Abstract
Taiwanofungus camphoratus has attracted much attention because it can abundantly produce various active substances that exhibit blood-sugar lowering, immunity improving, and antioxidant properties. Currently, T. camphoratus is cultured using four main methods: cutting wood culture, solid-state fermentation, submerged fermentation, and dish culture. T. camphoratus produces different metabolites under different culture methods. In this study, nontargeted metabolomics was used to compare the metabolites of T. camphoratus produced under these four culture methods. Principal component analysis and supervised partial least squares-discriminant analysis were used to analyze the differences in the metabolites. Moreover, in vitro hypoglycemic activity of T. camphoratus extracts produced under four culture methods was compared by assessing their ability to inhibit the activity of α-glucosidase, α-amylase, and sucrase. A total of 186 metabolites were identified. In total, 127 metabolites were common under the four culture methods. Under solid-state fermentation, submerged fermentation, and cutting wood culture, 12, 1, and 4 metabolites were unique, respectively. The differential metabolites produced by T. camphoratus under four culture methods were mainly triterpenoids, phenolic compounds, and fatty acid compounds. α-glucosidase, α-amylase, and sucrase activity inhibition was the best using T. camphoratus extract obtained under cutting wood culture; the inhibition rates were 55.97%, 51.96%, and 78.02%, respectively, which were comparable to those exhibited by 0.001, 3, and 12 mg/mL acarbose (positive control). The metabolites produced by T. camphoratus and α-glucosidase, α-amylase, and sucrase inhibitory activities were different under the four culture methods. Cutting wood culture exhibited the best enzyme inhibitory activity. This study provided a theoretical basis for further use and development of various culture methods for the rational production of active metabolites of T. camphoratus.
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Introduction
Taiwanofungus camphoratus (M. Zang & C.H. Su) Sheng H. Wu, Z.H. Yu, Y.C. Dai & C.H. Su, belongs to the division Basidiomycota, class Agaricomycetes, order Polyporales, family Incertae sedis, and genus Taiwanofungus. It is a famous, rare, edible, and medicinal fungus in China, mainly distributed in Taiwan Province of China. It is known as “forest ruby” [1, 2]. T. camphoratus is used since ancient times as a folk medicine. Taiwan aborigines used T. camphoratus as an effective cure for alcoholism and believed that regular consumption of T. camphoratus could maintain the vitality of body and prolong life [3]. Although T. camphoratus is being used since a long time, official literature reports on this fungus began to get published in the 1990s in China [4]. After years of research, it is currently confirmed that T. camphoratus, Antrodia cinnamomea T.T. Chang & W.N. Chou, and Antrodia camphorata Sheng H. Wu, Ryvarden & T.T. Chang can represent T. camphoratus [5,6,7]. T. camphoratus exhibits various therapeutic properties such as blood-sugar lowering, immunity improving, and antioxidant activities.
Cinnamomum kanehirae, an endemic plant species, in Taiwan Province of China is the only natural host of wild T. camphoratus. Therefore, obtaining the wild fruiting body of T. camphoratus is extremely rare. Moreover, in recent years, the resources of C. kanehirae are becoming increasingly scarce due to the wanton deforestation, making the wild fruiting bodies of T. camphoratus even harder to obtain. Therefore, the development and utilization of wild T. camphoratus resources is difficult. T. camphoratus resources can be obtained via artificial culture. At present, for the artificial culture of T. camphoratus, four methods are commonly used: cutting wood culture, solid-state fermentation, submerged fermentation, and dish culture.
For cutting wood culture, earlier, wood from C. kanehirae was used for the inoculation and culture of T. camphoratus. However, due to the scarcity and difficulty in obtaining this wood, researchers began to culture T. camphoratus on the wood of other trees. The composition and pharmacological activity of T. camphoratus cultured using cutting wood culture are the closest to those of wild T. camphoratus; however, it takes up to 2–3 years to obtain the fruiting body using this method. Therefore, it is difficult to meet the market demand for T. camphoratus only via cutting wood culture.
