Analysis of cultivation and bioactive compounds on Pleurotus florida (Fr.) Kumm and Hypsizygus ulmarius (Bull. ex. Fr.)

1 Govintharajan et al. Int. J. Biosci. 2024 RESEARCH RESEARCH RESEARCH RESEARCH PAPER PAPERPAPER PAPER OPEN ACCESS OPEN ACCESSOPEN ACCESS OPEN ACCESS Analysis of cultivation and bioactive compounds on Pleurotus florida (Fr.) Kumm and Hypsizygus ulmarius (Bull. ex. Fr.) Abirami Govintharajan * , Gomathi Selvam, Ambikapathy Varatharaju, Panneerselvam Annamalai PG and Research Department of Botany, AVVM Sri Pushpam College, Poondi, (Affiliated to Bharathidasan University, Trichy-24), Thanjavur (Dt), Tamil Nadu, India Key words: Edible mushroom, Bioactive compounds, Different solvents and various agricultural substrates http://dx.doi.org/10.12692/ijb/25.2.1-9 Article published on August 03, 2024 Abstract Mushroom cultivation is one of the most profitable business and environment friendly enterprises, with the various horticultural crops in India. In the current investigation focused on the cultivation of edible mushroom with two different agricultural wast e substrates were performed. The two types of raw materials such as paddy straw and sugar cane trash were used for cultivation of Pleurotus florida and Hypsizygus ulmarius. Growth and yield parameters of P. florida was initiated from I, II and III harvest ed stages were observed. The first harvest mushroom in both was excellent growth and weight when compared with the other two harvests. The total yield of edible mushrooms in paddy straw was maximum production than that of sugarcane trash respectively. Whereas H. ulmarius mushroom was moderate growth yield were performed. The biological efficiency of H. ulmarius grows with paddy straw was maximum yield found to be recorded than sugarcane trash substrate respectively. However, the paddy straw substrate was ex cellent for cultivation of mushroom when compared with sugarcane trash substrates. Maximum fruit bodies were harvested when P. florida were cultivated in paddy straw substrates Qualitative insights of bioactive compounds such as alkaloids, amino acids, co umarins, flavonoids, glycosides, phenols, phlobatannins, quinones, saponin, steroids, tannin and terpenoids were screened from P. florida and H. ulmarius with four different solvents were used for extraction. Among the four solvents extracts of the diethyl ether using P. florida showed maximum bioactive compounds like alkaloids, amino acids, coumarines, glycoside, phenols, phlobatannins, quinones, saponin, steroids, tannin and terpenoids recorded respectively. These bioactive compounds are responsible for m any biological properties in our day today life. Hence, these bioactive compounds are responsible for many nutraceutical properties are described in mushrooms for prevention of various diseases including hypertension for the human era. * Corresponding Author: Abirami Govintharajan  [email protected] International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print) 22225234 (Online) http://www .innspub.net Vol. 25, No. 2, p. 1-9, 202 4 2 Govintharajan et al. Int. J. Biosci. 2024 Introduction Research on the therapeutic properties of oyster mushrooms started in the late 20th century. First, their hypertensive properties were confirmed (Bajaj et al., 1997). Pleurotus mushrooms are regarded as being nutritious due to their abundance in proteins, fibre, vitamins, and minerals because of their tasty flavour, pleasant perfume, and medical benefits, pleurotus mushrooms are used as a functional food (Feeney et al., 2014). Because of their tasty flavour, pleasant perfume, and medical benefits, Pleurotus mushrooms are used as a functional food (Knop et al., 2015). They have been chiefly used in traditional medicine in China and other Asian countries (Singh 2011). The substrate used for their cultivation does not require sterilization, only pasteurization, which is less expensive. Growing