Production of theophylline via aerobic fermentation of pu-erh ...

Differences between aerobic and anaerobic fermentation

In a previous study [20], caffeine content was decreased significantly (p&#;<&#;0.05) during the solid-state fermentation of pu-erh tea. Aerobic and anaerobic fermentation was carried out at a natural condition to simulate the solid-state fermentation of pu-erh tea. Fungi count, caffeine and theophylline contents were determined in the fermentation. Results are presented in Fig. 1. There were significant (p&#;<&#;0.05) differences in fungi count, caffeine and theophylline contents between aerobic and anaerobic fermentation. Compared with the anaerobic fermentation, caffeine content was decreased significantly (p&#;<&#;0.05) in the aerobic fermentation; about 16.73&#;mg/g of caffeine of dry matter weight of tea leaves was degraded at the end of the fermentation. Furthermore, theophylline content was increased significantly (p&#;<&#;0.05) along with caffeine degradation and about 15.52&#;mg/g of theophylline was produced at the end of the fermentation. As shown in Fig. 1A, fungal colony count increased dramatically from day 0 to day 6 and maintained at a high level during the whole fermentation. The microbial metabolism in aerobic fermentation was more active than anaerobic fermentation. Based on the changes of caffeine and theophylline contents, we predicted that the fungi in the aerobic fermentation lead to the conversion of caffeine to theophylline. Therefore, samples collected from the aerobic fermentation were used to isolate potential starter strains and the dominant fungi were analyzed in this paper.

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Differences between aerobic and anaerobic fermentation in fungi count (a), caffeine (b) and theophylline (c).Data are presented as mean value ± SD of three independent tests. * indicated there is significant difference between aerobic and anaerobic fermentation (Duncan&#;s multiple range test: p&#;<&#;0.05)

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Identification results of fungi in aerobic fermentation

A total of 5 fungal strains were isolated from the aerobic fermentation of pu-erh tea. The received sequence (546&#;bp) of PET-1 is provided in Additional file 1: Fig. S1. The received sequence (516&#;bp) of PET-2 is provided in Additional file 1: Fig. S2. The received sequences (541&#;bp ITS sequence, 516&#;bp β-Tubulin sequence and 765&#;bp Calmodulin sequence, respectively) of PET-3 are provided in Additional file 1: Fig. S3. The received sequences (532&#;bp ITS sequence, 515&#;bp β-Tubulin sequence and 757&#;bp Calmodulin sequence, respectively) of PET-4 are provided in Additional file 1: Fig. S4. The received sequences (525&#;bp ITS sequence and 420&#;bp β-Tubulin sequence, respectively) of PET-5 are provided in Additional file 1: Fig. S5. After the molecular identification, 5 fungal strains were identified as A. niger (99.8% sequence identity with strain NCBT110A), A. sydowii (99.8% sequence identity with strain NRRL250), A. pallidofulvus (99.9% sequence identity with strain NRRL), A. sesamicola (99.8% sequence identity with strain CBS) and P. mangini (99.6% sequence identity with strain CBS253.31), respectively (Table 1). As shown in Fig. 2, all fungal strains were detected at various stages in the aerobic fermentation. The isolated fungi account for 86.67&#;97.14% in total colony count from day 3 to day 15, which could be considered as the dominant fungi in the fermentation.

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Changes in distribution of dominant fungi during pu-erh tea aerobic fermentation

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Effects of microorganisms on the conversion of caffeine to theophylline

In order to find out the effects of microorganisms on the conversion of caffeine to theophylline, the isolated fungal strains were inoculated into the sun-dried tea leaves and cultivated in the incubator at 30&#;°C for the aerobic fermentation, respectively. As shown in Fig. 3, different microorganisms affected on caffeine and theophylline contents differently. Compared with the sterilization treatment, A. niger PET-1, A. pallidofulvus PET-3, A. sesamicola PET-4 and P. mangini PET-5 increased caffeine content significantly (Fig. 3a), which should be attributed to microbial metabolism and nutrient loss. However, there was no significant increase of theophylline content in the aerobic fermentation of A. niger PET-1, A. pallidofulvus PET-3, A. sesamicola PET-4 and P. mangini PET-5, respectively (Fig. 3b). During the fermentation inoculated by A. sydowii PET-2, caffeine content was decreased significantly (p&#;<&#;0.05) and theophylline content was increased significantly (p&#;<&#;0.05) (Fig. 3). At the end of the fermentation (day 15), 28.85&#;mg/g of caffeine was degraded with an amplitude of about 86.80%. With the degrading of caffeine, 24.60&#;mg/g of theophylline was produced at day 15 and became the main methylxanthine in the aerobic fermentation. Therefore, A. sydowii PET-2 had the capability of converting caffeine to theophylline,