Solid-state fermentation does not exhibit the limitations of cutting wood culture to grow wild T. camphoratus (e.g., scarcity of C. kanehirae wood). In solid-state fermentation, metabolites of T. camphoratus with chemical characteristics and biological activities similar to those of wild T. camphoratus can be obtained [8]. In this method, mostly grains are used as raw materials. This method has the advantages such as high yield and less pollution. Antrodin C and Antroquinonol compounds are abundantly obtained in the solid-state fermentation of T. camphoratus [9]. Currently, solid-state fermentation is widely used to culture T. camphoratus; although it yields abundant active substances, it requires longer culture time than submerged fermentation [10].
In submerged fermentation, also known as deep fermentation, microorganisms are grown in a liquid medium containing the required nutrients [11]. This is the most commonly used method to culture T. camphoratus in factories, as well as laboratories [12]. Submerged fermentation method exhibits advantages such as short production cycle, high growth rate, and rapid maturation process; thus, more mycelia can be obtained in short time [13]. However, compared with other culture methods, the types of active substances from T. camphoratus obtained are few [14]. The mycelia obtained via conventional submerged fermentation mainly contain maleic acid and succinic acid derivatives, mainly Antrodin C, and some metabolites are differentially produced by different strains [15]. The content of Antroquinol in T. camphoratus is less when cultured using ordinary submerged fermentation. If the precursor of Antroquinol is added during the culture, submerged fermentation of T. camphoratus can be used to synthesize Antroquinol [16]. Many studies have reported the strategies to improve the biomass and content of active substances in the mycelium of T. camphoratus under submerged fermentation [17]. Liu et al. directly used the mycelial fermentation broth to study the pharmacological activity of T. camphoratus and reported that it could inhibit angiogenesis through HIF-1α, thus disrupting the characteristics of tumor stem cells and improving the radiosensitivity of esophageal cancer cells. This auxiliary effect can enhance the effect of radiotherapy in esophageal cancer. In addition, mycelial fermentation broth of T. camphoratus can inhibit the epithelial–mesenchymal transition in esophageal cancer cells [18,19,20].
Dish culture is a commonly used method to culture microorganisms in laboratory. Dish culture of T. camphoratus exhibits advantages such as convenience and moderate culture period and is now used in the production of T. camphoratus [3]. Under dish culture, T. camphoratus produces various metabolites including triterpenoids, which are similar to those found in wild T. camphoratus [21]. However, the limitations of this method include extensive use of material (dishes) and labor resources, increasing the cost, and the growth of T. camphoratus is limited because of small volume of the dish [3, 12].
Differences in the culture conditions under various culture methods result in differences in the composition of T. camphoratus [22]. Therefore, for the appropriate application of T. camphoratus and to rationally obtain various active ingredients, it is urgent to study and optimize different culture methods. Though T. camphoratus is rich in various active substances, current studies mainly focus on the characteristic components such as triterpenoids and androquinol. Only few studies are available on other components of T. camphoratus, such as phenolic compounds. In addition, the metabolites produced by T. camphoratus under various culture methods are different. Therefore, analysis of metabolites produced by T. camphoratus under various culture methods is important for the rational use of T. camphoratus.
In this study, nontargeted metabolomic analysis was used to compare the metabolites produced by T. camphoratus under four culture methods. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to analyze the differences in the metabolites under these culture methods. The ability of T. camphoratus to lower blood sugar level was assessed by studying inhibition of the activity of α-glucosidase, α-amylase, and sucrase by T. camphoratus extracts obtained under the four culture methods. This study laid a theoretical foundation for further development and use of various culture methods of T. camphoratus to obtain specific active ingredients.
Materials and methods
Material and reagent
Material
T. camphoratus was purchased from the Guangdong Microbial Strain Conservation Center (GIM No. 5.530) and preserved as a slant culture on PDA medium (composition: 200 g potato, 20 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 3 g yeast extract powder, and 20 g agar in 1 L distilled water). Its molecular identification was performed via amplification of the ITS gene.
Reagents
Anhydrous ethanol was purchased from Chengdu Cologne Chemicals Co., Ltd. (Chengdu, China). Oleanolic acid, vanillin, potassium sodium tartrate, 3,5-dinitrosalicylic acid, and 4-nitrophenyl-β-d-glucopyranoside (PNPG) were purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China). Glacial acetic acid, perchloric acid, methanol, sodium hydroxide, phenol, sucrose, sodium carbonate, and soluble sucrose were purchased from Sinopharm Chemical Reagent Co., Ltd. (Tianjin, China). All reagents were of analytical grade. Three enzymes, namely, α-glucosidase (100 U/mg), α-amylase (50 U/mg), and invertase (sucrase) (≥ 200 U/mg), were purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China).
