Influence of low dose of gamma radiation and storage on some vitamins and mineral elements of dried oyster mushrooms (Pleurotus ostreatus)

Abstract Mushrooms contain some of the most potent natural medicines on the planet. Vitamins A, C, D, Mineral elements, contents, as well as total soluble solids (Brixo) of dried composition of Pleurotus ostreatus were investigated after exposing to gamma radiation doses of 0 (control), 0.5, 1, 1.5, and 2 kGy at a dose rate of 1.7 kGy per hour in air from a Cobalt 60 source (SLL 515, Hungary) batch irradiator prior to storage (0 month) and after storage (12 months) at room temperature (28 ± 2°C). Results obtained showed some significant (p < .05) differences due to irradiation and storage. Before storage, vitamins A, C, and D contents of dried and irradiated mushrooms ranged 0.003 ± 0.08–0.014 ± 0.08, 0.042 ± 1.06–0.132 ± 1.06, and 0.040 ± 0.76–0.057 ± 0.76 mg/g, respectively. After 12 months, vitamin contents decreased and ranged 0.0029 ± 0.08–0.010 ± 0.08, 0.038 ± 1.06–0.125 ± 1.06, and 0.031 ± 0.76–0.05 ± 0.76 mg/g for vitamins A, C, and D, respectively. Total soluble solids recorded 1.5 Brixo, however, showed no significant difference (p > .05) and did not change in 12 months after gamma irradiation up to 2 kGy. Sodium ranged from 14.00 ± 0.7 to 14.90 ± 0.8 mg/100 g. Potassium content varied from 30.20 ± 0.5 to 33.10 ± 0.6 mg/100 g. Magnesium content ranged 1.27 ± 0.15–3.53 ± 0.04 mg/100 g. Calcium ranged 11.00 ± 0.4–12.53 ± 0.4 ± 0.03 mg/100 g. Phosphorus content ranged 6.11 ± 0.30–6.41 ± 0.35 mg/100 g, whereas Nitrogen content was found to be 3.00 ± 0.03–3.60 ± 0.25 mg/100 g. Microelements or heavy metals included Copper; detected ranged 0.00 ± 0.00–0.02 ± 0.001 mg/100 g, Zinc content ranged 0.01 ± 0.002–0.03 ± 0.001 mg/100 g. Iron content was found to be in the range 0.29 ± 0.01–0.37 ± 0.1 mg/100 g. Manganese content was found to be in the range 0.03 ± 0.001–0.04 ± 0.01 mg/100 g. Lead content was found to be 0.00 ± 0.00–0.03 ± 0.001 mg/100 g. Food processing and storage has the potential to slightly alter the stability of vitamins in foods. Pleurotus ostreatus showed appreciable levels of mineral elemental composition, essential vitamins A, C, and D, and can be endorsed as a natural medicinal food product in the food and pharmaceutical industries. The heavy metals detected were with also below the upper limits permissible by the WHO standards and is thus safe for human consumption.

Total Soluble Solids measure the sum of the solids which are in solution. As it increases, water activity is reduced and survival of microorganisms becomes less likely (The Science Dictionary, 2015. Data on mushroom polysaccharides have been collected from hundreds of different species of higher fungus; some specific carbohydrates with these properties have been quantified in different mushrooms: rhamnose, xylose, fucose, arabinose, fructose, glucose, mannose, mannitol, sucrose, maltose, and trehalose (Ferreira, Barros, & Abreu, 2009).
Food irradiation processes have been widely studied and are as well known as any other food processing method, such as dehydration and freezing (Arvanitoyannis, 2008;Crawford & Ruff, 1996).
Nutritional value of the foods subjected to various processing techniques, especially food irradiation, has been questioned by both the activists and consumers alike (Crawford & Ruff, 1996;Kilcast, 1994).
One of the main impedances for the development of this technique in many countries is the misconception consumers have with regards to excessive nutrient denaturation, along with the myth of food becoming radioactive and generation of toxic compounds (Kilcast, 1994).
However, results of research as far back as the 1950's have already shown the absence of radioactivity inducement in the food treated by ionizing radiations (Wiendl, 1984).
Gamma irradiation as a means of preservation of foods has received endorsements by several international bodies (FAO, OIEA, OPS, 1992;ICGFI, 1991;International Atomic Energy Agency, 1999) as an effective means of decontamination which produces minimal or no loss in sensory attributes. The main advantages of irradiation are the small alterations in food components (Kilcast, 1994).
Micronutrients, especially vitamins, can be susceptible to any food treatment method (Crawford & Ruff, 1996;WHO -World Health Organization, 1994), but the extent of losses must not exceed the limit required in food for therapeutic and nutritional benefit to the consumer.
This study investigated the effect of gamma irradiation and storage time on the properties of some essential vitamins (A, C, D), mineral elements, and total soluble solids of P. ostreatus.

