The parasitic fungus
The application of the oxidative stress induced by gamma radiation on the mycelium of
After the cultures were irradiated, each was used to seed five replicates of new PDB liquid cultures by transferring 80 mL of homogenized mycelium (a 20% v/v inoculum) to a total volume of 400 mL of new culture. Thus, the irradiation experiment had five doses with five replicates each, which were cultured for 16 days after being irradiated, under the same temperature and agitation parameters as before.
Irradiations were carried out using the Co-60 research irradiator GC-5000 (B.R.I.T., India), within the IRASM Radiation Processing Center, part of “Horia Hulubei” National Institute of Physics and Nuclear Engineering, Romania.
We used an alanine-EPR dosimetry system to evaluate doses. The irradiations were performed at an average dose rate of 0.8 Gy/s with an associated relative uncertainty of 3%. The dose uniformity ratio, defined as the ratio of the maximum to the minimum dose in a set of samples, was about 1.6. All doses are expressed as absorbed dose in water.
16-day old cultures were harvested by separating the mycelial biomass from the fermented broth through a 500 nm stainless steel sieve, followed by further filtration of the broth through a 180 mm Nylon Net (Merck Millipore), under vacuum. The mycelia were then washed three times with deionized water and frozen at −50°C before being freeze dried. Mycelial biomass was estimated by dry weight.
100 mg of each irradiated mycelium was milled to a powder, distributed to 2 mL microtubes, and extracted two times in cold methanol. The dried biomass was kept in 2 mL of 80% cold methanol for 24 h at −50°C, and then the tubes were agitated on a horizontal shaker at 1300 rpm for 30 min and centrifuged for 10 min at 14 000 rpm. The two supernatants were saved and reunited in the end.
To remove lipids, each extract was washed three times with an equal volume of hexane, discarding the organic phase between washes. After removing the last volume of hexane, the extracts were evaporated at 40°C and then redissolved in 1 mL of 80% methanol on a horizontal shaker at 1300 rpm. Ultimately, the extracts were then centrifuged for 10 min at 14 000 rpm to remove any undissolved particles.
Negative controls were prepared using distilled water or 80% methanol, depending on the sample solvent. Samples were reacted three times with the colorimetric reagents and quantified twice. Results were calculated as an average of these technical replicates.
The free radical scavenging potential of the mycelium extracts was determined using a modified DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) colorimetric method [9]. A 0.125 mM DPPH (TCI Chemicals) solution was prepared using a 4:1 (v/v) methanol-PBS mixture, of which 295 mL were reacted with 5 mL of each extract. The reaction mixture was kept at room temperature, in darkness, for 30 min. The absorbance of the reaction mixture was recorded at 517 nm using a SpectraMax i3x (Molecular Devices) plate reader.
The calibration curve (0–500 mg/mL) was plotted using ascorbic acid dissolved in 80% methanol as a standard. The free radical scavenging potential was expressed as milligram of ascorbic acid equivalent per milliliter.
Both extra- and intra-cellular total phenolic compounds (EPC and IPC) were quantified from culture broth and mycelium extract, respectively.
The total phenolic content was determined by colorimetric methods [10] with Folin–Ciocalteu reagent (Sigma-Aldrich). A 10-fold dilution of this reagent and a 7.5% (w/v) Na2CO3 solution were prepared using deionized water. A 200 mL sample was mixed with 1 mL of the diluted Folin–Ciocalteu reagent, followed by 1 mL of sodium carbonate solution. The reaction mixture was kept at room temperature, in darkness, for 30 min. The absorbance of the reaction mixture was recorded at 765 nm using a SpectraMax i3x plate reader.
The calibration curve (0–200 mg/mL) was plotted using gallic acid as a standard. The total phenols were expressed as microgram of gallic acid equivalent per milliliter.
The total flavonoid content of the whole crude fermentation (culture) broth was determined using a modified aluminium chloride colorimetric method [11]. A 2.5% (w/v) AlCl3 solution was prepared using 50% (v/v) ethanol, of which 1 mL was mixed with an equal volume of a fermented broth sample. The reaction mixture was kept at room temperature, in darkness, for 30 min. The absorbance of the reaction mixture was recorded at 420 nm using a SpectraMax i3x plate reader.
The calibration curve (0–50 mg/mL) was plotted using quercetin as a standard. The total flavonoids were expressed as microgram of quercetin equivalent per milliliter.