oyster mushrooms convert a high percentage of the substrate to fruiting bodies, increasing profitability. P. ostreatus demands few environmental controls and their fruiting bodies are not often attacked by diseases and pests and they can be cultivated in a simple and cheap way. All this makes P. ostreatus cultivation an excellent alternative for production of mushrooms when compared to other mushrooms (Guillamon et al., 2010). The majority of lignocellulosic substrates and other wastes produced by the agricultural, forestry and foodprocessing industries are capable of being colonised and degraded by edible mushrooms. Particularly when compared to other edible mushrooms, P. ostreatus requires a shorter growing period. The substrate used for their cultivation just needs to be pasteurised, which is less expensive, rather than sterilised (Carmen, 2010). Mushrooms have become well-known globally for their nutritional and therapeutic properties (Aditya et al., 2024). The substrates that have been used for mushroom production in previous studies include rice straw, rice bran, wheat straw, pulp, corncobs, cocoa shell waste, cotton waste, spent grain, sawdust, maize husks, and cassava peelings (Samuel, 2012; Shah et al., 2004). Other substrates are soybean straw, paddy straw, sun flower stalks, sugarcane bagasse, fruit waste, used tea leaves, bamboo leaves, and maize stalk (Diriba et al., 2013; Dehariya and Vyas, 2013). More recently, elaborated on the bioactivities of polysaccharides from Pleurotus species and the development of new extraction methods. The bioactive compounds identified in Pleurotus mushrooms can be divided into those with a high molecular weight and those with a low molecular weight (Chukwurah et al., 2012). Materials and methods Sample collection site In the present study Pleurotus florida and Hypsizygus ulmarius strain isolated samples obtained from fresh, healthy spawn MM spawn lab GSP mushroom farm, Karanthai, Thanjavur, Tamil Nadu, India. The mycelia form of colonies of different strains was maintained on PDA medium where inoculated in to 100 ml potato dextrose broth contained in 250 ml conical flask. After four weeks growth to the fungus in the liquid medium were observed. Another type of cultures were maintained from petriplates and test-tubes are used in the culture was maintained potato dextrose agar (PDA) medium. Mushroom cultivation Mushroom cultivation was adopted according to the Manimaran et al. (2017). Culture media The mushroom strains can be maintained in semi synthetic solid medium. Potato Dextrose Agar (PDA) is the most commonly used media for maintaining the inoculums and they showed good growth rate. PDA medium composition Dextrose 20.00g Peeled Potato pieces 250.00g Agar 18.00g Distilled water 1000 ml PH 7.0 to 7.5. About 250gms of Potato tubers were washed, peeled off, cut into small pice and taken in a 500 ml conical flask containing 300ml of water. It was boiled for about half an hour and the extract was decanted. About 18gm of agar shreds were weighted, taken in another conical flask and 500ml of distilled water was added. The agar was melted over a heater. The molten agar was then mixed with the potato extract and made up to 1 litter. Then 15gm of dextrose was added to the 3 Govintharajan et al. Int. J. Biosci. 2024 medium and mixed thoroughly. Since the growth of the fungus is flavoured by acidic range the pH was adjusted to 5-6. The medium was distributed in 250ml conical flask plugged with cotton and sterilized in an autoclave. To avoid bacterial growth by streptomycin sulphate was added. Culturing of Pleurotus florida and Hypsizygus ulmarius mycelium on Petriplates Inoculation technique The sterilized bottles are placed in the culture room. The UV lamp is switched on for 15 minutes to sterilize the air inside and then surface was cleaned using alcohol to ensure axenity before the use. The growing edge of the fungi from