Background: Pu-erh tea is a traditional Chinese tea and produced by natural solid-state fermentation. Several studies show that the natural microbiota influence caffeine level in pu-erh tea. Our previous research also found that the caffeine declined significantly (p < 0.05) in the fermentation, which suggested that the caffeine level could be influenced by specific strains. The purpose of this study was to isolate and identify microorganisms for caffeine degradation, and this research explored the degradation products from caffeine and optimal condition for caffeine degradation. Results: 11 Fungi were isolated from pu-erh tea fermentation and 7 strains could survive in caffeine solid medium. Two superior strains were identified as Aspergillus niger NCBT110A and Aspergillus sydowii NRRL250 by molecular identification. In the substrate tests with caffeine, A. niger NCBT110A could use caffeine as a potential carbon source while glucose is absent, A. sydowii NRRL250 could degrade 600 mg/L caffeine completely in a liquid medium. During the degradation product analysis of A. sydowii NRRL250, theophylline and 3-methlxanthine were detected, and the level of theophylline and 3-methlxanthine increased significantly (p < 0.05) with the degradation of caffeine. The single factor analysis showed that the optimum conditions of caffeine degradation were 1) substrate concentration of 1200 mg/L, 2) reaction temperature at 30 °C, and 3) pH of 6. In the submerged fermentation of tea infusion by A. sydowii NRRL250, 985.1 mg/L of caffeine was degraded, and 501.2 mg/L of theophylline was produced. Conclusions: Results from this research indicate that Aspergillus sydowii NRRL250 was an effective strain to degrade caffeine. And theophylline and 3-methlxanthine were the main caffeine degradation products. Keywords: Tea, Fungi, Biodegradation, Caffeine, Fermentation Background and coffee. Although caffeine has lots of benefits, such as Caffeine (1, 3, 7-trimethylxanthine or 3, 7-dihydro-1, 3, regulating the central nervous system, excess caffeine in- 7-trimethyl-1H-2, 6dione) belongs to a group of com- take could develop hypertension and induce osteoporosis. pounds known as purine alkaloids. Caffeine is a key fla- Based on the recent researches, caffeine content only vor substance in many popular drinks, especially in tea changes in different physiological metabolism of tea tree (Camellia sinesis (L.) O. Kuntze) , and caffeine content * Correspondence: email@example.com; firstname.lastname@example.org; is influenced by tea tree varieties , but caffeine level re- email@example.com; firstname.lastname@example.org mains stable among different kind of teas, which showed Binxing Zhou and Cunqiang Ma contributed equally to this work. that tea processing cannot impact caffeine content [3, 4]. State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, Anhui, China Pu-erh tea (pu-erh shucha) (PET) is produced though Henan Key Laboratory of Tea Comprehensive Utilization in South Henan, a natural solid-state fermentation (SSF) process with Xinyang Agriculture and Forestry University, Xinyang 464000, Henan, China sun-dried green tea leaves (Camellia sinensis var. College of Long Run Pu-erh Tea, Yunnan Agricultural University, Kunming 650201, Yunnan, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. BMC Microbiology (2018) 18:53 Page 2 of 10 assamica (JW Masters) Kitamura) as the raw material A solid-state fermentation [5–7]. PET has been produced and drank by some minor- In this study, PTSSF was manufactured in Tea Process- ities of the Yunnan people in China for centuries [8, 9]. Mi- ing Laboratory, College of Long Run Pu-erh Tea, Yun- croorganisms, involved in pu-erh tea solid-state nan Agricultural University, Kunming of Yunnan fermentation (PETSSF), have been mainly studied using province to simulate pu-erh tea industrial production. culture-based approaches and culture-independent ap- Sun-dried green leaves (400 g) were moistened with dis- proaches [6–12]. Many fungi and yeast have been isolated tilled water (220 mL) to achieve a final moisture content from PET, especially Aspergillus niger, A. tubingensis, A. of 35% (w/w) . SSF was carried out with the natural fumigatus, A. acidus, A. awamori, Penicllium sp., Rhizomu- microbiota exist on the leaves . The whole fermenta- cor pusillus, Rhizomucor tauricus, Blastobotrys adeninivor- tion process continued for 35 d in a nature condition. ans, Arxula adeninivorans, Pichia farinose and Candida The leaves were turned over with sprayed by moderate tropicalis, which have been reported widely [8–16]. sterile water for every 5 days to ensure a homogeneous Due to the participation of microorganisms, caffeine fermentation. Samples were collected periodically from content is changeable during PETSSF [17–20]. Cephalos- fermentation for chemical and microbial analyses . porium acremonium dramatically increases 60–70% caf- In addition, parallel tests were carried out to ensure the feine content during PETSSF, whereas Saccharomycetes data reliability. sp. and A. niger could potentially reduce caffeine content [19–21]. In addition, the level of caffeine content is rela- Isolation and identification of target strains tively stable with the effect of A. fumigatu and Lactoba- Fermented sample would be used to isolate the fungi cillus sp. . Therefore, microorganisms have a certain and they were counted by dilution plating method . effect on caffeine and other purine alkaloids . The colony forming units (CFU) was calculated by per In this paper, samples from PETSSF were used to gram dry weight of tea leaves after 2 days of cultivation select target strains with the capability of caffeine at 30 °C. The caffeine content of related samples was de- degradation. This report found that Aspergillus sydowii termined by HPLC . NRRL250 leads caffeine biodegradation. In addition, The target strains were inoculated and cultivated in this paper investigated the effect of Aspergillus niger the potato dextrose agar (PDA) and Czapek-Dox me- NCBT110A on caffeine degradation. diums at 30 °C, respectively. The colony morphological characteristics and conidia structure were observed after cultivation for 5 d. The target strains grew aerobically as Methods pure cultures in 20 mL of Czapek-Dox liquid medium in Ethics statement 125 mL shake flasks at 30 °C, 250 rpm, for 2 d. The No specific permits were required for the described fresh cells were obtained by centrifugation at 1700 g for study. No specific permissions were required for these 5 min and freeze-dried at − 80 °C [10, 23]. DNA was ex- locations/activities, because specimens used in this study tracted by using SP fungal DNA kit. The extracted DNA were manufactured in the laboratory. was subject to amplify the ITS region, the universal fun- gal primers ITS1 and ITS4 were used in the PCR [11, Material and reagents 12, 23]. The final volume of 50 μL, 1.0 μL of containing Assam sun-dried green tea leaves (C. sinensis var. template DNA, 5 μL of 10 x buffer, 5 μL of dNTPs assamica (JW Masters) Kitamura) with moisture (2.5 mM), 0.5 μL of Taq polymerase, 1.0 μL (10 μM) of content 6.25% by weight were obtained from Yunnan each primer, and 36.5 μL of sterile distilled water were province, China. Caffeine (purity about 95%), used in used to implement amplifications [12, 23]. The PCR culture medium, was purchased from Tianjin Guangfu reaction procedure was as follows. Pre-degeneration at fine chemical industry institute, China. Caffeine 95 °C for 5 min, degeneration at 94 °C for 1 min, anneal- (≥99%), theophylline (≥99%) and 3-methylxanthine ing at 54 °C for 1 min, extension at 72 °C for 1.5 min, (≥99%) standards were purchased form Sigma with 35 cycles, extension at 72 °C for 10 min . It was Company, USA. SP fungal DNA kit, DNA marker, stored at 10 °C in the end of the reaction process. polymerase chain reaction (PCR) spread reagent, The PCR mixtures were analyzed by using ABI3730 internal transcribed spacers (ITS): ITS1 (5`-TCCG automatic DNA sequencer (Applied Biosystems, USA). TAGGTGAACCTGCGG-3`) and ITS4 (5`-TCCT The received sequence was sent to Genbank of NCBI to CCGCTTATTGATAGC-3`) were purchased from seek semi-root sequence. ITS1–5.8S rRNA-ITS2 gene TaKaRa Company, Japan. High performance liquid sequence of related strains were transferred and com- chromatography (HPLC) grade of acetonitrile was pared with ClustalX 1.8. The evolution distance was cal- purchased from Beijing Mreda Biotechnology Company, culated through a Kimura2-parameter of the MEGA 4 China. Other reagents were of analytical grade. Soft. Neighbor-Joining method was used to establish Zhou et al. BMC Microbiology (2018) 18:53 Page 3 of 10 phylogenetic trees. 1000 random samples were taken to concentration, reaction temperature and pH on the calculate Bootstrap for evaluation of the phylogenetic kinetic parameters were investigated . confidence level. 1) Substrate concentration. The seed was inoculated Growth of tea-derived fungi in a solid medium into the sterile liquid medium by 10% (v/v) of above Qualitative screenings were carried out on Petri dishes noted inoculum with different initial caffeine containing a solid culture medium with glucose (2% w/ concentration (600, 1200 and 1800 mg/L, v) (control culture) and a selection medium with caffeine respectively). The flask was incubated in an orbital instead of glucose in three different concentrations: 600, shaker during 5, 10 and 15 d (130 rpm, 30 °C). 