SCIFED. Publishers. Abstract. Keywords Organic Nitrogen Supplements; Yield, Nutrient Content; Oyster Mushroom

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1 Research Article SCIFED Publishers Abiodun Olusola Salami,, 2018, 1:1 SciFed Journal of Mycology Open Access Effect of Organic Nitrogen Supplements on the Yield and Nutrient Content of Oyster Mushroom (Pleurotus florida) Cultivated on Corncobs * Abiodun Olusola Salami, Faith Ayobami Bankole, Ogbonnaya Chinyere Andrew * Faith Ayobami BANKOLE Ogbonnaya Chinyere ANDREW Department of Crop Production and Protection, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria Abstract This study investigated the response of Oyster mushroom (Pleurotus florida) on different organic nitrogen supplements, determined the best organic nitrogen supplemented substrate that well supported the growth of Pleurotus florida and evaluated the effect of organic nitrogen additives on the crude protein content of Pleurotus florida. This was with the aim to improving the production of Oyster mushroom using Agrowastes. Organic nitrogen supplemented substrates have not been optimally harnessed for the cultivation and nutrient content determination of Oyster mushroom. Corncob (CC) was used as the main substrate while soybean shell (SYB), rice bran (RB) and groundnut shell (GS) were used as additives. Spawns were produced by weighing 180 g of ground corncob powder treated with 2% gypsum and 3% limestone respectively and mixed properly with sterile water and were later transferred into mayonnaise cream bottles, sterilized at 121 o C for 30 minutes, allowed to cool and then inoculated. Three hundred grams of both the main substrate and the additives were weighed in ratio 80:20, 70:30 and 60:40 respectively. These were thoroughly mixed and filled into transparent nylons and subjected to the same treatment above for spawn production, inoculated with spawns and were kept in dark cupboards until complete ramification. Data were collected on days taken for complete mycelia ramification of substrates, days to pinhead formation, number of fruiting bodies, weight of harvested fruiting bodies, length of stipe of matured fruiting body, diameter of the Cap, diameter of the stipe and biological efficiency. Data collected were subjected to analysis of variance and significant means were separated using LSD at 0.05 level of probability. The result showed that combination of CC with the additives increased the growth rate and yield of Oyster mushroom and the yield of Oyster mushroom was highest on substrate CC+SYB. In addition, the crude protein content of Oyster mushroom increased with increasing percentage of the additives. Keywords Organic Nitrogen Supplements; Yield, Nutrient Content; Oyster Mushroom Introduction Mushrooms are known as macro fungi with distinctive fruiting bodies, which can be either epigenous or hypogenous. In addition, mushrooms are the fleshy, spore-producing fruiting bodies of a fungus called Basidiocarp typically produced above ground on the soil or on its food source. In a narrow sense, mushroom only refer to the fruity body. They belong to the kingdom fungi, division basidiomycota, class agaricomycetes and order agaricales; moreover, mushrooms have been part of the fungal diversity for around 300 million years ago. They * Corresponding author: Abiodun Olusola Salami, Faith Ayobami BANKOLE Ogbonnaya Chinyere ANDREW Department of Crop Production and Protection, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. sola1salami@yahoo.com; Tel: Received November 26, 2017; Accepted January 19, 2018; Published February 2, 2018 Citation: Salami, et al. (2018) Effect of Organic Nitrogen Supplements on the Yield and Nutrient Content of Oyster Mushroom (Pleurotus florida) Copyright: 2018 Abiodun Olusola Salami. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. page 1 of 9

2 are macro fungi have fruiting bodies large enough to be seen with the naked eye and to be picked up by hand. Mushrooms are saprophytes, lack chlorophyll and unlike other green plants, cannot get their food from the sun through photosynthesis. Instead, during their vegetative growth stage, mushroom mycelia secrete enzymes that break down compounds such as cellulose and lignin present in the substrate. The Mycelium is the vegetative part of the fungal organism. In nature, mycelium is seldom seen, as it will dehydrate if exposed to sun or wind. Oyster mushrooms are of the most edible mushrooms belonging to the genus Pleurotus and to the family Pleurotaceae. The common name Oyster mushroom comes from the white shell-like appearance of the fruiting body [1]. Mushroom cultivation has two phases, viz; spawn running phase (this is the period