Methods
Methods to culture T. camphoratus
T. camphoratus was cultured using cutting wood culture, solid-state fermentation, submerged fermentation, and dish culture at 26 °C. Raw materials were weighed using SQP electronic balance (Sartorius Scientific Instruments). The media for all culture methods were autoclaved prior to use.
For cutting wood culture, apple wood was used, inoculated with T. camphoratus, and cultivated for 2 years.
The medium for solid-state fermentation was oats with twice the amount of water. The T. camphoratus samples were collected at 30, 45, 60, 75, and 90 days of solid-state fermentation.
Submerged fermentation medium was prepared by mixing 200 g potato, 20 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, and 3 g yeast extract powder. Submerged fermentation was performed at 120 rpm. The T. camphoratus samples were collected at 10, 15, 20, 25, and 30 days of submerged fermentation.
For dish culture, the medium was the same as that used for submerged fermentation but added with 20 g agar. The T. camphoratus samples were collected at 20, 25, 30, 35, and 40 days of dish culture.
The obtained fruiting bodies and mycelia were separately dried in an oven (DHT-450A high-temperature blast drying oven, Shanghai Dao-han Industry Co., Ltd.) at 45 °C, crushed in a pulverizer (2500C pulverizer, Yongkang Red Sun Mechanical and Electrical Co., Ltd.), sieved (through a 0.425-mm sieve), and stored at 4 °C till further analysis.
Construction of standard curve of triterpenoids
The content of triterpenoids in the mycelia of T. camphoratus cultured using various methods was determined using vanillin–perchloric acid colorimetric method [23]. For constructing the standard curve for this experiment, 1 mg/mL oleanolic acid was used as the standard solution. In total, 25, 50, 75, 150, and 200 μL of the standard solution was taken in test tubes and evaporated to dryness in boiling water. To this, 0.1 mL of freshly prepared 5% vanillin–glacial acetic acid solution and 0.4 mg/mL perchloric acid solution was added, followed by heating in water bath (HHS-21-6 constant temperature water bath, Shanghai Boxun Industry Co., Ltd. Medical Equipment Factory) at 60 °C for 20 min and cooling to room temperature. To this, 5 mL glacial acetic acid solution was added and mixed well. The experiment included blank control and three replicates for each concentration. The absorbance was measured at 548 nm using T6 new Yue visible spectrophotometer, Beijing Persee General Instru-ment Co., Ltd.). The abscissa was concentration of oleanolic acid, and the ordinate was the absorbance value. The standard curve was constructed, and the regression equation was obtained as y = 6.9361x − 0.0815 (R2 = 0.9999).
Determination of triterpenoid content in T. camphoratus
To weigh 40 mg of T. camphoratus mycelium powder, 1 mL of 70% ethanol was added, and the mixture was placed in an ultrasonic extractor (KQ-500DE ultrasonic extractor, Kunshan Ultrasonic Instruments Co., Ltd.) for 40 min at 60 °C with power 400 W. The mixture was centrifuged (Neo15R high-speed refrigerated centrifuge, Shanghai Lishen Scientific Instruments Co., Ltd.) at 12,000 rpm for 5 min. The supernatant is hereafter referred to as the “crude extract.” Using the supernatant, the triterpenoid content of T. camphoratus mycelia cultured using solid-state fermentation, dish culture, and submerged fermentation for various culture times was determined according to the method described in “Construction of standard curve of triterpenoids” section. The triterpenoid content of fruiting bodies of T. camphoratus cultured via cutting wood method served as the control. The duration (days) of culture was determined at which the highest triterpenoid content was obtained under each culture method.
Detection of metabolite profile of T. camphoratus using liquid chromatography–electrospray ionization–tandem mass spectrometry
The suspension of 40 mg/mL T. camphoratus in 70% ethanol was centrifuged. After centrifugation, the supernatant was collected, evaporated, redissolved in methanol, mixed, and centrifuged. The supernatant was collected and used for the analysis of metabolite profile as given below.
Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) was used for evaluating metabolite profile using the extracts of T. camphoratus obtained from four culture methods. The analysis was conducted using Thermo Fisher UPLC-Q Exactive Focus (Symefeishier Technology Company) equipped with an electrospray ionization source (ESI). The C18 column (100 × 2.1 mm, 3 μm) was used for the chromatographic separation. The solvent flow rate was 0.4 mL/min, and 2 μL of all samples and standards was injected. The mobile phase was solvent A (water with 0.1% formic acid) and solvent B (methanol). The mobile phase gradient was programmed as follows: 65% A (v/v) from 0 to 10.0 min, 30% A (v/v) from 10.0 to 30.0 min, 5% A (v/v) from 30.0 to 35.0 min, 65% A (v/v) from 35.0 to 35.1 min, and 65% A (v/v) from 35.1 to 40.0 min. The total run time was 40 min, and the column temperature was maintained at 40 °C.
Inhibition of α-glucosidase activity
α-Glucosidase activity inhibition rate was assessed using a method by Masao Hattori with slight modifications [24]. Acarbose was used as the positive control. In total, 20 μL of the crude extract of T. camphoratus or 20 μL of 0.001 mg/mL acarbose, 20 μL of 5 U/mL α-glucosidase solution, and phosphate buffer (to make up the total volume to 90 μL) were added to 96-well plates, mixed, and incubated at 37 °C for 30 min. To this, 20 μL PNPG solution (15 mM) was added, mixed, and incubated at 37 °C for 60 min. Finally, sodium carbonate solution was added to terminate the reaction. The absorbance of the released p-nitrophenol was measured at 405 nm using a microplate reader. The sample blank group contained the sample (or acarbose) but phosphate buffer was added instead of α-glucosidase solution. The control group contained phosphate buffer instead of sample (or acarbose). The control blank group contained phosphate buffer instead of sample (or acarbose) and PNPG. The inhibition rate of α-glucosidase can be calculated using formula (1).
where A0, A, Ax, and Ay are the absorbance of control group, control blank group, sample group, and sample blank group, respectively.
Inhibition of α-amylase activity
The inhibition of α-amylase activity was assessed according to the method by Wang [25]. Acarbose was used as the positive control. First, DNS solution was prepared as follows. In total, 91 g of potassium sodium tartrate was dissolved in 250 mL distilled water. The mixture was heated at low temperature until potassium sodium tartrate was dissolved. Further, 10.5 g of sodium bicarbonate, 0.15 g of 3, 5-dinitrosalicylic acid, and 2.5 g of phenol were added and stirred to dissolve. The mixture was cooled, and the volume was adjusted to 500 mL using distilled water. The mixture was stored in dark for 1 week before using.
The crude extract of T. camphoratus (50 μL) or 50 μL of acarbose solution (3 mg/mL) were added to a centrifuge tube. To this, 50 μL of α-amylase solution (10 U/mL) was added and incubated at 37 °C for 10 min. To this, 100 μL of 1% soluble starch solution was added, mixed, and incubated at 37 °C for 10 min. Further, to terminate the reaction, 200 μL of DNS solution was added. The mixture was placed in boiling water bath for 5 min, taken out, cooled, and diluted with 1 mL distilled water. The absorbance was measured at 540 nm using a spectrophotometer. The inhibition rate of α-amylase was calculated using formula (1).
Inhibition of sucrase activity
The inhibition rate of sucrase was determined according to the method by Zhang with slight modifications [26]. Acarbose was used as the positive control. The crude extract of T. camphoratus (250 μL) or 250 μL of acarbose solution (12 mg/mL) was mixed with 250 μL of sucrase solution (200 U/mL) in a centrifuge tube and incubated at 37 °C for 10 min. To this, 500 μL of sucrose solution was added, mixed, and incubated at 37 °C for 20 min. Further, to terminate the reaction, 1 mL of DNS solution was added. The mixture was placed in boiling water bath for 5 min, taken out, and allowed to cool at room temperature. The absorbance was measured at 550 nm using a spectrophotometer. The inhibition rate of sucrase was calculated using formula (1).