1200 and 1800 mg/L per plate, respectively . Fungal 2) Reaction temperature. The seed was inoculated into mycelia from recent cultures were transferred to the sur- the sterile liquid medium by 10% (v/v) of above face of the agar plates with an inoculating loop. Strains noted inoculum with the initial caffeine were incubated at 30 °C for 5 d. Compared with the con- concentration of 1200 mg/L. The flask was trol culture, the strains utilized caffeine was estimated incubated in an orbital shaker with different by the size of the colony grown on the plates (Table 1). reaction temperature (25, 30, 35 °C, respectively) during 5, 10 and 15 d. Growth of tea-derived fungi in a liquid medium 3) pH. The seed and liquid medium were adjusted for Spore solutions of target strains were prepared by grow- different pH (5.0, 6.0,7.0, respectively) by phosphate ing the fungi for 5 d at 30 °C in dishes containing solid buffer. The seed was inoculated into the sterile culture medium with glucose . Two loopfuls of tar- liquid medium by 10% (v/v) of above noted get strains were transferred aseptically from a dish slant inoculum with the initial caffeine concentration of into 25 mL of a sterile liquid medium (per liter: potato 1200 mg/L. The flask was incubated in an orbital starch 4 g, dextrose 20 g, chloramphenicol 0.1 g) with shaker during 5, 10 and 15 d (130 rpm, 30 °C). 600 mg/L of caffeine in a 125 mL Erlenmeyer flask. The flask was incubated aerobically on an incubator shaker Fungal dry mass was determined. The final caffeine (250 rpm) at 30 °C for 48 h. The volume of the seed was and biodegraded products viz. theophylline and 10% (v/v) of total initial volume . The flask was incu- 3-methylxanthine were determined by HPLC . bated in an orbital shaker during 3, 6, 9, 12 and 15 d (130 rpm, 30 °C). The mycelium was collected after the A submerged fermentation (SMF) of tea infusion culture was filtered in a Buchner funnel, and rinsed in Sun-dried green tea leaves (1.0 g) were infused for 20 mL of water: ethyl acetate (1:1) . The mycelial 15 min in boiling distilled water (30 mL) and the tea mass was determined as fungal dry mass after drying at infusion was made up to 30 mL with distilled water after 35 °C for 24 h . Biodegraded products of caffeine filtration . Caffeine and other functional ingredients were analyzed by HPLC . The results were summa- (tea polyphenols and theabrownins) are relatively stable in rized in Table 2. high temperature condition. Based on the investigation of several thermal treatment methods (Additional file 1: Biodegradation in a liquid medium Table S1), including the control (no further treatment), Studies were performed during 15 d in the liquid high temperature sterilization at 121 °C for 5 min, medium to evaluate the kinetic parameters for biodeg- pasteurization at various conditions (65 °C, 30 min; 75 °C, radation reactions of caffeine. Effects of substrate 30 min and 80 °C, 30 min) and microwave heating Table 1 Growth of tea-derived fungi in agar solid medium (2% w/v) with glucose (2% w/v) (control culture) or presence of caffeine instead of glucose (30 °C, 5 d, pH 7.0) Tea- Strains growth (cm) derived Control culture 600 mg/L of caffeine 1200 mg/L of caffeine 1800 mg/L of caffeine fungi No. 1 3.5 × 3.5 1.0 × 1.0 2.0 × 1.5 2.0 × 2.0 No. 2 4.0 × 2.5 0.5 × 0.5 1.0 × 0.5 1.0 × 0.5 No. 3 3.0 × 2.5 1.0 × 0.5 1.0 × 1.0 1.0 × 1.0 No. 4 3.0 × 3.0 no growth 0.5 × 0.5 1.0 × 0.5 No. 5 4.0 × 2.5 3.0 × 2.5 2.5 × 2.5 3.0 × 2.5 No. 6 3.0 × 2.5 0.5 × 0.5 1.0 × 0.5 1.0 × 0.5 No. 7 3.0 × 3.0 no growth 0.5 × 0.5 1.0 × 1.0 Zhou et al. BMC Microbiology (2018) 18:53 Page 4 of 10 Table 2 Quantitative biodegradation of caffeine by A. sydowii and A. niger on 5, 10 and 15th day a a b Reaction time (d) Fungal dry mass (g) C (mg/L) C (mg/L) % of caffeine degraded caffeine theophylline A. sydowii NRRL250 (600 mg/L of caffeine) 5 0.23 ± 0.02 431.5 ± 39.7 40.4 ± 1.0 28.1 ± 6.6 10 0.24 ± 0.02 134.8 ± 6.5 209.9 ± 22.6 77.5 ± 1.1 15 0.22 ± 0.01 3.7 ± 0.8 262.6 ± 20.7 99.4 ± 0.1 A. niger NCBT110A (600 mg/L of caffeine) 5 0.28 ± 0.03 592.6 ± 3.1 NF 1.2 ± 0.5 10 0.29 ± 0.02 580.0 ± 2.9 NF 3.3 ± 0.5 15 0.27 ± 0.01 577.3 ± 6.0 NF 3.8 ± 1.0 C Concentration determined by HPLC % of caffeine degraded was estimated as follow: of caffeine degraded = (C -C )/C *100% (1) 0 t 0 In Eq. (1) C was the initial caffeine concentration (mg/L), C was the final caffeine concentration (mg/L) after the fermentation 0 t All data are presented as mean ± SD. NF not found (640 W, 2 min) , sterilization can kill viable microor- 1.2 × 10 CFU/g on day 20. Due to nutrient limitation in ganisms with minimal damage in main functional compo- the tea leaves, the colony count decreased after day 20. nents. Therefore, sterilization was selected as the With changing of fungi count, caffeine content (Fig. 1) reasonable treatment for SMF. declined significantly (p < 0.05) from 3.685 ± 0.1006% Two loopfuls of target