Petri plate was cut with the help of a cork borer and transferred to the spawn bottle in front of the flame. The bottles are incubated at room temperature. The white mycelium is observed in the entire bottle after 12 days of inoculation. This is known as ‘mother spawn’. Substrates Mushroom beds can be prepared using Paddy straw and sugarcane trash substrates. One spawn bottle can be used to prepare two beds. Size of the bed should be about 30×60 cm. Bed preparation Fresh substrates are chopped into pieces of 2-3 inches length and soaked in water for 10 hours. Water is then drained off from the substrates. Afterwards, the substrates are sterilized using vertical autoclave at 15 lbs pressure for 20 minutes. The sterilized substrates are placed on a wire mesh net for draining excess water. Polythene covers in the size of 30×60cm are procured and filled with the treated substrates. Before preparing mushroom beds, hands and all the instruments should be sterilized with a dilute solution of KMnO 4 / alcohol. A polythene bag is tied at one end and sterilized substrates are filled through the open end for about 5cm in length. A handful of spawn from the bottle is spread (15g) towards the periphery of this layer. Over the spawn some more substrates are put and pressed lightly. This process is repeated five times. The mouth of the bag is rolled and closed with stapler pins. Holes are made over the bag for aeration. Inoculated paddy straw bags are kept in a ventilation dark chamber. The mycelia will colonize the entire paddy straw bag within 15 days. Now the polythene cover is peeled off and the compact lump of paddy straw is placed in a cool shady room and sprayed with water 3-4 times per day. The young fruit bodies will come out from the bag. When the fruit bodies attain full growth, they could be harvested. Bioactive compounds screening of mushrooms Phytochemical analysis was carried out for solvents of mushrooms as per standard methods (Ebana et al., 2015) but with some little modifications. Preparation of extracts After collection, the mushroom samples were wrapped in newspaper and stored in moisture-free open spaces. The removal of all foreign matters was done. Then, they were ground using a metal mortar and pestle. The powder was collected and ground again at the end. The bioactive components of oyster mushrooms were determined using standard method (Khan et al., 2006; Garcha, 1994; Sofowora, 1982). Detection of bioactive components The bioactive component analysis was done using standard method (AOAC, 1984). Qualitative bioactive compounds analysis Freshly prepared different solvents extracts were tested for bioactive compounds using standard methods. The bioactive compounds such as alkaloids, amino acids, coumarins, flavonoids, glycoside, phenols, phlobatannins, quinones, saponins, steroids, tannins and terpenoids were analysed with the solvents of aqueous, ethanol, methanol and diethyl ether extracts (Harborne, 1973). Quantitative bioactive analysis Preliminary bioactive substances like alkaloids, aminoacids, coumarins (Harborne, 1973), flavonoids (Boham and Kocipai, 1994), glycoside, phenols, phlobatannins, quinines (Harborne, 1973), saponins (Obadoni and Ochuko, 2001), steroids (Harborne, 4 Govintharajan et al. Int. J. Biosci. 2024 1973), tannins (Van-Burden, 1981) and terpenoids (Harborne, 1973) were analysed by using standard methods. Statistical analysis Experiments were carried out in triplicate and the results are expressed as mean values with standard deviation. Results Grown on a variety of crop residues as substrates were oyster mushrooms, P. florida and H. ulmarius (Table 1 & 2). Spawn running phase The ideal primordial initiation days varied between species and substrates, ranging from 15-20 days for the full spawn run of P. florida on paddy straw to 1725 days on sugarcan trash for H. ulmarius. The least optimal growth period was recorded at 16 and 