The Expression Of Gene Cel9C In Engineered Yeast Strain And The Fermentation Of Konjac Kombucha

Konjac glucomannan(KGM),a linear non-ionic functional polysaccharide,has various physiological functions to human body.With the characteristics of high molecular weight,easy to swell,high viscosity,and difficult to digest and absorb,the nutrition effect of direct consumption of KGM is poor as a functional food.Konjac mannoligosaccharides(KMOS),which is low molecular weight,low viscosity,and easy to absorb and utilize by probiotics,can be prepared by physical,chemical and biological methods from KGM,and can make use of total nutritional value of KGM.Kombucha is a traditional fermented acidic tea beverage,and has various health protection functions.There are lots of fermentation researches about kombucha already,and new flavor kombucha has became a research hotspot in recent years.Kombucha together with KMOS can increase the content of oligosaccharides of kombucha,and enhance the health effects of kombucha,and can make use of total nutritional value of KGM.The primers were designed according to the sequences of gene cel9 C,and BamH I and Sma I restriction sites were introduced respectively with plasmid pET28a-cel9 C as templates,and the target gene cel9 C fragment about 2.0 kb was obtained by PCR.The recombinant plasmid pESP-cel9 C was constructed using target gene fragment and vector pESP-2-2,and transfered in Escherichia coli TOP10 strain.Plasmid pESP-cel9 C was abstracted from the recombinant strain,and was treated with double restriction endonuclease BamH I and Sma I,and the result showed that the recombinant plasmid pESP-cel9 C was constructed successfully.Plasmid pESP-cel9 C was transfered into Schizosaccharomyces pombe yAS56 strain,and there were 12 yeast transformants grown on EMM+U plate.Congo red dyeing showed that all these 12 yeast transformants expressed ?-glucanase activity.SDS-PAGE showed that the recombination ?-glucanase was a fusion protein with molecular weight about 99 kDa.The intracellular ?-glucanase activity of yeast transformants was determined by DNS method,and results showed that the No.10 yeast transformant had the hightest ?-glucanase activity of 169.5 U/mL.The No.10 yeast transformant,named S.pombe yAS56(pESP-cel9C),was used to study the fermentation of konjac kombucha.Gluconacetobacter hansenii CGMCC1671,Saccharomyces cerevisiae CGMCC1670 and S.pombe yAS56(pESP-cel9C)were mixed inoculated for kombucha fermentation,and the microbial growth and acid production were determined during kombucha fermentation.The mixed inoculations were very good for fermentation of kombucha;and konjac powder added in kombucha medium could enhance G.hansenii CGMCC1671 and S.cerevisiae CGMCC1670 cells proliferation,accelerated the growth rate of G.hansenii CGMCC1671;shortened the length of stationary phase of G.hansenii CGMCC1671 and S.cerevisiae CGMCC1670,and accelerated the accumulation of organic acids.The influence of time,green tea,sucrose,konjac powder,inoculum size,the ratio of wild strain to engineered strain of yeast and loading volume on total acid of kombucha were investigated by single factor experiments,and the results showed that time,green tea,inoculum size and loading volume had obvious influence on total acid of kombucha.According to the results of single factor experiments,the optimal level of factors were as the following: time 96 h,green tea 0.4%,sucrose 6%,konjac powder 0.2%,inoculum size 10%,the ratio of wild strain to engineered strain of yeast being 0.5,and loading volume 100 mL.Two significant factors,loading volume and time,were selected among seven factors by fractional factorial experiments.The fermentation conditions of konjac kombucha were optimized by central composite experiments,and the results were as following: time 124 h,green tea 0.4%,sucrose 7.5%,konjac powder 0.25%,inoculum size 10%,the ratio of wild strain to engineered strain of yeast being 0.5,loading volume 98.7 mL.The maximum total acid of konjac kombucha was(27.88±0.26)g/kg under the optimal conditions,which was increased by 41.45% compared with those of the control conditions.The results of the study showed that the application of engineered yeast with konjac powder in kombucha fermentation was feasible.