2.1. Chemicals

Standard of ergocalciferol (Vitamin D₂) was obtained from Sigma-Aldrich (St. Louis, USA). Sodium hydroxide, potassium hydroxide, sodium L-ascorbate, ethanol, methanol, acetonitrile, and n-pentane used in this study were of high purity (>99%) chemicals purchased from Merck (Mumbai, India). The potato dextrose agar and broth were purchased from HiMedia (Mumbai, India).

P. eryngii Mycelia Cultivation

P. eryngii (18.K07) mycelial culture was obtained from Haryana Agro Research Centre, India, and maintained in potato dextrose agar plates. The seed culture was prepared in 100 ml potato dextrose broth medium (24 g/l) by transferring two 8 mm punches of mycelia taken from a full grown fresh agar plate using a cork borer, properly crushed using an Ultra-Turrax (IKA, Germany) homogenizer for 20 s at 12,000 rpm and incubated under aerobic conditions for 4 days at 130 rpm and 28°C. The production media composition was kept as described in Singh et al. 2020 [11]. About 4% homogenized inoculum was used to inoculate production media. The mycelia production was accomplished in 10 days with complete consumption of carbon source. The produced mycelia were collected, washed thrice using deionized water, and filtered (Whatman paper no.1, Sigma-Aldrich, USA).

2.3. UV Irradiation Set-up

The UV exposure set-up includes three 1.75 m long (100 W) UV-B tubes (UV Narrowband TL 100, Phillips) having a definite band of 290–315 nm (311 nm peak) (Wittig et al., 2013), was used for exposure of mycelia. The UV exposure experiments were performed using fresh homogenized mycelial biomass (50 ml) in glass Petri dishes (ID = 15 cm diameter). The sampling was done by removing the whole set of Petri plates (n = 3) for analysis. The samples were lyophilized after UV treatment and kept at −20°C till further analysis. The sample without UV treatment was set as a control. All the calculations were done on a dry cell weight basis.

2.4. Analysis of Vitamin D₂ and Mycelial Nutritional Quality

Vitamin D₂ (present in mycelia) was extracted and analyzed as described by Singh et al., 2020. Precisely, to 0.5 g powdered (freeze-dried) mycelia, 4 ml of sodium ascorbate solution (17.5% w/v in 1 M NaOH), 50 ml ethanol, and 10 ml of 50% (w/v) KOH were added in a 250 ml round bottom flask. This mixture was refluxed at 80°C for 1 h and then cooled to 25°C. The refluxed mixture was extracted in a separating funnel with distilled water (50 ml), ethanol (15 ml), and n-pentane (50 ml, 50 ml, and 20 ml), respectively. The clear organic layer obtained was washed thrice with 50 ml of 3% (w/v) KOH in 5% (v/v) ethanol and, thereafter, neutralized with the deionized water. The organic layer was evaporated at 40°C to dryness in a rata vapor. The residue left was redissolved in 10 ml HPLC-grade methanol (ultrasonicate for 5 min) and filtered through a 0.45 μm filter. Vitamin D₂ concentration was determined using a reverse phase C18 column (Shimadzu) under a UV detector (264 nm) in an Agilent-1260 HPLC system. Acetonitrile/methanol mixture (75:25, v/v) was used as mobile solvent at a flow rate of 1.0 mL/min.

The β-glucan content in the freeze-dried mycelia was quantified using a yeast-mushroom β-glucan kit (Megazyme, Ireland). Reducing power was calculated using a method reported by Reis et al., 2012 [12]. Crude protein and crude fat were analyzed [13]. Micro-Kjeldahl method (AOAC-925.10) was used for calculating crude protein with a conversion factor of 6.25 [14], crude fat (AOAC-2003.05) was analyzed through Soxhlet extraction with hexane. All the calculations were made on a dry weight basis.

2.5. Selection of Irradiation Conditions

Preliminary experiments were performed to select an appropriate exposure time, UV-B intensity, and ambient temperature. First, a range of temperature (20–50°C) selected keeping exposure time and UV-B intensity constant at 20 min and 0.6 W/m² , respectively. Second, UV-B intensity varied from 0.3 to 1.2 W/m² , and the optimal temperature was taken as 30°C from the first step, keeping exposure time constant at 20 min. Finally, using optimal temperature and intensity obtained from the previous two steps, exposure time varied from 10 to 40 min. On the basis of these results, the upper, middle, and lower levels of the independent factors were selected for RSM.