Data analysis
The results of triterpenoid content and enzyme activity inhibition were analyzed using SPSS (IBM SPSS Statistics 26) software. Data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey’s post-hoc test (P < 0.05) was used to determine significant differences between the means. Ingredient testing data of the results were analyzed using Compound Discoverer software. Nontargeted analysis of the experimental results was performed using mz Cloud, mz Vault, and self-built databases [12]. Unsupervised PCA and supervised PLS-DA were conducted to explore the differences in the metabolites of T. camphoratus produced under four culture methods. The analysis of differential metabolites was performed using the free online data analysis platform OmicShare tool (https://www.omicshare.com/tools).
Results and analysis
Effect of different culture time on the triterpenoid content of T. camphoratus
Triterpenoids are the characteristic products of T. camphoratus. Our previous study reported that wild and artificially cultured T. camphoratus have some comparable characteristic triterpenoids with similar hepatoprotective activities [27].
The triterpenoid content of T. camphoratus mycelia cultured using solid-state fermentation, dish culture, and submerged fermentation for various culture times was determined. The triterpenoid content of T. camphoratus mycelia cultured via cutting wood method served as the control (Figs. 1, 2, 3). The result revealed that under solid-state fermentation, the triterpenoid content of T. camphoratus mycelia was the highest after 75 days of culture. Under submerged fermentation, it was the highest after 25 days of culture. Under dish culture, triterpenoid content increased with time and tended to be stable after 30 days. After comprehensive consideration including optimal time and high triterpenoid content, dish culture for 25 days was the best to obtain triterpenoids from T. camphoratus. For further analysis, the T. camphoratus samples collected at 75, 25, and 25 days under solid-state fermentation, submerged fermentation, and dish culture, respectively, were selected with three replicates in each group.
Effect of different culture time on the content of triterpenoids in the mycelia of Taiwanofungus camphoratus under solid-state fermentation
Effect of different culture time on the content of triterpenoids in the mycelia of T. camphoratus under submerged fermentation
Effect of different culture time on the content of triterpenoids in the mycelia of T. camphoratus under dish culture
The effect of different culture time on the content of triterpenoids of T. camphoratus in the fruiting bodies was not included in this part, it is because the fruiting bodies grows very slowly. The fruiting bodies of T. camphoratus grow very slowly, and the triterpenoids in them are still accumulating after 5 years, but the content change of triterpenoids in them is very little in a few months. Therefore, triterpenoids content was not determined regularly as the other culture methods. The optimum culture time can be obtained by the determination of triterpenoids content in the other culture methods, and for the fruiting body, it seems that the longer the culture time, the higher the content of triterpenoids. In the following study, we selected the fruiting bodies of T. camphoratus that had grown on apple wood for 3 years.
Metabolite profile of T. camphoratus cultured using various methods
The metabolite profile of T. camphoratus cultured using various methods was determined using LC–ESI–MS/MS (Thermo Fisher UPLC-Q Exactive Focus, Thermo Fisher Scientific Inc.).
For this experiment, 10 μL of various samples was mixed, which was termed as quality control (QC) sample, and 100 μL of QC sample was injected for analysis.
The metabolites in the samples were analyzed according to their precise molecular weight, and fragment ions were obtained. A total of 186 metabolites were detected in the positive ion mode. The total ion current spectrum of the QC sample of the extract of artificially cultured T. camphoratus is shown in Additional file 1. Under solid-state fermentation, dish culture, submerged fermentation, and cutting wood culture, 158, 161, 157, and 160 metabolites of T. camphoratus were obtained, respectively. Therefore, T. camphoratus cultured using various methods contained abundant metabolites (Fig. 4). In total, 127 metabolites were common among the four culture methods. Under solid-state fermentation, submerged fermentation, and cutting wood culture, 12, 1, and 4 metabolites were unique, respectively. These metabolites were classified according to the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and MeFSAT database (https://cb.imsc.res.in/mefsat/) (Table 1).
Venn diagram of the metabolites of T. camphoratus produced under various culture methods
PCA of metabolites of T. camphoratus produced under four culture methods
Unsupervised PCA was conducted to explore the differences in the metabolites of T. camphoratus produced under four culture methods. PCA can show the rules of data through a few principal components while retaining as much original data as possible. The results of PCA indicated that the data in the four groups were well duplicated, and differences existed among the four groups (Fig. 5). The four culture methods of T. camphoratus could be distinguished to a certain extent. In addition, the metabolites of T. camphoratus cultured using dish culture and submerged fermentation were more similar under principal component 1 and were clearly distinguishable from those of T. camphoratus cultured using solid-state fermentation and cutting wood culture. The mycelia of T. camphoratus cultured using solid-state fermentation and fruiting bodies of T. camphoratus cultured using cutting wood culture could not be completely distinguished under principal component 2, and the difference was small.
