Evaluation results of tea-derived fungi in theobromine utilization
To assess theobromine utilization capacity of tea-derived fungi, each microbial isolate was inoculated into different theobromine agar medias (TAM) and theobromine liquid mediums (TLM), respectively. Colony diameters and theobromine concentrations were determined after cultivation at 30 °C for 5 days. Colony diameters and sporulation time on TAM are recorded in Table 1, and theobromine concentrations in TLM are presented in Fig. 1. As shown in Table 1, apart from Aspergillus pallidofulvus PT-3 and Penicillium mangini PT-5, other microbial isolates had relatively high theobromine utilization capacity, such as Aspergillus niger PT-1, A. sydowii PT-2, Aspergillus sesamicola PT-4, Aspergillus ustus PT-6 and A. tamarii PT-7. Comparison of colony diameters on different TAM showed that dextrose or sucrose as carbon source could promote theobromine utilization partly. TAM-S with the maximal colony diameter was most suitable for theobromine utilization by candidate fungal strains.
Table 1 Colony diameter and sporulation time of tea-derived fungi on theobromine agar medias.
Tea-derived fungi
|
Colony diameter (cm)
|
Total diameter (cm)
|
Day of sporulation
|
TAM-D
|
TAM-N
|
TAM-S
|
TAM-T
|
TAM-D
|
TAM-N
|
TAM-S
|
TAM-T
|
A. niger PT-1
|
3.2 ± 0.1
|
2.9 ± 0.1
|
3.5 ± 0.1
|
1.0 ± 0.1
|
10.6 ± 0.3
|
4
|
-
|
5
|
-
|
A. sydowii PT-2
|
4.0 ± 0.2
|
3.3 ± 0.1
|
4.5 ± 0.1
|
1.8 ± 0.1
|
13.7 ± 0.4
|
5
|
-
|
4
|
-
|
A. pallidofulvus PT-3
|
2.2 ± 0.2
|
1.2 ± 0.2
|
2.6 ± 0.1
|
0
|
6.0 ± 0.4
|
-
|
-
|
-
|
-
|
A. sesamicola PT-4
|
2.2 ± 0.1
|
2.1 ± 0.5
|
2.4 ± 0.2
|
0.5 ± 0.1
|
7.2 ± 0.2
|
5
|
-
|
4
|
-
|
P. mangini PT-5
|
1.6 ± 0.1
|
1.5 ± 0.2
|
2.1 ± 0.1
|
0
|
5.2 ± 0.4
|
-
|
-
|
-
|
-
|
A. ustus PT-6
|
3.5 ± 0.1
|
2.7 ± 0.1
|
4.0 ± 0.2
|
1.8 ± 0.1
|
12.0 ± 0.2
|
4
|
5
|
4
|
-
|
A. tamarii PT-7
|
5.6 ± 0.2
|
4.6 ± 0.2
|
6.0 ± 0.2
|
3.0 ± 0.3
|
19.1 ± 0.4
|
3
|
4
|
2
|
4
|
TAM-D = theobromine agar media with dextrose as carbon source; TAM-N = theobromine agar media with ammonium sulphate as nitrogen source; TAM-S = theobromine agar media with sucrose as carbon source; TAM-T = theobromine agar media with theobromine as sole carbon and nitrogen source.
In this study, TLM-S, TLM-D, TLM-N, TLM-SN were prepared to select potential theobromine-degrading fungi and optimal medium in the liquid culture. As shown in Fig. 1, due to the difference in cultivation modes, A. pallidofulvus PT-3, A. sesamicola PT-4 and P. mangini PT-5 could not utilize theobromine completely in all given TLM. A. niger PT-1 just used the theobromine in TLM-S slightly. Only A. sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7 could utilize theobromine largely in the liquid culture. The additional carbon source promoted theobromine utilization capacity of A. sydowii PT-2 and A. tamarii PT-7 through enhancement of cell density in the liquid culture [19]. Particularly, the highest theobromine removal rate was found in TLM-S for the potential theobromine-degrading fungi, including A. niger PT-1, A. sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7 in the liquid culture. The composition of TLM-S was therefore chosen as the optimal medium to investigate theobromine degradation metabolites in the liquid culture.
Thebromine degradation characterization in liquid culture
A. niger PT-1, sydowii PT-2, A. ustus PT-6 and A. tamarii PT-7 were inoculated into TLM-S with an increasing theobromine concentration (100, 200 and 300 mg/L, respectively), and Tissue-culture bottles were incubated in an orbital shaker (130 rpm, 30 °C), respectively. The inoculated bottles were took every 24 h for the determination of theobromine and related metabolites by using high-performance liquid chromatography (HPLC). Theobromine concentrations (Additional file 1: Table S1) are presented in Fig. 2. Significant difference (p < 0.05) was found in theobromine concentrations between four candidate isolates. Theobromine decreased slightly (p > 0.05) in all concentrations inoculated by A. niger PT-1 and A. ustus PT-6, which showed a limited theobromine utilization capacity in the liquid culture. In time-course experiments over a period of 6 days, A. tamarii PT-7 could degrade almost all the theobromine in the liquid culture. However, theobromine degradation capacity of A. sydowii PT-2 was limited with theobromine removal rates about 61.92% and 73.12% in high substrate concentrations of 200 mg/L and 300 mg/L, respectively.
