Australian Journal of Basic and Applied Sciences, 6(7): 603-610, 2012 ISSN 1991-8178

Mycelial Biomass Production of Enoke Mushroom (Flammulina velutipes) by Submerged Culture

Hassan, F.R.H., Ghada M. Medany and El-Kady, A.T.M. Food Tech. Res. Inst., Agric. Res Center, Giza, Egypt.
Abstract: Enoke; winter; velvet stems; mushroom (Flammulina velutipes) is well known as a very popular, delicious, edible and medicinal mushroom in Asian countries specially China and Japan. Since this mushroom is highly prized for their culinary and medicinal value, it has recently been introduced to Egypt. This study tended to optimize the submerged culture conditions for the production of mycelial biomass of F.velutipes (two strains). Among studied carbon sources, glucose was the most favorable at level of 6% for Fv.19 and 7% for Fv.01 strains. Yeast extract was the most suitable nitrogen source for both strains at concentration of 0.6%. The optimum fermentation temperature was 25oC for 15 days and the most favorable initial pH value was 6.5 with inoculume size of 8-9 %(v/v). Applying the determined optimum condition together enhanced the mycelial biomass of the tested strains more than using each individual factor. The mycelial biomass of the tested F.velutipes strains contained relatively comparable chemical constituents to that of fruit bodies. The results of this study are promising and could be considered as a successful step for further studies on the nutritional and medicinal properties of this fungus. Key words: Enoke mushroom, Flammulina velutipes, submerged culture, optimum conditions. INTRODUCTION Many mushrooms have been valued throughout the world as food and medicine. Europeans have always appreciated their gastronomic value. In Japan pushcart venders sell medicinal mushrooms on the street which are regularly used in diet to maintain health and promote longevity (Smith and Sullivan, 2004). A variety of mushrooms have been used traditionally for the maintenance of health, and for prevention and treatment of diseases. Mushroom extracts may modulate the response of host immune system; in particular, various mushroom polysaccharides are likely to affect promotion and progression stages towards cancer. Other substances contained in mushrooms may be able to interfere with tumor initiation through a variety of mechanisms (Chatterjee et al, 2011). Flammulina velutipes (Curt.:Fr.) Sing is the second mushroom after Auricularia auricula (Bull.: Fr.) Wett. which has been grown since 800 A. D. (Chang and Miles, 1987). This mushroom is mainly cultivated for gourmet and tonic purposes. Flammulina ranks at fourth place in the category of edible mushrooms for production and consumption (Leifa et al, 2001). Flammulina velutipes has many desirable characteristics including a delicious taste, a highly notorious composition, a capacity to prevent illness, and an ability to promote growth of the young. In recent years, consumption has increased rapidly, and some countries have established factories for producing this mushroom (Wang et al, 2005). Flammulina (= Collybia) velutipes is one of the major cultivated mushrooms worldwide, although its popularity is largely restricted to Asia (Farr, 1983 & Tautorus, 1985). It is second only to Lentinus edodes (Shiitake) in volume produced in Japan. Because of its nutritional and medical properties, F. velutipes (Curtis) Singer is one of the six most actively cultivated mushroom species in the world with a production over 300,000 tons per year by the end of 20th century (Psurtseva, 2005). The Flammulina fungus is also known as winter mushroom or velvet stems with worldwide distribution (Chang and Miles, 2004). Different groups of bioactive compounds such as polysaccharides, protein- glucan complex, sterols, lectins, peroxidases, laccases, cellulases and proteases, with medicinal and pharmaceutical properties (immunomodulating, antitumor, anti-oxidant, thrombolytic, fibrinolotic, antibacterial, antifungal, antiviral, mitogenic were isolated from F. velutipes (Leung et. al., 1997). Enokipodins A, B, C, and D are cuparene-type sesquiterpenoids antimicrobial metabolites produced in the stationary stage of F. velutipes mycelia development in malt extract broth (Melo et al., 2009). Several medicinal properties of mycelial and fruiting body samples of F. velutipes have been established in in-vitro experiment such as immunomodulatory effect via induction of cytokines and antifungal, antibacterial, antiviral, antioxidant, antiprotozoal, mitogenic activities (Badalyan and Hambardzumyan, 2001). A significant antifungal/antagonistic activity of several strains of F. velutipes against filamentous pathogenic fungi for men and/or animal has been reported (Badalyan, 2004). Polysaccharides and a low-weight protein-bound polysaccharide with high antitumor activity were also isolated from this mushroom (Ikekawa, 2001). Flammulin, a basic simple protein from F. velutipes is able to markedly inhibit tumor cells (Komatsu et al., 1963). An epidemiological study in Nagano Prefecture, Japan showed that
Corresponding Author: Hassan, F.R.H., Food Tech. Res. Inst., Agric. Res Center, Giza, Egypt. E-mail:-fathym77@@hotmail.com

