Effect of Ultraviolet Irradiation on Vitamin D in Commonly Consumed Mushrooms in Thailand

This study examined the effect and stability of ultraviolet B (UV-B) irradiation and subsequent cooking on vitamin D content in commonly consumed mushrooms in Thailand. Eight varieties of mushrooms were exposed to two-sided UV-B lamps for up to 3 h in a patented cabinet, followed by vitamin D content analysis. Thereafter, the four mushroom varieties with the highest vitamin D content were exposed to UV irradiation, cooked, and analyzed for various forms of vitamin D using LC-MS-MS. The results showed that vitamin D2 in all varieties of mushrooms significantly increased (p < 0.05) after UV-B irradiation according to the exposure time. The highest level of vitamin D2 was found in enokitake mushrooms. In addition, 25-OH D2 and vitamin D4 contents increased after UV-B irradiation in enokitake mushrooms. The vitamin D2 true retention in all cooked mushrooms ranged from 53 to 89% and was highest in stir-fried mushrooms. With economic investment, the two-sided UV-B cabinet has the potential to increase the vitamin D content in commercial mushroom production.


Introduction
Vitamin D is a calciferol and a fat-soluble vitamin comprising two core forms, namely, cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2), which are structurally comparable secosteroids derived from the UV irradiation of provitamin D sterols [1]. A basic source of vitamin D3 in humans and many animals arises from the transformation of 7-dehydrocholesterol in the epidermis into vitamin D3 upon contact with ultraviolet (UV) irradiation in sunlight [2].
In human nutrition, vitamin D is essential for infants and young children to prevent rickets, while vitamin D deficiency in adults is related to osteoporosis. Vitamin D plays a crucial role in muscle growth and development and may help protect against neurodegenerative diseases, respiratory diseases in children, cardiovascular and autoimmune diseases, and both type 1 and type 2 diabetes, though current evidence for non-skeletal benefits is not yet conclusive [3,4]. Currently, very few diets contain naturally occurring vitamin D. Fish liver oils and the meat of fatty fish are among the greatest sources of vitamin D3. In Thailand, Tirakomonpong et al. (2019) [5] reported that Nile tilapia, common silver barb, and red Nile tilapia have high vitamin D content (19.8, 31.0, and 48.5 µg/100 g fresh weight (FW), respectively). More recently, Sridonpai et al. (2023) [6] reported that termite mushrooms and lung oyster mushrooms have high vitamin D content (7.15 ± 0.67 and 15.88 ± 7.31 µg/100 g edible portion, respectively). a food blender (MR-1268, MARA HOUSE Co., Ltd., Bangkok, Thailand. For freeze-d homogenized UV-B treated samples were put in freeze-dryer trays and kept in a fr at −20 °C for more than 4 h prior to their being transferred to the freeze-dryer ma Freeze-dried samples were dried in the freeze-dryer system (Heto Powerdry PL Freeze Dryer, Corston, UK) until completely dried (2-3 days). After that, the samples ground into homogenized samples (MR-1268, MARA HOUSE Co., Ltd., Bangkok, land) and kept at −20 °C until vitamin D analysis. The mushroom color was determined by comparing mushroom samples befor after UV-B irradiation and at different times to the Munsell color system (Munsell ® , more, MD, USA) and reported as the Munsell color value.

