Phytochemical characterization of the mushroom Agaricus bisporus and assessment of its nutritional ability in the place of fishmeal for survival and growth of the freshwater prawn Macrobrachium rosenbergii post-larvae

Prawn is nutritious delicacy to mankind. Fishmeal is a chief value protein ingredient in prawn feed, which is a depleting resource. The present study was conducted to assess whether edible Mushroom, Agaricus bisporus can partially be replaced the fishmeal to promote the growth of the freshwater prawn, Macrobrachium rosenbergii post-larvae (PL). In the methanolic extract of A. bisporus, the presence of terpenoids, flavonoids, polyphenols, saponins and cardiac glycosides were qualitatively detected. In the petrolium etheric extract of A. bisporus, the presence of alkaloids, terpenoids, tannins, polyphenols, cardiac glycosides and quinines were qualitatively detected. A. bisporus, containing 14 secondary phytochemicals, of which 9 compounds {Dodecanoic acid; Tetradecanoic acid; 2-Pentadecanone, 6, 10, 14-trimethyl; Hexadecanoic acid, methyl ester; 4-(6, 6-Dimethyl-2-methylenecyclohex-3-enylidene) pentan-2-ol; Tetradecanoic acid; Neophytadiene; Hexadecanoic acid; 9, 12, 15-Octadecatrienoic acid, ethyl ester, (Z, Z, Z)-} having bioactive properties. Isonitric diets were prepared by replacement of the fishmeal (25%, 50%, 75% and 100%) with A. bisporus powder. Diet prepared without replacement of fishmeal was served as control. These diets were fed to M. rosenbergii PL (1.00±0.20 cm in length and 0.07±0.02 g in weight) for 90 days, and 75% fishmeal replaced diet produced significant elevations in survival and growth rates, muscle total protein, amino acid, carbohydrate, lipid and ash contents, profiles of proteins, amino acids and fatty acids and activities of protease, amylase and lipase when compared with control. Thus, it is recommended that up to 75% of fishmeal can be replaced with A. bisporus for sustainable maintenance of M. rosenbergii seeds in the nursery. This would offer better nutrition and employment opportunity.


Introduction
The giant freshwater prawn Macrobrachium rosenbergii is commercially cultured in India and other south Asian countries as it is nutritious delicacy for mankind and have high market price with good export value [1]. This seafood yields quality protein for averting malnutrition. Although its culture is profitable, there are some constraints in the grow-out phase [2]. The availability of quality seeds and feeds are major constrains. The feed price plays a vital role in overall operational cost. Thus, feed formulations with locally available low cost agricultural, animal husbandry and industrial by-products are in practices [3,4]. Fishmeal is one of the chief valued ingredients for protein source in artificial feed formulation. Its stock is depleting day by day, whereas the cost is on the reverse. Therefore, there is an urgent need for finding out some alternatives.
The fishmeal can efficiently be replaced by alternative protein sources such as soybean protein and poultry by-products [5][6][7]. Mushroom by-product also considered as one of the alternative sources for replacing the fishmeal protein in fish. In fact, mushroom stalk itself contained a rich source of protein, polysaccharide and antioxidant. The stalk of mushroom, Pleurotus sajor caju when given as feed to replace fishmeal protein yields good growth in the fingerlings of Nile tilapia, Oreochromis niloticus [8]. Mushroom meal is more suitable and acceptable ingredient in the fish feed than fishmeal for the better growth of fingerlings of Labeo rohita and Hemigrammus caudovittatus [9]. According to Zhang et al. [10], when the dietary fishmeal (up to

Preparation of solvent extracts of A. bisporus
The powdered whole plant sample of A. bisporus (50 g) was packed in Whatmann No. 1 filter paper and put into soxhlet apparatus. The extracts were successively soaked with 300 ml (1:6 w/v) of non-polar solvent, petroleum ether (99.98% purity, SRL Pvt. Ltd. India), and a polar solvent, methanol (99.9% purity, Changshu Yangyuan Chemicals, China) individually and sequentially extracted for 6-9 h each (30 to 36 cycle). Repeated extraction was done until a clear colorless solution was obtained. The extracts were filtered by using double layered muslin cloth and concentrated at 40-50°C using rotary vacuum evaporator (ROTAVAP). The extracts obtained were vacuum-dried under 40°C and used for further investigation. The extracts obtained were appeared as dark green, gummy solid.