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cancer death rate among farmers producing F. velutipes was remarkably lower than that of other people in the Prefecture and other places in Japan (Ikekawa, 2001). Some drug or dietary supplement products have been made commercially for aniflammatory, antitumor and immunomodulating purpose (Wasser and Wies, 1999). Antagonistic activities of F. velutipes are reported against cereal pathogenic fungi such as Gaeumonnomyces graminis var. tritici, Bipolaris sorokiniana, Fusarium culmurum, Rhizoctonia cerealis (Badalyan et al., 2002). Traditionally, edible and medicinal mushrooms have been produced in solid cultures using composts or lignocellulosic wastes, such as straw or wood, a process that usually takes several months to produce fruiting bodies (Solomons, 1975). Submerged culture, in contrast to cultivation on solid media, gives rise to potential advantages of higher mycelial production in a compact space and shorter time with lesser chances of contamination (Yang and Liau, 1998). Moreover, possible uses of the biomass are food or feed in the form of protein supplement. It can also be used for the extraction of flavours and other metabolites, such as enzymes and polysaccharides (Jong and Birgmingham, 1993). Thus, investigators have found that growing mushroom mycelia by submerged fermentation in a defined medium is a rapid alternative method for obtaining fungal biomass of consistent quality (Kwon et al., 2009; Yang and Liau, 1998). In addition, poly-saccharides that have synergistic bioactive effects with mycelia can be produced simultaneously with this method (Kim et al., 2002; Kim et al., 2002). In order to improve the production of fungal bioactive polysaccharides, investigators have worked to optimize the fermentation conditions and medium composition of solid-state and submerged fermentation systems (Hwang and Yun, 2010; Kim et al., 2002; Kim et al., 2010; Kwon et al., 2009; Tang and Zhong, 2002). This study aimed to determine the optimum conditions of submerged culture for producing mycelial biomass of winter mushroom (Flammulina velutipes). Estimate the main chemical composition of the produced mycelial biomass. MATERIALS AND METHODS Fungal Strains: The edible fungal strains (Fv. 19 and Fv. 01) of Flammulina velutipes were kindly obtained from Fujian Agricultural &Forestry Univ., China. The cultures were maintained on Potato Dextrose Agar (PDA) medium and stored in refrigerator at 5 - 7 C after growth for routine culture and storage purposes. Submerged Culture Medium: The media used for seed cultures and fermentation cultures were prepared according to Hamedi, et al., (2007). Preparation Of F. Velutipes Inoculume: The seed culture medium composed from (g/l ): glucose, 10; yeast extract, 3; malt extract, 10; peptone, 1 and initial pH value was adjusted to 5.5. For 250 ml. flask contained 50ml. of sterilized seed culture, two pieces7mm. mycelia of each F. velutipes strain were transferred from 7days old slant to each flask. The inoculated seed culture flasks were incubated on shaker incubator (120 rpm) at 25C for 7 days. All basic fermentation cultures were performed in 500 ml. flasks containing 100ml. of fermentation medium, 5% (v/v) of the seed culture was used as inoculume and initial pH value was adjusted to 5.5. The fermentation was performed in a shaker incubator at 120 rpm, 25C for 15 days. Different carbon and nitrogen sources and concentrations, besides culturing conditions were examined individually (one factor at a time) for their optimal levels. Various and different medium composition (carbon and nitrogen sources), pH value and temperature were selected according to Hamedi, et al (2007). All experiments in this study were carried out in triplicates and results were expressed in mean values. Carbon Source Selection: Various carbon sources namely (maltose, sucrose, glucose, fructose and starch) were examined to find out the most favorable one for mycelium growth of tested F.velutipes strains. Culturing medium used in this experiment contained (g/l) carbon source, 10; yeast extract, 3; peptone, 1; MgSO47H2O, 1 and K2HPO4 .3H2O, 1. The medium that lacks any carbon compound (0%) served as the control. The most suitable carbon was chosen to estimate their optimum concentration for mycelial biomass production. Nitrogen Source Selection: Different nitrogen sources (urea, potassium nitrate, ammonium sulfate, yeast extract, beef extract and peptone) were tested to choose the best one for mycelial growth. Fermentation medium is similar to that used for carbon source selection, but without nitrogen sources. The tested nitrogen sources were supplemented individually to the medium at concentration of 0.3%. Starch (20 g/l) was used as carbon source in this