Principal component analysis of metabolites of T. camphoratus produced under four culture methods (G1: solid-state fermentation; G2: dish culture; G3: submerged fermentation; G4: cutting wood culture)
PLS-DA of the metabolites of T. camphoratus produced under four culture methods
Supervised PLS-DA was performed to explore the differences among the metabolites produced using four methods. PLS-DA more focuses on the analysis of differences among groups. The results revealed that the data from four groups did not overlap; the distinction was obvious, and the fitting accuracy of the model was good (Fig. 6). Compared with the metabolites of T. camphoratus cultured using cutting wood culture, those in the other three groups were significantly different. The metabolites of T. camphoratus cultured using solid-state fermentation and submerged fermentation were more similar.
Partial least squares-discriminant analysis of metabolites of T. camphoratus cultured using four different methods (G1: solid-state fermentation; G2: dish culture; G3: submerged fermentation; G4: cutting wood culture)
Analysis of significantly differential metabolites of T. camphoratus cultured using four different methods
Based on the PLS-DA model, 20 metabolites with significant differences were screened under the condition of P < 0.05 and VIP > 1. Through the cluster heat map (Fig. 7), the differences among the metabolites could be clearly seen. Under cutting wood culture and solid-state fermentation, the metabolites with significant differences were mostly triterpenoids and fatty acid compounds, respectively. The top-20 differential metabolites were analyzed in Table 2. These metabolites were observed to be important for production of food products, medicine.
Heat map analysis of 20 differential metabolites of T. camphoratus produced under four culture methods (G1: solid-state fermentation; G2: dish culture; G3: submerged fermentation; G4: cutting wood culture)
In addition, 166 metabolites produced by T. camphoratus were common under all four culture methods (see it in Additional file 1). These metabolites include some characteristic components of T. camphoratus, such as Antcin E and AntcinF. Further studies should assess whether these compounds can be obtained from T. camphoratus via culture methods (other than cutting wood culture) with short culture time and easy bulk production.
Analysis of metabolites with significant differences among the four groups
The differences in the metabolites of T. camphoratus produced under different culture methods were further analyzed using PLS-DA model (Fig. 8). The results revealed that the metabolites of T. camphoratus produced under different culture methods could be distinguished by pairwise comparison, which once again proved that there were differences in the metabolites of T. camphoratus under different culture methods.
Score plot of PLS-DA model for two groups (a G1 vs G2; b G1 vs G3; c G2 vs G3; d G1 vs G4; e G2 vs G4; f G3 vs G4; G1: solid-state fermentation; G2: dish culture; G3: submerged fermentation; G4: cutting wood culture)
Based on the PLS-DA model of pairwise comparison, with the threshold of P < 0.05 and VIP > 1, the metabolites with significant differences were screened to plot the heat map (Fig. 9). Compared with solid-state fermentation, the triterpenoid expression under dish culture was higher, and the main fatty acids and phenolic compounds produced by T. camphoratus were significantly different under these two methods. Compared with solid-state fermentation, the upregulated metabolites under submerged fermentation were mostly phenolic compounds. Compared with dish culture, the upregulated metabolites under submerged fermentation were mostly phenolic compounds. Compared with solid-state fermentation, dish culture, and submerged fermentation, the upregulated metabolites under cutting wood culture were mostly terpenes and mainly triterpenoids, which are the characteristic metabolites of T. camphoratus (Table 3).
