تولید اسیدگانودریک و اگزوپلی‌ساکارید توسط گانودرما لوسیدوم در کشت نیمه جامد و غوطه‌ور ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

Introduction

G. lucidum (Red Reishi) is currently the most popular among all mushroom species, because of its high medicinal values. Even today the basidiocarp (fruiting body) of G. lucidum is a popular remedy to treat many diseases. Various Ganoderma species are widely used in the synthesis of several drugs and also act as functional foods. G. lucidum produces several metabolites with biological activity, such as terpenoids and exopolysaccharides which might be responsible for medicinal properties (1-5). In recent years, submerged fermentation of G. lucidum has received great attention in Asia regions as a promising alternative for efficient production of its valuable metabolites, especially exopolysaccarides and Ganoderic acid. Ganoderic acid has been reported to possess hepatoprotective, anti-hypertensive, anti-HIV, hypocholesterolemic, anti-histaminic effects, anti-tumor, anti-engiogenic activity and many effects on platelet aggregation and complement inhibition. Exopolysaccharides have been known to possess anti-tumor effects through immunomodulation and anti-angiogenesis. In addition, polysaccharides have a protective effect against free radicals and reduce cell damage caused by mutagens (6-8).

In Asia regions basidiocarp production of G. lucidum is common. The advantages of submerged fermentation over traditional basidiocarp cultivation are the reduction in the time spent to obtain the product of interest. The production of basidiocarp takes at least 3 to 5 months, while reasonable amounts of GA and EPS can be obtained by submerged fermentation after only 2 to 3 weeks. Submerged fermentation advantages are less production with high quality, high control on environmental condition; and its important disadvantage is less GA production (9).

As mentioned, G. lucidum is traditionally cultivated in solid culture. In contrast, with submerged culture, in the traditional cultivation technique of G. lucidum, it takes at least several months until the fruit bodies are developed and this culture technique is used to obtain only basidiocarp. The advantage of solid-State fermentation (SSF) over other techniques is high production of GA; and its important disadvantages are the long time required to cultivate fruiting body, the difficulty of controlling the quality of the product, unsterile production process, and the ease with which the culture is affected by the environmental changes. There is a great need to supply the market with a large amount of high-quality G. lucidum products. In this studythe advantages of both submerged and Solid-State fermentation were accumulated in Semi-Solid-State fermentation. It is obviously necessary and important to develop a process for the production of these valuable metabolites. However, as far as we know, until now there are no reports on Semi-Solid-State fermentation for high production of GA and EPS in biomass instead of fruiting body (9, 10). In addition, present work aimed to evaluate the effect of different medium components on the production of GA and EPS.

Material and Methods

Microorganism preparation: G. lucidum (Curtis) P. Karst. Gilan, Gorganwas obtained from National Center for Genetic and biological reserves in Iran, It was characterized and identified as G. lucidum according to the key of Iranian Ganoderma species (11, 12).

Media: G. lucidum were maintained by an occasional transfer on PDA with 2.5% wheat bran extract and incubated at 30°C for 8 days.

Pre-culture medium: For inoculation in pre-culture medium, 1cm×1cm block of fresh growing mycelia on PDA were inoculated into 500mL cotton-plugged Erlenmeyer flasks that contained 150mL of pre-culture medium. It consisted of the following components (g/L): sucrose 35, peptone 5, yeast extract 2.5, KH₂PO₄.H₂O 1 and MgSO₄.7H₂O 0.5 with initial pH of 5.5 (13). Flask cultivation was carried out at 30°C±2, 150 rpm for two days.

.Experimental design of Submerged, Semi-Solid-State. and Solid-State fermentation medium: The experimental designs were applied to extract independently the main effects of the factors; the analysis of variance technique was then applied to determine which factors were statistically significant. All calculations were performed using Qualitek-4 software. Table1 presents the important variables to be optimized and their trial levels. Standard Taguchi orthogonal array of L₈ (2×4) for submerged fermentation and L₉ (3×3) for Solid-State and Semi-Solid-State fermentation were used (Table 2, 3 and 4), respectively, L and the subscript 8 represent Latin square and the number of experimental formulation runs. Concentrations of other ingredients in the medium and inoculation were fixed.

100mL fermentation medium was prepared in 500mL cotton-plugged Erlenmeyer flasks that were composed of the following components with some modification (g/L): glucose and sucrose with substitution 40, peptone and malt with substitution 5, yeast extract 5, KH₂PO₄.H₂O 1 and MgSO₄.7H₂O 0.5. Before sterilization, the medium pH was adjusted to 6.5 by adding 1N HCl or 1N NaOH. Fermentation medium was inoculated with 10mL pre-culture broth (with ca. 100mg dry weight of cells/L), then followed by a 7-day incubation at 30°C±2 on a rotary shaker with150 rpm and some flasks were incubated without agitation (14).