strains were transferred aseptic- (w/w) to 2.612 ± 0.1398% (w/w) during the fermentation. ally from a dish slant into 25 mL of sterile tea infusion According to the analysis above, it suggested that the in a 125 mL Erlenmeyer flask . The flask was fungal colonies cause the decrease of caffeine content. incubated aerobically at 30 °C for 48 h on an incubator Through separation and purification, 11 fungi were shaker (250 rpm). The volume of the seed was 10% isolated from PETSSF. (v/v) of total initial volume of the inoculation . Non-inoculation (control) and non-sterilization (natural treatment) were carried out. The flask was incubated in Screening result of tea-derived fungi in a solid medium an orbital shaker for 3, 6, 9, 12 and 15 d (130 rpm, 30 ° The screening was carried out in an agar solid C), respectively. Fungal dry mass, caffeine and theophyl- medium for selecting the tea-derived fungi able to line contents were determined. utilize caffeine. In order to evaluate the biocatalytic potential for degradation of caffeine, all investigated Results strains were inoculated into an agar solid medium Caffeine content and fungi count during PETSSF with presence of glucose and they were also inocu- The Fig. 1 shows that the fugal colony count dramatic- lated into a set of agar solid medium with presence ally increased from 0 to 10 days. Then, it increased of different concentration caffeine. The sizes of the slowly from 4.8 × 10 CFU/g dry weight of tea leaves to colonies were measured and showed in Table 1. Fig. 1 Changes in caffeine content and the fungal count of tea leaves during SSF. Data are presented as mean values ± SD. *,** and *** show the significant difference levels (p < 0.05) during the fermentation Zhou et al. BMC Microbiology (2018) 18:53 Page 5 of 10 Among 11 tea-derived fungi, 7 strains could survive in and 3-methylxanthine contents as well as caffeine degrad- the agar solid medium (2% w/v) with caffeine alone. ation rates were showed in Table 3. Degradation of Strains No. 1, No. 3 and No. 5 showed the best growth approximately 7.1, 33.0, 52.8, 68.7 and 90.1% were in all concentrations evaluated. And, stains No. 4 and observed in3,6,9,12and 15 d, respectively.Both No. 7 had no growth in low caffeine concentration, theophylline and 3-methylxanthine were detected in which showed that the utilization ratio of caffeine was the fermentation, which showed that theophylline and restricted. If fungi had a higher growth in a low caffeine 3-methylxanthine were the main degradation products concentration, it may indicate that fungi could use caf- from caffeine. Theophylline was first detected on day 3 and feine as a carbon source directly or indirectly. Because it increased obviously with the caffeine degradation, which strains No. 5 and No. 1 had a high growth rate at the showed that theophylline was a direct degradation product lowest caffeine concentration, they were considered as from caffeine through demethylation. 3-Methylxanthine the potential target strains. Colony characteristics and was not detected on day 3 and first detected on day 6, microscopic structure of strain No. 5 were showed in which indicated that 3-methylxanthine might be a direct Additional file 2: Figure S1 and S2, colony characteristics degradation product from theophylline instead of caffeine. and microscopic structure of strain No. 1 were showed As the secondary product, 3-methylxanthine content was in Additional file 2: Figure S3 and S4. Through molecu- far below theophylline and only 178.7 ± 10.8 mg/L was lar identification (Additional file 3: Figure S5), strain No. produced in 15 d. 5 was identified as Aspergillus sydowii NRRL250 with 99.8% homology, strain No. 1 was identified as Aspergil- Effects of substrate concentration, reaction temperature lus niger NCBT110A with 99.8% homology (Fig. 2). and pH on the kinetic parameters for caffeine degradation by A. sydowii NRRL250 Biodegradation of caffeine by A. sydowii NRRL250 and A. Microorganism metabolism and degradation capability niger NCBT110A were influenced by substrate concentration, reaction For the biodegradation reaction in a liquid medium, the temperature and pH. In this paper, effects of substrate con- selected strains (A. sydowii NRRL250 and A. niger centration, reaction temperature and pH on fungal dry NCBT110A) were inoculated into the nutrient medium mass and kinetic parameters of caffeine degradation by A. with the presence of caffeine. During the fermentation, sydowii NRRL250 were investigated (Tables 4, 5 and 6,re- fungal dry mass and caffeine content were determined. In spectively). A. sydowii NRRL250 was inoculated into a li- addition, the caffeine degradation rates were calculated by quid medium with increasing caffeine concentrations (600, the Eq. (1) to investigate