Changes in contents of caffeine (a) and theophylline (b) in aerobic fermentation inoculated by tea-derived strains, and sterilization treatment group. Data are presented as mean value ± SD of three replications. * indicated there is significant difference (p&#;<&#;0.05) between A. sydowii PET-2 fermentation group and other treatment groups according to Duncan&#;s test

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Ability of A. sydowii PET-2 converting caffeine to theophylline

A. sydowii PET-2 was inoculated into the sun-dried tea leaves for the aerobic fermentation and the kinetic parameters were established to study the conversion of caffeine to theophylline. As the fermentation progressed, tea polyphenols, and the main tea pigments including theaflavins, thearubigins and theabrownins contents were measured. The results are provided in Additional file 2:Table S1. During the aerobic fermentation, tea polyphenols content was decreased significantly (p&#;<&#;0.05), theabrownins content was increased significantly (p&#;<&#;0.05). Similar changes of tea polyphenols and theabrownins contents were found in the solid-sate fermentation of pu-erh tea. In the end of the fermentation, theabrownins content reached the national quality requirements of pu-erh tea. Therefore, A. sydowii PET-2 could be used in the pu-erh tea fermentation.

The kinetic parameters in the inoculated fermentation, including caffeine content (Ccaffeine,t), theophylline content (Ctheophylline,t), the yield of theophylline (Ytheophylline/caffeine), caffeine removal ratio (%) and theophylline production (mg/g) are listed in Table 2. As shown in Table 2, approximately 7.25, 15.52, 34.47, 63.93, and 83.88% of caffeine were degraded at day 3, 6, 9, 12 and 15, respectively. Moreover, theophylline was detected and produced with the degrading of caffeine. At the end of the fermentation, 24.60&#;±&#;1.18&#;mg/g of theophylline was produced, the yield of theophylline stayed at 85%, and about 93.18% of degraded caffeine was converted to theophylline. The production of theophylline was consistent with the tendency of caffeine degradation. A. sydowii PET-2 could lead caffeine degradation and convert most of the degraded caffeine to theophylline in the inoculated fermentation, which indicated that A. sydowii PET-2 had the most capability of converting caffeine to theophylline.

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Due to the caffeine conversion characteristic, A. sydowii PET-2 had broad application prospects in the production of theophylline via an aerobic fermentation. The influence factors of theophylline production such as moisture content, inoculation quantity and incubation temperature were discussed later in this paper.

Selection of optimal condition in moisture content with inoculation quantity of 4% and incubation temperature at 30&#;°C

A. sydowii PET-2 was inoculated into the sun-dried tea leaves with increasing initial moisture contents (25, 30, 35, 40 and 45%, respectively) and cultivated in the incubator at the temperature of 30&#;°C. Caffeine and theophylline contents were determined by HPLC during the fermentation. Caffeine removal ratio (%) and theophylline production (mg/g) were calculated to investigate the influence of moisture content on caffeine conversion. As shown in Fig. 4, caffeine removal ratio and theophylline production increased steadily with the fermentation. The microbial metabolism was influenced by moisture content, thus moisture content had significant impacts on caffeine degradation and theophylline production. The experiments indicated that the fermentation group with the initial moisture content of 35% (w/w) had the highest caffeine removal ratio and theophylline production. In addition, caffeine removal ratio and theophylline production decreased significantly (p&#;<&#;0.05) in lower moisture content. Only about 49.78% of caffeine was degraded and 13.34&#;mg/g of theophylline was produced in 25% (w/w) of moisture content at day 15. The single factor analysis showed that 35% (w/w) was the appropriate initial moisture content for the conversion of caffeine to theophylline.