| Mushroom material
Pleurotus ostreatus mushroom samples were grown on composted sawdust as described by Kortei et al. (2014) and harvested at maturity from the cropping house of the Mycology Unit, Food Research Institute, Accra, between the periods of February and May, 2014.

| Drying of mushroom samples and storage
Drying was carried out by using a solar dryer at a temperature of 50-60°C to reduce moisture content to about 12% for an average period of 12 days as prescribed by , Kortei, Odamtten, Ayim-Akonor, and Akonor (2016), and Akonor and Tortoe (2014). The dried samples of the mushroom were stored in polythene and polypropylene packs before and after at room temperature.

| Irradiation of mushroom materials
Forty (40) grams of dried oyster mushrooms (Pleurotus ostreatus) were packed and irradiated at doses of 0 (control), 0.5, 1, 1.5, and 2 kGy at a dose rate of 1.7 kGy per hour in air at 28 ± 2°C from a Cobalt 60 source (SLL 515, Hungary) batch irradiator. Doses were confirmed using Fricke's dosimetry system which is a reference chemical dosimeter based on the chemical process of oxidation of ferrous ions (Fe 2+ ) in aqueous sulfuric acid solution to ferric ions by ionizing radiation at the Radiation Technology Centre of the Ghana Atomic Energy Commission, Accra, Ghana.

| Provitamin A or beta carotene (standard preparation)
A small amount of the pure trans-beta-carotene was dissolved in petroleum ether and its concentration was spectrophotometrically determined using the formular below.

| Samples preparation
Extraction was done with 50-ml cold acetone in mortar using a pestle.
It was repeated until the mushroom sample was devoid of color. The extracts were pooled and filtered. The filtrate was partitioned on a 20-ml petroleum spirit in a 500-ml separating funnel. It was washed several times with distilled water until the aqueous layer became clear. The petroleum spirit was dried by passing it through anhydrous sodium sulfate sitted on cotton wool at the base of the funnel. The total volume of the extract was recorded. The total volume, which is a representative of the sample weight, was evaporated under a stream of Nitrogen gas, reconstituted with 1 ml of the mobile phase and, finally, 20 μl was injected into the High Performance Liquid Chromatoghraph (HPLC) (Shimadzu SPD-6A UV spectrophotometric detector, Japan).
The standard was injected thrice and the average of the three (3) corresponding areas was calculated.
The average standard Area = 141282 Therefore, 1.3442 μg/ml = 141282 The samples were injected and the respective areas were obtained using the formular below from which the respective concentrations were calculated:

| Analysis of ascorbic acid
Ascorbic acid was analyzed by reversed-phase chromatography with tetrabutylammonium added as an NH 2 column.

Extraction
Extraction was done by ultrasonication and was diluted with the mobile phase. It was filtered through 0.45-μm membrane filters and 20 μl was injected into the HPLC column.

Standard vitamin C
A known standard concentration (100 mg/Tab.Vitro C, Kinapharma, Ghana) was used to calibrate the instrument which in turn gave a factor, upon which all samples concentration was calculated.

| Analysis of vitamin D
This was carried out using the method prescribed by Wallace, Gibson, De La Hunty, Lamberg-Allardt, and Ashwell (2010).

| Determination of total soluble solids
Estimation was done by dissolving 1 g of dried mushroom sample in 10-ml distilled water and content of sample detected by a hand-held optical refractometer (RF30, Extech Instruments, U.S.A).

| Determination of moisture content
The moisture content was determined by the gravimetric method of AOAC (1995).