the degrading capability of 1200 and 1800 mg/L, respectively), and the flasks were in- selected strains. For A. niger NCBT110A, caffeine was no cubated in an orbital shaker for 15 d (130 rpm, 30 °C). Fun- significantly degraded with about 1.2, 3.3, 3.8% of caffeine gal dry mass and the kinetic parameters, including final degraded and theophylline was not detected in the caffeine concentration (C ), final theophylline con- caffeine,f fermentation. Caffeine degradation capability of A. niger centration (C ), final 3-methylxanthine concentra- theophylline,f NCBT110A was limited in the liquid medium, which tion (C ), the volumetric rate of caffeine 3-methylxanthine,f showed that A. niger NCBT110A could use caffeine as a degradation (Q ), the volumetric rate of theophylline caffeine potential carbon source when glucose and other nutrients production (Q ), the yield of theophylline (Y theophylline theophyl- were limited or absent. For A. sydowii NRRL250, caffeine ) and caffeine degradation rate (% of caffeine de- line/caffeine was almost completely degraded at 600 mg/L of caffeine graded) in different substrate concentrations were showed (Table 2). In 15 d, caffeine had been degraded completely. in Table 4. There was no significant difference in fungal dry As the degradation product, theophylline was determined mass (p > 0.05). The final concentrations of theophylline by HPLC and increased observably from 40.4 ± 1.0 mg/L and 3-methylaxthine increased significantly (p <0.05), caf- on 5 d to 262.6 ± 20.7 mg/L on 15 d. As A. sydowii feine degradation rate decreased significantly (p < 0.05) in NRRL250 has ability to degrade caffeine, it will be used to higher initial caffeine concentrations. Only about 62.9% of conduct the biodegradation products analysis of caffeine caffeine was degraded in 1800 mg/L caffeine concentration. in below. By comparing the results, 1200 mg/L was an appropriate substrate concentration with the high caffeine degradation Biodegradation products analysis of caffeine by A. sydowii rate and the high theophylline production. NRRL250 In order to compare the kinetic parameters in different A. sydowii NRRL250 was inoculated into a liquid reaction temperatures, A. sydowii NRRL250 was inocu- medium with 1200 mg/L of caffeine for analysis of its lated into an identical liquid medium with the initial caf- biodegraded products. And caffeine, theophylline and feine concentration of 1200 mg/L, and the flasks were 3-methylxanthine were determined by HPLC during 3, 6, incubated in an orbital shaker with different reaction tem- 9, 12 and 15 d in the fermentation. Caffeine, theophylline peratures (25, 30 and 35 °C, respectively) for 15 d. The Zhou et al. BMC Microbiology (2018) 18:53 Page 6 of 10 Fig. 2 Phylogenetic tree of the target strains (strains No. 5 and No.1) Table 3 Biodegradation products analysis of caffeine by A. sydowii NRRL250 in a liquid medium with 1200 mg/L of caffeine a a a Reaction time (d) C (mg/L) C (mg/L) C (mg/L) % of caffeine degraded caffeine theophylline 3-methylxanthine 3 994.9 ± 35.2 62.8 ± 11.3 NF 7.1 ± 2.9 6 804.1 ± 26.5 201.2 ± 8.4 28.4 ± 1.5 33.0 ± 2.2 9 566.3 ± 16.5 274.7 ± 14.7 38.4 ± 3.4 52.8 ± 1.4 12 375.3 ± 15.3 426.3 ± 20.8 68.1 ± 6.9 68.7 ± 1.3 15 105.0 ± 16.9 549.4 ± 29.3 178.7 ± 10.8 90.1 ± 3.0 C Concentration determined by HPLC All data are presented as mean ± SD. NF not found Zhou et al. BMC Microbiology (2018) 18:53 Page 7 of 10 Table 4 Comparison of the kinetic parameters for caffeine degradation in different substrate concentrations (30 °C, 15 d, pH 7.0) Substrate Fungal dry C C C Q Q Y % of caffeine caffeine,f theophylline,f 3-methylxanthine,f caffeine theophylline theophylline/ concentration (mg/L) mass (g) (mg/L) (mg/L) (mg/L) (mg/L d) (mg/L d) degraded caffeine A A A A A A A A 600 0.22 ± 0.02 3.7 ± 0.8 262.6 ± 20.7 115.8 ± 10.1 39.8 ± 0.5 17.5 ± 1.4 0.44 ± 0.04 99.3 ± 0.1 A B B B B B B B 1200 0.22 ± 0.03 105.0 ± 16.9 549.4 ± 29.3 178.7 ± 10.8 73.0 ± 1.1 36.6 ± 2.0 0.50 ± 0.03 91.3 ± 1.4 A C C B B C C C 1800 0.23 ± 0.02 668.2 ± 37.3 643.8 ± 25.3 191.2 ± 4.5 75.5 ± 2.5 42.9 ± 1.7 0.57 ± 0.01 62.9 ± 2.1 All kinetic parameters were calculated according to Sirisansaneeyakul and others (2013)  A-C All data are presented as mean ± SD, p < 0.05 in the same column Concentrations of caffeine, theophylline and 3-methylxanthine determined by HPLC C the final caffeine concentration (mg/L), C the final theophylline concentration (mg/L), C the final 3-methylxanthine concentration caffeine,f theophylline,f 3-methylxanthine,f (mg/L), Q the volumetric rate of caffeine degradation (mg/L d), Q the volumetric rate of theophylline production (mg/L d), Y caffeine theophylline theophylline/caffeine theophyline yield on caffeine (mg/mg) fungal dry mass and the kinetic parameters were showed high-theophylline tea through an inoculated fermenta- in Table 5. In the temperature range between 25 and 30 ° tion. In this paper, A. sydowii NRRL250 was inoculated C, there were no significant differences in fungal dry mass, into the sterile tea infusion for SMF, caffeine and theo- the final caffeine concentration, the volumetric rate of caf- phylline contents were determined by HPLC during 0, 3, feine degradation, and caffeine degradation rate (p >0.05). 