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experiment. The medium did not contain any nitrogen source used as control. The superior nitrogen source that produces the highest mycelial biomass was selected to estimate their optimum concentration for the production. Fermentation Temperature and Initial pH Value: Fermentation process was carried out in wide range of temperature (15, 20, 25, 30, 35and 40C), and pH values (4.0, 4.5,8.0) to select the most favorable level for mycelial growth. The fermentation medium used in this experiment composed of (g/l): starch, 20; yeast extract, 4; MgSO47H2O, 1 and K2HPO4 .3H2O, 1. Incubation Period And Inoculume Sizes: The best incubation period was selected from different periods (5, 10, 15, and 20 days) of fermentation. Also, a wide range of inoculume sizes (1-10 %, v/v) was carried out to estimate the most suitable one. The fermentation media used in these experiments were similar to that used for temperature and pH value selection. The most suitable culturing conditions estimated were applied together in further experiment to assay their combined effect on mycelial biomass of F. velutipes. The mycelial biomass of tested F. velutipes gained through this experiment was chemically analyzed. The mycelial biomass produced in each treatment was harvested by vacuum filtration to separate the culture broth. The fungal biomass was washed several times with distilled water, and then oven dried at 55C until constant weight and represented as dry cell weight (DCW). Analytical Methods: Moisture content, ether extract, protein and ash content were estimated according to the methods described in A.O.A.C. (1995). Total carbohydrates were calculated by differences. Statistical Analysis: The obtained data were statistically analyzed using ANOVA procedure of the SPSS statistical package at confidence level of 5% (0.05) (SPSS, 1990). RESULTS AND DISCUSSION Effect Of Incubation Time And Temperature: Submerged culturing of F.velutipes strains for 15 days at different temperatures showed the ability of such mushroom to grow on wide range of temperatures (15- 31 C) with distinctly degrees (Fig, 1). Both F. velutipes strains ( Fv. 19 and Fv. 01) exhibited maximum mycelium growth at 25oC being 5.65, 5.19 g/l DCW, respectively which differed significantly than those grown on other temperatures. A very low amount of DCW was obtained by culturing tested strains on 34 C. Moreover, no mycelium growth was recorded at 37 C or above. No significant differences in DCW produced at 21 and 28 C for Fv.01 strain or between those obtained at 15 and 31 C for either Fv.19 or Fv.01 strain.