2.6. Design of Experiments and Statistical Optimization using RSM

The RSM was used for the statistical optimization of process parameters – experimental temperature (A, °C), duration of exposure time (B, min), and intensity of UV-B irradiation (C, W/m² ) for ergosterol to Vitamin D₂ conversion. A central composite design was applied for three variables having replicates at the midpoint. A total of 20 experimental trials were designed and the Vitamin D₂ concentration (μg/g, dry biomass) was set as a response variable with a mean of triplicate values. For experimental design and validation of a model for the experimental conditions – Design Expert (12^th version; Stat-Ease Inc., USA) was employed in the present study.

3.1. Selection of Parameter for Vitamin D₂ Synthesis

The effect of the type of UV radiation (A, B, and C) has been studied by many authors [15-17] and it is evident from these studies that the maximum Vitamin D₂ formation from ergosterol present on fungal mushrooms is obtained by UV-B irradiation. Hence, UV-B irradiation was selected for Vitamin D₂ synthesis in P. eryngii mycelia.

Preliminary experiments have been performed to estimate the upper, middle, and lower levels of each factor and the outcomes are shown in Table 1. From the results, it was clear that the Vitamin D₂ content increased until the surrounding temperature reached to 30°C. The study conducted by Jasinghe and Perera, 2006, on mushrooms fruiting bodies reported that the optimum temperature for Vitamin D₂ was 35°C while Lee and Aan, 2016, conducted a study on Agaricus bisporus mushroom powder and found a temperature of 26.3°C for maximum Vitamin D₂ synthesis. It has also been observed that as the temperature increases, mycelial surface turns brown which could also negatively affect the Vitamin D₂ synthesis process [18].

Thus, for experimental design, 30°C was taken as temperature midpoint and lower and upper values were set at 20 and 40°C, respectively. To study the effect of exposure time, the temperature and intensity of UV irradiation were fixed at 30°C and 0.6 W/m² with exposure time varying from 10 to 40 min. The results showed [Table 1] that Vitamin D₂ content enhanced as the exposure time increased and accordingly the three levels of exposure time were chosen for RSM as 10, 20, and 30 min, respectively. The analysis showed that with increasing exposure time, Vitamin D₂ content increases initially up to 20 min but later on, no significant conversion occurred possibly because of pigmentation on the mycelial surface. A rapid increase in Vitamin D₂ was noticed with an increase of irradiation intensity [Table 1]. The preliminary experiments [Table 1] have shown that after an optimum intensity, the rate of conversion begins to decline as higher doses of intensity could possibly induce photodegradation reactions [19], causing a reduction in Vitamin D₂ content. Furthermore, it can negatively affect the mycelial moisture content, color, and nutritional quality [20]. Hence, irradiation intensities for RSM were taken as 0.6, 0.9, and 1.2 W/m² .

3.2. RSM Model Fitting

A central composite rotatable design was used to determine the effect of physical parameters, that is, irradiation intensity, ambient temperature, and exposure time for conversion of ergosterol present on the cell membrane of P. eryngii mycelia into Vitamin D₂. The design of experiments and obtained outcomes are given in Table 2. A multiple regression analysis was conducted, and the concentration of Vitamin D₂ in P. eryngii mycelia was assessed with the help of a second-order polynomial equation stated below:

Y = 324.41 - 24.61A + 19.67B + 28.46C - 12.68AB - 7.92AC - 6.83BC - 28.11A² –26.46B² – 20.51C² (1)

Table 1: Effect of different parameters on Vitamin D2 formation in Pleurotus eryngii mycelia. [Click here to view]

The significance of fitting of the second-order polynomial equation was determined using the analysis of variance (ANOVA) for the assessment of experimental values, as depicted in Table 3. The “F-value” of the model is 6.42 and P = 0.0077 indicates the significance of the model. In this model, values for individual variables and their interactions are significant, as shown in Table 3. It is clearly visible from Table 2 that irradiation intensity (C) is the most significant factor affecting the ergosterol conversion into Vitamin D₂ in P. eryngii mushroom mycelia. The determination coefficient (R² ) of the model is 0.8785 which indicates that the variability of this model can be 87%. The “lack of fit F-value” of 5.34 represents not a significant lack of fit compared to the pure error. Thus, the projected model fits satisfactorily to the data obtained through experiments. The “Adeq. Precision” is utilized to determine the ratio between response and deviation, and a ratio beyond 4 indicates an acceptable signal. In the present investigation, the ratio was 7.072 indicating that the signal was adequate and thus the above model could be further utilized to direct the design space.

