PROXIMATE AND CHEMICAL PROPERTIES OF SOME UNDERUTILIZED NIGERIAN WILD MUSHROOMS

This investigation aims at determining the nutritional value of twenty-three underutilized wild macrofungi from a biodiversity forest in Southwest Nigeria. The mushroom species collected across the ligneous (woody) and terrestrial (soil) habitats were analysed for proximate (moisture, protein, fibre, lipid, ash and carbohydrate), minerals (potassium, sodium, phosphorus, magnesium, calcium, iron and zinc) and vitamins A and C content following standard analytical procedures. Interestingly, all the mushrooms had high moisture (>80.91%) and those harvested from soil debris in the terrestrial habitat contained significantly high protein content (26.80 48.68%). Dietary fibre was in the range of 0.20 and 42.37%; low lipid (0.12 9.89%) and ash (1.25 14.08%) were also recorded. Furthermore, all the samples contained high carbohydrate except Macrolepiota procera (2.01%). Minerals varied across the habitats and ranged as follows: potassium (268.13 8972.00 mg. 100 g), sodium (89.36 425.92 mg. 100 g), phosphorus (0.32 375.51 mg. 100 g), magnesium (9.39 19.32 mg. 100 g) and calcium (7.98 37.82 mg. 100 g). Low iron (0.55 1.32 mg. 100 g) and zinc (2.21 4.98 mg. 100 g) were obtained. While vitamin A ranged between 0.41 and 1.41 mg. 100 g, vitamin C was from 4.68 to 6.93 mg. 100 g. Conclusively, the mushrooms investigated are a good source of nutrients and thus, can be exploited as foods or food supplements.

following the manufacturer's instruction. Determination of sodium (Na) and potassium (K) were done using flame photometry (Jenway, PFP7, UK) following the manufacturer's guide. The spectrophotometry method using Yellow Vanado molybdate was used in the determination of phosphorus in the mushroom samples (AOAC, 2016).

Determination of vitamins A and C
The chemicals and reagents used included HPLC-grade. Analytical reagent-grade acetonitrile and methanol (Tedia, USA) and vitamins A and C standards (Sigma, Dorset) were used for the analysis.

Vitamin A
One gram (1 g) of pyrogallic acid, 70 ml ethanol and 30 ml (50 %) KOH were added to 10 g mushroom powder, stirred, and refluxed for 40 min using a water bath (50 ± 2 ℃). Extracts were obtained three times using 50, 30 and 20 ml ether. Double-distilled water was used to neutralize the extract, anhydrous sodium sulphate added to dehydrate it and further concentrated to 5 ml using a water bath (50 ± 2 ℃). Methanol was added to the concentrate to 10 ml mark, filtered using a 0.45 m membrane, and finally subjected to HPLC analysis. Reversed-phase (RP) HPLC analysis was performed with the Agilent 1100 series HPLC system (Agilent; USA), including a diode array detector. The column was made of stainless steel Agilent Eclipse XDB-C18 column (5 m, 4.6 × 150 mm) and methanol and UV detection was recorded at 325 nm for vitamin A. Separation was based on isocratic elution and the solvent flow rate was maintained at 1 ml. min -1 . Twenty microlitre of mushroom extract was directly injected into the HPLC column. Identification was done by comparing their retention times with those of known standards. All procedures were carried out under subdued light conditions. Standard solutions of vitamin A were prepared from stock stored in the dark at -20 ℃ by serial dilution to concentrations of 0.1, 1, 2, 5, and 10 mg per litre of vitamin A. Twenty microlitre of the standard solution was injected, and peak areas were determined to generate standard curves.

Vitamin C
Ten grams (10 g) of mushroom powder was mixed with extracting solution prepared by dissolving 15 g of metaphosphoric acid with 40 ml acetic acid and made up to 500 ml with distilled water. The mixture was filtered through Whatman No. 1 filter paper and samples were extracted in triplicate. Ascorbic acid standard solution was prepared by weighing 100 mg ascorbic acid, dissolved and made up to 100 ml with metaphosphoric acidacetic solution. The calibration line was converted to a line arrange based on four measured concentration levels. Quantification of ascorbic acid content was performed on an Agilent HPLC system. Chromatographic separation was achieved on an RP-HPLC column through the isocratic delivery of a mobile phase (A/B 33/67; A: 0.1M potassium acetate, pH = 4.9, B: acetonitrile: water [50:50]) at a flow rate of 1 ml. min -1 . UV absorbance was recorded at 254 nm at room temperature.

