The use of gamma irradiation to stimulate bioactive compound synthesis in Inonotus obliquus submerged cultures

I. obliquus (Ach. ex Pers.) Pilát (CBS 314.39) mycelium was purchased from Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands) and grown on potato dextrose agar for seven days at 23.5°C. Subsequently, 1 cm × 1 cm agar sectors were each transferred to potato dextrose broth (PDB) seed culture medium. The mycelium was cultured at the same temperature, in 500 mL bottles with polytetrafluoroethylene (PTFE) membrane screw caps, agitated at 100 rpm for 28 days, until it occupied the entire volume of the culture vessels. In order to ensure identical inoculum for all replicates, five such fully grown colonies were brought together in a sterile 3000 mL Erlenmeyer flask and triturated; and 400 mL of the homogenized mycelium was distributed back into the culture vessels.

The application of the oxidative stress induced by gamma radiation on the mycelium of I. obliquus, in a liquid medium, was performed separately on each culture vessel. Sublethal doses of 0, 100, 200, 300, and 400 Gy were given to the triturated mycelium, according to the experimental design.

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 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 (F = 12.520, and P <0.001).

To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer post-hoc test was performed. The result showed very significant (P <0.010) differences between the following groups: 0 Gy vs. 200, 300, and 400 Gy; 100 Gy vs. 300 Gy and 400 Gy.

The distribution of dry mycelium weight within and between irradiated groups is represented in Fig. 1.

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 (F = 13.286 and P <0.001).

To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer post-hoc test was performed. The result showed very significant (P <0.010) differences between the following groups: 0 Gy vs. 200, 300, and 400 Gy. Significant (P <0.050) differences were also found between the 0 Gy vs. 100 Gy groups; and 100 Gy vs. 300 Gy groups.

The distribution of dry mycelium weight within and between irradiated groups is represented in Fig. 2.

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 (F = 23.466 and P <0.001).

To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer post-hoc test was performed. The result showed very significant (P <0.010) differences between the following groups: 0 Gy vs. 100, 200, 300, and 400 Gy.

The distribution of free radical scavenging potential of the mycelium extracts within and between irradiated groups is represented in Fig. 3.

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 (F = 10.167 and P <0.001).

To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer post-hoc test was performed. The result showed very significant (P <0.010) differences between the following groups: 0 Gy vs. 200, 300, and 400 Gy.

The total phenolic content of the mycelium extracts within and between irradiated groups is represented in Fig. 4.

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 (F = 15.102 and P <0.001). Results showed an increase by 40.04–62.987%, depending on the dose, in comparison with 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 post-hoc test was performed. The result showed very significant (P <0.010) differences between the following groups: 0 Gy vs. 300 Gy and 400 Gy; 100 Gy vs. 200, 300, and 400 Gy. Significant (P <0.050) differences were found between the 0 Gy vs. 200 Gy groups.

The total phenolic content of the fermented broths within and between irradiated groups is represented in Fig. 5.

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 (F = 14.089 and P <0.001).

To identify the groups between which these differences are found, and to ascertain the degree of significance for each comparison, the Tukey–Kramer post-hoc test was performed. The result showed very significant (P <0.010) differences between the following groups: 0 Gy vs. 200, 300, and 400 Gy; 100 Gy vs. 200, 300, and 400 Gy.

The total flavonoid content of the fermented broths within and between irradiated groups is represented in Fig. 6.

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 (F = 1.404 and P >0.100) linking the given radiation dose and increases in broth concentration of naringenin.

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 (F = 2.044 and P >0.100) linking the given radiation dose and increases in broth concentration of isoquercitrin.

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 (F = 0.004 and P >0.100) linking the given radiation dose and increases in broth concentration of chlorogenic acid.

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 (F = 2.409 and P >0.100) linking the given radiation dose and increases in broth concentration of luteolin.

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 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 post-hoc test was performed. The result showed very significant (P <0.010) differences between the 0 Gy vs. 400 Gy groups. Significant (P <0.050) differences were also found between the 0 Gy vs. 200 Gy and 300 Gy; 100 Gy vs. 400 Gy groups.

The total sinapinic acid content of the fermentation broths within and between irradiated groups is represented in Fig. 7.

Fig. 7