Evaluation results of theobromine-degrading fungi on agar solid mediums and in liquid culture
To assess theobromine utilization capacity of tea-derived fungi, each isolate was inoculated into different theobromine agar mediums (TAM) and theobromine liquid mediums (TLM), respectively. Colony diameters and theobromine concentrations were determined after cultivation at 30 °C for 5 days. Colony diameters on solid mediums were recorded in Table 1 and theobromine concentrations in liquid culture were presented in Fig. 1. Based on the colony diameter values set for categorizing theobromine utilization on agar medium, apart from Aspergillus pallidofulvus and Penicillium mangini, other isolates such as A. niger, A. sydowii, Aspergillus sesamicola, Aspergillus. ustus and A. tamarii had relatively high theobromine utilization capacity on all given agar mediums. Through comparisons, dextrose or sucrose as carbon source could promote theobromine utilization partly. TAM-S with the maximal colony diameters was most suitable for theobromine utilization by tea-derived fungi on agar mediums.
Table 1. Colony diameters of tea-derived fungi on theobromine agar mediums.
Tea-derived fungi
|
Colony diameter(cm)
|
Total diameter(cm)
|
TAM-D
|
TAM-N
|
TAM-S
|
TAM-T
|
A. niger
|
3.2 ± 0.1
|
2.9 ± 0.1
|
3.5 ± 0.1
|
1.0 ± 0.1
|
10.6 ± 0.3
|
A. sydowii
|
4.0 ± 0.2
|
3.3 ± 0.1
|
4.5 ± 0.1
|
1.8 ± 0.1
|
13.7 ± 0.4
|
A. pallidofulvus
|
2.2 ± 0.2
|
1.2 ± 0.2
|
2.6 ± 0.1
|
0
|
6.0 ± 0.4
|
A. sesamicola
|
2.2 ± 0.1
|
2.1 ± 0.5
|
2.4 ± 0.2
|
0.5 ± 0.1
|
7.2 ± 0.2
|
P. mangini
|
1.6 ± 0.1
|
1.5 ± 0.2
|
2.1 ± 0.1
|
0
|
5.2 ± 0.4
|
A. ustus
|
3.5 ± 0.1
|
2.7 ± 0.1
|
4.0 ± 0.2
|
1.8 ± 0.1
|
12.0 ± 0.2
|
A. tamarii
|
5.6 ± 0.2
|
4.6 ± 0.2
|
6.0 ± 0.2
|
3.0 ± 0.3
|
19.1 ± 0.4
|
TAM-D = theobromine agar medium with dextrose as carbon source; TAM-N = theobromine agar medium with ammonium sulphate as nitrogen source; TAM-S = theobromine agar medium with sucrose as carbon source; TAM-T = theobromine agar medium 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 liquid culture. As shown in Fig. 1, although all isolates could survive with a high growth, due to the difference in cultivation modes, A. pallidofulvus, A. sesamicola and P. mangini could not utilize theobromine completely in all liquid mediums. A. niger just used the theobromine in TLM-S slightly. Only A. sydowii, A. ustus and A. tamarii could utilize theobromine largely in given mediums. The additional carbon source promoted theobromine utilization capacity of A. sydowii and A. tamarii through enhancement of cell density in liquid culture. Particularly, theobromine-sucrose medium had a highest theobromine removal rates for the potential theobromine-degrading fungi including A. niger, A. sydowii, A. ustus and A. tamarii in liquid culture. The composition of TLM-S was therefore chosen as the optimal medium to investigate theobromine degradation metabolites in liquid culture inoculated by elect isolates.