| Determination of macro-and microelements (heavy metals)
This procedure was carried out according to a modified method of Obodai et al. (2014). Approximately 0.3 gram was weighed into labeled digestion tubes and dissolved in 2 ml concentrated HNO 3 . The solution was heated at 450°C for 4 hr and later dissolved in 1 ml concentrated H 2 SO 4 , 1 ml HNO 3 , and 1 ml H 2 O 2 , and then diluted with double deionized water up to a volume of 25 ml. A blank digest was carried out by following the above procedure. Contents of macroelements and microelements (heavy metals) in the mushroom samples were determined by using Atomic Absorption Spectrophotometer (Perkin Elmer precisely A Analyst 400).

| RESULTS AND DISCUSSION
According to Murano (1995), when food is irradiated, there is a reaction of ionizing radiation and water in the food causing the release of electrons and the formation of highly reactive free radicals. The free radicals interact with vitamins in ways that can alter and degrade their structure and/or activity. The results obtained from the analysis of total vitamins are presented in Tables 1 and 2. Initial vitamin A contents of mushroom stored ranged from 0.0038 ± 0.0007 to 0.012 ± 0.0008 mg/g. After 12 month of storage, it ranged 0.0031 ± 0.0009-0.010 ± 0.002 mg/g. Low gamma radiation doses and storage time had significant (p < .05) effect on vitamin A content of P. ostreatus. The extent to which vitamin loss occurs can vary based on a number of factors, including the type of food, temperature of irradiation, and availability of oxygen. Nonetheless, vitamin loss almost always increases with increasing doses of radiation (Kilcast, 1994). Wiafe-Kwagyan, 2015). The major antioxidants found in mushrooms are phenolic compounds, whereas other potential antioxidants, for example, vitamin C, β-carotene, and γ-tocopherols, have been found in small quantities (Yang, Lin, & Mau, 2002). Generally, irradiation dose of 0.5 kGy increased production of phenolics in P. ostreatus and caused a significant (p < .05) higher contents of phenolics , Kortei, Odamtten, Ayim-Akonor, et al. (2016). On the other hand, 2 kGy recorded the least phenols in ethanol, methanol, and aqueous extracts of P. ostreatus. This may partly explain the decline in vitamin C with increase in dose from 0 to 2 kGy (Tables 1 and 2).
Vitamin C acts as the first-line natural antioxidant and also serves as a free radical scavenger (Maxwell, 1995). The nonirradiated mushrooms recorded higher values. Previous studies by Kumari and Achal (2008) reported vitamin C values of 0.277 ± 0.0015 mg/g dry fruit body and 0.363 ± 0.0025 mg/g fresh fruit body when they studied the effect of different substrates on the production and nonenzymatic antioxidant activity of P. ostreatus. Obodai (1992) (Kilcast, 1994). However, it has been noted that when reporting vitamin C levels in irradiated food, many workers have not taken into consideration the fact that ionizing radiation can cause a partial conversion of ascorbic acid into dehydroascorbic acid (Kilcast, 1994) reflecting in a lower content of ascorbic acid after irradiation.
Vitamin D content of mushrooms initially ranged from 0.040 ± 0.0090 to 0.054 ± 0.008 mg/g. Gamma radiation and storage time had no significant (p > .05) effect. After 12-month storage, values ranging from 0.036 ± 0.007 to 0.046 ± 0.008 mg/g were detected. T A B L E 2 Effect of gamma irradiation on vitamin A, C, and D (mg/g) contents and total soluble solids (Brix o ) of mushrooms stored for 12 months in polypropylene materials during postharvest storage (Hammond & Nichols, 1975), and steady decreases in the soluble solids concentration were previously reported in fruit bodies stored at cold temperatures (Tseng & Mau, 1999).
Nonetheless, radiation effects on TSS in mushrooms have not been reported exhaustively. This is the first report of effect of gamma irradiation on the TSS content of P. ostreatus cultivated in sawdust in Ghana. Total soluble solids did not change with increasing dose up to 2 kGy and storage for up to 12 months.
