Production of theophylline via aerobic fermentation of pu-erh tea using tea-derived fungi

Abstract

BackgroundCaffeine is one of the most abundant methylxanthines in tea, and it remains stable in processing of general teas. In the secondary metabolism of microorganism, theophylline is the main conversion product in caffeine catabolism through demethylation. Microorganisms, involved in the solid-state fermentation of pu-erh tea, have a certain impact on caffeine level. Inoculating an appropriate starter strain that is able to convert caffeine to theophylline would be an alternative way to obtain theophylline in tea. The purpose of this study was to isolate and identify the effective strain converting caffeine to theophylline in pu-erh tea, and discuss the optimal conditions for theophylline production.ResultsCaffeine content was decreased significantly (p < 0.05) and theophylline content was increased significantly (p < 0.05) during the aerobic fermentation of pu-erh tea. Five dominant fungi were isolated from the aerobic fermentation and identified as Aspergillus niger, Aspergillus sydowii, Aspergillus pallidofulvus, Aspergillus sesamicola and Penicillium mangini, respectively. Especially, A. pallidofulvus, A. sesamicola and P. mangini were detected in pu-erh tea for the first time. All isolates except A. sydowii TET-2, enhanced caffeine content and had no significant influence on theophylline content. In the aerobic fermentation of A. sydowii TET-2, 28.8 mg/g of caffeine was degraded, 93.18% of degraded caffeine was converted to theophylline, and 24.60 mg/g of theophylline was produced. A. sydowii PET-2 could convert caffeine to theophylline significantly, and had application potential in the production of theophylline. The optimum conditions of theophylline production in the aerobic fermentation were 1) initial moisture content of 35% (w/w), 2) inoculation quantity of 8%, and 3) incubation temperature at 35 °C.ConclusionsFor the first time, we find that A. sydowii PET-2 could convert caffeine to theophylline, and has the potential value in theophylline production through aerobic fermentation.

Isolation, characterization and application of theophylline-degrading Aspergillus fungi

“…We found that theophylline content was increased significantly (p < 0.05) and Aspergillus sydowii caused caffeine degradation in SSF [40]. After further research, A. sydowii had a significant (p < 0.05) impact on caffeine metabolism and potential value in theophylline production through aerobic fermentation [41,42].…”

“…The colony forming units (CFU) were calculated by per gram of the dry weight after 2 days of cultivation at 30 °C. Caffeine and theophylline contents were determined by HPLC described by Zhou et al [40,41] using an Agilent 1200 series system and an Agilent C 18 Chromatogram column (250 mm × 4.6 mm, 5 μm). Samples collected on days 20 and 25 were stored at 4 °C and selected for further isolation and identification of theophylline-degrading fungi.…”

“…Fresh cells were obtained by centrifugation at 1700g for 5 min after cultivation in 20 mL of Czapek Dox medium at 30 °C for 2 days on an incubator shaker (250 rpm) and freeze-dried at − 80 °C [40,41]. DNA was extracted by using SP fungal DNA kit.…”

“…The fungal primers ITS1 and ITS4, Bt2a and Bt2b, and CF1L and CF4 were used in PCR to amplify ITS, β-tubulin and calmodulin regions, respectively [43]. The final volume of 50 μL, 1.0 μL of containing template DNA, 5 μL of 10 × buffer, 5 μL of dNTPs (2.5 mM), 0.5 μL of Taq polymerase, 1.0 μL (10 μM) of each primer, and 36.5 μL of sterile distilled water were used to implement amplifications [40,41]. The PCR reaction procedure was as follows.…”

“…The collected mycelium was filtered in a Buchner funnel, and rinsed in 20 mL of water: ethyl acetate (1:1) [46]. The mycelial mass PT-5 Circular Rough Greyish-green centre with yellow patches Red pigment [41] PT-6 Circular Rough Iron gray bulge with milk white edges None Additional file 1: Figure S1 PT-7 Irregular Rough Hazel green with gray back None Additional file 1: Figure S2 Zhou et al Microb Cell Fact (2020) 19:72 was determined as fungal dry mass after drying at 35 °C for 24 h and results were summarized in Additional file 2: Table S1 [46]. Theophylline concentration was determined by HPLC [21].…”