24 days for P. florida and H. ulmarius, respectively, when using paddy straw as the substrate. This difference was found to be statistically significant, indicating that the specific substrate used had an effect on the primordial initiation period. Additionally, the difference between the two species was also found to be statistically significant, suggesting that there are differences between them in terms of their optimal growth periods. Pin head formation Among sugarcane trash combinations, the maximum number of days between pinhead formation and fruiting body formation varied significantly (21 and 28 days for P. florida and H. ulmarius, respectively). On paddy straw, the shortest growth periods for the development of fruiting bodies were observed (19 - 25 days for P. florida and H. ulmarius respectively). Table 1. Effect of different agricultural waste substrates and biological efficiency of P. florida Substrates Size of the bag Spawn run (days) Pin head formation (days) Yield per harvest (g/kg) Total Yield (g/kg) I II III Paddy straw 60 × 30 cm 16 19 420 360 260 1,040 Sugarcane trash 18 21 380 260 220 860 Table 2. Effect of different agricultural waste substrates and biological efficiency of H. ulmarius Substrates Size of the bag Spawn run (days) Pin head formation (days) Yield per harvest (g/kg) Total Yield (g/kg) I II III Paddy straw 60 × 30 cm 22 25 360 273 220 853 Sugarcane trash 24 28 340 240 190 770 Table 3. Qualitative analysis of bioactive compounds of Pleurotus florida Bioactive compounds Different solvents Aqueous Diethyl ether Ethanol Methanol Alkaloids + + + + Amino acids + + + + Coumarins + + - - Flavonoids + - + + Glycosids + + - + Phenols + + + + Phlobatannins + + + + Quinones + + - - Saponin - + - - Steroids + + + + Tannin + + - - Terpenoids - + - + (+)Present (-) Absent 5 Govintharajan et al. Int. J. Biosci. 2024 Table 4. Quantitative analysis of bioactive compounds of P. florida Bioactive compounds Quantity (mg/g) Aqueous Diethyl ether Ethanol Methanol Alkaloids 1.05±0.03 1.02±0.06 1.10±0.03 1.09±0.06 Amino acids 2.02±0.23 2.31±0.00 1.63±0.36 1.36±0.30 Coumarins 1.03±0.00 1.03±0.23 - - Flavonoids 1.10±0.07 - 1.69±0.00 1.49±0.09 Glycosids 1.03±0.23 1.26±0.20 - 1.00±0.00 Phenols 1.51±0.01 1.26±0.08 1.38±0.03 1.44±0.43 Phlobatannins 1.66±0.22 1.30±0.02 1.00±0.00 1.02±0.36 Quinones 1.00±0.02 1.25±0.63 - - Saponin - 1.16±0.03 - - Steroids 1.45±0.00 1.75±0.05 1.74±0.03 1.17±0.09 Tannin 1.36±0.00 1.65±0.63 - - Terpenoids - 1.63±0.02 - 1.36±0.36 Standard deviation ± error Fresh weight of fruiting bodies On paddy straw, P. florida and H. ulmarius harvested mushrooms had a maximum fresh weight of 420g and 360g, respectively, and a minimum fresh weight of 380g and 340g, respectively, from the sugarcane waste. On paddy straw, harvested mushrooms had a maximum fresh weight of 360g and a minimum fresh weight of 240g for P. florida and H. ulmarius, respectively. The highest fresh weight of mushrooms from the third harvest (260g and 220g of P. florida and H. ulmarius) was recorded on paddy straw and the lowest (220g and 190 g of P. florida and H. ulmarius) was recorded from the sugarcane trash respectively. Paddy straw recorded the maximum fresh weight of harvested mushrooms (P. florida and H. ulmarius) (420 g and 360 g), whereas sugarcane trash recorded the minimum fresh weight (380 g and 340 g) (Table 3 & 4). Table 5. Qualitative analysis of bioactive compounds of H. ulmarius Bioactive compounds Different solvents Aqueous Diethyl ether Ethanol Methanol Alkaloids + + + + Amino acids + + + + Coumarins + + - - Flavonoids + - + + Glycosids - + - - Phenols + + + + Phlobatannins + - + - Quinones + + - - Saponin - - - - Steroids + + + + Tannin + + - - Terpenoids - + - + (+) Present (-) Absent Bioactive compounds of P. florida and H. ulmarius The present study was carried out using a preliminary bioactive compound analysis of P. florida and H. ulmarius with four