Qualitative analysis of primary phytochemical substances
The extracts were subjected to detection of the presence of primary phyto-molecules, such as alkaloids, terpenoids, flavonoids, tannins, polyphenols, saponins, and cardiac glycosides using the standard qualitative procedures [33].

Gas Chromatography-Mass Spectrum (GC-MS) analysis for secondary phytochemical compounds
The petroleum etheric and methanolic extracts of A. bisporus were subjected to GC-MS analysis (Thermo GC-trace ultra ver-5.0; Thermo MS-DSQ-II; ZB 5-MS capillary standard non-polar column (30 mts, 0.25 mm id, 0.25 μm film) for identification of different phytochemical compounds at South India Textile Research Association (SITRA), Coimbatore, Tamil Nadu, India. The relative percentage constituent was expressed as percentage with peak area normalization. Peaks resolved with relative abundance of 0-100 were considered as major compounds. To show the minor peaks, the chromatogram was magnified. Identification of various components present in these extracts were assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures. National Institute Standard and Technology (NIST4) and WILEY9 [34] on-line library source was also used for matching the identified components.

Analysis of the proximate composition of A. bisporus
A. bisporus powder was subjected to proximate composition analysis following the methodology of Castell and Tiews [35] as given in AOAC [36], and the values are presented in Table 1.

Diet preparation with A. bisporus powder
Diets were formulated with the following branded feed basal ingredients (BI). The fish meal, groundnut oilcake and soybean meal as protein sources, wheat bran as a carbohydrate source, sunflower oil as lipid source, and tapioca flour and egg albumin were used as binding agents. The fishmeal, groundnut oilcake, soybean meal, wheat bran, and tapioca flour were thoroughly mixed, dough was prepared with sterilized water, then it was steam cooked and cooled at room temperature. Then the Sunflower oil and egg albumin were added to the dough and mixed well. A. bisporus powder was incorporated with the dough of BI at four different concentrations, 25%, 50%, 75%, and 100% by replacing the right quantity of fishmeal, and in order to prepare iso-nitric diets, Button mushroom contains moisture (90-93%), good quality protein (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42).5%) with lysine and tryptophan that are normally lacking in cereals, fibre (8.3-16.2%), ash (9.4-14.5%), carbohydrates (59.4%) with glycogen, chitin, and hemicellulose instead of starch, a very low fat (3.1%) with rich in linoleic acid, and cholesterol is absent and in its place ergo-sterol is present. 100 g (dry wt.) button mushroom also contains several minerals including, calcium (71.0 mg), phosphorous (912 mg), sodium (106 mg), iron (8.8 mg), and potassium (2850 mg). Mushrooms are fairly good source of vitamin C and vitamin B complex, particularly thiamine, riboflavin, niacin, biotin and pantothenic acid. Mushrooms also contains folic acid and vitamin B12 which are absent in most vegetables [20].
In India, Agaricus bisporus is the most popular variety which fetches high price [21]. It is a good source of trace elements like sodium, potassium, and phosphorus, conjugataed linoleic acid and antioxidants [22]. A. bisporus extracts can be potentially applied in Alzheimer's disease since it has acetylcholinesterase and butyrylcholinesterase inhibition activity [23]. The extracts of A. bisporus and its bioactive compounds are used as antioxidant, anti-cancer and anti-inflammatory against coronary heart diseases, diabetes mellitus, bacterial and fungal infections, and disorders of human immune system [24][25][26][27][28][29][30]. A. bisporus contain three main polysaccharides α-glucan, β-glucan and galactomannan [31], and fatty acids mainly linoleic, palmitic and stearic acids [32].
In above views, the present study was conducted by partially replacing the fishmeal with edible mushroom, A. bisporus. First, its proximate biochemical composition was assessed, and then its survival and growth promoting potential has been evaluated on the late aged post-larvae (PL) of M. rosenbergii by replacing the fishmeal in its artificial diet. Contents of basic biochemical constituents such as total protein, amino acid, carbohydrate and lipid, profiles of protein, amino acids and fatty acids, and activities of digestive enzymes such as protease, amylase and lipase were studied in this economically important prawn.