Mushroom Color Determination
The mushroom color was determined by comparing mushroom samples before and after UV-B irradiation and at different times to the Munsell color system (Munsell ® , Baltimore, MD, USA) and reported as the Munsell color value.
A UHPLC (ultimate 3000 system, ThermoFisher, Waltham, MA, USA), connected with a reversed-phase column (Water HSS T3 2.1 × 150 mm, and 1.8 µm particle size) (Acquity uplc, Waters Corporation, Milford, MA, USA), was used to separate the various forms of vitamin D. The mobile phase comprised 2 mM ammonium formate in methanol and 2 mM ammonium formate in water. The flow rate of the mobile phase was 500 µL/min, the temperature of the column was set at 60 • C, and the injection volume was 20 µL. TSQ Quantis tandem mass spectrometry (ThermoFisher, Waltham, MA, USA) was used for quantitative measurement. Atmospheric pressure chemical ionization (APCI) was used as the ionization method for mass spectrometry. Quantitative analysis was performed by MS-MS mode for various forms of vitamin D. The following MS-MS parameters were used: 350 • C of vaporizer temperature, 325 • C of iron transfer tube temperature, 45 Arbitrary units (Arb) of sheath gas, 5 Arb of auxiliary gas, 1 Arb of sweep gas, and 6.5 µA of a positive ion discharge current. Seven vitamers of vitamin D (Vitamin D2, Vitamin D3, Ergosterol, 7-DHC, 25-OH D2, 25-OH D3, and Vitamin D4) and 2 H 3 D 2 (internal standard) were separated and detected by UHPLC-MS-MS). As an example, the chromatograms of the vitamin D components are shown in Figure 2.
forms of vitamin D. The mobile phase comprised 2 mM ammonium formate in methanol and 2 mM ammonium formate in water. The flow rate of the mobile phase was 500 µL/min, the temperature of the column was set at 60 °C, and the injection volume was 20 µL. TSQ Quantis tandem mass spectrometry (ThermoFisher, Waltham, MA, USA) was used for quantitative measurement. Atmospheric pressure chemical ionization (APCI) was used as the ionization method for mass spectrometry. Quantitative analysis was performed by MS-MS mode for various forms of vitamin D. The following MS-MS parameters were used: 350 °C of vaporizer temperature, 325 °C of iron transfer tube temperature, 45 Arbitrary units (Arb) of sheath gas, 5 Arb of auxiliary gas, 1 Arb of sweep gas, and 6.5 µA of a positive ion discharge current. Seven vitamers of vitamin D (Vitamin D2, Vitamin D3, Ergosterol, 7-DHC, 25-OH D2, 25-OH D3, and Vitamin D4) and 2 H 3 D2 (internal standard) were separated and detected by UHPLC-MS-MS). As an example, the chromatograms of the vitamin D components are shown in Figure 2.

Effect of Cooking on High-Vitamin-D Mushrooms
Four mushroom varieties having the uppermost vitamin D content after UV-B irradiation under the optimum conditions were selected to study the effect of cooking. Samples were cooked by stir-frying (with a small amount of palm oil), boiling, and grilling according to common household cooking methods ( Table 2). Yield factor, weight loss, and moisture contents of cooked mushrooms were assessed. Raw and cooked mushrooms were blended in a food blender. Thereafter, the samples were prepared for freeze-drying, ground into fine powder, and kept at −20 °C until vitamin D analysis.

Yield Factor Determination
Yield factor data were calculated from the weight of edible portion before and after cooking according to the formula below.

Effect of Cooking on High-Vitamin-D Mushrooms
Four mushroom varieties having the uppermost vitamin D content after UV-B irradiation under the optimum conditions were selected to study the effect of cooking. Samples were cooked by stir-frying (with a small amount of palm oil), boiling, and grilling according to common household cooking methods ( Table 2). Yield factor, weight loss, and moisture contents of cooked mushrooms were assessed. Raw and cooked mushrooms were blended in a food blender. Thereafter, the samples were prepared for freeze-drying, ground into fine powder, and kept at −20 • C until vitamin D analysis.

Yield Factor Determination
Yield factor data were calculated from the weight of edible portion before and after cooking according to the formula below.
% Yield factor = Weight of edible portion of cooked sample (g) Weight of edible portion of raw sample (g) × 100

Weight Loss Determination
Weight loss data were calculated from the weight of edible portion before and after cooking according to the formula below.
% Weight loss = Weight of raw sample (g) − weight of cooked sample (g) Weight of edible portion of raw sample (g) × 100

True Retention of Vitamin D
Samples were weighed (to at least 2 significant digits) before and after cooking. The data gained, combined with the amounts of vitamin D in raw and cooked mushrooms, were applied to calculate true retention [20].

