| With the incessant development of the food industry,the proportion of the beverage market is gradually increasing.High sugar content in food and beverage has become the cause for many diseases,the food industry needs to be low-sugary.Zymomonas mobilis is a natural facultative anaerobic gram-negative microorganism,which is commonly identified in plant juices,fruit juices and wines with high sugar concentration.It can ferment glucose,fructose and sucrose at high concentrations to produce ethanol efficiently,and can tolerate high concentrations of sugar and ethanol.Z.mobilis is generally recognized as safe(GRAS),and has been reported to substitute yeast in food industry production in recent years.In this study,we compared the effectiveness of lowering sugar to produce low-sugar beverages in three commercial sugary products(Chine Traditional Wine and yellow or green pear paste)and two fruits(pear and persimmon)using Z.mobilis and Saccharomyces cerevisiae,a classical industrial ethanol producer.The results showed that Z.mobilis can consume nearly all sugars with the ethanol yields of 81.83～96.97%in 50～70%CTW,and60%dilution(the initial concentrations of glucose and alcohol were 70 g/L and 60 g/L)was the best ratio.In the diluted yellow and green pear paste,Z.mobilis fermented nearly all glucose(about 60 g/L)and most fructose(110 g/L)within 100 hours,the yield and fermentation efficiency of ethanol were 0.33～0.37 g/g and 64.5～73.1%.Z.mobilis produced about 20 g/L ethanol and less than 5 g/L sorbitol in pears within 22 hours,and about 45 g/L ethanol and 30 g/L sorbitol within 34 hours in persimmon.The yield and fermentation efficiency of Z.mobilis in these two fruits were similar to those in pear paste,which were0.30～0.37 g/g and 57～72%.The fermentation performance of Z.mobilis was better than S.cerevisiae with higher sugar conversion rate and ethanol yield for sugar reduction under the conditions of this study.To address the problem that wild-type Z.mobilis utilizes fructose less efficient than glucose in high sugar products with a prolonged fermentation time,we used the strategy of Adaptive Laboratory Evolution(ALE)to select mutant strains that can ferment fructose effectively even at high concentration.Two mutants with improved fructose utilization were selected,and named as ZMF~+-73 and ZMF~+-74.When fructose was the only carbon source,the biomass of mutants was 23～40%higher than that of wild-type strain,and the ethanol titer of mutants was increased by 11～15%.The mutants had advantage on sucrose utilization with fermentation time reduced to 27 h,ethanol titer increased by 9～13%and the productivity increased by 114～116%reaching to 2.4 g/L/h.In summary,this work explored the potential of sugar reduction using Z.mobilis in sugary products,and demonstrated that Z.mobilis can be used as an industrial strain for sugar reduction with the advantages of high sugar conversion efficiency,fast growth and simple fermentation conditions.Meanwhile,two efficient fructose utilization mutants ZMF~+-73 and ZMF~+-74 with improved fructose and sucrose fermentation performance were selected through ALE.These mutants have no lag phase in high sucrose conditions,and can help increase ethanol productivity and reduce the fermentation cost.This study laid a foundation for the development of low-sugar alcoholic drinks using Z.mobilis to reduce the sugar content in high-sugar commodities and fruits.In addition,the efficient fructose-utilizing mutants obtained in this study can be used to replace wild-type Z.mobilis as industrial strains for the development of sugar-free drinks in the future. |
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