Procurement of the mushroom A. bisporus
The mushroom, A. bisporus was procured from JPR Agro Farms, Porthiyada, the Nilgiris, Tamil Nadu, India ( Figure 1). The species was authenticated by Dr. G. V. S. Murthy, Scientist 'G' , Southern Regional Center, Botanical Survey of India (BSI), Coimbatore, India. The procured mushroom was cleaned by rubbing, scraping and brushing to Figure 1. The mushroom, Agaricus bisporus the protein level was maintained by adjusting the groundnut oilcake and soybean meal ( Table 2). Sterilized water was adequately added for maintaining the dough in moist and paste form. Then it was pelletized in a manual pelletizer (Kolkata, India) fixed with 3 mm diameter mesh. The pellets were dried in a thermostatic oven (M/s Modern Industrial, Mumbai, India) at 40ºC until they reached a constant weight and stored in airtight jars at room temperature. The pelletized feeds were subjected to proximate composition analyses (AOAC) [36] and the results are also presented in Table 2.

Procurement and acclimation of M. rosenbergii PL
The post-larvae (PL-15) of the freshwater prawn, M. rosenbergii (1.00±0.20 cm and 0.07±0.02 g) length and weight respectively) were procured from the Nursery pond at Kanathur, Chennai, Tamil Nadu, India. They were transported to the laboratory in polythene bags filled with oxygenated water. The prawns were acclimatized to ambient laboratory conditions for 2 weeks in large cement tank (1000 L) with groundwater (Temperature, 23±1.0°C; pH, 6.8±0.20; total dissolved solids (TDS), 0.94±0.06 g L -1 ; dissolved oxygen (DO), 6.40±0.10 mg L -1 ; salinity, 0.59±0.02 mg L -1 ; biochemical oxygen demand (BOD), 31.10±2.82 mg L -1 ; chemical oxygen demand (COD), 108.58±5.41 mg L -1 ; ammonia 0.027±0.004 mg L -1 ), APHA [37]. During acclimatization, the prawns were fed with boiled egg albumin and artificially prepared feed of our own laboratory feed. More than 50% of tank water was routinely changed every day in order to maintain a healthy water environment and aeration was also provided. The unfed feed, feces, moult, and dead prawns if any were removed by siphoning without disturbing the prawns.

Feeding trial
M. rosenbergii (PL-30) (1.30±0.11 cm in length and 0.12±0.02 g in weight) was starved for 24 h before commencing the feeding trial ( Figure 2). Five groups, each with 30 PL were maintained in 30 L plastic tanks under a triplicate experimental set-up. One group served as control and fed with feed formulated without incorporation of A. bisporus and the other four groups were fed with experimental feeds prepared by incorporation of A. bisporus (at 25%, 50%, 75%, and 100% respectively by replacing the right quantum of fishmeal). The feed was allocated to the prawns for two times a day (7:00 am and 7:00 pm) at 10% of body weight. The experiment was extended for a period of 90 days, by this time it reached the juvenile stage. The unfed feed, feces, and moult (if any) were collected on a daily basis by siphoning method causing minimum disturbance to the prawns during renewal of the water medium. For morphometric and nutritional analysis, 10 prawns from each group were randomly measured and the mean was considered as a single value (mean of 10 individual measurements=one observation), and three such measurements were made to fulfill the triplicate analysis.

Calculation of survival and growth parameters
On the 90 th day of feeding trial, the growth parameters, such as survival rate (SR), length gain (LG), weight gain (WG), specific growth rate (SGR) and food conversion rate (FCR) were determined by adopting the formulae of Tekinay and Davies [38].
i. Survival (%)=Total No. of live animals/Total No. of initial animals×100 ii. Weight gain (g)=Final weight (g) -Initial weight (g) iii. Specific growth rate (%)=log W 2 -log W 1 /t×100 Where, W 1 & W 2 =Initial and final weight respectively (g), and t=Total number of experimental days iv. Food conversion ratio (g)=Total feed intake (g)/Total weight gain of the prawn (g) v. Protein efficiency ratio (g)=Total weight gain of PL (g)/Total protein consumed (g)

Estimations of basic biochemical constituents
The concentrations of total protein, amino acid and carbohydrate in experimental PL were estimated by adopting the methodology of Lowry et al. [39], Moore and Stein [40] and Roe [41], respectively. The total lipid was extracted by Folch et al. [42] gravimetric method, and estimated by Barnes and Blackstock [43] spectrophotometric method. The contents of ash and moisture were analyzed by following AOAC [36] methodology.