The fermentation broths were subjected to a solid phase extraction (SPE) procedure to obtain concentrated extracts in polyphenols. Chromabond cartridges (Macherey–Nagel, 500 mg) were used. The stationary phase of the cartridges was activated by successive washes with methanol and water, after which 30 mL of each sample was added. The elution of the polyphenols from the cartridges was performed with 10 mL of methanol. The extracted samples were evaporated to dryness in a turbovap (Eppendorf) at 30°C, and the residue thus obtained was dissolved in 100 mL methanol, centrifuged, and injected into the capillary electrophoresis apparatus. The results are given as the average of three injections.
Polyphenols separation was performed on an Agilent CE system with a diode array detector. A standard bare fused-silica capillary (I.D. 50 mm, effective length of 72 cm) was used for separation. Before each sample migration, the capillary was washed with 0.1 M NaOH, ultrapure water, and migration buffer. The sample was injected in hydrodynamic mode (3500 Pa, 12 s) and the capillary was maintained at 30°C. The migration buffer consisted of a 50 M sodium tetraborate and 0.9 mM sodium dodecyl sulfate (SDS) solution, with the pH adjusted to 9.15 using 1 M HCl. The applied voltage was 30 kV and the detection was performed in the 200–360 nm range [12]. The quantification of the compounds was performed by measurements at a 280 nm wavelength. The data have been processed using the ChemStation (Agilent) application for data analysis.
The gamma irradiation of mycelial cultures and subsequent biomass analysis were performed in duplicate, each experiment containing 25 shake flask cultures (five doses with five replicates each). Results from both experiments are shown separately.
For the first experimental run, after irradiation and 16 days of shake flask culture, the dry mycelial biomass presented an increase of 4.572–19.764% in the irradiated experimental variants, depending on the dose, compared to the non-irradiated controls.
The statistical significance between the irradiated groups in terms of dry mycelium weight was evaluated by analysis of variance (Table 1). The result demonstrates that the differences are statistically highly significant (
The one-way ANOVA of dry mycelium weight after irradiation and culture of the first experimental run
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 6.78 | 1.356 | 0.00468 | 0.068411 | 0.030594 |
100 Gy | 5 | 7.09 | 1.418 | 0.00342 | 0.058481 | 0.026153 |
200 Gy | 5 | 7.73 | 1.546 | 0.00753 | 0.086776 | 0.038807 |
300 Gy | 5 | 8.07 | 1.614 | 0.00863 | 0.092898 | 0.041545 |
400 Gy | 5 | 8.12 | 1.624 | 0.00433 | 0.065803 | 0.029428 |
ANOVA single factor (α = 0.01) | ||||||
Source of variation | SS | df | MS | |||
Between groups | 0.286376 | 4 | 0.071594 | 12.52081147 | 0.000029182 | 4.430690161 |
Within groups | 0.114360 | 20 | 0.005718 | |||
Total | 0.400736 | 24 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The distribution of dry mycelium weight within and between irradiated groups is represented in Fig. 1.
For the second experimental run, after irradiation and 16 days of shake flask culture, the dry mycelial biomass presented an increase of 8.586–16.630% in the irradiated experimental variants, depending on the dose, compared to the non-irradiated controls.
The statistical significance between the irradiated groups in terms of dry mycelium weight was evaluated by analysis of variance (Table 2). The result demonstrates that the differences are statistically highly significant (
The one-way ANOVA of dry mycelium weight after irradiation and culture of the second experimental run
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 9.20 | 1.840 | 0.00535 | 0.073144 | 0.032710 |
100 Gy | 5 | 9.99 | 1.998 | 0.00452 | 0.067231 | 0.030066 |
200 Gy | 5 | 10.49 | 2.098 | 0.00352 | 0.059330 | 0.026532 |
300 Gy | 5 | 10.73 | 2.146 | 0.00843 | 0.091815 | 0.041060 |
400 Gy | 5 | 10.35 | 2.070 | 0.00490 | 0.07 | 0.031304 |
ANOVA single factor (α= 0.01) | ||||||
Source of variation | SS | df | MS | |||
Between groups | 0.284016 | 4 | 0.071004 | 13.28667665 | 0.000019303 | 2.866081402 |
Within groups | 0.106880 | 20 | 0.005344 | 0.10688 | ||
Total | 0.390896 | 24 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The distribution of dry mycelium weight within and between irradiated groups is represented in Fig. 2.
The DPPH free radical scavenging activity of the irradiated mycelium extracts showed a 51.99–79.83% increase, depending on the dose, in comparison with non-irradiated control.