solvents such as aqueous, diethyl ether, ethanol and methanol were performed. Diethyl ether and aqueous extracts of P. florida contain bioactive compounds with medicinal uses. Both aqueous and diethyl ether extracts of P. florida contained alkaloids, amino acids, coumarins, glycosides, phenols, phlobatannins, quinones, and steroids. Flavonoids are not present in diethyl ether but they are present in aqueous solutions. Diethyl ether contains saponin and terpenoids that are absent from the aqueous extract. P. florida ethanol and methanol extracts were found to be devoid of some bioactive compounds, such as alkaloids, amino acids, flavonoids, phenols, phlobatannins and steroids as well as coumarins, saponins, quinones and tannins which are usually absent. 6 Govintharajan et al. Int. J. Biosci. 2024 Table 6. Quantitative analysis of bioactive compounds of H. ulmarius Bioactive compounds Quantity (mg/g) Aqueous Diethyl ether Ethanol Methanol Alkaloids 1.05±0.13 1.00±0.15 1.13±0.13 1.19±0.16 Amino acids 4.00±0.03 3.69±0.55 1.13±0.16 2.16±0.10 Coumarins 1.01±3.00 1.36±0.15 - - Flavonoids 1.00±0.09 1.36±0.69 2.19±0.10 2.09±0.19 Glycosids 1.03±0.33 1.23±0.32 - - Phenols 1.01±0.11 1.60±0.58 1.08±0.13 1.04±0.43 Phlobatannins 1.06±0.02 - 1.10±0.00 2.12±0.26 Quinones 1.10±0.12 1.87±0.00 - - Saponin - - - - Steroids 1.05±1.00 1.63±0.36 1.04±0.13 1.10±0.00 Tannin 1.06±3.00 1.47±0.26 - - Terpenoids - 1.69±0.26 - 1.06±0.00 Standard deviation ± error Diethyl ether and aqueous extracts of H. ulmarius revealed the presence of medicinally essential bioactive components. The aqueous and diethyl ether extracts of H. ulmarius have been located to comprise alkaloids, amino acids, coumarins, flavonoids, glycosides, phenols, quinones, steroids and tannin. These are commonly present and saponin is generally absent. Phlobatannins are present in aqueous solutions but are absent in diethyl ether. Terpenoids are found in diethyl ether but are absent from the aqueous extract. H. ulmarius ethanol and methanol extracts have been found to contain a few bioactive compounds that are commonly found with alkaloids, amino acids, flavonoids, phenols, phlobatannins, and steroids, but coumarins, glycosides, quinones, and saponins are usually absent. The ethanol extract of H. ulmarius, which turned into found to be without bioactive compounds, including terpenoids, the methanol extract carries them (Table 5 & 6). Discussion The oyster mushroom (Pleurotus ostreatus) was successfully grown on substrates used for cultivation. (Obadoni and Ochuko, 2001) reported that agricultural wastes for substrate production and nearly all types of agricultural waste can be used to grow mushrooms. Oyster mushrooms (P. ostreatus) could be grown on corncob, finger millet straw, bamboo waste, and their combinations with varying growth performance. The lowest biological efficiency and fresh weight were recorded from a mixture of corncob and bamboo waste substrates. Finger millet straw was the best substrate in terms of yield and biological efficiency (Van-Burden and Robinson, 1981). The four Pleurotus species, P. citrinopileatus, P. eryngii, P. ostreatus, and P. sapidus showed different mycelial growth rates and colonisation on the different substrates used. It has been indicated that P. ostreatus recorded the shortest colonisation time of the two substrates followed by P. sapidus (Brazkova et al., 2022) However, P. eryngii had the most extensive mycelial colonization followed by P. citrinopileatus. Coffee parchment gave the fastest mycelia colonisation time as compared to the mycelia colonisation on coffee husks. However, this may also be due to variations in the structure, chemical composition and nutrient content of the coffee parchment substrate (2022). The time it took for pinheads (primordials) to form after the spawn run varied between the four mushroom species. P. ostreatus recorded the earliest pinhead formation (20 days), followed by P. sapidus (22 days) and lastly P. citrinopileatus (23 days) and P. eryngii (26 days). This may be due to the variations in extracellular enzyme production and the prevailing mushroom growing conditions since each Pleurotus species requires different environmental conditions of CO 2 concentration, relative humidity, and temperature (Bulti et al., 2021). They could be able to variations in cultivation and storage conditions (Liuzzi et al., 2023). 