Assays of digestive enzymes activities
Activities of digestive enzymes (protease, amylase and lipase) were assayed at 90 th day of feeding trial. The digestive tract of three prawns from each replicate were carefully dissected out and homogenized in ice-cold distilled water and centrifuged at 9000 g under 4ºC for 20 min. The supernatant was used as a source of crude enzyme. Total protease activity was determined by casein-hydrolysis method of Furne et al. [44], where one unit of enzyme activity represented the amount of enzyme required to liberate 1 μg of tyrosine per minute. Amylase activity was determined according to Bernfeld [45], the specific activity of amylase was calculated as milligrams of maltose liberated per gram of starch per hour (mg/g/h). Lipase activity was assayed by the method of Furne et al. [44], one unit of lipase activity was defined as the amount of free fatty acid released from triacylglycerol per unit time.

Analysis of protein profile
SDS-PAGE profile of the muscle samples of prawns fed with control and 75% of fishmeal replaced with A. bisporus (the best concentration) diets were performed. The muscle tissue sample was first defrosted in phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH-7.4), homogenized under ice-cold condition and centrifuged at 1500 rpm under 4ºC for 5 min. The soluble protein content in the supernatant was determined Lowry et al. [39]. SDS-PAGE was performed on vertical slab gel with 4% stacking and 10 % separating gels Laemmli [46] along with protein markers (β-galactosidase-116 kDa, bovine serum albumin-66 kDa, ovalbumin-45 kDa, carbonic anhydrase-29 kDa, soybean trypsin inhibitor-20 kDa and lysozyme-14 kDa) of Medox-Biotech Pvt. Ltd., Chennai, India. The polypeptides banding patterns between control and test prawns were compared by using the information on apparent molecular masses of bands and their intensities.

Analysis of amino acid profile
The profiles of amino acids in the muscle samples of prawns fed with control and 75% of fishmeal replaced with A. bisporus (the best concentration) diets were performed using High-Performance Thin Layer Chromatographic (HPTLC) method of Hess and Sherma [47]. TLC for four groups of standard amino acids: lysine, asparagine, glutamine, glutamic acid and methionine (group-I); proline, serine, cysteine, tyrosine and tryptophan (group-II); histidine, arginine, aspartic acid, threonine and leucine (group-III); and glycine, alanine, valine, isoleucine and phenylalanine (group-IV) were also performed simultaneously. The peak area of the sample was compared with standard amino acids and quantified.

Analysis of fatty acid profile
The profiles of fatty acid in the muscle samples of prawns fed with control and 75% of fishmeal replaced with A. bisporus (the best concentration) diets were performed using Gas Chromatographic (GC) method of Nichols et al. [48]. Fatty acid samples were obtained from lipid by saponification using NaOH dissolved in methanol-H 2 O mixture (hydrolysis with alkali). They were methylated into fatty acid methyl ester using methanol-HCl mixture. The fatty acid methyl ester was separated using hexane-anhydrous diethyl ether mixture. For the organic phase, aqueous NaOH was used as a base wash and the upper organic layer was separated. 2 μL of the sample was injected and analyzed using Chemito 8610 Gas Chromatography, with BPX70 capillary column and flame ionization detector. Nitrogen was used as a carrier gas. Standard fatty acids were analyzed simultaneously. Based on the retention time of the standard fatty acids, each fatty acid in the unknown sample was identified. The peak areas of standard and unknown were compared and quantified

Statistical analysis
The data between control versus experiment, and between experiments were subjected to statistical analysis through one-way ANOVA and subsequent post-hoc multiple comparison with DMRT by adopting the SPSS v16. All the details of statistical analyses were given in respective tables. The P values less than 0.05 were considered statistically (95%) significant.

Primary phytochemicals of A. bisporus
The primary phytochemicals present in A. bisporus are presented in Table 3. The petroleum etheric extract of A. bisporus contained luxurious presence of alkaloids and tannins, moderate presence of polyphenols and quinines, and terpenoids and cardiac glycosides are poorly present. The methanolic extract of A. bisporus contained luxurious presence of saponins, moderate presence of terpenoids, polyphenols and cardiac glycosides, and poorly presence of flavonoids. Similar to the present study, presence of alkaloids, flavonoids, phenols, steroids, terpenoids, saponins and glycoside have been reported in methanolic extract of A. bisporus [49,50]. Petroleum ether, chloroform, acetone and ethanol have also been used to screen the phytochemicals of A. bisporous [49]. Plant derived flavonoids, anthrax quinines, and terpenens stimulate glucose uptake and exhibit hypoglycaemic activity [51], and also known for their ability of beta cell regeneration in pancreas [52].