Table 2 Theobromine degradation metabolites detected in the liquid culture of four candidate isolates.
Candidate isolates
|
Metabolites
|
A. niger PT-1
|
Not found
|
A. sydowii PT-2
|
3-Methylxanthine and xanthine
|
A. ustus PT-6
|
3,7-Dimethyluric acid and 3-methyluric acid
|
A. tamarii PT-7
|
3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine and uric acid
|
TLM-S inoculated by candidate isolates were analyzed by HPLC for 3,7-dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, 7-methyluric acid, xanthine and uric acid.
Theobromine catabolic intermediates were identified by HPLC using internal standard method (Table 2). 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively in the liquid culture. The detected metabolites showed that both N-demethylation and oxidation were found in theobromine catabolism. Quantitative analysis indicated that 3-methylxanthine was common and main demethylated metabolite through N-demethylation at the N-7 position of theobromine in A. sydowii PT-2 and A. tamarii PT-7 culture. 7-Methylxanthine was inferred as the demethylated product through the N-3 demethylation in A. tamarii PT-7 culture. Xanthine was a further demethylated metabolite found in A. tamarii PT-7 culture through N-3 demethylation of 3-methylxanthine or N-7 demethylation of 7-methylxanthine. In A. ustus PT-6 and A. tamarii PT-7 culture, 3,7-dimethyluric acid, 3-methyluric acid and uric acid were direct oxidation products from theobromine, 3-methylxanthine and xanthine, respectively.
Production of 3-methylxanthine through theobromine biodegradation
3-Methylxanthine and other methylxanthines have been shown various biomedical effects as adenosine receptors and inhibitors of Primary Amine Oxidase [39, 40]. Due to high accumulation of 3-methylxanthine, 3-methylxanthine concentrations were determined by HPLC in sydowii PT-2 and A. tamarii PT-7 culture, respectively. 3-Methylxanthine concentrations are recorded in Additional file 1: Table S2 and presented in Fig. 3. The accumulation of 3-methylxanthine increased along with theobromine degradation since it was detected in the liquid culture after cultivation for 24 h. Over a 6-day period cultivation of A. sydowii PT-2 (Fig. 3a), 71.84 ± 4.44 mg/L, 92.81 ± 2.86 mg/L and 177.12 ± 14.06 mg/L of 3-methylxanthine were produced and increased significantly (p < 0.05) with an increasing initial theobromine concentration, respectively, showing a linear relationship between theobromine degradation and 3-methylxanthine accumulation. However, the accumulation of 3-methylxanthine maintained at a low level about 66.31 ± 5.68 mg/L in A. tamarii PT-7 culture with 300 mg/L theobromine, which was far below that in A. sydowii PT-2 culture. Generally, A. sydowii PT-2 showed its advantage in the production of 3-methylxanthine with 300 mg/L theobromine as feedstock in the liquid culture.
The non-linear relationship between theorbromine degradation and 3-methylxanthine accumulation in A. tamarii PT-7 culture indicated that as the main intermediate of theobromine degradation, 3-methylxanthine might be degraded by A. tamarii PT-7 and other candidate isolates in the liquid culture. To investigate 3-mehylxanthine metabolism, four candidate isolates were inoculated into a linearly increasing concentration of 3-methylxanthine from 100 mg/L to 300 mg/L, 3-methylxanthine and related metabolites were determined by HPLC (Fig. 4). Compared with other isolates, A. sydowii PT-2 and A. tamarii PT-7 could reduce 3-methylxanthine significantly (p < 0.05) in all given concentrations. Particularly, A. tamarii PT-7 degrade almost all 3-methylxanthine in a low substrate concentration (100 mg/L 3-methylxanthine), and maintained a relatively high removal rate about 34.97% in 300 mg/L substrate concentration after cultivation for 5 days. Through the analysis of related metabolites with 3-methylxanthine degradation (Additional file 1: Table S3), 3-methyluric acid, xanthine and uric acid were detected in the liquid culture, respectively. Associated with the metabolites detected in theobromine degradation, xanthine was demethylated product from 3-methylxanthine through N-3 demethylation. Alternatively, 3-methyluric acid and uric acid were direct oxidative products from 3-methylxanthine and xanthine at the C-8 position, respectively.
Effects of pH and metal ions on 3-methylxanthine production
The lower degradation capacity of 3-methylxanthine in liquid culture (Fig. 4) confirmed that A. sydowii PT-2 had application potential in production of 3-methylxanthine with theobromine as feedstock. Metal ions and pH were principal factors influencing theobromine biodegradation and 3-methylxanthine production. Two series of experiments, such as a pH range from 3 to 7 and various metal ions, including Fe2+, Ca2+, Mg2+, Mn2+, Cu2+ and Zn2+, were prepared in TLM-S to investigate the influences of pH and metal ions, respectively. A. sydowii PT-2 exhibited a high sensitivity to pH, showing the best theobromine degradation and 3-methylxanthine production at pH 5 (Fig. 5a). Cu2+ and Zn2+ restrained theobromine degradation and 3-methylxanthine production significantly (p < 0.05), only Fe2+ promoted 3-methylxanthine production significantly (p < 0.05) compared with the control (Fig. 5b).