Access the full text.
Sign up today, get DeepDyve free for 14 days.
Annals of Microbiology, 57 (3) 389-393 (2007) Optimisation of submerged culture conditions for the production of mycelial biomass and exopolysaccharide by Pleurotus nebrodensis Jia LE , Shunzhen HU, Mei XU Department of Microbiology, College of Life Science, Shandong Agricultural University, Taian, Shandong, 271018, China Received 13 March 2007 / Accepted 22 June 2007 Abstract - The optimisation of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) from Pleurotus nebrodensis. The optimal temperature and initial pH for both mycelial growth and EPS pro- duction in shake flask cultures were 25 °C and 8.0, respectively. Maltose was found the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract was favourable nitrogen source for both mycelial biomass and EPS production. Optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media -1 -1 -1 -1 constituents for mycelial growth and EPS production was as follows: 200 g l bran, 25 g l maltose, 3 g l yeast extract, 1 g l -1 -1 -1 KH PO , 1 g l MgSO 7H O. Under the optimal conditions, the mycelial biomass (4.13 g l ) and EPS content (2.40 g l ) of Pleurotus 2 4 4 2 nebrodensis was 2.3 and 3.6 times compared to the control with basal medium respectively. Key words: Pleurotus nebrodensis, biomass, exopolysaccharide, optimisation. INTRODUCTION easy to harvest mycelia and extract useful material from fermentation broth (Zhang et al., 2002), especially, it facilitated the release of polysaccharide. Therefore, liquid The wild species of Pleurotus nebrodensis is a precious, edi- ble and medicinal mushroom, it only distributes in the arid culture of Pleurotus nebrodensis is worthy of investigating desert of Xinjiang area in China. In 1983, wild Pleurotus greatly. nebrodensis was cultivated successfully on wood chips of In recent years, submerged cultivation for the produc- spruce, cottonseed hulls and brans by Chinese scientists for tion of mycelial biomass from Pleurotus nebrodensis has the first time (Chen, 1986). been investigated widely (Li et al., 2003; Dong et al., Pleurotus nebrodensis is a tasting crisp and delicious 2004a, 2004c), while submerged cultivation for production with pleasant aroma functional mushroom; it is rich in pro- of exopolysaccharide (EPS) has not been reported yet up to tein, amino acids, polysaccharide, edible fibre and mineral now. In this study, Pleurotus nebrodensis was cultivated in elements. Various experiments have demonstrated that liquid media, and then the optimum nutritional require- Pleurotus nebrodensis had physiological effects, such as ments of media and environmental conditions of culture for preventing cardiovascular disease and rachitis (Lin, 2000). producing EPS were investigated. A number of pharmacologic studies have shown that poly- saccharide of Pleurotus nebrodensis had immunological activities (Gan and Lv, 2001; Deng and Lv, 2002), antiox- MATERIALS AND METHODS idative and antitumor effects (Zheng et al., 2005). In fact, the importance of the medicinal properties of Pleurotus Microorganism and culture conditions. J3 (conserved nebrodensis polysaccharides had been realised all over the in our laboratory), a strain of Pleurotus nebrodensis, was world by now. used in this experiment. This strain was incubated on PDA Polysaccharide of Pleurotus nebrodensis was normally slant (20% w/v potato, 2% w/v dextrose, 2% w/v agar) at extracted from its fruiting bodies (Dong et al., 2004b). 