Secondary phytochemicals of A. bisporous
The GC-MS analysis of A. bisporus showed overall presence of 14 compounds, of which 9 compounds possessed bioactive properties ( Table 4)
Protein is the major constituent of prawn feed as larvae and juveniles have greater requirement than adults for growth and metabolism [66]. Carbohydrates are the most economical and inexpensive source of energy. It together with proteins and lipids form a dietary source of energy and are required to synthesis of glycogen, chitin, steroid, and fatty acids [67,68]. Actually, dietary protein supplies amino acids required to build body tissues, essential for growth and production of hormones, antibodies, enzymes, etc. [66].

Survival, growth, contents of basic biochemical constituents and activities of digestive enzymes, and profiles of amino acid and fatty acid in fishmeal replaced diets fed prawns
The morphometric parameters (length and weight gains), nutritional indices (survival rate, specific growth rate and protein efficiency ratio), concentrations of basic biochemical constituents (total protein, carbohydrate, lipid, amino acid and ash) and activity of digestive enzymes (protease, amylase and lipase) were found to be gradually and significantly increased (P < 0.05) from 25% to 75% fishmeal replaced diets fed prawns when compared with control, whereas the 100% fishmeal replaced diet showed a decreasing trend when compared to that of other experimental diets (Table 5) ( Figure  5). The reverse trend in FCR was found, which represented the quality of diets prepared.

Amino acid and fatty acid profiles of 75% fishmeal replaced diet fed prawns
The content of all the essential (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, arginine, asparagines, and glycine) and non-essential (glutamic acid, proline, alanine, tyrosine, aspartic acid, cysteine, and glutamine) amino acids were found to be significantly elevated (P<0.05) in 75% fishmeal replaced diet fed prawns when compared with control (Table 6). Similarly, the contents of saturated fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid), monounsaturated fatty acids (palmitoleic acid, and oleic acid) and polyunsaturated fatty acids (linoleic acid, EPA and DHA) were found to be significantly increased (P<0.05) in 75%     (Table  7). The elevated profiles of amino acid and fatty acid determined the high survival and growth of M. rosenbergii PL.
The result recorded clearly indicated the fact that 75% of fishmeal replaced diet with A. bisporus produced better survival and growth, contents of protein, essential amino acids, unsaturated fatty acids, and activities of protease, amylase and lipase. Therefore, replacement of fishmeal with mushroom contributes to an increase in protein assimilation and feed utilization. The improved essential amino acids involved in energy metabolism, protein synthesis, osmoregulation and neurotransmitter [69][70][71][72][73]. Improved survival, growth and nutritional indices, contents of protein, carbohydrate and lipid, activities of protease, amylase and lipase, and profiles of amino acid and fatty acid have been reported in M. rosenbergii PL fed with 50% replacement of

Profile of proteins of 75% fishmeal replaced diet fed prawns
The general comparison between control and experiment revealed that the polypeptide bands were clearly resolved in fishmeal replaced diet fed prawns. The 119, 117, 84, 34, 28, 24 and 23 kDa protein bands were newly appeared in the muscle of M. rosenbergii PL fed with 75% fishmeal replaced diet with A. bisporus. The 19 and 17 kDa protein bands were deeply stained in experimental prawns when compared to that of control ( Figure 6). The results observed indicated the fact that the muscle quality of M. rosenbergii PL has improved through incorporation of A. bisporus protein.

Conclusion
Fish meal is an excellent source of protein and other essential nutrients for aquaculture feed industry. Up to 75% of this has successfully and partially been replaced with A. bisporus. The recorded better growth and survival of M. rosenbergii PL by better feed conversion ratio indicated that the formulated feeds were well served and fully utilized. A. bisporus powder led to increased digestive enzymes activity, which offered better digestion, absorption and assimilation. Hence, the quality of muscle protein was improved. Thus, sustainable culture of Macrobrachium is possible.

Conflict of interest
There is no conflict of interest among authors.