of mycelia growth) and fruiting phase (this is the growth of the fruiting bodies) which are both dependent on temperature and humidity. They can be grown anywhere as long as the conditions for their growth and cultivation are provided. Its cultivation is affected by environmental factors like temperature, oxygen, carbon dioxide, humidity, light, moisture and ph have been reported to affect mycelia growth in the spawn preparation [2]. Oyster mushrooms (Pleurotus florida) are highly edible, nutritious and rank second among the commercially cultivated mushrooms in the world [3]. The nutrient content of food is primarily determined by their protein content and reserve of carbohydrates (or fats). Edible mushrooms are highly nutritious and can be compared with eggs, milk and meat [4, 5]. [6] Confirmed that edible mushrooms have high nutritional attributes and potential applications in industries. Oyster (Pleurotus) mushrooms are considered one of the most efficient producers of food protein, producing 30% of its dry weight [6]. One of the values of commercial cultivation of mushrooms, especially in a developing economy like Nigeria, is the availability of large quantities of several agro-industrial wastes, which can serve as substrate for the cultivation of mushrooms [7]. A substrate is any substance that can facilitate mycelia growth. High concentrations of carbohydrate and nitrogen sources are usually needed in order to achieve a high yield (dry weight) of mycelium. Mushroom is not grown directly on soil as other crops but on organic substrate either raw or composted. These substrates are mostly materials from farm, plantations or factories. During an investigation of the cultivation of mushroom on agricultural residues, it was found that rice husk sorghum stover, saw dust, cotton waste, cocoa bean shell, and sawdust - Gliricidia mixture are suitable substrates for the cultivation of edible mushroom [8], while, rice straw, water lilly and banana leaves are equally implicated [4]. Efficiency of protein production and flavour formation vary with different carbon and Nitrogen sources. Protein content of the mushroom mycelium can be controlled by the amount of nitrogen supplied in the growth media. The range of carbon: nitrogen (C:N) ratio is important. This also influences the yield and efficiency of the production of mushroom mycelium. Nitrogen supplementation increases yield and quality of Oyster mushroom. Given the dietary importance of Oyster mushroom, its awareness and production is still below the expected demand. Although some experiments have been carried out using Agro-wastes as substrates, yet the full potential of cheaply available organic nitrogen supplements have not been optimally exploited for the preparation of substrates for the cultivation and nutrient determination of Oyster mushroom. Some studies show that supplementation with nitrogen source increase the biomass and mushroom s productivity. The most common method of estimating the nutrient quality in food materials is the Weede System also called Proximate Analysis. It measures the chemical content in food materials in six parameters moisture, ash, crude-protein, crude fibre, crude fat and Nitrogen- free extract (carbohydrate). The objectives of this study were to estimate the response of Oyster Mushroom on different organic nitrogen supplemented substrates, select the best nitrogen source supplemented substrate that well supported the growth of Oyster mushroom (Pleurotus florida), evaluate the effect of organic nitrogen additives on the crude protein content of Pleurotus florida. Materials and Methods Experimental Location This study was carried out at the Mycology Laboratory, Department of Crop Production and Protection, Faculty of Agriculture, Obafemi Awolowo University, Ile- Ife, Osun State. Substrate Composition The substrates used in the study are the following; Corncobs + soybean shell (CC + SYB), Corncobs + Rice bran (CC + RB) and Corncobs + groundnut shell (CC + GS). Each of the substrate combinations above were in ratio 80:20, 70:30 and 60:40 respectively. page 2 of 9