Fig. 1: The results illustrated in Fig (2) show that maximum mycelial biomass of F. velutipes strain(Fv. 19 and Fv. 01) was obtained when grown at 25 C for 15 being 5.56 and 5.19 g/l , respectively. Increasing fermentation time to 20 days did not produce significant increment in biomass yield than those obtained at 15 days for both strains. Significant low amounts of DCW for both strains were obtained either at 10 or 5 days, so 15 days fermentation at 25 C were considered the best conditions for mycelial biomass production. These results for time and temperature of incubation coincide with the data obtained by many authors through their work on F. velutipes,(1.0, 2.4, 4.2, 6.1, 9.4, 13.5 and 13.9 g/l) for 2, 4, 6, 8, 10, 12 and 14 days fermentation at 25oC, Mau & Ma (2001); 4.86 g/l after 7 days to be 8.28 g/l after 14 days, Maziero et al, (1999) ; ( 0.92-1.01g/l 605

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after 5 days), (3.37- 4.4g/l after 10 days) and (4.16- 4.82 g/l after 15 days) at 25C, Kim et. al., (2002); and Diamantopoulou et. al., (2012) found that the mycelial biomass of F.velutipes increased from 7.0 g/l after 12 days to be 10.9 g/l after 16 days at 26oC.

Fig. 2: Effect of Initial pH Value and Inoculume Size: The results in Table (1) show that F.velutipes has the ability to grow on a wide range of pH values (4-8). The DCW increased significantly by progressively increase of pH value from 4 and reached the maximum (6.37 g/l) at pH value 6, then turned to decline for Fv. 19 strain. The same pattern was observed with Fv 01 strain but the maximum biomass yield was obtained at pH values range (6.5 7.5) and the most favorable one is pH value 7. Xu and Yun (2003) stated that, the optimum pH value will depend upon several factors including strain of the organism used. Moreover, Shin et. al., (2007) reported that the optimum pH value for F. velutipes was 6.5. Hameidi et. al., (2007) stated that optimal initial pH value for A. blazei biomass production was 7.0; Jonathan et. al., (2009) recorded that; very good biomass of A. polytricha was produced at pH 6.5 while no biomass was produced at pH values 4.0 and 9.0. Kozhemyakina et al., (2010) found that F. velutipes could grow in a wide range of initial pH values (from 3.0 to 7.5). However the maximum output of mycelium biomass took place at the initial values of pH 5.0- 6.5. Increasing inoculume size progressively increased the mycelial biomass of both strains of F.velutipes (Table, 1). The maximum DCW was gained at 7 and 8% (v/v) inoculume size for Fv.19 strain with no significant differences, while for Fv 01 it was at 9 and 10 %without significant differences. It is not worthily to increase the inoculume size over 8% for Fv 19 and 9% for Fv.01, since the resulting increments in DCW were not significant. Different levels of inoculume size were cited by many authors, 4% for nineteen strains of mushroom (Kim et. al., 2002), 4% for F. velutipes (Shin et al., 2007); 10% for L. mylittae (Zhou et al, 2009) and 7% for A polytricha (Jonathan et al, 2009).
Table 1: Effect of pH value and inoculume size on dry cell weight (DCW)g/l for F.velutipes. pH value DCW g/l Inoculume size%(v/v) Fv. 19 Fv. 01 4.0 3.73 g 3.24 e 1 4.5 4.12 f 3.88 d 2 5.0 4.94 de 5.11 c 3 5.5 5.56 b 5.19 c 4 6.0 6.37 a 5.93 b 5 6.5 5.41 bc 6.32 ab 6 7.0 5.01 cd 6.47 a 7 7.5 5.21 bcd 6.21 ab 8 8.0 4.59 e 5.32 c 9 10 Means within the same column have different small superscript are significantly different. DCW g/l Fv. 19 2.66 g 3.28 f 3.76 e 4.11 e 5.56 d 6.84 c 6.93 bc 7.34 ab 7.75 a 7.25 bc Fv.01 2.24i 3.10 h 3.85 g 4.27 f 5.19 e 5.97 d 6.49 c 7.05 b 7.51 a 7.60 a

Effect of Carbon and Nitrogen Sources on DCW: Five carbon sources namely maltose, sucrose, glucose, fructose and starch were evaluated individually as a sole carbon source for promoting mycelial biomass of F. velutipes strains (Figure 3). The results show that maltose, sucrose and starch did not exhibit considerable high amounts of Fv. 19 strain mycelial biomass. The least amount of DCW was gained from control medium for both strains. Glucose as a sole carbon source stimulates the DCW for Fv.19 and produced 3.81 g/l and differed significantly than the other carbon sources followed by fructose (3.31g/l). Moreover, glucose and fructose were the most suitable carbon source for Fv.01 strain, which produced the maximum DCW being 3.67 and 3.25 g/l, respectively without significant differences. It seems that monosaccharides are most favorable for mycelial biomass production compared to di or polysaccharides. These results are similar to those reported by Kim et al (2002) who get 6.4 mg/ ml DCW of F. 606