Thebromine degradation characterization and metabolites in liquid culture
A. niger, sydowii, A. ustus and A. tamarii were inoculated into TLM-S with increasing theobromine concentrations (100, 200 and 300 mg/L, respectively), and Tissue-culture bottles were incubated in an orbital shaker (130 rpm, 30 °C). The inoculated bottles were took every 24 h for the determinations of theobromine and related metabolites by using high-performance liquid chromatography (HPLC). Theobromine concentrations are presented in Fig. 2. There was significant difference (p < 0.05) in theobromine concentrations between four candidate isolates. Theobromine decreased slightly (p > 0.05) in all concentrations inoculated by A. niger and A. ustus, which showed the limited theobromine utilization capacity in liquid culture. In time-course experiments over a period of 6 days, A. tamarii could degrade almost all the theobromine in given substrate concentrations. However, in high substrate concentrations, theobromine degradation capacity of A. sydowii were limited with theobromine removal rates about 61.92% and 73.12% in the 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
|
Not found
|
A. sydowii
|
3-Methylxanthine and xanthine
|
A. ustus
|
3,7-Dimethyluric acid and 3-methyluric acid
|
A. tamarii
|
3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine and uric acid
|
TLM-S mediums 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 position N-7 of theobromine in A. sydowii and A. tamarii culture. 7-Methylxanthine was inferred as the demethylated product through N-demethylation at the position N-3 of theobromine in A. tamarii culture. Xanthine was a further demethylated metabolite found in A. tamarii culture through N-demethylation at the position N-3 of 3-methylxanthine or N-7 of 7-methylxanthine. In A. ustus and A. tamarii 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 [33, 34]. Due to theobromine degradation characterization that 3-methylxanthine accumulated largely, 3-methylxanthine concentrations were determined by HPLC in sydowii and A. tamarii culture, respectively, and the results are presented in Fig. 3. 3-Methylxanthine was accumulated largely along with theobromine degradation science it was detected in the culture after cultivation for 24 h. Over a 6-day period cultivation of A. sydowii (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 increasing initial theobromine concentrations, respectively, showing a linear relationship between theobromine degradation and 3-methylxanthine accumulation. However, despite a significant (p < 0.05) increasing of theobromine degradation in a high initial substrate concentration (300 mg/L theobromine), the accumulated concentration of 3-methylxanthine maintained at a low level about 66.31 mg/L in A. tamarii culture, which was far below that in A. sydowii culture. Generally, A. sydowii showed the advantage in the production of 3-methylxanthine with 300 mg/L of theobromine as feedstock in liquid culture.
The non-linear relationship between theorbromine degradation and 3-methylxanthine accumulation in A. tamarii culture indicated that as the main intermediate of theobromine degradation, 3-methylxanthine might be degraded by A. tamarii and other candidate isolates in liquid culture. To investigate 3-mehylxanthine 3, four candidate isolates were inoculated into linearly increasing concentrations of 3-methylxanthine from 100 mg/L to 300 mg/L, 3-methylxanthine and related metabolites were determined by HPLC. As shown in Fig. 4, compared with other isolates, A. sydowii and A. tamarii could reduce 3-methylxanthine significantly (p < 0.05) in all given concentrations. Particularly, A. tamarii degrade almost all 3-methylxanthine in low substrate concentration (100 mg/L 3-methylxanthine), and maintained a high removal rate of 3-methylxanthine about 34.97% in a substrate concentration of 300 mg/L after cultivation for 5 days. Through the analysis of related metabolites with 3-methylxanthine degradation (Additional file 1: Table S1), 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-demethylation at N-3 position. 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 theobromine biodegradation and 3-methylxanthine production
Due to the lower degradation capacity of 3-methylxanthine in liquid culture, A. sydowii had application potential in production of 3-methylxanthine with theobromine as feedstock. pH value and metal ion species were principal factors influencing theobromine biodegradation and 3-methylxanthine production. Summarized the achievements in previous studies [35, 36] , series of pH within the range from 3 to 7 in increments of one unit and metal ions including Fe2+, Ca2+, Mg2+, Mn2+, Cu2+ and Zn2+ were prepared in TLM-S medium with 300 mg/L of theobromine, respectively. Theobromine and 3-methylxanthine were determined by HPLC, and the results are demonstrated in Fig. 5. A. sydowii 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).