3 Production of Spawn A bottle of fully ramified grain spawn of Pleurotus florida was collected from the Mycology section of the OAU, Ile-Ife, Osun State Nigeria. This spawn was multiplied using ground corncobs powder. One hundred and eighty grams of the ground corncobs were soaked in sterile water and CaSO 4 and CaCO 3 were added in the ratio of 2:3% respectively to regulate the ph of the ground corncob powder [9]. Labeled sterilized mayonnaise cream bottles were filled with these corncob powder to about ¾ of the size of the container used. This was corked with aluminum foil and autoclaved at 121 C for about 30 minutes after which the container was allowed to cool for 24 hours at room temperature. After 24 hours of cooling, grain to grain spawning was done to multiply the corncob powder spawn. Grain to Grain Spawning An axenic environmental condition was maintained to reduce contamination and this was achieved with the use of a sterile Laminar flow hood. Hand gloves and nose mask were also worn to avoid contamination. The grain spawn obtained from the Mycology section of Department of Crop Production and Protection was scooped out of the bottle into a clean bowl with the aid of spatula; small quantity of the grain spawn was introduced into each of the bottles containing the pasteurized ground corncobs and covered immediately with foil paper and the foil paper was made firm on the bottles with rubber band. The bottles of the newly spawned grains were incubated at room temperature in complete darkness for 9 days to allow mycelia colonization of the corncob powder spawns [9]. Substrate Preparation Dried Corncobs and each of the soybean shell and groundnut shell were shredded into bits of about 2 cm long and the dry weight of each was measured and recorded. The corncobs as well as the supplements (groundnut shell, soybean shell and the rice bran) were poured into separate clean bowls and about 2% Ca (OH) 2 and 3% CaCO 3 were added to each bowl for optimization and to adjust the ph for optimum mycelia colonization. Hot water was poured to submerge the corncobs as well as the supplements and were allowed to soak for 15 minutes. These were drained and allowed to cool. Three hundred grams of both the main substrate (corncob) and the additives were weighed according to the ratio 80%, 70% and 60% of the weight of the main substrate and 20%, 30% and 40% of each of the additives (groundnut shell, soybean shell and rice bran) respectively. These were thoroughly mixed and packed into transparent nylons properly labeled for easy identification. The substrates were sterilized using the autoclave at 121 o C for 30 minutes. Substrate Inoculation The ramified spawn of Pleurotus florida was inoculated into the transparent nylons containing the prepared substrates. The inoculated bags of substrates were transferred into the dark chamber for incubation and were monitored until full ramification of the substrate by the mushroom mycelia. The fully ramified bags were brought out from the dark chamber to the fruiting body chamber for pinhead formation. At this stage, the fully ramified substrates were exposed to light and wet with clean water. These were set in the mushroom house for wetting and close observation was made for pinheads formation leading to fruiting bodies. The ph of the substrates was maintained at a range of Harvest was done with hand after maturity. Data Collection The following parameters were collected as the growth occurred: days taken for complete mycelia ramification of substrates (DTCSR), days to pinhead formation (DPHF), number of fruiting bodies (NOFB), weight (g) of harvested fruiting bodies (WTFB) using an electronic weighing balance, length of stipe (LOS) of harvested fruiting bodies using a meter rule, diameter of the Cap (DOC) and diameter of the stipe (DOS) using a thread and a meter rule respectively. Yield and Biological Efficiency Total weight of all the fruiting bodies harvested from all the three pickings were measured as total yield of mushroom. The biological efficiency (yield of mushroom per kg substrate on dry wt. basis) was calculated by the formula below according to [5]. Yield of mushroom B.E(%) = X100 Weight of substrate Experimental Design A split plot design was used with two factors in this investigation. page 3 of 9

4 Proximate and Mineral Analysis The mushroom fruiting bodies were analyzed for proximate parameters: moisture, protein, ash, crude fiber, fat, carbohydrate (Nitrogen Free Extract); the mineral contents of the substrates were determined by atomic absorption spectrometry, flame photometry and spectrophotometer according to the methods of AOAC (2010) at the Department of Food Science and Technology, Obafemi Awolowo University, Ile-Ife. Statistical Analysis The data obtained were subjected to ANOVA using Statistical Analysis Software (SAS) version 9.1 and significant means were separated using Fischer s least significant difference at 0.05 level of probability. Microsoft excel 2013 was also used to plot the graphs with standard error bar. Results and Discussion Full ramification of the substrates was observed days after inoculation of the substrates with the spawn of P. florida. Sequel to the full ramification of the substrates were pinheads formation (Plate 1a) which later developed into matured