6, 9, 12 and 15 d. The final fungal dry mass was also In 35 °C, there was no significant decline (p >0.05) in determined. In addition, the SMF inoculated by A. niger fungal mass. And the final caffeine concentration, the NCBT110A, natural treatment and sterile treatment volumetric rate of caffeine degradation and caffeine (control) were carried out to explore the influence of degradation rate declined significantly (p < 0.05). However, microorganism on caffeine. Changes of caffeine and the theophylline concentration, the volumetric rate of theophylline contents were showed in Fig. 3. Fungal dry theophylline production and the yield of theophylline mass and the kinetic parameters of caffeine degradation increased significantly (p < 0.05) in 35 °C. The optimal were showed in Table 7. temperature for caffeine degradation was 30 °C. And There were no significant changes of caffeine and theo- higher temperature promotes theophylline production. phylline contents in sterile treatment (control) (p >0.05). In order to compare the kinetic parameters in different During SMF by A. niger NCBT110A, caffeine increased pH, phosphate buffer was used to adjust the pH of liquid significantly (p < 0.05), which showed that the caffeine medium. The fungal mass and the kinetic parameters degradation capability of A. niger NCBT110A was limited were showed in Table 6. pH had remarkable effects on in the presence of carbohydrate and other nutriment. Dur- fugal dry mass and the kinetic parameters. In pH of 5, ing SMF by A. sydowii NRRL250, most of caffeine the growth and caffeine catabolism of A. sydowii (985.1 mg/L) was degraded (Fig. 3a, Table 7). As a main NRRL250 were restricted. Through comparisons, pH of degradation product, theophylline increased sharply from 6 was the optimum pH for caffeine degradation with the 24.7 mg/L to 501.2 mg/L in the SMF by A. sydowii highest fugal dry mass, caffeine degradation rate and NRRL250 (Fig. 3b, Table 7). Because the existence of A. theophylline production. sydowii NRRL250, in natural treatment, caffeine decreased significantly with about 256.3 mg/L of caffeine degraded Applications of A. sydowii NRRL250 and A. niger and theophylline increased significantly with 74.7 mg/L in NCBT110A in SMF of tea infusion 15 d (p < 0.05) (Fig. 3). Therefore, A. sydowii NRRL250 Due to the caffeine degradation characteristic, A. sydowii was appropriate for the production of decaffeinated tea or NRRL250 was suitable to produce decaffeinated and high-theophylline tea through an inoculated fermentation. Table 5 Comparison of the kinetic parameters for caffeine degradation on different reaction temperatures (1200 mg/L of caffeine,15d. pH 7.0) Reaction Fungal dry C C C Q Q Y % of caffeine caffeine,f theophylline,f 3-methylxanthine,f caffeine theophylline theophylline/ temperature (°C) mass (g) (mg/L) (mg/L) (mg/L) (mg/L d) (mg/L d) degraded caffeine A A A A B A A B 25 0.22 ± 0.02 121.6 ± 14.4 478.8 ± 20.2 196.6 ± 7.5 71.9 ± 1.0 31.9 ± 1.3 0.44 ± 0.02 89.9 ± 1.2 A A B B B B B B 30 0.22 ± 0.03 105.0 ± 16.9 549.4 ± 29.3 178.7 ± 10.8 73.0 ± 1.1 36.6 ± 2.0 0.50 ± 0.03 90.5 ± 1.4 A B C B A C C A 35 0.19 ± 0.02 202.0 ± 15.7 618.4 ± 18.8 149.8 ± 13.2 66.5 ± 1.0 421.2 ± 1.3 0.61 ± 0.02 83.2 ± 1.3 All kinetic parameters were calculated according to Sirisansaneeyakul and others (2013)  A-C All data are presented as mean ± SD, p < 0.05 in the same column Concentrations of caffeine, theophylline and 3-methylxanthine determined by HPLC Ccaffeine,f the final caffeine concentration (mg/L), Ctheophylline,f the final theophylline concentration (mg/L), C3-methylxanthine,f the final 3-methylxanthine concentration (mg/L), Qcaffeine the volumetric rate of caffeine degradation (mg/L d), Qtheophylline the volumetric rate of theophylline production (mg/L d), Ytheophylline/caffeine theophyline yield on caffeine (mg/mg) Zhou et al. BMC Microbiology (2018) 18:53 Page 8 of 10 Table 6 Comparison of the kinetic parameters for caffeine degradation in different pH (1200 mg/L of caffeine, 30 °C,15d.) pH Fungal dry mass (g) C C C Q Q Y % of caffeine degraded caffeine,f theophylline,f 3-methylxanthine,f caffeine theophylline theophylline/caffeine (mg/L) (mg/L) (mg/L) (mg/L d) (mg/L d) A C A A A A A A 5 0.14 ± 0.01 508.5 ± 45.4 245.3 ± 17.5 87.8 ± 12.5 46.1 ± 3.0 16.4 ± 1.2 0.35 ± 0.01 57.6 ± 3.8 B A C B C C C C 6 