.Solid medium contained 100g of dry .substrate that were dispensed equally into each cotton-plugged Erlenmeyer flasks with 500mL capacity. Flasks were sealed with cotton plugs to facilitate air transfer, autoclaved for 20min at 121°C. Flasks contained wheat bran and oak sawdust as carbon source and tea slag as an inducer. Mentioned flasks were inoculated with 10mL pre-culture broth (with ca. 100mg dry weight of cells/L), then followed by 7-weeks incubation at 30°C±2 without agitation (Table 1 and 2).

Semi-Solid-State fermentation was conducted in 500mL cotton-plugged Erlenmeyer flasks contained oak chips and wheat bran according to Table 1 and 3. The composition of the culture medium was same as Solid-State fermentation but with duplicate volumes of submerged medium without carbone source. Then flasks were incubated in static and agitated conditions at 30°C±2 for 21 days. All experiments were happened in triplicates.

Table 1- Definition and trial levels of factors

Table 2- Design and experimental formulation according to Taguchi

Table 3- Design and experimental formulation according to Taguchi

Table 4- Design and experimental formulation according to Taguchi

Crude GA and EPS Extraction: For extraction of crude GA, 50­mL of distilled water were added to flasks that contained fermented solids for 24h on rotary shaker at 160rpm. Then broth was filtrated with Whatman paper (Ashless, 42, 12.5 cm, England). After that, filtrates were further separated by centrifugation at 6000×g for 20min. Submerged and Semi-Solid-State fermentation broth also were filtrated with Whatman paper. All filtrates were lyophilized and powders were used to determine the amounts of GA and EPS production.

.Analytical methods

.Determination of crude GA by Gravimetric technique: Previous extraction step filtrates, contained crude GA. Fang and Tang´s GA determination method with some modification was used: The lyophilized powder of samples (2±0.1g) was subjected to extraction using 75% (v/v) ethanol for 4 to 5 days at room temperature. The suspension which contained G. lucidum powder and ethanol was shook for overnight using a mechanical shaker (Model IRC-1-U kühner, German). After the removal of the solid particles by centrifugation at 7000×g for 10min, the supernatants were dried at 50^oC under vacuum (150 mbar) in a rotary evaporator (Lab Tech, EV311 series, Beijing, China) until all ethanol was vaporized. The residues were then suspended in distilled water (10mL) and later extracted with 10mL chloroform for 1 to 2 days. After chloroform removal by evaporation (at 50^oC for 35min.), GA in water extract were further extracted with 5% (w/v) NaHCO₃ for 12 hours. HCl (2N) was added to the solution to adjust the pH of the NaHCO₃ phase to lower than 3.0. Again GA in NaHCO₃ layer was further extracted by chloroform for 24 hours. After removal of chloroform by evaporation at 50^oC, crude GA was dissolved in absolute ethanol (99.4% v/v). Moreover, the absorbance was measured at 245nm in a spectrophotometer (Biochrom, Libra S12 Biowave II, UK) andthe average readings were obtained(14).

Standard curve for 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.1mg/mL thymol (Merck, 108167, crystal) concentration was obtained and absolute ethanol was used as blank solution. Then crude GA content of each sample was analyzed according to standard curve.

TLC technique:In order to compare terpenoids band patterns between fruit body and mycelium in three kinds of fermentation formulation strategies, fruit body and mycelium of G. lucidum were used. Fruit body of G. lucidum finely were powdered, in 50mL of alcohol for 15min, centrifuged, withdrawer the supernatant, and evaporated at 50°C. The residues were dissolved in 2.0mL of alcohol, centrifuged, and supernatant were collected. Fruit body and mycelia extract were spotted on the plates as 2mm bands. For normal-phase TLC examination, pre-coated silica-gel TLC plates (Kieselgel 60 F254, DC-Alufolien, and Merck) were used. The developing reagents were composed of: chloroform, methanol and water in a volumetric ratio of 30:4:1. Visualization proceeded by spraying 10% sulfuric acid in alcohol and then heating sample to 105°C in order to evaporate solvents. Samples were initially examined under a UV lamp with a wavelength of 366nm and plates were further heated to 105°C for visualization(15).

Preparation of standard glucose curve: Lu´s gravimetric method with some modification was used: D-glucose anhydrous (0.1g) was accurately weighed and then dissolved in 100mL of distilled water, to produce corresponding stock standard solution (1mg/mL). Accurately glucose control solution 0.1, 0.2, 0.3, 0.4, 0.5, 1mL was draw to the test tubes and water was added to the volume of 20mL. Anthrone sulfuric acid solution (0.1g of anthrone was dissolved in 50mL concentrated sulfuric acid) was prepared. 3mL Anthrone solution was added to 1.5mL of D-glucose dilutions and mixture was heated for 10min, then was removed and put in room temperature for 15min, with the corresponding reagent as control. Absorbance was determined in 490nm wavelength and was made it as the ordinate, concentration as abscissa to establish a standard curve. For sample solution the method of establishing the standard curve was followed, as the “precisely Anthrone sulfuric acid 3mL was added” and 1.5mL of diluted sample solution was mixed then absorbance was determined; after that the content of the polysaccharide according to the standard curve was calculated (16).