25 °C for about 12 days. However, solid-cultivation would not only take a long peri- od of time, but also get low products. Therefore, Inoculum preparation and flask cultures. Pleurotus researchers had tried to obtain polysaccharide of nebrodensis was initially grown on PDA medium in a Petri Pleurotus nebrodensis from its mycelia (Yang et al., 2004; dish for about 12 days, and then transferred to liquid media Li et al., 2005). Actually, liquid culture had a lot of advan- by punching out approximately 0.5-cm mycelial block tages for gaining mass production, such as it is relatively from the agar plate culture. The fermentation culture was grown in a 250-ml Erlenmeyer flask containing 100-ml tested medium and shaken continuously at 160 rpm, 25 °C * Corresponding author. Phone: +86-538-8242908; for 6 days after being laid still for 24 h. Fax: +86-538-8248696. E-mail: firstname.lastname@example.org 390 J. Le et al. The tested medium used for all flask culture experi- the optimal carbon and nitrogen sources and environmen- ments was derived from the basal medium with natural tal conditions of producing EPS from Pleurotus nebrodensis initial pH. The composition of the basal medium was have not been reported. The biomass and content of EPS 10% w/v bran, 1% w/v sucrose, 0.3% w/v peptone, were observed in liquid media containing five different car- 0.1% w/v KH PO , 0.05% w/v MgSO 7H O (Jiang et al., bon sources (Table 1). The optimum carbon source of pro- 2 4 4 2 -1 -1 2005). ducing maximum biomass (1.9 g l ) and EPS (1.7 g l ) was maltose. In the liquid medium containing maltose, the Optimisation of the composition of fermentation yield of biomass and EPS was obviously higher than medium. The selection of carbon and nitrogen sources is sucrose (47 and 42%), dextrose (38 and 45%), lactose (6 the first step in the course of media optimisation. Sucrose and 30%), fructose (62 and 35%) respectively. By ANOVA, (1.500 g), dextrose (1.737 g), maltose (1.579 g), lactose the analysis of variance showed the different effect of the (1.579 g) and fructose (1.579 g) were provided with the five carbon sources on biomass (p < 0.01) and on EPS (p proportion equal to the carbon content of 1.5 g sucrose < 0.05). (0.632 g) in the basal medium separately. During in the course of screening for the nitrogen sources, peptone (0.300 g), yeast extract (0.623 g), beef extract (0.335 g), TABLE 1 - Effect of carbon and nitrogen sources on production of ammonium nitrate (0.125 g), ammonium sulphate (0.206 biomass and EPS by Pleurotus nebrodensis g), sodium nitrate (0.265 g) and ammonium chloride Biomass Content of EPS (0.166 g) were tested with the proportion equivalent to the -1 -1 nitrogen content of 0.3 g peptone (0.044 g). The orthogo- (g l )(g l ) nal test was applied to determine the optimum combination Carbon sources of carbon and nitrogen sources after finding out the optimal kind of carbon and nitrogen sources. Sucrose 1.01 0.992 Dextrose 1.19 0.947 Optimisation of environmental conditions. The sin- Maltose 1.91 1.710 gle-factor test was used to determine the optimal envi- Lactose 1.79 1.200 ronmental conditions respectively. Inoculation volume, Fructose 0.73 1.120 incubation temperature, revolution speed, initial pH value, culture time and the volume of liquid media in ** * 250 ml-sized flask were tested in this experiment which was conducted in the optimum fermentation medium Nitrogen sources obtained above. Peptone 1.65 0.686 Measurement of the mycelial biomass and EPS. At the Yeast extract 1.93 1.040 end of liquid culture course, the original inoculated mycelial Beef extract 1.97 0.792 block was discarded and then the fermentation broth was Ammonium nitrate 0.49 0.798 centrifuged (4000 rpm, 20 min. Dry weight of mycelia was Ammonium sulfate 1.23 0.864 measured after rinsing the mycelial pellet with distilled Sodium nitrate 1.08 0.771 water for three times and then drying at 60 °C to a con- Ammonium chloride 1.21 0.867 stant weight. Five millilitres of supernatant fluid was mixed ** * with triplicate volume of 95% ethanol, stirred vigorously and kept at 4 °C for 18 h. After centrifugation (4000 rpm, *: p < 0.05, **: p < 0.01. 