The developmental stages of mushrooms (stage -2,-1,0,+1,+2 day) were determined according to their sizes (cap diameter, stalk diameter, cap: stalk diameter and cap edge), cap color and their wet weight (Table 2). Mushroom optimal physiological stage for harvest was set at 0 days, mushroom harvest prior or after that time set on minus or plus. Shiitake cap color strengthened from light to dark brown with developmental stages advance. In Shiitake early developmental stage (two days' prior harvest, -2) the cap edge were attached to stalk, and as an advanced developmental stage, the cap edge disengaged from stalk and the cap had concave shape. The more advanced physiological stage beyond the stage 0, there is a transition from concave cap shape to convex shape appears second opening at the edge of the cap. As Oyster mushrooms develop, the cap edge turning to wrinkled and torn from smooth and continuous edge (Fig 15).

As shown in Table 2, the most effective physiological development stage determination is according to cap: stalk diameter ratio. Shiitake cap: stalk diameter ratio was significant higher in advanced developmental stage (+2) compared to earlier developmental stages (-2,-1,0, +1;p<0.01). There was no cap: stalk diameter ratio significant difference between physiologic developmental stage 0 and +1 in Oyster mushrooms, but the ratio was significantly higher than early developmental stage (-2,-1). Additionally, in both mushroom species as physiological developmental stage increases the cap diameter: wet weight ratio increases linearly (Fig. 16).

First, we wanted to determine what is the most optimal developmental stage to induce vitamin D formation from ergosterol. Therefore, shiitake and oyster mushrooms harvested in various development stages.

At different physiological developmental stages, Shiitake ergosterol remain relatively constant. The measured Shiitake ergosterol content ranged from 0.84 - 2.05 mg\gr DM, (perhaps because of the focus on the stalk) (Fig 17A). Exposure of various development stages of shiitake to 10 UV-B pulses from each cap sides did not effect on ergosterol content (similar to the information reported in various studies), except -2 shiitake developmental stage in which the concentration of ergosterol decreased 3.3 times (p <0.05). At early development stages of Oyster mushrooms (-2 and -1) the ergosterol content were significantly higher than optimal developmental stage or later development stage. Similar to Shiitake results, Oyster ergosterol content significantly decreased following irradiation exposer at early development stage. Ergosterol concentration at -2 Oyster developmental stage decreased significantly 2.8 times (Fig. 17B; p<0.05 ).

Moreover, we examined whether the combination of specific developmental stage and radiation exposure can affect the content of mushroom's ergosterol. Shiitake mushroom picking at different stages do not affect the content of ergosterol, but exposure to UV-B radiation led to a significant change in ergosterol concentration (p<0.05), with no significant interaction between those two parameters (Fig 17C). On the other hand, Oyster ergosterol content influenced significantly from physiologic development stage (p<0.01) and UV-B exposure (p<0.05). In addition, there is an interaction between these two parameters (p<0.01) in Oyster mushrooms (Fig. 17D)
In both control mushroom species group, vitamin D2 concertation were under HPLC detection (Fig 18 A-B). Because ergosterol is a precursor of vitamin D, we assumed that mushrooms with high ergosterol concertation will produce high amount of vitamin D following UV-B exposure. In contrast to our hypothesis, following 10 pulses of UV-B from each cap side, Shiitake vitamin D2 concertation were higher significantly at late development stages compered to optimal and earlier development stages (p< 0.05; Fig 18A)., although ergosterol concertation were similar at all development stages. +2 Shiitake development stage produce 3.7 significantly higher vitamin D2 compared to -2 development stage (17.86 vs 66.88 µg/ gr DM; Fig 18A). Nevertheless, although Shiitake +1 development stage tend to produce higher vitamin D2 amount compared to +2 development stage (Fig 18A), it is financially worthwhile to prefer late development stage due to higher mushroom weight. Same phenomenon seems at Oyster mushrooms. Despite early development stages had higher ergosterol content, following 10 pulses of UV-B exposure from each cap side, vitamin D2 concertation were similar at all development stages (Fig 18B).

To evaluate the combination between development picking stages and irradiation exposure we conducted two-way ANOVA test. Regarding Shiitake mushrooms, there's significant interaction (p.< 0.001) between those two parameters. So, for higher vitamin D2 production it's should prefer +2 development stage (Fig 18C). In contrast, Oyster vitamin D2 concertation influenced only by irradiation exposure (p< 0.001; Fig 18D), so we choose to use +1 development stage for further studies duo to from financial reasons.