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velutipes using 2%glucose as carbon source. They also, recorded that in general, monosaccharides were better carbon sources than di- and polysaccharides for the mycelial growth of F. velutipes. The addition of glucose, fructose and mannose resulted in maximum mycelial growth. Also, these results are confirmed by other investigators who gained higher amount of mycelial biomass using glucose as carbon source, Zhou et al., (2009) stated that, dextrose, as a monosaccharide, is favorable for fast mycelia growth ,Medany (2011) with different L. edodes strains (6.73 -9.18 g / l) and Ding et al., (2012) with Coprinus comatus (10.25 g / l). This may be due to that, poly and disaccharides will be hydrolyzed to monosaccharides before they will enter the respiratory pathways Mahier and Cordes (1971); the ease with which this sugar (glucose) was metabolized to produce cellular energy (Garraway and Evans, 1984; Jandaik and Kapoor, 1976).

Fig. 3: The results illustrated in figure (4) show that, all tested nitrogen sources were utilized by F. velutipes resulted in different amounts of mycelial biomass. Organic nitrogen sources favored F.velutipes DCW than the inorganic ones. For both strains yeast extract stimulated significantly the mycelium growth resulted in the maximum DCW being 4.85 and 4.47 g/l for Fv. 19 and Fv. 01, respectively. While, beef extract and peptone were ranked the second and third order. Meanwhile, control medium that has not any nitrogen source produced the lowest yield of biomass (0.29, 0.48 g/l) for Fv19 and Fv.01, respectively. These results are in accordance with the data obtained by many authors. Stimulatory effect of yeast extract on the growth may be due to their carbohydrate and protein composition as reported by Alberghina, (1973). Zhou et al, (2009) tested various nitrogen sources for L. mylittae. They found that among the various nitrogen sources tested neither NH4Cl nor urea was beneficial for biomass or protease production as inorganic nitrogen sources. For mycelia growth, yeast extract was the best nitrogen source followed by peptone, both of which are simple organic nitrogen sources. The stimulatory action of yeast extract on biomass yield may be linked with its high carbohydrates, amino acids and vitamins composition (Gbolagade et al., 2006).

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Effect Of Glucose And Yeast Extract Concentration: According to the results gathered from figures (3 and 4), glucose and yeast extract are the best carbon and nitrogen sources for stimulating mycelyial growth of F.velutipes strains. Complementary experiments were applied to determine the most suitable concentration of glucose and nitrogen (Table, 2). Increasing glucose concentration significantly increased the DCW until 6% glucose for Fv. 19 strain. It is not worthily to increase glucose over 6% since, no significant increment in Fv. 19 mycelial biomass was obtained. The same trend was observed with Fv. 01 strain, while the increment DCW peak was obtained with 7% glucose, meanwhile no significant increment was gained by increasing glucose over such concentration up to 10%. Medany (2011) stated that, mycelial biomass of L. edodes strains increased significantly by increasing glucose concentration to get the maximum at 7%. Also, Ding et al (2012) recorded that, the cultures average growth rates ranged from 2.18 per day to 2.54 per day, with the maximum rate being observed with an initial concentration of 40 g/l. A higher glucose concentration was generally favorable for the production of more biomass in the fermentation of C. comatus. The highest amount of DCW for Fv.19 strain was achieved by using 0.6 and 0.7% yeast extract being 7.93 and 8.16 g/l , without significant differences. Also, Fv. 01 reached its DCW peak (7.22 g/l) at 0.6% yeast extract and no significant increment was gained in DCW for both strains by increasing yeast extract concentration over 0.6 % up to 1%. ). Xu and Yun (2003) declared that, yeast extract was the most effective nitrogen source for DCW of A. polytricha at 1% level. Also, Medany (2011) found that, yeast extract at 0.4% level was the most suitable nitrogen source for L. edodes mycelial biomass.
Table 2: Effect of glucose and yeast extract concentration on DCW of F.velutipes strains. Glucose conc.% DCW g/l Yeast extract conc.% Fv. 19 Fv.01 1 3.81 d 3.67g 0.3 2 5.26 c 3.34f 0.4 3 6.63 b 4.85e 0.5 b d 4 7.01 5.27 0.6 5 8.01 b 6.39 c 0.7 6 8.69 a 6.87 b 0.8 7 8.93 a 7.85 a 0.9 8 8.35 ab 7.44 a 1.0 9 8.62 ab 7.49 a 10 8.66 ab 7.51 a Means within the same column have different small superscript are significantly different. DCW g/l Fv. 19 4.85 f 5.63 e 6.72 d 7.93 ab 8.16 a 7.67 b 7.13 c 7.60 b Fv.01 4.47e 5.97 d 6.83 bc 7.22 a 6.80 bc 6.64 c 6.77 bc 7.00 ab