Data analysis
All experiments were carried out in triplicates. Data analyses were performed using SPSS IBM version 23 software. All data were expressed as mean ± standard deviation. The difference in means was evaluated using ANOVA and Duncan multiple tests employed in case of variance heterogeneity. Values with a probability level of less than 0.05 were considered significant. Nutritional distance and similarity among mushrooms were determined and dendrogram obtained using a complete linkage approach.

Presumptive identification of mushroom samples
Mushrooms play significant roles in biogeochemical recycling of elements in the environment, human nutrition and dietetics, and medicines (Odeyemi et al., 2014). In this study, a total of 23 different mushroom samples were collected between April and October, 2018 at ENPOST farm, Ido-Ijesa, Ilesa, Southwest Nigeria, and classified into 17 genera, namely: Amanita, Auricularia, Cantharellus, Ganoderma, Hydnellum, Hydnum, Inonotus, Lentinus, Macrolepiota, Marasmiellus, Oxyporus, Pleurotus, Polyporus, Stereum, Termitomyces, Trametes and Tricholoma. Representative pictures of the mushrooms are shown in figure 1. All the genera had one species each except Auricularia (2), Termitomyces (4), Trametes (2) and Tricholoma (2). These mushrooms had been previously reported (Zoberi, 1973 . While some grow on soil or wood substrates, others exist in mycorrhizal relationship with trees (Onuoha and Obi-Adumanya, 2010). In the current study, eight (8) of the mushrooms were from terrestrial habitats whereas the remaining fifteen (15) were of the ligneous origin. This corroborates Adeniyi et al. (2018a) who detected higher mushroom species from the ligneous habitat than the terrestrial. This could probably be attributable to the abundance of lignocellulose substrates, which support the growth of mushrooms on the farm. All the terrestrial mushrooms grew directly on soil debris except Pleurotus tuber-regium, whereas the ligneous mushrooms grew on woody substrates including decaying Cola nitida, Mangifera indica, Bambusa vulgaris leaves, Cordyline australis, and Mangifera indica, decaying Elaeis guineensis and unidentified burnt tree associated with termite nest (Tab 1).

Edibility of the mushroom samples
All the mushrooms from the terrestrial habitat were fleshy, as against those from the ligneous habitat that were either fleshy, woody or tough. Presumptively, nine of the mushrooms (

Lipids
Lipids provide the major caloric value in foods and excess intake can consequentially lead to coronary heart disease and other health issues (dos Passos et al., 2013). Mushrooms are low in lipids dominated by unsaturated fatty acids, contributing less to the energy required in human diet and serving as a means of combating obesity (Wani et al., 2010). Generally, the current investigation ranged between 0.12 and 9.89% (Tab 2). This tally with the observation of Maftoun et al. (2015). Difference in mushroom species may account for the varied lipid contents observed in the study.

Ash
Ash contents indicate the presence of mineral contents in any given food sample (Kassegn, 2018

Potassium
Potassium controls the water and minerals in the blood and tissues, and significant in the transmission of electrical impulses in the heart (Kowey, 2002). Among all the nutrients analysed, potassium predominates other minerals (Tab 3). This is in line with some studies conducted elsewhere ( .

Sodium
Sodium is the main electrolyte and major cation outside the cell (Knochel, 1999;Wardlaw and Kessel, 2002

Magnesium
Magnesium ranged from 9.39 to 19.32 mg. 100 g -1 ) (Tab 3) and its lower compared to many common legumes (178 -197 mg. 100 g -1 ), whole-grain cereal  et al., 2015). It plays an important role in nerve transmission and neuromuscular conduction and protection against excessive excitation that can lead to excitotoxicity (Kirkland et al., 2018).