Heat map analysis of differential metabolites between two groups (a G1 vs G2; b G1 vs G3; c G2 vs G3; d G1 vs G4; e G2 vs G4; f G3 vs G4; G1: solid-state fermentation; G2: dish culture; G3: submerged fermentation; G4: cutting wood culture)
Inhibitory activity of α-glucosidase, α-amylase and sucrase
α-Glucosidase is one of the key components affecting postprandial blood glucose and is considered an important target for the treatment of diabetes [60]. The inhibition of α-glucosidase activity by the crude extracts of T. camphoratus cultured using various methods was assessed (Fig. 10A). Acarbose was used as the positive control. Under solid-state fermentation, submerged fermentation, dish culture, and cutting wood culture, the inhibition rates of α-glucosidase activity by T. camphoratus crude extracts were 23.86%, 17.37%, 26.56%, and 55.97%, respectively. The inhibition rate of α-glucosidase activity by 0.001 mg/mL acarbose was 59.87%. The rates of α-glucosidase activity inhibition by acarbose and T. camphoratus extract under cutting wood culture were not significantly different.
A Inhibition rate of α-glucosidase activity by T. camphoratus extracts; B inhibition rate of α-amylase activity by T. camphoratus extracts; C inhibition rate of α-amylase activity by T. camphoratus extracts. (T. camphoratus crude extract: 40 mg/mL; acarbose: 0.001 mg/mL)
The inhibition rates of α-amylase activity by T. camphoratus crude extract obtained under solid-state fermentation, submerged fermentation, dish culture, and cutting wood culture were 39.43%, 28.98%, 47.52%, and 51.96%, respectively (Fig. 10B). The rate of α-amylase activity inhibition by 3 mg/mL acarbose was 51.17%. The rates of α-amylase activity inhibition by acarbose and T. camphoratus extract under dish culture and cutting wood culture were not significantly different.
The inhibition rates of sucrase activity by T. camphoratus crude extracts obtained under solid-state fermentation, submerged fermentation, dish culture, and cutting wood culture were 49.96%, 41.61%, 62.23%, and 78.02%, respectively (Fig. 10C). The inhibition rate of sucrase activity by 12 mg/mL acarbose was 76.92%. The rates of sucrase activity inhibition by acarbose and T. camphoratus extract under cutting wood culture were not significantly different.
Although the above experiments have confirmed that the extract of T. camphoratus has the inhibitory activity to the target proteins related to diabetes, the specific substances that play the main role still need to be further verified. According to the current research results, triterpenoids are mainly involved, and the content and types of triterpenoids directly affect the enzyme inhibitary activity. In the future, we will continue to conduct network pharmacology studies, and to isolate and purify the active substances of T. camphoratus to further verify their enzyme inhibition efficacy.
Discussion
In recent years, studies on T. camphoratus focused on its characteristic metabolites such as triterpenoids and androquinol and their activities. However, T. camphoratus produces abundant active metabolites and produces different metabolites under different culture methods. Most studies have only used liquid chromatography–tandem mass spectrometry or high-pressure liquid chromatography to compare the characteristic components of T. camphoratus under different culture methods. However, no study has compared the metabolome of T. camphoratus under different culture methods, and this is the first study to perform nontargeted metabolome analysis of T. camphoratus cultured using cutting wood culture, dish culture, solid-state fermentation, and submerged fermentation.
In this study, the metabolites of T. camphoratus produced under four culture methods were statistically analyzed in detail. First, the content of total triterpenoids in T. camphoratus under solid-state fermentation, dish culture, and submerged fermentation was measured and compared with that in the fruiting bodies of T. camphoratus cultured using cutting wood culture. The metabolites produced under four methods were analyzed using LC–ESI–MS/MS. A total of 186 compounds were detected including 61 terpenoids, 22 nitrogen-containing compounds, 42 phenolic compounds, 2 glycoside compounds, 3 amino acid compounds, 1 vitamin compound, 26 fatty acid compounds, and 29 other compounds. These compounds were classified according to the PubChem database and the MeFSAT database. T. camphoratus produced abundant metabolites under four culture methods.
Multivariate statistical analysis can simplify and reduce the dimension of complex data while retaining the original information to the greatest extent. PCA and PLS-DA were used to explore the similarities and differences in the metabolites produced via four culture methods. Multivariate statistical analysis revealed that the differences in the metabolites of T. camphoratus under four culture methods were mainly in terms of some terpenes, phenolic compounds, and fatty acid compounds. The metabolites without significant differences were mainly nitrogen-containing compounds, glycosides, some terpenes, and some phenolic compounds.