10 min), the supernatant was discarded, and the precipi- tate, i.e. the crude EPS, was dissolved with distilled water heated to 60 °C. The EPS content was determined by phe- nol-sulphuric acid method with dextrose as the standard (Chaplin and Kennedy, 1994). The EPS content (Table 1), in the medium containing -1 yeast extract, was the highest (1.04 g l ), while the bio- -1 -1 Statistical analysis. In shake-flask experiments, all treat- mass was 1.93 g l . The biomass was 1.97 g l in the ments were run in triplicate and all experiments were medium containing beef extract, a little higher than that in repeated at least twice. Data from each treatment were the medium containing yeast extract, but the EPS content -1 subjected to DPS for ANOVA (analysis of variance), and was only 0.792 g l , significantly lower than that in the Duncan’s multiple range test was used to determine signif- medium containing yeast extract. The yield of biomass and icant differences (p < 0.05) among the treatments. EPS in medium containing yeast extract was higher than that in medium containing peptone (14.5 and 34.0%), ammonium nitrate (74.6 and 23.3%), ammonium sulphate RESULTS AND DISCUSSION (36.3 and 16.9%), sodium nitrate (44.0 and 25.9%), ammonium chloride (37.3 and 16.6%) respectively. On Effects of carbon and nitrogen sources on biomass account of EPS content being the main examined object, and EPS content yeast extract was then determined as the optimal nitrogen Although some researchers have reported the effect of liq- source. Similar to the experiment screening for the optimal uid media component on the content of EPS from Pleurotus carbon sources, ANOVA revealed the diverse effect of ostreatus (Rosado et al., 2003), Boletus edulis (Deng and seven nitrogen sources on biomass (p < 0.01) and on EPS Chen, 2005) and Ganoderma lucidum (Wang et al., 2005), (p < 0.05). Ann. Microbiol., 57 (3), 389-393 (2007) 391 TABLE 2 - Effect of carbon and nitrogen sources on production of (p < 0.01). The degree of the influence of all the factors on biomass and EPS by Pleurotus nebrodensis biomass and on EPS came in this turn: B > C > E > D > A and B > A > E > C > D, respectively. Finally, we deter- -1 Levels Factors (g l ) mined the optimal composition of culture medium: 20% w/v bran, 2.5% w/v maltose, 0.3% w/v yeast extract, AB C D E 0.1% w/v KH PO , 0.1% w/v MgSO ·7H O. 2 4 4 2 1 100 15 2 1 0.5 2 150 20 3 2 1.0 Effects of environmental conditions on biomass and 3 200 25 4 3 1.5 EPS content Pleurotus nebrodensis could grow at initial pH value ranging A: bran, B: maltose, C: yeast extract, D: KH PO , E: MgSO · 2 4 4 from 5 to 10 and its mycelial biomass came to a head at ini- 7H O. tial pH 6 (Table 4), in accordance with the results reported by some researchers (Dong et al., 2004c). However, the -1 EPS content reached its maximum (1.84 g l ) at initial pH Optimisation of culture medium by orthogonal test 8, significantly higher than that at the others (Table 5). A five-factor-three-level orthogonal design was adopted as Initial pH value had a remarkable influence not only on shown in Table 2 and the results of the orthogonal test mycelial biomass, but also on EPS by ANOVA analysis (p < were summarised in Table 3. By ANOVA, only maltose was 0.01). So we chose initial pH 8 as the optimum in view of found to have a significant influence on the content of EPS EPS content. TABLE 3 - Results of the orthogonal test No. 12345 Biomass EPScontent -1 -1 AB C D E (g l ) (g l ) 1 11111 2.30 0.638 2 11112 2.15 0.683 3 11113 2.75 0.552 4 12221 3.10 0.846 5 12222 5.10 0.950 6 12223 2.30 0.944 7 13331 4.90 1.230 8 13332 2.10 1.440 9 13333 6.70 1.020 10 21231 3.80 0.670 11 21232 2.45 0.683 12 21233 2.20 0.795 13 22311 3.45 0.859 14 22312 3.90 0.832 15 22313 3.10 0.910 16 23121 3.70 0.766 17 23122 3.15 1.670 18 23123 4.55 1.590 19 31321 3.50 0.824 20 31322 2.50 1.380 21 31323 3.65 0.936 22 32131 2.10 0.800 23 32132 3.05 0.790 24 32133 3.10 0.859 25 33211 2.30 0.872 26 33212 3.40 3.410 27 33213 4.50 3.250 K 3.49 2.81 2.98 3.09 3.24 K 3.37 3.24 3.24 3.51 3.09 K 3.12 3.92 3.76 3.38 3.65 ∑=89.8 R 0.37 1.11 0.78 0.42 0.56 Significance test K ’ 0.922 0.796 0.928 1.330 0.834 K ’ 0.975 0.866 1.380 1.100 1.320 K ’ 1.460 1.690 1.05 0.921 1.210 ∑=30.2 R’ 0.538 0.894 0.452 0.413 0.481 Significance test ** A: bran, B: maltose, C: yeast extract, D: KH PO , E: MgSO ·7H O. **: p < 0.01. 