0.22 ± 0.02 41.7 ± 5.9 776.5 ± 35.8 125.1 ± 10.9 77.2 ± 0.4 51.8 ± 2.4 0.67 ± 0.03 96.5 ± 0.5 B B B C B B B A 7 0.22 ± 0.03 105.0 ± 16.9 549.4 ± 29.3 178.7 ± 10.8 73.0 ± 1.1 36.6 ± 2.0 0.50 ± 0.03 91.3 ± 1.4 All kinetic parameters were calculated according to Sirisansaneeyakul and others (2013)  A-C All data are presented as mean ± SD, p < 0.05 in the same column Concentrations of caffeine, theophylline and 3-methylxanthine determined by HPLC C the final caffeine concentration (mg/L), C the final theophylline concentration (mg/L), C the final 3-methylxanthine concentration caffeine,f theophylline,f 3-methylxanthine,f (mg/L); Q the volumetric rate of caffeine degradation (mg/L d), Q the volumetric rate of theophylline production (mg/L d), Y caffeine theophylline theophylline/caffeine theophyline yield on caffeine (mg/mg) Discussion lowest caffeine concentration, which suggested that those Although several effective strains were selected from the 2 strains used caffeine as a carbon source directly or soil of tea and coffee gardens to degrade caffeine [26–28], indirectly. The two superior strains were identified as A. the functional strain selected from tea was not reported. niger NCBT110A and A. sydowii NRRL250 by molecular In this paper, 11 fungi were isolated from PETSSF, and 7 identification method. strains could survive in a solid medium with caffeine The substrate tests in the liquid medium with caffeine alone. But only 2 strains had a high growth rate at the found that the caffeine degradation capability of A. niger Fig. 3 Changes in concentrations of caffeine (a) and theophylline (b) in shake flask fermentation with various pure culture. Data are presented as mean ± SD. Control was no inoculation treatment Zhou et al. BMC Microbiology (2018) 18:53 Page 9 of 10 Table 7 Comparison of the kinetic parameters for caffeine degradation in tea infusion fermentation (30 °C, 15d, natural pH) Strains or Fungal dry mass C C C Q Q Y % of caffeine caffeine,0 caffeine,f theophylline,f caffeine theophylline theophylline/ treatments (g) (mg/L) (mg/L) (mg/L) (mg/L d) (mg/L d) degraded caffeine A A A C B B B A. sydowii 0.19 ± 0.02 1082.9 ± 65.8 157.8 ± 10.2 501.2 ± 13.5 61.7 ± 31.8 ± 0.8 0.52 ± 0.05 85.4 ± 1.7 NRRL250 5.0 B A D A A. niger 0.24 ± 0.01 1085.3 ± 64.8 1248.1 ± 30.5 27.2 ± 0.8 ND ND ND ND NCBT110A A A B B A A A Natural treatment 0.20 ± 0.02 1073.9 ± 78.6 817.6 ± 8.6 74.7 ± 3.3 17.1 ± 3.3 ± 0.1 0.21 ± 0.06 23.6 ± 5.3 5.14 A C A Control ND 1101.6 ± 89.5 1096.4 ± 33.2 25.0 ± 2.1 ND ND ND ND All kinetic parameters were calculated according to Sirisansaneeyakul and others (2013)  A-D All data are presented as mean ± SD, p < 0.05 in the same column Concentrations of caffeine, theophylline and 3-methylxanthine determined by HPLC C initial caffeine concentration (mg/L), C the final caffeine concentration (mg/L), C the final theophylline concentration (mg/L) Q the caffeine,0 caffeine,f theophylline,f caffeine volumetric rate of caffeine degradation (mg/L d), Q the volumetric rate of theophylline production (mg/L d), Y theophyline yield on theophylline theophylline/caffeine caffeine (mg/mg), ND not determined NCBT110A was limited in the presence of glucose and A. sydowii could be used in biodegradation of methyl para- other nutrients. A. niger NCBT110A could use caffeine thion . Due to the caffeine degradation characteristic, A. as a potential carbon source when the absence of sydowii NRRL250 would be applied in the production of de- glucose. A. sydowii NRRL250 could degrade caffeine caffeinated tea or high-theophylline tea. In SMF, 985.1 mg/L completely in a liquid medium with 600 mg/L of of caffeine was degraded, and 501.2 mg/L of theophylline caffeine. Therefore, A. sydowii NRRL250 was a potentially was produced in 15 d. Further research could be con- effective strain to degrade caffeine. ducted in related to the caffeine degradation pathway and In theperspectiveof physiology of teatree(C. sinesis (L.) productive technology of decaffeinated tea by A. sydowii O. Kuntze), caffeine is synthesized in the root. Theobromine NRRL250. (3, 7-dimethyxanthine) is a direct precursor of caffeine anabolism and a major rate-limiting step in caffeine Conclusions synthesis . Theophylline (1, 3-dimethyxanthine) and The purpose of this research was to screen and identify the 3-methylxanthine are the main degradation products in strains which able to degrade caffeine during the PET caffeine catabolism . In addition, theophylline is a fermentation process. The results of the research show that rate-limiting step of caffeine catabolism in the physiology strain Aspergillus sydowii NRRL250 and strain A. niger of tea tree (C. sinesis (L.) O. Kuntze) and coffee tree NCBT110A could use caffeine as a potential carbon source (Coffea arabica L.). And demethylase is an important when glucose and other nutrients were limited or absent. enzyme which catalyzes the reaction from caffeine to A. sydowii NRRL250 was an effective strain to degrade theophylline. In microbial secondary metabolites, the caffeine, which could be applied in the production of degradation products and degradation pathways of decaffeinated or high-theophylline tea. In addition, theo- caffeine were not completely clear. In the substrate tests phylline and 3-methlxanthine were the main degradation with caffeine of A. sydowii NRRL250, theophylline and products from caffeine in secondary metabolites of A. 3-methlxanthine were detected. And theophylline and sydowii NRRL250. 