Nitrogen content was found to be 3.00 ± 0.03-3.60 ± 0.25 mg/100 g. There were statistical differences (p > .05) observed with the varying treatment. The body utilizes nitrogen for promoting protein synthesis, the creation of compounds and amino acids influence growth, hormones, brain functions, and .10 mg/100 g. As RDI of P is 0.7 g, P. ostreatus is high in P content, and can therefore contribute to human nutrition as good source of phosphorus (Çağlarirmak, 2007).
Sodium contents in this study ranged from 14.00 ± 0.7 to 14.90 ± 0.8 mg/100 g. There were significant differences (p < .05) with increasing dosage. The preponderance of mineral elements in the fruit T A B L E 3 Effect of irradiation on the elemental composition of P. ostreatus before storage (0 months)  bodies could be attributed to the varying degrees of stimulatory effect of doses on dry matter due to activation of cellular and extracellular metabolic enzymes (Dawoud & Abu Taleb, 2011). Oyetayo and Ariyo (2013) reported values of range 4.03 ± 0.02-4.39 ± 0.012 mg/kg in P. ostreatus. Regula and Siwulski (2007)  Heavy metal concentration in mushrooms is considered higher than those in agricultural crop plants, vegetables, and fruits. This connotes that mushrooms have a very effective mechanism which enables them to readily take up some heavy metals from the environment (Zhu et al., 2011) due to their dense mycelia system which infiltrates the substrate (García, Alonso, & Melgar, 2005).
Zinc content was found to be in the range 0.01 ± 0.002-0.03 ± 0.001 mg/100 g (Tables 3 and 4). There was significant differences (p < .05) observed with doses applied. Zinc levels obtained in this study were within the RDI of trace elements reported by Indian Council of Medical Research (ICMR) (1990). Soylak, Saracoglu, Tȕzen, and Mendli (2005) recorded values of range 45.2-173.8 mg/kg, Tuzen (2003) recorded a range 33. 5-89.5 mg/kg, and Isiloglu, Yilmaz, and Merdivan (2001) also recorded a range of 29.3-158 mg/kg. Zn is an essential micronutrient associated with a number of enzymes, especially in the synthesis of ribonucleic acids and DNA polymerases (Sadiq, Bhatti, & Hanif, 2008).
Manganese content was found to be in the range 0.03 ± 0.001-0.04 ± 0.01 mg/100 g. Manganese plays an important role in enzymatic catalysis and is crucial to virtually all biochemical and physiological process (Sadiq et al., 2008). Ahmed et al. (2013) Sesli and Tüzen (1999) also obtained results ranging from 14.5 to 63.6 mg/kg in macrofungi in Turkey.
Results obtained in this study were within the RDI of trace elements reported by Indian Council of Medical Research (ICMR) (1990) and was also found to be below toxicity levels of 400-1000 mg/kg (World Health Organization, 1982a,b).
Lead concentrations of P. ostreatus were found to be nil (0.00 ± 0.00-0.03 ± 0.001 mg/100 g). Results obtained agreed with levels reported by Regula and Siwulski (2007) who did not find lead in Pleurotus ostreatus and Lentinus edodes. Tuzen (2003) and Tuzen, Özdemir, and Demirbas (1998)  According to FAO/WHO (2001) tolerable weekly intake of lead is 0.025 mg/kg body weight. Lead (Pb) is toxic even at trace levels (Dobaradaren, Kaddafi, Nazmara, & Ghaedi, 2010) and the impairment related to Pb toxicity in humans includes abnormal size and hemoglobin content of the erythrocytes, hyperstimulation of erythropoisis and inhibition of hemoglobin synthesis. Lead concentrations detected in P. ostreatus in this study were very low and is considered safe for human consumption.

| CONCLUSION
The primary effects of radiation on vitamins at low and medium doses are not considerable. Studies showed that after low-dose gammairradiation, vitamin losses in the food were minimal in most cases.
Mineral elements found in this study were below the WHO prescribed safe limits and so are safe for human consumption. Although the fruit bodies contained heavy metals like Zn, Fe, Mn, Pb, and Cu, their concentrations were below safe limits set by the WHO and render P. ostreatus safe for human consumption.
A regular and judicious consumption of this mushroom will be beneficial as nutrients and natural medicine giving a healthy diet to Ghanaians and most West African countries where these mushrooms are consumed on a regular basis.