The statistical significance between the irradiated groups in terms of free radical scavenging potential of the mycelium extracts was evaluated by analysis of variance (Table 3). The result demonstrates that the differences are statistically highly significant (
The one-way ANOVA of free radical scavenging potential of the mycelium extracts
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 1.3562 | 0.2712 | 0.0019 | 0.044382 | 0.019848 |
100 Gy | 4 | 1.6489 | 0.4122 | 0.0011 | 0.034140 | 0.017070 |
200 Gy | 5 | 2.1722 | 0.4344 | 0.0008 | 0.029030 | 0.012983 |
300 Gy | 5 | 2.4386 | 0.4877 | 0.0025 | 0.050210 | 0.022455 |
400 Gy | 5 | 2.3953 | 0.4790 | 0.0014 | 0.038588 | 0.017257 |
ANOVA single factor (α = 0.01) | ||||||
Source of variation | SS | df | MS | |||
Between groups | 0.152096309 | 4 | 0.038024077 | 23.466281 | 0.0000003965 | 4.500257699 |
Within groups | 0.030787046 | 19 | 0.001620371 | |||
Total | 0.182883355 | 23 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The distribution of free radical scavenging potential of the mycelium extracts within and between irradiated groups is represented in Fig. 3.
The total phenolic content of the mycelium methanolic extract increased by 28.4–55.7%, depending on the dose, in comparison with non-irradiated controls.
The statistical significance between the irradiated groups in terms of total phenolic content of the mycelium extracts was evaluated by analysis of variance (Table 4). The result demonstrates that the differences are statistically highly significant (
The one-way ANOVA of total phenolic content of the mycelium extracts
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 439.4212 | 87.8842 | 98.0936 | 9.9042 | 4.4293 |
100 Gy | 4 | 451.3970 | 112.8492 | 124.0126 | 11.1361 | 5.5680 |
200 Gy | 5 | 632.8615 | 126.5723 | 76.2878 | 8.7343 | 3.9061 |
300 Gy | 5 | 684.2167 | 136.8433 | 417.0659 | 20.4222 | 9.1331 |
400 Gy | 5 | 667.4257 | 133.4851 | 238.70581 | 15.4501 | 6.9095 |
ANOVA single factor (α = 0.01) | ||||||
Source of variation | SS | df | MS | |||
Between groups | 7 904.44478 | 4 | 1 976.111195 | 10.16779312 | 0.00014191 | 4.500257699 |
Within groups | 3 692.651123 | 19 | 194.3500591 | |||
Total | 11 597.0959 | 23 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The total phenolic content of the mycelium extracts within and between irradiated groups is represented in Fig. 4.
Furthermore, the extra-cellular total phenolic content was also estimated by the same method for the crude fermentation broth. As with the mycelium extracts, the statistical significance between the irradiated groups was evaluated by analysis of variance (Table 5). The result demonstrates that the differences are statistically highly significant (
The one-way ANOVA of total phenolic content of the fermented broths
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 73.6308 | 14.72610 | 4.2755 | 2.0678 | 0.9247 |
100 Gy | 5 | 72.1087 | 14.42170 | 0.4407 | 0.6639 | 0.2969 |
200 Gy | 5 | 103.1155 | 20.62311 | 7.9304 | 2.8161 | 1.2594 |
300 Gy | 5 | 111.8099 | 22.36199 | 3.9634 | 1.9908 | 0.8903 |
400 Gy | 5 | 120.0085 | 24.00170 | 15.6380 | 3.9545 | 1.7685 |
ANOVA single factor (α = 0.01) | ||||||
Source of variation | SS | df | MS | |||
Between groups | 389.6157862 | 4 | 97.403946550 | 15.10220151 | 0.000007715 | 4.430690161 |
Within groups | 128.9930431 | 20 | 6.449652157 | |||
Total | 518.6088293 | 24 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The total phenolic content of the fermented broths within and between irradiated groups is represented in Fig. 5.
The total flavonoid content of the crude culture broth increased by 26.6–934.678%, depending on the dose, in comparison with non-irradiated controls.
The statistical significance between the irradiated groups in terms of total flavonoid content of the fermented broths was evaluated by analysis of variance (Table 6). The result demonstrates that the differences are statistically highly significant (
The one-way ANOVA of total flavonoid content of the fermented broths
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 0.5825 | 0.1165 | 0.00007 | 0.008539 | 0.003819 |
100 Gy | 5 | 0.9134 | 0.1826 | 0.00974 | 0.098740 | 0.044158 |
200 Gy | 5 | 5.7376 | 1.1475 | 0.26596 | 0.515721 | 0.230637 |
300 Gy | 5 | 5.9381 | 1.1876 | 0.10480 | 0.323743 | 0.144782 |
400 Gy | 5 | 6.0272 | 1.2054 | 0.18652 | 0.431886 | 0.193145 |
ANOVA single factor (α = 0.01) | ||||||
Source of variation | SS | df | MS | |||
Between groups | 6.392584426 | 4 | 1.598146107 | 14.08987153 | 0.000012734 | 4.430690161 |
Within groups | 2.268503447 | 20 | 0.113425172 | |||
Total | 8.661087873 | 24 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The total flavonoid content of the fermented broths within and between irradiated groups is represented in Fig. 6.