2 4 4 2 392 J. Le et al. TABLE 4 - Effect of environmental conditions on production of biomass by Pleurotus nebrodensis a b c Initial Biomass Temp. Biomass Time Biomass Mv Biomass Iv Biomass Rs Biomass -1 -1 -1 -1 2 -1 -1 pH (g l )(°C)(g l )(d)(g l )(ml)(g l )(cm )(g l )(rpm)(g l ) 5.0 4.37 20 4.43 2 0.32 50 2.74 0.25 1.07 130 1.70 6.0 4.83 25 5.13 3 0.57 100 1.55 0.5 1.43 160 2.33 7.0 4.43 30 4.17 4 0.89 150 0.87 1.0 2.73 190 3.13 8.0 4.13 5 0.98 200 0.59 1.5 3.03 9.0 3.93 6 1.13 10.0 3.60 7 1.17 82.90 ** ** ** ** ** a b c Mv: medium volume, Iv: inoculation volume, Rs: revolution speed. **: p < 0.01. The maximal biomass and EPS yield were both obtained speed of 160 rpm, due to the lower wastage of electricity, highly when the culture temperature was 25 °C (Tables 4, the smaller damage to shaker and the higher security in 5), consistent with some reports in 25 °C optimum for fungi large scale production, was finally decided. growth (Li et al., 2003; Dong et al., 2004c). But it was unknown that the effect of culture temperature on biomass Validity experiment and EPS content had no statistical significance (p > 0.05). The control group was cultured in basal media under the With the prolongation of culture time, the biomass remark- conditions of natural initial pH, 25 °C incubation tempera- ably increased until day 8 (p < 0.01) and the maximal EPS ture, 160 rpm revolution speed, 100-ml liquid media in a -1 2 content (2.38 g l ) occurred at day 5 (p < 0.01), similar to 250 ml-sized flask, 0.50-cm mycelial block of inoculation the value reported by Yang et al. (2006) in Pleurotus feru- volume and 7 days culture time. The tested group was con- -1 lae. The EPS yield (2.36 g l ) of day 4 was only a little ducted under the optimised conditions by orthogonal test -1 lower than that of day 5, but we still chose day 4 as the and single-factor test. The mycelial biomass (4.13 g l ) -1 optimal culture time for the sake of shortening total fer- and EPS content (2.40 g l ) of Pleurotus nebrodensis in mentation period. tested group were 2.3 and 3.6 times compared to the con- The capacity of liquid media in a 250-ml Erlenmeyer trol, respectively. flask had a significant effect on both biomass and EPS con- The optimal conditions of producing biomass and EPS tent (p < 0.01) (Tables 4, 5). Despite of the highest biomass by Pleurotus nebrodensis in submerged culture could offer in 50-ml liquid media, accordant with the report by Li et al. references to large-scale cultivation, but further research of (2003), the EPS yield reached the maximum when the industrial fermentation and exploitation should be done capacity of liquid media was 100 ml. The optimal inoculation continuously. volume of producing EPS was 0.5-cm mycelial block (Table 5). With the revolution speed increasing, the biomass of Acknowledgements Pleurotus nebrodensis increased gradually (Table 4), but the This work was supported by Chinese Shandong education- EPS yield came to a head at 160 rpm (Table 5). To our sur- al project. We should like to express our sincere thanks to prise, the effect of revolution speed on biomass and EPS Prof. Du for reviewing the manuscript and Prof. Ding for content also displayed significantly (p < 0.01). Revolution advice on the statistics. TABLE 