7 Govintharajan et al. Int. J. Biosci. 2024 Pleurotus mushrooms are a good source of bioactive substances. Despite the fact that the number of studies on the health-promoting effects of Pleurotus mushrooms has increased dramatically in recent years (Guta, 2022), P. ostreatus and P. florida revealed the presence of major bioactive components such as flavonoids, polyphenols, saponins, triterpenoids and steroids. This result is similar to that of (Getachew et al., 2019). The bioactive compound such as alkaloids, glycosides, resins and tannins were absent from the methanol extracts. Bioactive compounds found in edible mushrooms are known to play a vital role in promoting health. The absence of alkaloids and glycosides confirms the report (Wona et al., 2018). Preliminary bioactive compounds analysis of P. florida. Were determined the ethanol extract of P. florida revealed the presence of medicinally important bioactive ingredients. The aqueous extract of P. florida was found that the alkaloids, flavonoids, terpenoids, steroids and cardiac glycosides whereas ethanol extract of P. florida showed, the presence of alkaloids, flavonoids, terpenoids, saponins, and steroids. The aqueous extract of P. florida was found to be some phytochemical compounds such as phenols, saponins, tannins, quinines, phlobatannins and anthroquinones. The bioactive characters of P. florida investigated in water and ethanol extract were summarised (Iwalokun et al., 2007). Conclusion It can be concluded that the yield of P. florida and H. ulmarius grown on paddy straw and sugarcane trash as substrates. The results revealed that P. florida yielded the maximum biological efficiency on the paddy straw substrates followed by H. ulmarius which produced the least biological efficiency on the when compared with sugarcane trash substrates. Paddy straw was the best substrate in terms of yield and biological efficiency. However, the paddy straw substrate was excellent substrate candidature for the growth of mushroom growers. The bioactive component analysis of edible mushrooms P. florida and H. ulmarius revealed the presence of major bioactive components such as alkaloids, amino acids, coumarins, glycosides, phenols, phlobatannins, quinones and steroids. The aqueous and diethyl ether solvents are the most bioactive compounds present in both mushrooms. Acknowledgments The authors sincerely acknowledge the services rendered by the management and Principal of A. V. V. M. Sri Pushpam College (Autonomous), Poondi, Thanjavur for the successful completion of Research work. References Association of Official Analytical Chemists (AOAC). 1984. Official Methods of Analysis, 13th ed. AOAC, Washington D.C. 987-1012. Bajaj M, Vadhera S, Brar A, Soni G. 1997. Role of oyster mushroom (Pleurotus florida) as hypocholesterolemic/antiatherogenic agent. Indian Journal of Experimental Biology 35(10), 1070-1075. Boham BA, Kocipai AR. 1994. Flavanoids and condensed tannins from leaves of Hawaiian Vaccinium reticulatum and V. calycinum. Pacific Science 48, 458-463. Bulti KF, Belsti AT, Mestawot MT. 2021. Cultivation of Pleurotus ostreatus on agricultural wastes and their combination. International Journal of Agronomy 2021, 1-6. Chukwurah NF, Eze SC, Chiejina N. 2012. Performance of oyster mushroom (Pleurotus ostreatus) in different local agricultural waste materials. African Journal of Biotechnology 11(37), 8979-8985. Dehariya P, Vyas D. 2013. Effect of different agrowaste substrates and their combinations on the yield and biological efficiency of Pleurotus sajor-caju. IOSR Journal of Pharmacy and Biological Sciences 8(3), 60-64. 