The concentrated fermentation broths were assessed by capillary electrophoresis using 14 standards, such as were rutin, naringenin, isoquercitrin, umbelliferone, cinnamic acid, chlorogenic acid, sinapinic acid, ferulic acid, kaempferol, luteolin, coumaric acid, quercetol, caffeic acid, and gallic acid.
Electrophoresis of the samples showed the presence of several compounds in the culture broths. However, not all of the samples could be analysed due to a clogging issue with the capillaries when migrating the extracts.
Naringenin was found at a concentration ranging from 4.94 mg/100 mL to 15.16 mg/100 mL of broth, across all the irradiated groups. After applying an analysis of variance, no statistical significance was found (
Isoquercitrin was found at a concentration ranging from 18.76 mg/100 mL to 131.27 mg/100 mL of broth, across all the irradiated groups. After applying an analysis of variance, no statistical significance was found (
Chlorogenic acid was found at a concentration ranging from 3.96 mg/100 mL to 7.21 mg/100 mL of broth, across all the irradiated groups. After applying an analysis of variance, no statistical significance was found (
Luteolin was found at a concentration ranging from 2.15 mg/100 mL to 19.75 mg/100 mL of broth, across all the irradiated groups. After applying an analysis of variance, no statistical significance was found (
Sinapinic acid was found at a concentration ranging from 1.41 mg/100 mL to 55.23 mg/100 mL of broth, across all the irradiated groups. After applying an analysis of variance, we found differences that are statistically highly significant (Table 7).
The one-way ANOVA of the sinapinic acid content of the fermentation broths
Groups | Count | Sum | Average | Variance | Std. deviation | Std. error |
---|---|---|---|---|---|---|
0 Gy | 5 | 40.0005 | 8.0001 | 25.7390 | 5.073365 | 2.536682 |
100 Gy | 3 | 51.3022 | 17.1007 | 26.4171 | 5.139765 | 3.634363 |
200 Gy | 2 | 58.7856 | 29.3928 | 304.6469 | 20.619987 | 14.580533 |
300 Gy | 3 | 91.1330 | 30.3776 | 18.1002 | 4.254438 | 3.008342 |
400 Gy | 1 | 55.2323 | 55.2323 | 0 | 0 | 0 |
ANOVA single factor (α= 0.01) | ||||||
Source of variation | SS | df | MS | F | F crit. | |
Between groups | 2466.721606 | 4 | 616.6804015 | 11.17539311 | 0.001532718 | 6.422085458 |
Within groups | 496.6378863 | 9 | 55.18198737 | |||
Total | 2963.359492 | 13 |
SS, sum of squares; df, degrees of freedom; MS, mean square.
The sinapinic acid content of culture broth showed a 113.756–590.395% increase, depending on the dose, compared to the non-irradiated control.
To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer
The total sinapinic acid content of the fermentation broths within and between irradiated groups is represented in Fig. 7.
In this study, oxidative stress induced by gamma irradiation on the mycelial inoculum of the medicinal fungus
The fermentation broth of the cultures has shown a very significant increase in levels of total polyphenols and total flavonoids, corresponding to larger radiation doses. The optimal dose, in these instances, is 400 Gy. The broths were further assayed using capillary electrophoresis. Naringenin, isoquercitrin, chlorogenic acid, luteolin, and sinapinic acid were found to be present, but only the latter showed statistically significant increases correlated to higher radiation doses; in this case, the optimal dose was 300 Gy.
The total dry weight of the submerged cultured mycelia also showed a very significant increase after irradiation, with an optimal dose at 300 Gy in one experiment, and 400 Gy in another. Both experiments show a trend of higher mycelial mass correlated with increasing irradiation.
To summarize, the 300–400 Gy dose interval proved optimal for inoculum pre-treatment in a biotechnological process of
Other authors used oxidative stress to trigger the enhanced synthesis of diverse secondary metabolites synthesis. Zheng
To our knowledge, this is the first work showing the use of sublethal doses of gamma irradiation as a pre-treatment for increasing biomass yield and for upregulating synthesis of biologically active metabolites in medicinal fungi. Since the treatment is applied to a small mycelium mass that is subsequently multiplied, such treatment is of particular interest for both fundamental biological processes and industrial potential. Medically potent compounds synthesis is yet to be investigated through this novel production system. The technique is both economically and environmentally effective in exploitation of endangered species and satisfying nutraceutical and pharmaceutical markets’ demands for higher amounts of bioactive ingredients.