5 - Effect of environmental conditions on production of EPS by Pleurotus nebrodensis a b c Initial EPS Temp. EPS Time EPS Mv EPS Iv EPS Rs EPS -1 -1 -1 -1 2 -1 -1 pH (g l )(°C)(g l )(d)(g l )(ml)(g l )(cm )(g l )(rpm)(g l ) 5.0 1.21 20 2.55 2 2.13 50 1.35 0.25 1.53 130 1.89 6.0 1.21 25 2.62 3 2.15 100 2.77 0.50 1.72 160 2.88 7.0 1.53 30 2.53 4 2.36 150 1.58 1.00 1.35 190 2.41 8.0 1.84 5 2.38 200 1.56 1.50 1.30 9.0 1.43 6 2.08 10.0 1.23 7 1.97 81.66 ** ** ** ** ** a b c Mv: medium volume, Iv: inoculation volume, Rs: revolution speed. **: p < 0.01. Ann. Microbiol., 57 (3), 389-393 (2007) 393 REFERENCES Li Z.P., Cai Y.P., Lin Y., Li T.C., Fan H.H. (2005). The extraction of polysaccharide from submerged fermentation of Pleurotus ferulae. Packaging and Food Machinery, 23: 28-30. Chaplin M.F., Kennedy J.F. (1994). Carbohydrate Analysis, A Lin J. 2000). The key technology for cultivation of Pleurotus fer- nd Practical Approach, 2 edn., IRL Press Ltd, USA. ulae. Edible Fungi of China, 19: 28-29. Chen Z.C. (1986). The experiment on cultivation of Pleurotus Rosado F.R., Germano S., Carbonero E.R., Da Costa S.M., ferulae. Edible Fungi, 2: 10-11. Iacomini M., Kemmelmeier C., (2003). Biomass and Deng C.S., Lv Z.Z. (2002). Study on immunological activities of exopolysaccharide production in submerged cultures of water soluble polysaccharides from Pleurotus ferulae Lanzi. Pleurotus ostreatoroseus Sing. and Pleurotus ostreatus “flori- Journal of Huazhong Agricultural University, 21: 447-449. da” (Jack.: Fr.) Kummer [J]. Journal of Basic Microbiology, 43: 230-237. Deng B.W., Chen W.Q. (2005). Studies on optimal conditions of producing exopolysaccharide from Boletus edulis [J]. Food Wang X.F., Wang Y.L., Liang J.G. (2005). Study on optimization and Fermentation Industries, 31: 60-63. of media for Genoderma lucidum polysaccharide and its fer- mentation [J]. Amino Acid and Biotic Resources, 27: 23-26. Dong H.X., Liu J.J., Wang K. (2004a). Initial studies on the fer- mentation conditions of Pleurotus ferulae. Biotechnology Yang L.H., Lin J., Huang Q.R., Cai D.H., Lang X.C. (2004). Bulletin, 5: 48-51. Optimization of polysaccharide extraction from mycelia of Pleurotus ferulae Lanzi prepared by liquid submerged fer- Dong H.X., Liu X.H., Xu Z.L., Cai D.H. (2004b). A study on the mentation. Industrial Microbiology, 3: 28-31. extraction technology of polysaccharide from Pleurotus nebrodensis fruiting body. Acta Edulis Fungi, 11: 22-25. Yang M., Chen L.H., Shi L., Wang L.Y. (2006). A study on sub- merged culture technology of Pleurotus ferulae and the Dong H.X., Ren G.T., Huang Q.R., Wang S.F. (2004c). Study on analysis of the composition. Journal of Fujian Normal submerged culture technology of Pleurotus ferulae. Journal University (Natural Science Edition), 22: 77-81. of Microbiology, 24: 22-24. Zhang J.H., Zhao J.Z., Hao Z.B. (2002). Submerged culture of Gan Y., Lv Z.Z. (2001). Studies on physical and chemical prop- edible and medicinal fungi. Forestry Science and Technology, erties and immunological activities of polysaccharides from 27: 61-63. Pleurotus ferulae. Mycosystema, 20: 228-232. Zheng L., Huang Y.C., Gao Q.X., Pu X., Li S.F., Bi Y.R. (2005). Jiang Y.H., Jiang X.L., Wang P., Wang S.Y. (2005). A study on Antioxidative and antitumor activities of the crude extracts of submerged fermentation conditions of Pleurotus nebrodensis Pleurotus eryngii var. nebrodensis. Mycosystema, 24: 71-78. and the cultivation. Acta Edulis Fungi, 12: 37-41. Li Y.Q., Wu J., Yang Q.Z., Qin W.P., Shen D.H., Wu Y.W. (2003). Study on submerged fermentation technology of Pleurotus nebrodensis. Journal of Northwest University for Nationalities (Natural Science), 24: 14-18.
Annals of Microbiology – Springer Journals
Published: Nov 20, 2009
Access the full text.
Sign up today, get DeepDyve free for 14 days.