Moisture Determination
The moisture contents of fresh samples were determined in duplicate according to the Association of Official Analytical Chemists (AOAC) method no. 925.45, 2019 [21]. Moisture content was calculated based on weight loss after drying at 100 + 1 • C.

Statistical Analysis
The vitamin D content of each mushroom sample was presented as mean ± SD. Analysis of variance (two-way ANOVA) and Duncan's multiple range test were used to compare mushroom varieties by UV time and cooking methods to determine the significance of differences (p ≤ 0.05). Correlation analysis was used to identify the relationship of increasing change between vitamin D2 and vitamin D4 contents in mushrooms after exposure to UV-B irradiation. Statistical analysis was performed by using SPSS Statistics for Windows, Version 19.0.

Physical Characteristics of Raw Mushroom Samples
Most mushrooms of the same species including oyster mushrooms (Bhutan and lung oyster mushrooms) and shimeji mushrooms have similar characteristics (size of the cap, stalk, and height). However, they are visually different in color, which is a factor in identifying and confirming different varieties of mushrooms even in the same species (e.g., white shimeji and brown hon shimeji mushrooms) (Tables 3 and 4).     The vitamin D2 contents of the different varieties of mushrooms exposed to UV-B irradiation at different times are shown in Tables 5-8. The control mushroom (unexposed to UV-B) that showed the highest vitamin D2 content was the white shimeji mushroom, 83.73 ± 12.79 µg/100 g dried weight (DW) (7.96 ± 1.22 µg/100 g fresh weight, FW), while the lowest contents were in wood ear and straw mushrooms, which were undetectable and found to be less than 1 µg/100 g DW in enokitake, shiitake, and brown hon shemeji mushrooms. Most vitamin D2 content levels of mushrooms in this study were found in the same range of raw cultivated mushrooms purchased from retail shops sold in the UK, Europe, North America, Australia, and New Zealand, which commonly reported less than 1 µg/100 g FW [2,12,13,22,23].   Note: C refers to the mushroom cap, while S refers to the mushroom stalk/stem. Different letters in the same column indicate significant differences (p < 0.05). Note: C refers to the mushroom cap, while S refers to the mushroom stalk/stem. Different letters in the same column indicate significant differences (p < 0.05).  Note: C refers to the mushroom cap, while S refers to the mushroom stalk/stem. Different letters in the same column indicate significant differences (p < 0.05).
After UV-B irradiation, the overall amount of vitamin D2 in mushrooms increased significantly (p < 0.05) after exposure, which agrees with a previous study that reported that vitamin D2 content in mushrooms increased after UV exposure [16]. In addition, Keegan et al. (2013) [24] reported that mushrooms exposed to UV-B radiation contain a significant amount of vitamin D2. For the enokitake mushroom, the amount of vitamin D2 increased significantly (p < 0.05) to the highest amount, from 6.68 ± 1.42 to 1880.22 ± 197.76 µg/100 g DW (0.77 ± 0.16 to 271.59 ± 28.57 µg/100 g FW), after UV-B irradiation from 0 to 3 h, followed by Bhutan oyster, lung oyster, and brown hon shimeji mushrooms. While not being detected for both wood ear and straw mushrooms before UV-B irradiation, the amount of vitamin D2 in these mushrooms increased significantly (p < 0.05) after exposure to irradiation for 3 h. This finding is similar to a study by Hu et al. (2020) [24], which reported that the amount of vitamin D2 increased from an undetectable amount to 23.71 ± 5.72 µg/g DW in dry oyster mushroom powder upon UV irradiation.
The mushrooms with the highest levels of vitamin D2 were the enokitake mushroom, brown hon shimeji mushroom, Bhutan oyster mushroom, and lung oyster mushroom. Consequently, these varieties were selected for studying the effect of cooking on vitamin D retention. In addition, the optimum condition for UV-B irradiation (one hour at a distance of 15 cm from the UV-B lamp) was identified based on three main factors: color change, increasing vitamin D2 content, and the price of the mushrooms.