8 Govintharajan et al. Int. J. Biosci. 2024 Diriba M, Gume B, Abate D. 2013. Evaluation of locally available substrates for cultivation of oyster mushroom (Pleurotus ostreatus). African Journal of Microbiology Research 7(20), 2228-2237. Dissasa G. 2022. Cultivation of different oyster mushroom (Pleurotus sp.) on coffee waste and determination of their relative biological efficiency and pectinase enzyme production, Ethiopia. International Journal of Microbiology 2022, 1-10. Ebana RUB, Etok CA, Edet UO. 2015. Phytochemical screening and antimicrobial activity of Nypa fruticans harvested from Oporo River in the Niger Delta Region of Nigeria. International Journal of Innovation and Applied Studies 10(4), 1120-1124. Feeney MJ, Dwyer J, Hasler-Lewis CM. 2014. Mushrooms and health summit proceedings. Journal of Nutrition 144(7), 1128S-1136S. Garcha HS. 1994. A manual of mushroom growing. PAU, Ludhiana. Getachew A, Keneni A, Chawaka M. 2019. Production of oyster mushroom (Pleurotus ostreatus) on substrate composed of wheat straw, waste paper, and cotton seed waste. International Journal of Microbiology and Biotechnology 4(2), 38-44. Guillamon E, Garcia-Lafuente A, Lozano M, D’Arrigo MA, Rostagno MA, Villares A. 2010. Edible mushrooms: role in the prevention of cardiovascular diseases. Fitoterapia 81, 715-723. Harborne JB. 1973. Phytochemical methods: A guide to modern techniques of plant analysis. 2nd ed. London, New York. Iwalokun BA, Usen UA, Otunba AA, Olukoya DK. 2007. Comparative phytochemical evaluation, antimicrobial and antioxidant properties of Pleurotus ostreatus. African Journal of Biotechnology 6, 1732-1739. Khan SM, Nazir J, Zahoor HK, Sultan MK. 2006. Yield performance of oyster mushroom. Pakistan Journal of Phytopathology 18, 89-93. Knop D, Yarden O, Hadar Y. 2015. The ligninolytic peroxidases in the genus Pleurotus: divergence in activities, expression, and potential applications. Applied Microbiology and Biotechnology 99(3), 1025-1038. Manimaran K, Murugesan S, Laksmikanth S. 2017. In vitro studies on preliminary phytochemical screening of Pleurotus florida cultivated on paddy straw. Indo American Journal of Pharmaceutical Research 7(7), 1-10. Obadoni BO, Ochuko PO. 2001. Phytochemical studies and comparative efficacy of the crude extracts of some homeostatic plants in Edo and Delta states of Nigeria. Global Journal of Pure and Applied Sciences 8, 203-208. Samuel AA, Eugene TL. 2012. Growth performance and yield of oyster mushroom (Pleurotus ostreatus) on different substrate compositions in Buea, South West Cameroon. Science Journals Publication 2012, 1-6. Sánchez C. 2010. Cultivation of Pleurotus ostreatus and other edible mushrooms. Applied Microbiology and Biotechnology 85, 1321-1337. https://doi.org/10.1007/s00253-009-2343-7. Shah ZA, Ashraf M, Ishtiaq C. 2004. Comparative study on cultivation and yield performance of oyster mushroom (Pleurotus ostreatus) on different substrates (wheat straw, leaves, sawdust). Pakistan Journal of Nutrition 3(3), 158-160. Singh MP, Singh VK. 2011. Yield performance and nutritional analysis of Pleurotus citrinopileatus on different agro-wastes and vegetable wastes. Paper presented at: The 7th International Conference on Mushroom Biology and Mushroom Products; Oct 4-7; Arcachon, France. 9 Govintharajan et al. Int. J. Biosci. 2024 Sofowora EA. 1982. Medicinal plants and traditional medicine in Africa. John Wiley and Sons Ltd., Hoboken, 64-79. Van-Burden TP, Robinson WC. 1981. Formation of complexes between proteins and tannin acid. Journal of Agricultural and Food Chemistry 1981, 77. Wona G-S, Alina K, Tomasz S, Marek S, Krzysztof S. 2018. Bioactive compounds and medicinal properties of oyster mushrooms (Pleurotus sp.). Folia Horticulturae 30(2), 191-201.