fruiting body (plate 1b). These matured fruiting bodies were harvested by excision from the substrates. It was observed that the number of days to complete substrate ramification was not significantly different for the substrate combinations (CCSYB and CCGS) as it took P. florida 16 days to completely ramify the substrates (CCSYB and CCGS) but was significant for CCRB which was fully ramified by the organism 14 days after inoculation. The number of days taken for pin head formation on the substrates were significantly different from each other as pinheads were observed on the substrates (CCSYB, CCGS, and CCRB) after 22, 21, and 20 days respectively of inoculation with completely ramified spawns of P. florida (Table 1). The differences in the growth parameters was as a result of the different nutrient composition of the substrates used in this study. The highest weight of matured fruiting body was observed on the CCSYB substrate which recorded g, this was followed by CCGS and CCRB with corresponding values of g and g. It was obvious that the recorded weight of the matured fruiting bodies of P. florida were significantly different from each other at 0.05 levels of probability. The average number of matured fruiting bodies of P. florida harvested on CCSYB, CCGS and CCRB substrates were 3.00, 3.00 and 3.30 respectively. These values were observed not to be significantly different from each other at 0.05 level of probability. The highest BE of P. florida was observed on CCSYB substrate which recorded 9.76% followed by CCGS and CCRB whose values were 5.73% and 4.90%. The average length of the stipe of P. florida harvested from the CCSYB substrate was 4.82 cm and was obvious to be longer than the length of stipe of P. florida harvested from other substrates which was found to be 3.85 cm and 3.99 cm for CCGS and CCRB respectively. The diameter of the stipe of P. florida harvested from CCRB was found to be significantly lower than the diameter of stipe of P. florida harvested from other substrates used for this investigation. The P. florida fruiting bodies harvested from CCSYB were found to have the highest average diameter of cap (6.35 cm) and was followed by P. florida harvested from CCGS and CCRB which had average diameters of 5.80 cm and 4.50 cm respectively (Table 1). Table 1: Effect of Different Combinations of Organic Nitrogen Supplements on the Growth Parameters of P.florida Combination DTCSR (DAYS) DPHF (DAYS) WTFB (g) NOFB LOS (cm) DOS (cm) DOS (cm) BE (%) CCSYB CCGS CCRB LSD DTCSR: Days to complete substrate ramification, DPHF: Days to pinhead formation, WTFB: Total weight of fruiting body, NOFB: Number of fruiting body, LOS: length of stipe, DOS: Diameter of stipe, DOC: Diameter of cap, BE: Biological efficiency, CCSYB: corncobs + soybean shell, CCGS: corncobs +Groundnut shell, CCRB: corncobs + rice bran page 4 of 9

5 Plate 1a: Pin Heads Formation of P. florida Plate 1b: Matured Fruiting Bodies of P.florida The effect of the different ratio of combination (80:20, 70:30, and 60:40) of the main substrates (CC) to the additives (SYB, GS and RB) on the growth parameters of Pleurotus florida was presented in Table 2. Pleurotus florida grown on the substrate combinations CCSYB, CCGS, and CCRB with 60% main substrates to 40% additive combination for the fruiting bodies of P. florida was discovered to completely ramify 15 days after inoculation with the spawns (Table 2). Complete ramification of the CCSYB, CCGS, and CCRB with 80% main substrates to 20% additives and 70% main substrate to 30% additive respectively were found not to be significantly different from each other as it took them the same number of days (16 days) for full colonization. It took 22 days for pinheads of P. florida to emerge from the combination with 80:20 of CCSYB, CCGS, and CCRB. For substrate combination of CCSYB, CCGS, and CCRB with ratio 70:30 and 60:40 respectively, it was evident that complete ramification of the substrate combination occurred 21 and 20 days after inoculation with spawns of P. florida (Table 2). The highest weight (24.66 g) of matured P. florida was observed on the substrate combination with 80:20 of CCSYB, CCGS, and CCRB followed by substrates with 70:30 and then 60:40 which were observed to have average weight of g and g respectively. It was also noticed that as the amount of additives increased, the weight of the fruiting bodies of P. florida decreased. This concurred with the findings made by [10]. This was also observed for the BE of the substrate combination as CCSYB, CCGS, and CCRB with 80% main substrate and 20% additive had the highest recorded BE of 8.22% followed by substrate combination with 70:30 and 60:40 (7.45% and 5.47%) respectively. The