Abstract: Submerged fermentation of higher fungi Ganoderma is increasingly, studied for its potential advantages particularly, improving the production biomass and bioactive metabolites. The present study mainly focuses on the qualitative assessment of triterpenoids and steroids in chloroform extracts of harvested mycelia from submerged cultures of three different strains of Ganoderma sp. by using a low cost complex medium. For the screening of these bioactive substances we employed a High Performance Liquid Chromatography (HPLC) system based on a gradient of acetonitrile acidified with formic acid, coupled to a Quadrupole Time-of-Flight Mass Spectrometer (QToF-MS) for a tentative determination of Ganoderic Acids (GA) and sterols. The LC-QToF-MS approach was successfully, used in the qualitative analysis of bioactive metabolites. The results show a considerable diversity of produced metabolites in the three samples and indicate the presence of putative target components, such as; GAA, GAH, GA theta, GA beta, GAK, GAE, GAC2, GAR, GAX and ergosterol. The comparison of the compounds according to their retention time and QToF-MS spectra reveals nine shared metabolites produced by the three different strains of Ganoderma sp., GAAm1, GAY, GAS, GAT-Q and GAT.
INTRODUCTION
Higher fungi represent an immense source of a huge range of structurally diverse compounds with health promoting properties. Ganoderma a well-known genus of the Ganodermataceae (Basidiomycetes) (Schmidt, 2006). The species of Ganoderma have been currently used for medicinal purposes for thousands years. Over the last decades, the bioactive compounds of these mushrooms were increasingly studied and several pharmacological affects have been revealed. The bioactive properties of Ganoderma, such as; antiviral and antibacterial (El-Mekkawy et al., 1998; Zjawiony, 2004), anti-androgenic (Liu et al., 2010), anti-inflammatory (Ko et al., 2008), lowering cholesterol (Berger et al., 2004; Hajjaj et al., 2005), anti-oxidant (Saltarelli et al., 2009), immuno-modulatory (Chen et al., 2004; Kuo et al., 2006) and anti-tumor activities (Calvino et al., 2010; Gao et al., 2006; Ma et al., 2013; De Silva et al., 2012) are mainly due to triterpenoids and polysaccharides and sterols (Boh et al., 2007; Petrova et al., 2008).
The bio-valuable metabolites have been widely isolated from fruiting bodies and spores (Keypour et al., 2010; Seo et al., 2009), whilst the field cultivation and the solid state fermentation are laborious, time-consuming and expensive processes. Therefore, submerged fermentation has gained plentiful attention as a promising alternative for producing high biomass yield and bioactive compounds in a compact space, in relatively short time and with lesser chance for contamination (Saltarelli et al., 2009; Zhong and Xiao, 2009). Now-a-days, serious efforts are being made to enhance the production of metabolites in submerged fermentation by optimizing the composition of culture media (Fang and Zhong, 2002b; Xu et al., 2008; Tang and Zhong, 2002), physicochemical conditions (Fang and Zhong, 2002a; Tang and Zhong, 2003; Zhang et al., 2010) and studying biosynthesis pathways and regulation of genes expression (Liang et al., 2010; Ren et al., 2013; Shi et al., 2010).
Moreover, investigators have successfully extracted and characterized GA and sterols from Ganoderma species (Hirotani et al., 1987; Seo et al., 2009; Yuan et al., 2007). The use of powerful methods of analysis is crucial to obtain reliable results. Hence, several researches were carried out for developing the analysis techniques of the bioactive components. Liquid Chromatography Combined with Mass Spectrometry (LC-MS) has become the method of choice for screening and characterizing complex mixtures (Liu et al., 2011; Wang et al., 2006; Yang et al., 2009, 2007). However, providing the internal standards for such analysis still being a challenging obstacle to identify all the constituents of complex mixtures (Gao et al., 2004; Liu et al., 2011).
In this present study, an investigation was carried out on the production of triterpenoids and sterols by Ganoderma isolates collected from Kala National Park, El Tarf (Algeria) and grown in submerged fermentation, using a low-cost complex media. The metabolites were assessed with Reversed-Phase High Performance Liquid Chromatography Coupled to Electro-Spray Ionization Quadrupole Time of Flight Mass Spectrometry (RP-HPLC-ESI-QToF-MS).
MATERIALS AND METHODS
Chemicals: Chloroform R.P. Normapur analysis grade was purchased from Prolabo (Fontenay-sous-Bois, France). Formic acid (analytical reagent grade) was purchased from Merck (Darmstadt, Germany). Acetonitrile LC/MS grade and methanol absolute LC/MS grade were purchased from Biosolve (Valkenswaard, The Netherlands). Pure water was prepared from a Milli-Q SP Regent Water system (Millipore, Bedford, MA, USA).
