| Pu-erh tea(pu-erh shucha)is produced though a natural solid-state fermentation process with sun-dried green tea leaves(Camellia sinensis var.assamica(JW Masters)Kitamura)as the raw material.Solid-state fermentation(pile fermentation)is key process to form the specific qualities of pu-erh shucha tea.Microorganisms,existed in the solid-state fermentation of pu-erh tea,especially multifarious fungi,have profound effect on component contents and quality of pu-erh shucha tea.Over the years,caffeine content,which is stable in general tea,changed significantly in the samples from large-scale solid-state fermentation of pu-erh tea.Therefore,it is speculated that one or more microorganisms existed in the solid-state fermentation with caffeine-degrading capability.Although few kinds of bacterial and fungal strains with caffeine-degrading capability have been selected from the soil of tea and coffee gardens,directional selection of caffeine-degrading strain from fermented tea is the first time.In this thesis,caffeine-degrading strains were selected by using liquid and solid selection medium from solid-state fermentation of pu-erh tea.The dominant fungi and caffeine-degrading strain were identified by using molecular biological method.Based on UPLC-QTOF-MS metabonomics and Label-free proteomics,caffeine degradation pathways and related proteins with caffeine degradation under the effect of caffeine-degrading strain were detected and analyzed in the paper.The main findings were declared as follows.1.Changes of purine alkaloids contents and fungi amount in a natural solid-state fermentation of pu-erh tea.Referring to the industrial production of pu-erh tea,the natural solid-state fermentation was carried out to simulate pu-erh tea processing.Caffeine,theobromine and theophylline contents were determined by using HPLC(high performance liquid chromatography)method,and fungi amount was determined according to the method of mold and yeast count.The results were showed as follows.Caffeine content declined significantly(p<0.05)with a decreasing amplitude about 29.12%and theophylline content increased drastically(p<0.05)from 0.43±0.052mg/g to 9.18±0.755mg/g in the natural solid-state fermentation.Fungi amount changed significantly(p<0.05),and maintained a level of 5×104 CFU/g～1.2×106 CFU/g.Hence,the species and quantity of fungi in the solid-state fermentation had profound influence on caffeine and theophylline contents.2.Selection and identification of caffeine-degrading strains from pu-erh tea solid-sate fermentation.The caffeine-degrading strain was selected by using liquid and solid selection mediums from the purified fungi of pu-erh tea for the first and second time.Strain PE-5 was confirmed with caffeine degradation capability and identified as Aspergillus sydowi NRRL250 by using molecular biological method,which could degrade 87.70%of caffeine within 48 hours.The 5 dominant fungal strains,separated and purified from the natural solid-state fermentation of pu-erh tea,were identified as Aspergillus niger NCBT110A,A.pallidofulvus NRRL4789,A.sesamicola CBS 137324,Aspergillus sydowii NRRL250 and Penicillium mangini CBS253.31 by using molecular biological method,respectively.The dominant fungal strains occupied 86.67-97.14%in total colony amount.In the inoculated fermentation,A.niger NCBT110A,A.pallidofulvus NRRL4789,A.sesamicola CBS 137324 and P.mangini CBS253.31 had no significant(p>0.05)effect on caffeine,theobromine and theophylline metabolisms.Only in the inoculated fermentation of Aspergillus sydowii NRRL250,caffeine decreased significantly(p<0.05)with an amplitude about 83.89%,theophylline increased drastically(p<0.05)and 25.03 ±1.172mg/g of theophylline was produced at the end of the fermentation.Therefore,Aspergillus sydowii NRRL250 was confirmed as caffeine-degrading effective strain selected from pu-erh tea solid-sate fermentation,had the potential ability converting caffeine to theophylline.3.Study on caffeine-degradation pathways under the effect of Aspergillus sydowii NRRL250.To investigate the caffeine degradation products,Aspergillus sydowii NRRL250 and A.niger NCBT110A were inoculated into liquid nutrient medium with different concentrations of caffeine(600,1200 and 1800 mg/L of caffeine,respectively),and related caffeine degradation products were detected by using HPLC method.The results showed that caffeine degradation capability of A.niger NCBT110A was limited,A.niger NCBT110A could not degrade caffeine.At different caffeine concentrations,Aspergillus sydowii NRRL250 could degrade 99.3%,91.3%and 62.9%of caffeine,respectively.And theophylline and 3-methylxanthine were detected consecutively in the inoculated medium of Aspergillus sydowii NRRL250.Theophylline and 3-methylxanthine concentrations were 549.4±29.3 mg/L and 178.7±10.8 mg/L in the end fermentation of Aspergillus sydowii NRRL250,respectively.Therefore,theophylline and 3-methylxanthine were the main caffeine degradation products under the effect of Aspergillus sydowii NRRL250.In addition,the effects of substrate concentration,reaction temperature and pH on caffeine-degrading capability were investigated in this study.The optimum conditions of caffeine degradation were 1)substrate concentration of 1200 mg/L;2)reaction temperature at 30℃,3)pH of 6.With raw material and sterilization treatment groups as contrast,the metabolites in inoculated fermentation was detected and analyzed based on UPLC-QTOF-MS metabonomics.9 related metabolites with caffeine catabolism were detected from the inoculated fermentation group.Theophylline,3-methylxanthine,paraxanthine,7-methylxanthine,1-methylxanthine,1-methyluric acid and hypoxanthine increased significantly(p<0.05).2 degradation pathways were found in Aspergillus sydowii NRRL250 through N-demethylation.The main pathway was caffeine→theophylline→3-methylxanthine,and the secondary pathway was caffeine→paraxanthine→7-methylxanthine.Several degradation products formed 1-methyluric acid and 1,7-dimethyuric acid through oxidation.4.Analysis of related enzymes in caffeine catabolism based on Label-free proteomics.Aspergillus sydowii NRRL250 was inoculated into sterile tea infusion for submerged fermentation.And caffeine,theobromine and theophylline contents were determined by using HPLC method.The results showed that caffeine content decreased significantly(p<0.05),theophylline content increased radically(p<0.05)and theobromine content keep stable in the submerged fermentation of Aspergillus sydowii NRRL250.Hence,Aspergillus sydowii NRRL250 could convert caffeine to theophylline.Based on Label-free proteomics,proteins were investigated in the submerged fermentation of Aspergillus sydowii NRRL250.Compared with basic liquid cultivation,149 differential proteins were found in the submerged fermentation of Aspergillus sydowii NRRL250.140 identical proteins and 232 differential proteins were found from different stages of the submerged fermentation(CT4d:CT7d).Among 140 identical proteins,94 proteins had significant difference in protein expression,only about 46 proteins had no significant change in protein expression.According to GO annotation,several enzymes that were associated with polyphenols,carbohydrate,amino acids and caffeine metabolisms in the submerged fermentation were identified,which provides reference for the application and the further research of Aspergillus sydowii NRRL250. |
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