Ergosterol Content
High amounts of ergosterol in the cultivated mushrooms were found (Tables 5-8). The ergosterol contents in several varieties of cultivated mushrooms were not significantly different (p < 0.05) before and after UV-B irradiation including wood ear, Bhutan oyster, white shimeji, brown hon shimeji, and lung oyster mushrooms. For the enokitake mushroom, the ergosterol content appeared to decrease by 19% from 140.58 ± 3.97 to 114.29 ± 11.92 mg/100 g DW (16.30 ± 0.46 to 14.29 ± 1.49 mg/100 g FW) after exposure to UV-B for 1 h. The amount of ergosterol in the lung oyster mushroom decreased slightly when exposed to UV-B for 3 h. Further, the level of ergosterol in the shiitake mushroom decreased by 40% from 211.48 ± 9.91 to 137.16 ± 18.00 mg/100 g DW (34.91 ± 1.64 to 27.58 ± 3.62 mg/100 g FW) after exposure to UV-B for 3 h. This finding agreed well with the results of the study of Hu et al. (2020) [25], which reported that the amount of ergosterol decreased with an increase in vitamin D2, whereas most of the ergosterol was probably UV-degraded. Jasinghe and Perera (2005) [16] and Perera (2003) [26] reported that, in shiitake mushrooms (fresh), the amount of ergosterol was highest in the gills, followed by the cap and stalk, with the gills having twice the amount of ergosterol compared to the cap. Moreover, the amounts of vitamin D2 in common mushroom varieties generally depend on several factors, such as time of day, season, latitude, weather conditions, surface area, exposure time, and the morphology of each mushroom variety.

Vitamin D4 Content
A study by Phillips et al. (2012) [2] confirms recent research findings that have identified vitamin D4 and provitamin D4 in various edible mushroom species. In the present study, vitamin D4 content in the cultivated mushrooms varied in both the control (unexposed UV-B) and the UV-B exposed samples (Tables 5-8). After irradiation, the amount of vitamin D4 in all types of mushrooms increased according to the time of UV-B exposure from 0.25 to 3 h. The highest concentration of vitamin D4 was found in the enokitake mushroom, which increased (2301%) significantly (p < 0.05), from 282.59 ± 0.34 to 6504.61 ± 21.74 µg/100 g DW, which was 29 times higher after being exposed to UV-B from 0 to 3 h, followed by lung oyster and Bhutan oyster mushroom. In addition, vitamin D4 content in brown hon shimeji mushrooms increased (552%) significantly (p < 0.05), from 261.23 ± 10.60 to 1702.47 ± 0.93 µg/100 g DW. A study by Krings and Berger (2014) [29] reported that UV-B lamp irradiation led to an increase in vitamin D4 content in oyster mushrooms from 0 to 20 µg/g DW after only 30 min of exposure. The 22,23-dihydroergosterol (provitamin D4) in mushrooms is converted to vitamin D4. All commonly consumed mushrooms contain provitamin D4, making them a probable source of vitamin D4 if exposed to UV radiation.

The Correlation between Vitamin D2 and Vitamin D4 Contents in Mushrooms after Exposure to UV-B Irradiation
The same trend of increasing vitamin D2 and vitamin D4 contents when exposed to UV-B irradiation for 0-3 h is shown in Tables 5-8. Statistical analysis of the correlation, vitamin D2, and vitamin D4 contents showed a statistically significant linear relationship (r = 0.893, n = 96, p < 0.01). The direction of the relationship is positive, meaning that vitamin D2 and vitamin D4 in all types of mushrooms tend to increase together after exposure to UV irradiation for 0-3 h. This finding agrees well with those of previous studies on vitamin D4 in mushrooms after exposure to UV-B irradiation that showed a positive correlation between vitamin D2 and vitamin D4 [2,30].

Edible Portion
Mushrooms tend to contain a high percentage of edible portion (over 80%). The percentages of edible portions in the cultivated mushrooms differed depending on variations within mushroom species (Table 9). The results showed that the percentages of edible portion for the cultivated mushrooms were within the same range of 80-95%. Hence, whole mushrooms were prepared as an edible portion and used for moisture and vitamin D analyses.