected, the cell form,size and colony property of F-20-7 were similar to S.ellisoideus 1450.