Theabrownins Production From Fresh Old Tea Leaves By Pu-erh Tea-derived Microbes

Theabrownins have been confirmed to have multiple pharmacological activities,such as hypoglycemic,hypolipidemic,anticancer,antimicrobial,antioxidant,and metabolic syndrome prevention.Normally,the raw materials for theabrownins production were fresh young tea after fixation.However,the mature leaves are unused,causing a waste of resources.Moreover,the endogenous enzymes in tea leaves were not utilized due to deactivation of enzymes in fixation process.Considering the effective integration of resources,therefore,a novel method was established for efficient theabrownins production using endogenous enzymes from fresh old tea leaves and suspension fermentation by Pu-erh tea-derived dominant strains in this work.The method could simplify the production process and make full use of tea resources to improve the tea economy.Firstly,effects of microorganisms on the transformation of chemical substances were explored.Differences in bacterial and fungal community structures and chemical compounds in tea leaves were analysed,including fresh young tea,green tea,black tea,ripen Pu-erh tea and low-grade ripen Pu-erh tea.The results showed that microbial community composition exhibited high similarity in ripen Pu-erh tea and low-grade ripen Pu-erh tea.And microbial community from fresh young tea,black tea and green tea showed similar.Compared with fresh tea leaves,the bacteria abundance in two ripen Pu-erh teas increased,while bacterial diversity,fungal abundance and diversity decreased.The dominant fungal genera in ripen Puerh tea included Blastobotrys,Thermomyces,Candida,Rasamsonia,Aspergillus,Rhizopus,Rhizomucor and so on.The dominant bacterial genera were Brevibacterium,Staphylococcus,Kocuria,Saccharopolyspora,Brachybacterium,Bacillus and so on.The tea polyphenol content of fresh old tea leaves was 28.07%,which was significantly higher than that of young tea leaves.What’s more,the content of total soluble polysaccharides,free amino acids,total flavones were higher than other teas.It was used to produce theabrownins with remarkable advantages.The total soluble polysaccharides content of green tea,black tea,ripen Pu-erh tea and low-grade ripen Pu-erh tea decreased gradually,while theabrownin content increased successively.Secondly,three dominant strains with the ability of efficiently convert tea polyphenols into theabrownins,were isolated from ripe Pu-erh tea by using plate primary screening and liquid re-screening methods.Further,the effects of different fermentation materials and methods for theabrownins production were explored.The three strains were Aspergillus fumigatus,Candida blankii and Aspergillus tubingensis,respectively.Among them,Aspergillus tubingensis was able to tolerate high substrate content,and Candida blankii had high products yield.As for different fermentation materials and methods,the experiments showed that fresh old tea leaves as fermentation substrates had obvious advantages,and suspension fermentation using a combination of endogenous enzyme catalysis in fresh old tea leaves and the dominant microbial of Pu-erh tea fermentation could enhance the conversion efficiency of tea polyphenols to theabrownins.Compared with sing-strain fermentation without endogenous enzyme,the endogenous enzyme catalysis with inoculation of Aspergillus fumigatus,Candida blankii and Aspergillus tubingensis at the end of fermentation increased the teabrownins yield by 6.96%,8.76% and 3.55%,respectively.Effects of fermentation temperature,aeration rate and stirring rate on theabrownins production by Candida blankii were investigated.The optimum temperature,aeration rate and stirring speed for production of theabrownins by pure suspension fermentation were 40°C,1.5 vvm and 250 rpm,respectively.Finally,the interaction of the three dominant pure cultures was investigated.It was found that the co-culture of Candida blankii and Aspergillus tubingensis had significant advantages than others.What’s more,the addition of Aspergillus tubingensis could improve the substrate tolerance of Candida blankii,and the higher theabrownins concentration could be achieved if the fermentation was conducted using a microbial community at volume ratio of Aspergillus tubingensis and Candida blankii of 3:1.Effects of fermentation temperature,aeration rate and stirring speed on the production of theabrownins by co-culture were investigated,and the optimum co-culture were established: 40°C,1.5 vvm and 250 rpm.Comparing the ability of converting tea polyphenols into theabrownins between pure fermentation and co-culture,it was found that co-culture was better than pure fermentation during 0-33 h,but pure fermentation had higher theabrownins yield.Compared with co-culture,pured fermentation had obvious advantages in theabrownin production(24.81 g/L and 19.56 g/L,respectively)and yield(58.90% and 46.46%,respectively).Comparing the antioxidant and antibacterial activities among pure fermentation tea(PFT),co-culture fermentation tea(CPT),commercialized ripen Pu-erh tea(RPT)and commercialized low-grade ripen Pu-erh tea(LRPT),it was found that the tea samples by dominant strains suspension fermentation had strong antibacterial and antioxidant activities compared with traditional pile fermented Pu-erh tea,in addition to retaining higher tea polyphenol and theabrownins content.In conclusion,this study established a new method for production of highly biologically active theabrownin by pure and co-culture in suspension at the bioreactor scale,which provided an experimental basis for industrial production of theabrownins.