Generally, it could be observed that Fv.19 is more efficient in producing DCW than Fv.01. The most favorable conditions determined through the course of this study for the two tested strains of F. velutipes were applied together in a complementary experiment. Applying the estimated optimal conditions increased the yield of DCW to be (11.86, 11.35 g/l for Fv.19 and Fv.01, in succession) compared to those obtained for each individual optimal condition factor. These results are in accordance with the findings of Kim et al (2002), 6.4 - 10.9 g/l according to the type of carbon and nitrogen sources; 9.2 13.6 g/l according to the strain of F.velutipes,(Wang et. al., 2005); 7.0 10.9 g/l according to incubation time, (Diamantopoulou et. al., 2012). The chemical analysis of DCW obtained from this experiment was determined (Table, 3).
Table 3: Main chemical constituents of mycelial biomass of F.velutipes (d.wt.). Constituents % Fv.19 Moisture content* 88.03 Crude protein 18.65 Ether extract 1.78 Ash 3.97 Total carbohydrates** 75.60 * On fresh weight. ** Total carbohydrates were calculated by differences. Fv.01 89.15 17.38 2.92 4.85 74.85

Nutritional Constituents Of F. Velutipes DCW: The data presented in Table 3 declare that moisture contents of mycelial biomass of F. velutipes were 88.03 and 89.15% for Fv 19 and Fv 01, respectively. Moreover, crude protein, ether extract, ash and calculated total carbohydrate contents were (18.65, 17.38%); (1.78, 2.92%); (3.97, 4.85%) and (75.60, 74.85%) for Fv 19 and Fv 01, consecutively. Kozhemyakina et al., (2010) analyzed the biomass of F velutipes. They found that, it contained 56.0% reducing substances, 17.5% protein and 1.4% ash content. These data confirmed our present results. Since, a very little data about chemical composition of F. velutipes mycelium was available, so the present data may be compared to that of fruit bodies.