Fungal isolates: Three Ganoderma sp. strains were isolated from Ganoderma basidiocarps harvested from different host trees from El Kala National Park, El Tarf (Algeria). Small pieces from each fruiting body were suspended in sterile distilled water, after shaking (2 min), 0.1 mL of suspension was aseptically transferred into Petri dishes containing Potato Dextrose Agar (PDA), or PDA supplemented with Rose Bengal (50 mg L–1). The incorporation of Rose Bengal in the medium was to inhibit the rapidly spreading of fungal colonies (Jarvis, 1973; King et al., 1979) to suppress most of bacteria (Ottow, 1972). The plates were incubated at 30°C until fungal growth. Then the three isolates of Ganoderma sp. designated G1, G2 and G3 were identified on the basis of their macroscopic and microscopic features.
Maintenance of fungi and media: The stock cultures were maintained on a PDA slants and routinely subcultured every month and the slants were incubated at 30°C for a week and then stored at 4°C. The submerged cultivation process was performed as described by Xu et al. (2008). The seed medium consisted of the following components, glucose 40 g L–1, peptone 4 g L–1, KH2PO4 1.5 g L–1, MgSO4.7H2O 1.0 g L–1 and vitamin B1 0.01 g L–1. The fermentation medium contained, glucose 16 g L–1, peptone 2.93 g L–1, corn flour 20.93 g L–1 and soybean powder 6.44 g L–1. Corn and soybean were purchased from a local market then ground to fine powders.
Inoculum and liquid shake cultivation: Actively growing mycelia obtained from a newly prepared agar-plate culture, after it was incubated for 5-7 days at 30°C, around 5-6 of 0.5 mm diameter disks were punched out and then transferred into a 250 mL Erlenmeyer flask with 50 mL of the seed medium. Cultures were incubated at 30°C for 7 days with shaking at 150 rpm. The seed medium in pre-culture flasks was poured carefully, while keeping most of pellets and then it was replaced by 100 mL of the fermentation medium, the culture flasks were maintained at 30°C and 125 rpm for 4 days. All experiments were carried out at least in duplicate to ensure reproducibility.
Sample extraction and preparation: The fermented broths were filtered through layers of sterile gauze. Then the pellets were dried at 50°C to a constant weight after repeated washing with distilled water. The amount of formed mycelia was determined by measuring the dry weight. Previous method reported by many authors (Keypour et al., 2010; Wang et al., 2006; Yang et al., 2007), which was modified and used for extraction of triterpenoids and sterols from the three samples (G1, G2 and G3). Two grams of powdered dried mycelia was extracted with 40 mL of chloroform with shaking for 20 min. The extraction process was repeated twice. The extracts were filtered, combined and evaporated to dryness at room temperature. A small amount of each dry residue was dissolved in 400 μL methanol in an ultrasonic bath for 5 min, after addition of 400 μL of Milli-Q water, the mixture was filtered through a 0.45 μm Millipore filter unit. A 20 μL aliquot of each sample was analyzed by LC-QToF-MS.
LC-QToF-MS conditions: Chromatographic analysis were performed on the agilent 1290 Infinity LC system equipped with a binary pump, an autosampler, a column oven and a Diode-Array Detector (DAD) coupled to an Agilent 6538 QToF mass spectrometer equipped with a dual Electro-spray Ionization (ESI) source. Twenty microliter of each sample was separated on Phenomenex Luna C18(2) 100 Å column (3 μm, 150×4.6 mm i.d.). The mobile phase consisted of 0.2% formic acid in water (A) and acetonitrile (B) using, a linear gradient program of 32% B over the first 15 min, 32-82% B in 15-40 min, 82-100% B in 40-42.50 min and held at 100% B in 42.50-48 min. The flow rate was 0.85 mL min–1 and column temperature was set at 20°C. The DAD was monitored at 220, 252 and 280 nm for acquiring chromatograms, the on-line UV spectra were recorded in the range of 190-400 nm.
The ESI MS spectra were acquired in both positive and negative ion modes, the conditions for ESI operation were as follows: Drying gas flow, 12 L min–1; nebulizing pressure, 45 psi; drying gas temperature, 350°C; capillary voltage, 3.8 kV and fragment or voltage, 180 V. Mass spectra were collected at a frequency of 4 GHz and scanned over a mass range of m/z 50-1700 with a scan rate of 1.5 spectra sec–1.
RESULTS AND DISCUSSION
Submerged fermentation of fungi: The growth of Ganoderma isolates in the seed medium was important. The fungal isolates have grown vigorously in the production complex medium and in the end of the fermentation process, the turbid broth culture became more and less clear, with quite similar amount of biomass.
RP-HPLC analysis: Obtained chromatograms showed a very different composition among the three extracts, The peaks were severely overlapping, by other means, each peak included several compounds with close retention times (Rt) on the analytical chromatography (Tang et al., 2006) (Fig. 1). This usually occurs in separation of complex mixture of natural products with very similar chemical structure, which require a longer elution time to achieve better separation (Gao et al., 2004; Liu et al., 2011). In such cases, the ESI-MS spectra provide additional information on the chromatographic peaks.