The significant upregulation of fatty acid compounds under solid-state fermentation may be related to the rich active substances such as oleic acid, linolenic acid, and palmitic acid in oat medium [61]. The significant upregulation of phenolic compounds in solid-state fermentation of T. camphoratus may also be related to the use of oats as a medium, which is consistent with the results of Drzymaa et al. [62]. Therefore, culturing T. camphoratus with oats as a medium can provide more abundant fatty acids and phenolic compounds.
The significant upregulation of phenolic compounds under submerged fermentation is related to the culture cycle and liquid medium. Under submerged fermentation, the accumulation of phenolic substances is more obvious, and because the culture cycle is relatively short, the generated phenolic substances are degraded less. At present, the process of obtaining phenolic substances in edible and medicinal fungi by submerged fermentation is well developed, and several phenolic acids can be obtained by submerged fermentation [63,64,65]. The significant upregulation of triterpenoids under cutting wood culture in this study was consistent with the results of other studies [66, 67]. This may be because the culture conditions of T. camphoratus under cutting wood culture are closest to those in natural habitat, providing suitable culture conditions and substrates for the accumulation of triterpenoids.
The inhibition of α-glucosidase, α-amylase, and sucrase by T. camphoratus extract was studied. The α-glucosidase and α-amylase inhibitory abilities of the crude extracts were different under the four culture methods, and they were the best under cutting wood culture. This could be because of the differences in the active substances produced by T. camphoratus under different culture methods, and the unique triterpenoids produced by T. camphoratus under cutting wood culture may play an important role in this. The α-amylase inhibitory activity of the same concentration of T. camphoratus crude extracts under dish culture and cutting wood culture was better than that of the extracts under other two culture methods. This could be because the content of active ingredients with α-amylase inhibitory activity was higher under dish culture and cutting wood culture. PCA and PLS-DA analysis revealed that these active substances with good α-amylase inhibitory ability were mainly triterpene compounds. Therefore, the extract of T. camphoratus obtained under cutting wood culture exhibited superior enzyme inhibitory ability than that obtained under other culture methods, which may be related to rich triterpenoid content produced under cutting wood culture.
In summary, all four methods, namely, solid-state fermentation, submerged fermentation, dish culture, and cutting wood culture of T. camphoratus provided valuable metabolites. The differences in the metabolites under different culture methods lead to differences in enzyme inhibitory activity. Further studies are needed to explore whether this difference in enzyme inhibitory activity is related to the content of characteristic triterpenoids or other compounds of T. camphoratus. In addition, for the metabolites with no significant difference among various culture methods, it is feasible to use the other three culture methods instead of cutting wood culture to obtain these compounds more quickly and in abundance considering the limitations of cutting wood culture. Therefore, In addition to the compounds with significant differences in T. camphoratus cultured in cutting wood culture, further studies should verify pay more attention to the activity of these non-significant differences metabolites.
The mycelium of T. camphoratus is reported to contain rich active substances. However, studies are needed to further explore the active ingredients in the mycelium of T. camphoratus, especially small molecular active substances. As a “treasure” of China, T. camphoratus is rich in various active substances. Although T. camphoratus is being studied since several years, further studies are needed to develop strategies to obtain quick and extensive yield of active metabolites of T. camphoratus. This study provided a theoretical basis for further rational use of different culture methods for T. camphoratus.
Data availability
The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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This study was funded by Shandong Province TCM Science and Technology Project (M-2023035) and Edible Fungus Industrial System Post Expert of Modern Agricultural Industrial Technology System of Shandong Province, Genetic Breeding Post, Lunong Technology (2021No. 26).
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R. Z. and X. C. designed this study and led the project. Y. M. (Yongfei Ming), Y. L., J. C., X. Z. and Y. H. performed the experiments, analyzed the data, and wrote the manuscript. S. Y., Y. M. (YueJun Mu) and L.W. edited and revised the manuscript. All authors have read and approved the final version of this manuscript.
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Additional file 1: Figure S1.
Total ion current spectrum for the extract of artificially cultured T. camphoratus. Table S1. No significantly different metabolites.
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Ming, Y., Li, Y., Chu, J. et al. Comparative analysis of metabolites and in vitro hypoglycemic activity of Taiwanofungus camphoratus cultured using various methods. Appl Biol Chem 67, 40 (2024). https://doi.org/10.1186/s13765-024-00890-x
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DOI: https://doi.org/10.1186/s13765-024-00890-x