highest number of fruiting bodies of P. florida was observed on the substrate combination (CCSYB, CCGS, and CCRB) with ratio 70:30 (4.00) while substrate combination with 80:20 and 60:40 ratio had 3.00 and 2.50 number of fruiting bodies respectively. The average LOS of P. florida (4.64 cm) was observed to be highest on the substrate combination (CCSYB, CCGS, and CCRB) with ratio 60:40 followed by the 70:30 and 80:20 ratio which had 4.40 cm and 3.82 cm respectively. Similar trend was also observed for the DOC and DOS as the substrate combination with 60% main substrate and 40% additive was highest for both growth parameters compared to other ratio (70:30 and 80:20) used in this study. It can be inferred that as the proportion of the additives increased, there was a corresponding increase for the LOS, DOS and DOC respectively (Table 2). Figure 1 shows the fresh weight of the combined ratio of additives used in this study. It was observed that each of the substrate combination produced yield at different levels. The fresh weights of the Oyster mushroom were observed to be proportional to the amount of mineral elements possessed by each organic nitrogen source used in the experiment. The highest weight was observed from the substrate combination CCSYB (102.0 g) while the least came from the substrate combination CCRB (57.3 g). The yield of CCSYB was not significantly different from CCGS (84.7 g) but was significantly different for the substrate combination CCRB (Figure 1). This was because both soybean and groundnut are major leguminous crops that have the capacity of fixing Nitrogen, thus containing page 5 of 9

6 high amount of nitrogen than rice bran. Proximate analysis of P. florida cultivated on different organic nitrogen source supplemented substrates revealed that the P. florida is rich in nutrients such as carbohydrates, crude protein, crude fats, crude fibre, and ash. In addition to these essential nutrients, it was also confirmed that P. florida possess high moisture content. The percentage composition of the essential nutrients varied with the different substrate combination used in this investigation. Figure 2 shows the Crude protein, Crude fat, and Carbohydrate content of P. florida. The relative high carbohydrate content for all the substrate combination, which revealed high food energy values in mushroom, suggests that they are good source of energy food items [5]. Substrates CCSYB with 80:20, and CCGS, CCRB with ratio 80:20, 70:30 and 60:40 respectively had similar carbohydrate content but that of the substrate combination CCSYB with 60:40 was significantly different from other substrates (Figure 2). The carbohydrate content ranged from to 54.19%. The maximum carbohydrate was recorded on the substrate combination CCRB with ratio 80:20 (54.19%) while the least carbohydrate content was found on the substrate combination CCRB with ratio 60:40 (49.60%). Table 2: Effect of Different Ratio of Combination of Organic Nitrogen Supplements On Growth Parameters of P. florida Combination DTCSR (DAYS) DPHF (DAYS) WTFB (g) NOFB LOS (cm) DOS (cm) DOS (cm) BE (%) 80: : : LSD DTCSR: Days to complete substrate ramification, DPHF: Days to pinhead formation, WTFB: Total weight of fruiting body, NOFB: Number of fruiting body, LOS: length of stipe, DOS: Diameter of stipe, DOC: Diameter of cap, BE: Biological efficiency, CCSYB: corncobs + soybean shell, CCGS: corncobs + Groundnut shell, CCRB: corncobs + rice bran Figure 1: The Average Fresh Weight of the Organic Nitrogen Supplemented Substrate CCSYB CCGS CCRB SUBSTRATE COMBINATION page 6 of 9

7 Figure 2: The Crude Protein, Crude Fat and Carbohydrate Contents of P. florida This study revealed that the P. florida fruiting bodies harvested from the substrate combination used in this investigation possess crude protein content although at varied levels. Protein content was discovered to fall within the range of 9.87% to 28.78%. The crude protein content of the harvested P. florida for the following substrate combination CCGS and CCRB with ratio 80:20, 70:30 and 60:40 respectively was not significantly different from each other. The substrate combination CCSYB with ratio 60:40 possessed the highest protein content of 28.78% followed by CCSYB with ratio 80:20 (24.12%) and the least was on CCRB with 80:20 (9.87%) (Figure 2). It was also observed that the protein content of the substrates increased gradually with increase in the percentage of the organic nitrogen supplements added to the individual substrates; this is because the nitrogen content for each substrate combination with ratio 60:40 was found to increase across all substrate used. The CCSYB substrate combination, which showed high crude protein