Calcium
Calcium is one of the foremost microminerals in the bone and teeth of human beings and animals and reduces the chance of having cardiac disorder or any heart-related challenge (Titilawo et al., 2018). The obtained values (7.98 -37.82 mg. 100 g -1 ) (Tab 3) were comparable to many common kinds of cereals (12 -51 mg. 100 g -1 ), starchy food (11 -43 mg. 100 g -1 ), meat and poultry products (4 -69 mg. 100 g -1 ) (FAO, 2012). However, Chye et al. (2008) obtained a range (77 -144.7 mg. 100 g -1 ) higher than ours. Consumption of a very high concentration of calcium may adversely affect the absorption of the essential elements (Nova Scotia Environment, 2005), nonetheless, its paucity could result in osteoporosis and osteomalacia. Hypertension has also been linked to low calcium in the body (Kožĺšek, 2003; Titilawo et al., 2018).

Iron
Iron is required for the synthesis of haemoglobin in red blood corpuscles and also aids growth and metabolic processes in humans and animals ( When zinc is lacking in the diet, it may lead to a reduction in fertility, forfeiture of taste, stunted growth and hypogonadism. Low levels of zinc can as well weaken the body's immunity (Edward et al., 2013; Titilawo et al., 2018).

Vitamin A
Mushrooms are non-animal sources of vitamin, contributing a very small percentage in human diet and crucial in disease prevention and lengthening of life span (Olaniyi, 2000). The outcome of our work revealed that all the wild mushrooms analysed contained vitamin A (Tab 3). This is contrary to Afiukwa et al. (2013) who detected vitamin A only in Agaricus bisporus among other varieties and with a value higher (38.36 mg. 100 g -1 ) than what was observed in our case (0.14 -1.41 mg. 100 g -1 ). Likewise, Musieba et al. (2013) investigated vitamin A in P. citrinopileatus and reported a low value <10 µg/100 g. Our findings agree with vitamin A level reported for some animal and plant materials including butter (0.59 mg. 100 g -1 ), cheese (0.39 mg. 100 g -1 ), egg (0.28 mg. 100 g -1 ), milk (0.04 mg. 100 g -1 ) and salmon (0.041 mg. 100 g -1 ) (Souci et al., 2000). Vitamin A role has been established to include apt sight, reproduction, growth and development, cellular differentiation and immune function (Bowman, 2001).

Vitamin C
The range of vitamin C obtained in this work was from 5.22 to 6.93 mg. 100 g -1 .
While the highest vitamin C content was found in Pleurotus tuber-regium, the lowest was detected in T. pubescens (Tab. 3). The values indicated that the mushrooms are considered good sources of ascorbic acid. Earlier studies by   , 2001). The nutrition hierarchy cluster shows that there is dietary relatedness among the mushroom species irrespective of their habitat, texture or edibility (figure 2). For instance, the fleshy, edible P. tuber-regium and T. inocybeoides in the terrestrial habitat closely associated with woody/tough, inedible G. applanatum and S. hirsutum in the ligneous habitat respectively. Likewise, the fleshy and edible T. striatus and C. cibarius in the ligneous habitat closely related to fleshy, edible T. bulborhizus (terrestrial habitat); and the fleshy, inedible M. candidus in the ligneous habitat found clustered with fleshy, edible M. procera, T. ustale and T. letestui in the terrestrial habitat ( figure 2). Also, the nature of the nutrient-rich organic substrate on which M. candidus, C. cibarius, and T. striatus grew may be responsible for dietary relatedness with mushrooms from terrestrial habitat. Likewise, the presence of sclerotium in P. tuber-regium and decaying debris where Tricholoma inocybeoides were harvested may be the reason their nutrient content is comparable with mushrooms from the ligneous habitat.

CONCLUSION
The nutritional composition of twenty-three underutilized Nigerian wild mushrooms was studied. While eight of the mushrooms were from the terrestrial habitat, fifteen were of the ligneous source. Interestingly, all the mushroom species are rich in moisture, protein, dietary fibre, ash, carbohydrate and minerals including sodium, potassium, calcium, magnesium, zinc, and phosphorus, and vitamins A and C, but low in lipid and iron, an indication that they are highly nutritious; although other sources of sodium must be checked when consuming these mushrooms. Similarly, the nutritive hierarchical cluster analysis shows that the dietary composition of all the mushrooms is related irrespective of their habitat, texture or edibility. Thus, they are potential foods and food supplements and stand to meet the diverse human dietary needs if objectively exploited. Further study is however encouraged to ascertain the safety of these mushrooms for consumption.

Conflicts of interest:
The authors declare no conflict of interest.