QToF-MS data analysis: The separated ions were detected and reported from accurate-mass scan data using, Agilent Mass Hunter Qualitative Analysis software. The putative assignment of acquired QToF-MS spectra was based on matching between the accurate measured and the theoretical masses using in-house molecular formula database of known triterpenoids and sterols previously isolated from Ganoderma. The match score and the mass accuracy (Δm) were calculated for each retrieved chemical formula in different samples. The identification of detected peaks could not be confirmed owing to the unavailability of standard compounds. For more credibility, the tentative assignments were compared by published data (Chen et al., 2008; Keypour et al., 2010; Yang et al., 2007).
Numerous target compounds were identified with very narrow mass tolerance (<10 ppm) and several unknown compounds were detected in the survey scan in the extracts of the Ganoderma samples.
In G1 extract, the compound 6 showed an accurate mass of [M+H]+ ion at m/z 530,2877, corresponding to the molecular formula C30 H42O8, compound 6 was tentatively assigned to be GA theta. Another compound 10 gave rise to a quasi-molecular ions [M+H]+ at m/z 574,3139, the molecular formula was defined as, C32H46O9, compound 10was tentatively identified to GAK.
The mass spectrum of compound 9 had a molecular ion peak at m/z 517,3165 [M+H]+, the molecular formula was defined as, C30H44O7. However, compound 9 (Rt = 19,343 min) is an isomer of compound 15 detected in extract G3 (Rt = 23,781 min) and compound 22 in G2 (Rt = 26,749 min), the three components were assigned to GAA.
The compounds 8,11 and 16 detected in G2 extract, were plausibly identified to be GAH, GAE and GAC2, respectively (8 at m/z 572,2996 [M+H]+ calcd. C32 H44 O9, 11 at m/z 512,275 [M+H]+ calcd, C30 H40O7 and 16 at m/z 518,3234[M+H]+ calcd. C30 H46 O7).
Compound 53 appeared in G2 at Rt = 40,859 min, has a molecular formula of C28H44O, which is an ergosterol isomer.
The molecular formula of compounds 24 and 46 was determined to be C32 H46 O5 (m/z 510,3339 [M+H]+) and C30 H40O7 (m/z 512,2799 [M+H]+), which were tentatively assigned to be GAT-Q and GAE, respectively.
Unfortunately, the identification based on chemical formula or accurate mass alone is insufficient, even for components expected to be present.
| Fig. 1(a-c): | LC-DAD-QToF-MS chromatograms at 252 nm of the three extracts (a) G1, (b) G2 and (c) G3, eluted with water with 0.2% of formic acid (A) and acetonitrile (B). The gradient and ESI parameters are described in materials and methods |
The MS data matching and the manual retention time alignment allowed the comparison of same metabolites across the different samples (Table 1). Overall, nine common peaks were observed among the three Ganoderma samples.
The detected ion peaks in the three Ganoderma extracts at Rt = 18.8 min corresponding to the same metabolite compound 7 have a molecular formula of C30H42O7, which was assigned as, GAAm1.
At Rt = 31,1 min, compound 33 gave rise to the following ion peaks [M+H]+ at m/z 454.3447 (G2), m/z 454.3454 (G1) and m/z 454.3445 (G3), the molecular formula was established to be C30 H46 O3 which was tentatively identified as GAY. The compound 42 observed at Rt = 35.6 min in the three Ganoderma extracts is an isomer of compound 33 and it was also assigned to be GAY.
The component 39 is produced by the three Ganoderma samples at slightly different retention times (G1 and G3 at Rt = 33.604 min, G2 at Rt = 34.055 min), this compound was assigned to be GAT-Q.
Similarly, another common component 48 between the three Ganoderma samples (Rt = 38.0 min) with the chemical formula of C36H52O8 was tentatively identified as GAT.
From the above results, the production of metabolites differed significantly from a strain to another, which is almost explained by the genetic differences between these fungi isolates, considering that they had the same cultivation conditions (Wang et al., 2006). The screening revealed the presence of similar components in the analyzed extracts.
| Table 1: | Interpretation of acquired QToF-MS data of the three analyzed extracts |
CONCLUSION
Production of bioactive metabolites by submerged fermentation of higher fungi has gained popularity over the past years and many papers about optimization of the fermentation process for more efficient production have been published. Besides, relevant researches have been made in isolation and structural elucidation of active constituents.
This study reports for the first time the production of triterpenoids and sterols by Algerian Ganoderma sp. strains. The used culture medium was suitable for mycelial growth and many compounds were produced in the submerged fermentation process. In the attempt to screen triterpenoids and sterols by using a LC-QToF-MS method, many targeted compounds were successfully assigned. Conclusive identification of targeted compounds was constrained by the unavailability of authentic standards. Besides unknown compounds that could not be identified through database searching or the literature. The obtained results highlight a varied production of metabolites, however, few compounds are commonly produced by the three strains.
These findings show the need that future studies in screening approaches of bioactive metabolites require complementary analytical techniques to improve accuracy and confidence in compound identification.