Figure 1a shows the interaction effects of temperature (20–40°C) and exposure time (10–30 min) along with a set UV-B intensity of 0.9 W/m² . It indicates the quadratic effect of two variables on the Vitamin D₂ synthesis.

Figure 1b indicates that under lower values of temperature, the irradiation intensity resulted in a linear rise in the Vitamin D₂ synthesis while exposure time was fixed at 20 min. Furthermore, the ambient temperature shows a quadratic effect on the outcome. Figure 1c indicates the effects of the interactions between exposure time and UV-B intensity with a similar tendency.

Table 2: Experimental design and central composite design results. [Click here to view]

Figure 1: Vitamin D2 contour plots for synthesis in Pleurotus eryngii mycelia. A: Impacts of temperature and exposure time on Vitamin D2 production in P. eryngii mycelia at a fix intensity of 0.9 W/m2 ; B: Impacts of temperature and intensity on Vitamin D2 production in P. eryngii mycelia at a fix time of 20 min; C: Impacts of exposure time and intensity on Vitamin D2 production in P. eryngii mycelia at a fix temperature of 30°C. [Click here to view]

The optimum operating conditions for Vitamin D₂ synthesis are 24.29°C, 23.88 min, and 1.099 W/m² for ambient temperature, exposure time, and UV-B intensity, respectively, and the predicted Vitamin D₂ synthesis using these optimized parameters was 346.84 μg/g, with a confidence level of 95%.

Ergosterol, when exposed to UV irradiation first, converts into unstable pre-Vitamin D after the opening of its “B-ring” and then gets rearranged into Vitamin D₂ along with various conformational isomeric forms of photoproducts such as tachysterol, lumisterol, Vitamin D₄, suprasterol, and toxysterol [21]. The rate of formation of Vitamin D₂ is a function of temperature, type of UV, and the intensity of UV, thus, it is imperative to expose fungal mycelia with the optimum doses of the same. Ergosterol to Vitamin D₂ conversion is also limited due to its further conversion into hydroxylated forms possibly by monooxygenases or through oxidative reactions [22]. The ergosterol to Vitamin D conversion efficiency increases when mushrooms are homogenized before UV exposure possibly because of increases in the surface area exposed [18].

3.3. Validation of the Model

The experiments for validation were conducted at the predicted conditions obtained through RSM analysis. The Vitamin D₂ concentration in P. eryngii mushroom mycelia was 329.43±5.32 μg/g with a confidence level of 95% for the projected maximum concentration (346.84 μg/g). The satisfactory correlation in the experimental and predicted values validates the effectiveness and applicability of the model suggested in the present study.

3.4. Impact of UV-B Irradiation on the Nutritional Quality of P. eryngii Mycelia

Contents of β-glucans and reducing power were determined in model optimized UV-B treated mushroom mycelia of P. eryngii and the corresponding values were compared with the untreated (control) mycelia [Table 4]. β-glucan in UV-B treated mycelia 29.43±0.78% was found to be increased in comparison to untreated mycelia 26.02±0.42%, which indicates the elicitation effect of ultraviolet irradiation on the contents of β-glucan. The cell wall of mycelia contains glucans as a major component and the UV irradiation has a stimulating effect on enhancing β-glucan content in mushrooms [23,24].

Table 3: ANOVA results for the response surface quadratic model. [Click here to view]

Table 4: Effect of ultraviolet exposure on nutraceutical content of Pleurotus eryngii mycelia. [Click here to view]

Reducing power of UV irradiated mycelia (0.98±0.22 mg/ml) was slightly improved than non-irradiated control samples (1.18±0.18 mg/ml). No significant changes were found in the contents of protein, fat, and carbohydrate after mycelial UV treatment [Table 4]. Similar observations in UV irradiated oyster mycelia were also reported by Huang et al. (2015) and Simon et al. (2011). Our results in this regard are corroborated by their findings. A detailed study is still to be done for effects of UV-B irradiation on other bioactive compounds in the submerged cultivated mushroom mycelia.