| Glucuronic acid?Glucuronic acid,GlcUA?is an important pharmaceutical material,chemical intermediate,or ingredient of functional foods.Current method of industrially production of GlcUA is mainly a chemical synthesis method,which causes serious environmental pollution and gets low yield of product.Production of GlcUA by microbial fermentation is green and more efficient.Therefore,development of method to produce GlcUA by microbial fermentation is of great significance for the replacement of chemical synthesis and expansion of its industrial production methods.In this study,a Zygosaccharomyces rouxii ZSR2with the ability to produce GlcUA was isolated from Kombucha and the titer was significantly increased by optimization of fermentation conditions.The results might lay the foundation of industrial production of GlcUA by microbial fermentation.The main results were described as follows:?1?Screening of strains for production of GlcUA.Seven strains with activity of myo-inositol oxygenase?MIOX,the key enzyme in the GlcUA synthesis pathway?were obtained from Kombucha and the laboratory culture collection.They were identified and designated as Bacillus cereus H3,H4 and H7,Zygosaccharomyces rouxii ZSR2 and ZQ01,Candida orthopsilosis Y3 and Aspergillus oryzae NRRL 3448.It has been verified that Zygosaccharomyces rouxii ZSR2 showed the ability to produce the highest level of glucuronic acid(1.13 g·L-1).Thus,it was selected as the production strain for optimization of fermentation conditions.?2?Optimization of GlcUA production by single factor and response surface analyses.The optimized fermentation conditions based on single factor analysis for production of GlcUA by Zygosaccharomyces rouxii ZSR2 were determined to be:fermentation medium was composed of 80 g·L-1 sucrose,30 g·L-1 soy peptone;The initial pH of medium was 5.0;The inoculum size was 3%and the inoculum age was 9 h.The titer of GlcUA under this conditions was detected to be 14.68 g·L-1,which was 3.8 times of that before optimization.In addition,the interaction of two extremely significant factors?the concentration of soy peptone and the medium initial pH?on the production of GlcUA was revealed by the response surface method.Production of GlcUA by Zygosaccharomyces rouxii ZSR2 reached 15.62 g·L-1,using the predicted optimal conditions(the soy peptone concentration was 34.1 g·L-1 and the medium initial pH was 4.94).?3?Enhancement of GlcUA production by other optimization strategies.Our research showed that addition of black tea powder or its functional ingredients such as chlorogenic acid and tea saponin,or to enhance MIOX activity promoted production.The titer of GlcUA reached18.06 g·L-1 when 3%black tea powder was added to the medium;and it raised to 19.32 g·L-1and 20.68 g·L-1 when 2 mmol·L-1 ferrous ion and L-cysteine was added to the medium,respectively.Moreover,addition of nitrogen source at the late stage of fermentation stimulated GlcUA production.With the addition of ammonium citrate at 14 h,GlcUA produced by Zygosaccharomyces rouxii ZSR2 was increased to 22.36 g·L-1,which was 1.5 times of that without addition of nitrogen source.?4?Verification of the optimization strategies for GlcUA production by Zygosaccharomyces rouxii ZSR2 at the 3 L fermenter fermentation level.The titer of GlcUA raised to 26.79 g·L-1 by combination of the optimization strategies such as addition of compounds that can enhance the MIOX activity and the supplementation of nitrogen source during fermentation.The highest titer of GlcUA produced by the Zygosaccharomyces rouxii ZSR2 in the 3 L fermenter was determined to be 35.26 g·L-1 at 20 h of fermentation. |
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