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Lin & Lin (2011) revealed that, each 100 g of dried F.velutipes fruit bodies contained 13.9 16.2g protein, 1.7 1.8g fat, 3.6 3.9g ash, 6.3 7.4g crud fiber, 60.2 62.2g carbohydrates, 280 -342 mg phosphorus, 6176 mg calcium, 0.16 mg thiamine, 1.59 mg riboflavin, 23.4mg nicotinic acid. The results obtained through this investigation are very promising and could be consider a successful important step towards getting important bioactive substances (food, pharmacological industries) from this edible and medicinal fungus through just two weeks. More studies in this area will be performed. REFERENCES Alberghina, F.A.M., 1973. Growth relation in Neurospora crasa .Effect of nutrients and temperature. Arch.Microbiol., 89: 83-94. AOAC., 1995. Official methods of analysis (16th ed.). Arlington VA, USA: Association of Official Analytical Chemists. Badalyan, S.M., 2004. Screening of antifungal activity of several Basidiomycetes mushrooms. Probl Med Mycol., 6: 18-26. Badalyan, S.M., G. Innocenti and N.G. Garibyan, 2002. Antagonistic activity of xylographic mushroom against pathogenic fungi of cereal in dual culture. Phytopathol Mediterr., 41: 220-225. Badalyan, S.M. and L.A. Hambardzumyan, 2001. Investigation of immune modulation activity of medicinal mushroom Flammulina velutipes in vitro cytokines induction by fruiting body extract. Int J Med Mushr., 3: 110. Chang, S.T. and P.G. Miles, 1987. Historical record of the early cultivation of Lentinus in China. Mush. J. Tropic, 7: 31-37. Chang, S.T. and P.G. Miles, 2004. Mushrooms, cultivation, nutritional value, medicinal effect and environmental effect. Sec. Edit., pp: 451. Chatterjee, S., G. Biswas, S.K. Basu and K. Acharya, 2011. Antineoplastic effect of mushrooms: a review. Australian J.Crop Sci., (7): 904-911. Diamantopoulou, P., S. Papanikolaou, M. Kapoti, M. Komaitis, G. Aggelis and A. Philippoussis, 2012. Mushroom polysaccharides and lipids synthesized in liquid agitated and static cultures. Part I: screening various mushroom species. Appl Biochem Biotechnol, 167: 536-551. Ding, Z., W. Wang, F. Wang, Q. Wang and K. Zhang, 2012. Polysaccharides production by submerged fermentation of Coprinus comatus and their inhibitory effects on non-enzymatic glycosylation. Journal of Medicinal Plants Research, 6(7): 1375-1381. Farr, D.F., 1983. Mushroom industry: diversification with additional species in the United States. Mycologia, 75: 351-360. Garraway, O.M. and C.R. Evans, 1984. Fungal nutrition and physiology. New York : John Wiley. Gbolagade, J., A. Sobowale and D. Adejoye, 2006. Optimization of sub-merged culture conditions biomass production in Pleurotus florida (Mont) Singer), a Nigerian fungus. Afr. J. Biotech., 5(16):1464-1469. Hamedi, A., H. Vahid and F. Ghanati, 2007. Optimization of the medium composition for production of mycelial biomass and exo-polysaccaride by Agaricus blazei Murill DPPh 131 using response surface methodology. Biotechnology, 6(4): 456-464. Hwang, H.S.and J.W. Yun, 2010. Hypoglycemic effect of polysaccharides produced by submerged mycelial culture of Laetiporus sulphureus on streptozotocin induced diabetic rats. Biotechnol. Bioprocess. E., 15: 173-181. Ikekawa, T., 2001. Beneficial affects of edible and medicinal mushrooms on health care. Int J Med Mushr., 3: 291-298. Jandaik, C.L. and J.N. Kapoor, 1976. Effect of carbon and nitrogen nutrition on growth of Pleurotus sajorcaju. Indian Phytopathol., 29: 326-327. Jonathan, S.G., D.O. Bawo, Adejoye and O.F. Briyai, 2009. Studies on Biomass Production in Auricularia polytricha Collected from Wilberforce Island, Bayelsa State, Nigeria. American Journal of Applied Sciences, 6 (1): 182-186. Jong, S.C. and J.M. Birgmingham, 1993. In: Mushroom Biology and Mushroom Products. Chang, S.T., J.A. Buswell and W. Chius (Eds.). The Chinese University Press, Hong Kong, pp: 345-366. Kim, S.S., J.S. Lee, J.Y. Cho, Y.E. Kim and E.K. Hong, 2010. Effects of C/N ratio and Trace elements on mycelial growth and exo-polysaccharide production of Tricholoma matsutake. Biotechnol. Bioprocess. Eng., 15: 293-298. Kim, S.W., H.J. Hwang, J.P. Park, Y.J. Cho, C.H. Song and J.W. Yun, 2002. Mycelial growth and exobiopolymer production by submerged culture of various edible mushrooms under different media. Letters in Applied Microbiology, 34: 56- 61.

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