FTIR Spectroscopy: FTIR spectroscopy of G. lucidum mycelia in submerged fermentation was tested in order to show glucans with using Nicolet FTIR Spectrometer (Nicolet 6700, Thermo Scientific). One part of sample was mixed with 99% of dried potassium bromide (KBr) powder and compressed to prepare a salt disc of 3mm diameter. These discs were subjected to FTIR spectrum measurement in the frequency range of 400 to 4000 cm^-1 (17, 18).

Results

Production time: Results showed that, 21 days for growth in Semi-Solid-State fermentation was more appropriate in comparison with Solid-State fermentation growth time (49 days). However, submerged fermentation took place only in 7 days.

.Optimized medium composition for GA production: In each formulation effect of absence and presence of main effects were analyzed. The first formulation shows control level of medium that hadn’t gotten any main effects. Fig. 1, 2 and 3 shows all medium formulation in submerged, Solid and Semi-Solid-State fermentation. As the histogram showed in formulation 1, GA wasn’t detected. Medium formulation 6 in Solid-State fermentation and medium formulation 6 and 9 in Semi-Solid-State fermentation showed the highest amount of GA production. In Solid-State formulation 6 with 18g oak chips and 26g wheat bran 0.19±0.004 mg/g GA was detected and in Semi-Solid-State formulation 6 and 9 with 9g and 13g oak chips and 13g wheat bran respectively, 0.256±0.006 and 0.253±0.003 mg/g GA was detected.

Fig.1- Ganoderic acid productionbysolid-state fermentation formulations

Fig.2- Ganoderic acid productionbySemi-Solid-State fermentation formulations

Fig. 3- Ganoderic acid productionbysubmerged fermentation formulations

Analysis of data for determination of significant parameters on GA production was performed and results are shown in ANOVA Table 5. ANOVA Table of obtained results showed that, changing the carbone content of medium has been the most important factor in causing differences in obtained results. By the same way, the least important factor was the inducer. The experimental results suggested that these factors at optimum level strongly support the production of GA. In three kinds of fermentation formulation media, factors including: wheat bran, oak chips and glucose with P value ≤0.050 were significant factors for GA production.

Table 5- ANOVA (Analysis of Variance) for Ganoderic acid production by G. lucidum

1. Degrees of Freedom  2. Sum of Squares

Optimized medium composition for EPS production: In each formulation, the effect of absence and presence of main effects were analyzed. The first formulation shows the control level of medium that hadn’t gotten any main effects. Fig. 4, 5 and 6shows all medium formulation in submerged, Solid and Semi-Solid-State fermentation. As histogram showed in formulation 1, EPS wasn’t detected. Medium formulation 3 and 8 in submerged fermentation showed the highest amount of EPS production.

Analysis of data for determination of significant parameters on EPS production was performed and results are shown in ANOVA Table 6. ANOVA Table of obtained results showed that, carbone content of medium and aeration have been the most important factors in causing differences in obtained results. By the same way, the least important factor was inducer. The experimental results suggested that these factors at optimum level strongly support the production of EPS. The production of 98.33±3.78mg/g EPS was observed in submerged fermentation with a combination of malt 20%, glucose 4%, sucrose 2% and with aeration. It can be observed that wheat bran and oak chips in Semi-Solid-State and Solid-State fermentation, and glucose in submerged fermentation respectively contributing of 67.506, 32.673 and 47.628%, have shown the highest positive impact on the EPS production. In three kinds of fermentation formulation media, factors including: wheat bran, glucose and aeration with P value ≤0.050 were significant factors for EPS production.

Fig.4- Exopolysaccharide productionbysolid-state fermentation formulations

 Fig.5-Exopolysaccharide productionbySemi-Solid-State fermentation formulations

Fig. 6- Exopolysaccharide productionbysubmerged fermentation formulations

Table 6- ANOVA (Analysis of Variance) for Exopolysaccharide production by G. lucidum

1. Degrees Of Freedom        2. Sum of Squares

TLC technique: Results in TLC technique were shown in Fig. 7. In red rejoin of TLC chromatogram, terpenoids bands were shown according to Ganoderic acids R_f (19). In comparison, between three kinds of fermentation formulation strategy, bands that were related to terpenoids region in Semi-Solid-State fermentation mycelia were noticeable than fruiting body and other fermentation formulation.

FTIR spectroscopy:A prominent band in the range of 1200 to 800 cm^-1 indicated the presence of exopolysaccharides in G. lucidum mycelia. In the current study, peak in region 891cm^-1 showed β-Dglucan (Fig. 8).

Fig. 7- TLC chromatogram of G. lucidum (Red rejoin in chromatogram shows Ganoderic acids band according to R_f) (19).

Lane 1, 7: G. lucidum mycelium biomass in submerged fermentation

Lane 2, 3: G. lucidum mycelium biomass in Semi-Solid-State fermentation

Lane 4, 5: G. lucidum mycelium biomass in Solid-State fermentation

Lane 6: G. lucidum Fruiting body

Fig. 8- FTIR spectra (4000-500 cm^-1 region) of exopolysaccharides of Ganoderma lucidum obtained under submerged fermentation formulation.