Effect of moisture content in an aerobic fermentation inoculated by A. sydowii PET-2 on caffeine degradation (a) and theophylline production (b).Data are presented as mean value ± SD of three replications. Caffeine removal ratio&#;=&#;(Ccaffeine,0-Ccaffeine,t)/Ccaffeine,0*100%; Theophylline production&#;=&#;Ctheophylline,t-Ctheophylline,0.Ccaffeine,0 was the initial caffeine content (mg/g), Ccaffeine,t was the caffeine content (mg/g) detected in the fermentation. Ctheophylline,t was the theophylline content (mg/g) detected in the fermentation, Ctheophylline,0 was the initial theophylline content (mg/g)

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Selection of optimal condition in inoculation quantity with initial moisture content of 35% (w/w) and incubation temperature at 30&#;°C

A set of different inoculation quantities was built to find out the optimal inoculation quantity for caffeine conversion. Caffeine removal ratio (%) and theophylline production (mg/g) were calculated and showed in Fig. 5. In low inoculation quantity (1%), the caffeine conversion was inhibited, only about 51.54% of caffeine was degraded and 15.72&#;mg/g of theophylline was produced at day 15. With the increasing inoculation quantity applied (from 1 to 8%), the caffeine conversion capability increased obviously. However, in high inoculation level (8, 12, and 16%, respectively), there were no significant (p&#;>&#;0.05) changes in caffeine removal ratio and theophylline production. Through comparison and analysis, 8% was the optimal inoculation quantity with the higher caffeine removal ratio and theophylline production.

Effect of inoculation level in an aerobic fermentation inoculated by A. sydowii PET-2 on caffeine degradation (a) and theophylline production (b). Data are presented as mean value ± SD of three replications.Caffeine removal ratio&#;=&#;(Ccaffeine,0-Ccaffeine,t)/Ccaffeine,0*100%; Theophylline production&#;=&#;Ctheophylline,t-Ctheophylline,0.Ccaffeine,0 was the initial caffeine content (mg/g), Ccaffeine,t was the caffeine content (mg/g) detected in the fermentation. Ctheophylline,t was the theophylline content (mg/g) detected in the fermentation, Ctheophylline,0 was the initial theophylline content (mg/g)

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Selection of optimal condition in incubation temperature with initial moisture of 35% (w/w) and inoculation quantity of 4%

Incubation temperature had a significant impact on microbial metabolism. In order to compare the caffeine conversion level from caffeine to theophylline in different incubation temperatures, A. sydowii PET-2 was inoculated into the sun-dried tea leaves with an initial moisture content of 35% (w/w) and an inoculation quantity of 4%, and cultivated in the incubator with different incubation temperatures (25, 30, 35, 40 and 45&#;°C, respectively) for 15&#;days. Caffeine and theophylline contents were determined, and caffeine removal ratio (%) and theophylline production (mg/g) are showed in Fig. 6. In the temperature range between 25 and 35&#;°C, caffeine removal ratio and theophylline production increased significantly (p&#;<&#;0.05). Between 40 and 45&#;°C, because the growth and metabolism of microorganism were inhibited, caffeine removal ratio and theophylline production declined obviously. Therefore, 35&#;°C was the optimal temperature for the conversion of caffeine to theophylline.

Effect of incubation temperature in an aerobic fermentation inoculated by A. sydowii PET-2 on caffeine degradation (a) and theophylline production (b). Data are presented as mean value ± SD of three replications.Caffeine removal ratio&#;=&#;(Ccaffeine,0-Ccaffeine,t)/Ccaffeine,0*100%; Theophylline production&#;=&#;Ctheophylline,t-Ctheophylline,0.Ccaffeine,0 was the initial caffeine content (mg/g), Ccaffeine,t was the caffeine content (mg/g) detected in the fermentation. Ctheophylline,t was the theophylline content (mg/g) detected in the fermentation, Ctheophylline,0 was the initial theophylline content (mg/g)

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