content, confirmed the findings of [6] that mushrooms are rich in protein content. Fresh mushrooms usually contain less fat, the amount being 1-8% of dry weight [9]. From this investigation, the fat content on dry weight basis ranged between 1.57 to 4.46% which is similar to the findings of [9, 11]. The fat content for the different substrate combination CCGS and CCRB with ratio of 80:20, 70:30 and 60:40 were not significantly different from each other but significantly different for CCSYB with 80:20 and 60:40. This study revealed that P. florida possess low fat content irrespective of the of the supplements added, thus indicating that it is low in caloric value and good for diet due to its low cholesterol content. This is in agreement with the findings of [4]. The crude fibre, percentage ash and percentage moisture content of P. florida were shown in figure 3 Mushrooms are valuable source of dietary fibres [12]. The amount of crude fibre was maximum on the substrate combination CCRB with 80:20 (15.26%) while the least was recorded on the CCSYB with ratio 80:20 (7.32%). The crude fibre content of CCRB with 80:20 is not significantly different from the substrates CCRB with ratio 70:30, 60:40 and CCGS with ratio 80:20, 70:30 and 60:40 respectively but was different for the CCSYB ratio. These results confirmed the findings of [9] that mushroom, a food of high quality flavor and nutritive value has high content of protein, low in fat and high in crude fiber content. The maximum ash content was found on CCSYB (8.7%) while the least was on CCRB with 80:20 (6.13%). The moisture content of P. florida followed a similar trend like its crude fibre content. The maximum moisture content was observed on CCGS with ratio 80:20 (11.40%), and the least was on substrate CCSYB with ratio 80:20 (7.07%). page 7 of 9

8 Figure 3: The Crude Fibre, % Ash and % Moisture Content of P. florida Conclusion It can be concluded from the experiment, that the supplementation of Corncobs with different sources of organic nitrogen supported the growth of Pleurotus florida. For the fruiting body production of Pleurotus florida, the different supplemented substrates have significant effect on improving the production of Pleurotus florida. The substrate with the additive, rice bran was observed to ramify faster than any other substrate. The best substrate that well supported the growth of Oyster mushroom in this study was CCSYB with ratio 80:20 as it contained the highest wet weight (88.8 g). The substrate CCSYB with ratio 80:20 was also discovered to have the highest biological efficiency while the least was from CCRB with ratio 60:40. The protein content of Oyster mushroom increased with increasing quantity of organic nitrogen sources. The highest protein content was discovered on the substrate CCSYB with ratio 60:40 followed by the substrate CCSYB with ratio 80:20 while the least protein content was found on CCRB with ratio 80:20. This investigation revealed that Agro-wastes are useful raw materials that could be utilized in mushroom production rather than constituting a challenge to waste management problem encountered in the agricultural industry. This research recommends that agro-waste conservation should be enhanced and supported in that nothing within the cycle of agricultural production should be wasted. Also, that mushroom should be incorporated into our diets more frequently in order to improve the quality of our diet, as well as our overall health and general well-being as mushroom have been discovered to contain with high protein which can be a substitute for expensive fish and meat. Also, the knowledge of mushroom production should be conveyed from our laboratories and universities to the outside world. This is to enable people become enlightened on how to produce mushroom in homes as mushrooms have been established to be substitute for egg and meat. This should be done because the world population is currently increasing at a faster rate and shortage of food and the diminishing quality of human health has been growing concerns due to increase in urbanization and a concomitant reduction in arable land. Arousing people s interest to venturing into mushroom production will arrest the problem of food insecurity and unemployment as they are liable to becoming entrepreneurs. References 1. Stanley RP (2011) Enumerative combinatorics. Cambridge University press Nwanze PI, Khan AU, Ameh JB et al. (2005) The effect of the interaction of various spawn grains with different culture mediums on carpophores dry weights and stipe and pileus diameters of lentinus squarrosulus (Mon.) singer. Afr J Biotechnol 4: page 8 of 9

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