Screening Of Strains Secreting Cellulase And Pectinase Of Sichuan Dark Tea During Post-fermentation And Optimization Of Conditions Producting Cellulase

Sichuan Dark Tea is an important life necessity for the ethnic groups living in the border regions of southern or western China.The fermentation process always called "post-fermentation" is the most important procedure for the manufacture of Sichuan Dark Tea.And its hot and humid environment is conducive to microbial growth and reproduction.The cellulase,pectinase and other hydrolases secreted by microorganisms are contributed to the quality of Sichuan Dark Tea.Microorganisms of Dark Tea during post-fermentation have been reported by large number,but the cellulose and pectinase secreted by microorganisms of Sichuan Dark Tea during post-fermentation and its related properties have not been studied. In this work,strians secreting cellulase and pectinase and its properties were analyzed by using the molecular biology,bioinformatics method,a various kinds of microbial tablet technologys.Production conditions of cellulase was also optimizated.In addition,liquid fermentation was performed to investigate the effects of microbes secreting cellulase and pectinase on the quality ingredients of Sichuan Dark Tea.The major results were as follows:(1)18 microbial strains decomposing cellulose were screened from Sichuan Dark Tea during post-fermentation,among that,l strains belonged to yeast,6 strains belonged to mildew,7 strains belonged to Streptomyces of actinomycetes;4 strains belonged to bacterium.And its ability to break down the cellulose was in the order of: actinomycetes>bacterium>yeast>mildew.Fermentation rescreening found that activity of actinomycetes secreting cellulase as a whole was higher than bacterium and fungi.Integrated results of screening and rescreening,strains SDT18,SDT53 and SDT95 were selected as high enzyme activity strains secreting cellulase.Strains SDT18, SDT95and SDT53 were identified as Aspergillus niger strain FCBP648,Bacillus subtilis strain ZZ120,Streptomyces diastaticus subsp.ardesiacus strain HDJZ-zwm-45 by molecular biological analysis respectively.Analysis of cellulase characteristic of strains SDT95 found that the strain biomass showed a gradual increasing trend with incubation time,while the CMCase and FPA activity of strain SDT95 did not show significant changes during shake flask culture.The CMCase activity of strains SDT95 achieved maximum when enzyme reaction was at pH 5,and high CMCase activity produced by strains SDT95 was appeared when enzyme reaction temperature was at 45℃,55℃,40℃ or 50℃.(2)Enzyme production conditions of strains SDT95 secreting cellulase were optimized. Concentration and components of the liquid medium were optimized by second rotation design experiments based on the single factor experiments.And the components ratio of liquid medium in favor of producting CMCase by strains SDT95 was:glucose 1.68%,beef extract 0.82%,NaCl 0.32%,K2HPO4.3H2O 0.22%,the components ratio of liquid medium in favor of producting FPA by strains SDT95was:glucose 1.63%,beef extract 0.86%,NaCl 0.37%,K2HPO4.3H2O 0.16%.Shaking culture conditions were optimized by second rotation design experiments based on the single factor experiments.And the shaking culture conditions in favor of producting CMCase by strains SDT95 was:strain inoculation 2ml,shaker time 6d,shaker temperature 29℃.The impact of three factors investigated in experiments on FPA activity was all small,FPA activity may also was affected by other factors not taken account to the experimental program.(3)19 microbial strains secreting pectinase obviously were screened from Sichuan Dark Tea during post-fermentation,among that,l strains belonged to yeast,6 strains belonged to mildew,9 strains belonged to Streptomyces of actinomycetes;3 strains belonged to bacterium.Shake flask screening experiments measured the activity of various types of groups of microorganisms secreting pectinase and the results showed that activity of actinomycetes secreting pectinase was higher sequentially than bacterium,mildew and yeast.Integrated results of screening and rescreening,strains SDT22,SDT60 and SDT87 were selected as high enzyme activity strains secreting pectinase.Strains SDT22,SDT87 and SDT60 were identified as Penicillium commune strain G20-23, Streptomyces thermoviolaceus,Streptomyces pactum strain 173612 by molecular biological analysis respectively.(4)Strains secreting cellulase and pectinase screened in single strain liquid fermentation experiments based on pure culture of tea could result in lower levels of TP in tea.Compared with the control,excepting strains SDT93,all strains could result in higher levels of TF in tea.The impact of the tested strains on TR and TB content was relatively complicated.Excepting strains SDT4,SDT 87 and SDT93,all test strains could make AA content in tea improved compared with the control.Compared with the control,strains SDT52,SDT53,SDT59,SDT60,SDT87,SDT93,SDT95 and SDT102 increased WSS content in tea,While strains SDT4,SDT10,SDT13,SDT18,SDT 22,SDT29,SDT40 and SDT 64 made WSS content in tea decreased.Excepting strains SDT93,WSP content in tea all appeared increasion compared with the control.