3-methlxanthine increased significantly (p < 0.05) with the degradation of caffeine. Caffeine catabolism in secondary Additional files metabolites of A. sydowii NRRL250 was similar to the metabolites in the physiology of tea tree (C. sinesis (L.) O. Additional file 1: Table S1. Heating method effects on microbial count Kuntze), theophylline and 3-methlxanthine were the main and main chemical components of tea infusion. Note: All date are A-B presented as mean ± SD, p < 0.05 in the same column, ND: not degradation products from caffeine by demethylation. detectable, TPs is the abbreviation of tea polyphenols. (DOCX 15 kb) In this study, the optimum substrate concentration, reac- Additional file 2: Figure S1. Colony characteristics of strain No. 5 on tion temperature and pH of A. sydowii NRRL250 were in- culture medium. Figure S2. Conidia structure of strain No.5 under optical vestigated. The optimum conditions of caffeine degradation microscope. Figure S3. Colony characteristics of strain No.1 on culture medium. Figure S4. Conidia structure of strain No.1 under optical were 1) substrate concentration of 1200 mg/L, 2) reaction microscope. (DOCX 3679 kb) temperature at 30 °C, and 3) pH of 6. The optimum condi- Additional file 3: Figure S5. ITS sequences data of the target strains. tions provided the relevant information for the application (DOCX 16 kb) of A. sydowii NRRL250 in caffeine degradation. In previous researches, A. sydowii is an important indus- Abbreviations trialand medicalmicroorganism,which couldproduce CFU: Colony forming units; DNA: Deoxyribonucleic acid; HPLC: High performance monosaccharide and indole alkaloids [31–33]. In addition, liquid chromatography; ITS: Internal transcribed spacer; MEGA: Molecular Zhou et al. BMC Microbiology (2018) 18:53 Page 10 of 10 evolutionary genetics analysis; NCBI: National Center for Biotechnology 11. Zhao ZJ, Tong HR, Zhou L, Wang EX, Liu QJ. Fungal colonization of pu-erh tea in Information; PCR: Polymerase chain reaction; PDA: Potato dextrose agar; PET: Pu- Yunnan. J Food Safety. 2010;30:769–84. erh tea; PETSSF: Pu-erh tea solid-state fermentation; RNA: Ribonucleic acid; 12. Abe M, Takaoka N, Idemoto Y, Takagi C, Imai T, Nakasaki K. Characteristic rRNA: Ribosomal ribonucleic acid; SMF: Submerged fermentation; SSF: Solid- fungi observed in the fermentation process for Puer tea. Int J Food state fermentation Microbiol. 2008;124:199–203. 13. Zhao M, Zhang DL, Su XQ, Duan SM, Wan JQ, Yuan WX, Liu BY, Ma Y, Pan Acknowledgements YH. An integrated metagenomics/metaproteomics investigation of the We thank Kunming Dapu Tea CO., LTD, and Yunnan Institute of Microbiology for microbial communities and enzymes in solid-state fermentation of pu-erh their assistance in sample collection and microorganism identification. tea. Sci Rep. 2015;5:10117. http://www.nature.com/articles/srep10117. 14. Qin JH, Li N, Tu PF, Ma ZZ, Zhang L. Change in tea polyphenol and purine Funding alkaloid composition during solid-state fungal fermentation of post- This work was supported by Modern Agricultural Industry Technology System fermented tea. J Agr Food Chem. 2012;60:1213–7. of China (CARS-23) and the national natural science found of China (C161104). 15. Haas D, Pfeifer B, Reiterich C, Partenheimer R, Reck B, Buzina W. The funding bodies had no role in the design of the study, in data collection, Identification and quantification of fungi and mycotoxins from Pu-erh tea. analysis or interpretation, or in writing the manuscript. Int J Food Microbiol. 2013;166:316–22. 16. Wang WN, Zhang L, Wang S, Shi SP, Jiang Y, Li N, Tu PF. 8-CN-ethyl-2- Availability of data and materials pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese The data that support the findings of this study are available from the dark teas formed in the post-fermentation process provide significant corresponding author upon reasonable request. antioxidative activity. Food Chem. 2014;152:539–45. 17. Zhang L, Li N, Ma ZZ, Tu PF. 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Published: Jun 5, 2018
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