Solid-state fermentation of cornmeal with the basidiomycete Ganoderma lucidum for degrading starch and upgrading nutritional value

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1 Journal of Applied Microbiology 2005, 99, doi: /j x Solid-state fermentation of cornmeal with the basidiomycete Ganoderma lucidum for degrading starch and upgrading nutritional value J.R. Han 1, C.H. An 2 and J.M. Yuan 2 1 School of Life Science and Technology, Shanxi University, Taiyuan, and 2 Biotechnology Center of Shanxi University, Taiyuan, China 2004/1400: received 2 December 2004, revised 12 March 2005 and accepted 13 March 2005 ABSTRACT J. R. H A N, C. H. A N A N D J. M. Y U A N Aims: The objective of this research was to study the ability of the basidiomycete Ganoderma lucidum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation (SSF). Methods and Results: On the basal medium that consisted of cornmeal and salt solution, a-amylase activity of G. lucidum reached its maximum value of 267 U g )1 of culture on day 20 after inoculation. Prolongation of fermentation time from 10 to 25 days increased significantly the degradation rate of starch and ergosterol yield (a kind of physiologically active substances of G. lucidum, also as an indicator of mycelial biomass) (P < 0Æ01). Supplementation of glucose, sucrose or maltose to the basal medium also caused a significant increase in either the degradation rate of starch or the ergosterol yield as compared with control (P <0Æ01). Among five kinds of nitrogen sources supplemented, yeast extract, casamino acid and peptone were more effective than (NH 4 ) 2 SO 4 and NH 4 NO 3, and yeast extract gave the highest degradation rate of starch and ergosterol yield, followed by peptone. Through orthogonal experiments, the theoretical optimum culture medium for SSF of this fungus was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 7Æ5 g glucose per litre. Conclusions: Under the optimum culture condition, the degradation rate of starch reached its maximum values of 70Æ4%; the starch content of the fermented product decreased from 64Æ5 to25æ3%, while the reducing sugar content increased from 4Æ2 to 20Æ6%. SSF also produced a significant increase (P < 0Æ01) from 11Æ0 to 16Æ5% in protein content. Significance and Impact of the Study: After SSF by G. lucidum, the digesting and absorbing ratio of cornmeal was strikingly increased and some active substances originated from G. lucidum remained in the fermented product. This implied that cornmeal could be processed into many kinds of special functional foods by SSF of G. lucidum. Keywords: amylase, cornmeal, Ganoderma lucidum, mycelial biomass, solid-state fermentation, starch. INTRODUCTION Ganoderma mushrooms are well known as traditional Chinese medicine and commonly used for pharmaceutical purposes and as health foods. For centuries in the Orient, Correspondence to: Jian-Rong Han, School of Life Science and Technology, Shanxi University, Taiyuan , China ( hjr@sxu.edu.cn). these medicinal mushrooms have been used to treat various human diseases, such as hepatitis, hypertension and gastric cancer (Jong and Birmingham 1992). Recently, most of the research has been related to Ganoderma lucidum, and much attention has been paid to the fungus regarding its physiological effects when used as a home remedy (Mizuno et al. 1995). As G. lucidum is very rare in nature, the amount of wild mushroom is not sufficient for commercial ª 2005 The Society for Applied Microbiology

2 SOLID-STATE FERMENTATION OF CORNMEAL BY GANODERMA LUCIDUM 911 exploitation. Its cultivation on solid substrate, stationary liquid medium or, in the last time, by submerged cultivation has become essential to meet the increasing demands in the international markets (Hsieh and Yang 2004). Of a white-rot fungus, the nutritional supply for fruitbody development of G. lucidum come from the degradation of lignin and cellulose by the extracellular enzymes, such as lignin peroxidase, laccase and cellulase (Perumal and Kalaichelvan 1996). Previous studies have mainly concentrated on delignification caused by G. lucidum (Adaskaveg et al. 1990). However, the studies on the degradation of starch by G. lucidum were reported in few papers so far. Do and Kim (1988) studied the extracellular amylase from the filtrate of the submerged culture of G. lucidum. Wang and Wang (1990) reported that four Ganoderma species could produce relatively weak amylase in sawdust medium. However, studies on solid-state fermentation (SSF) of G. lucidum on amylaceous substrates, such as cornmeal and potato meal, have not been reported. Solid-state fermentation is now being used for upgrading to feed values of waste cellulosic materials, for enzyme production and upgrading the values of existing foods, especially oriental foods (Pandey et al. 2000). SSF is a process whereby an insoluble substrate is fermented with sufficient moisture but without free water. SSF, unlike that of slurry state, requires no complex fermentation controls and has many advantages over submerged liquid fermentation (SLF). SSF requires less energy input than SLF, making its potential application of interest (Pandey et al. 2000). Filamentous fungi seem to be more suitable for SSF than single-celled micro-organisms like bacteria and yeasts. During previous studies (Han 2003), we found that cornflour could be degraded by the basidiomycete Hericium erinaceum, and nutritional value of cornmeal was upgraded strikingly. Encouraged by these results, we envisaged a new approach to develop a more efficient and dependable method for the culture of G. lucidum. We have focused on the SSF of cornmeal by G. lucidum. The aim of this research was to study the ability of the basidiomycete G. lucidum to degrade starch and upgrade nutritional value of cornmeal during SSF. The present report described a series of experiments on the SSF of G. lucidum in cornmeal medium and analysed the amylase activity, mycelial biomass and main nutritional component differences between the fermented product and nonfermented control. MATERIALS AND METHODS Organisms Ganoderma lucidum AS5.65 was obtained from Institute of Microbiology, Chinese Academy of Sciences, and routinely maintained on potato dextrose agar (PDA) slants. Chemicals Casamino acid and peptone were purchased from Sigma (St Louis, MO, USA). Ergosterol (Sigma) was recrystallized from ethanol. All other chemicals were reagent grade. Methanol was of HPLC grade. Preparation of medium The basal medium for SSF consisted of 100 g of dry cornmeal, which were ground to 20-mesh powder, moistened with 67 ml of nutrient salt solution which contained the following per litre of distilled water (g): KH 2 PO 4 1, MgSO 4 Æ7H 2 O 1, FeSO 4 Æ7H 2 O 0Æ5, CaClÆ2H 2 O 0Æ1 (a modified formula previously described by Yoon et al. 1994). The moisture content at this stage was about 40% (w/w). The initial ph of the salt solution was adjusted to 6Æ5. The saccharides and nitrogen sources being added to the basal medium were described in the text. Solid-state fermentation The solid medium containing 100 g of dry substrate was dispensed into each Erlenmeyer flask of 250 ml capacity. The flasks were sealed with cotton plugs to facilitate air transfer, autoclaved twice for 30 min each at 121 C. After sterilization, each flask was surface inoculated with five discs of mycelium and agar, 6 mm in diameter, from agar plate cultures of the organisms on PDA medium. Three replicate were prepared for each treatment, and an uninoculated flask served as control. All of the flasks were incubated at 25 C in the dark. The entire contents of flasks were harvested at 10, 15, 20 and 25 days for the assay of a-amylase activity. Then the samples were dried to constant weight at 70 C and used for the assay of main nutritional components and ergosterol. Enzyme extraction and assay One gram of the culture of SSF from each flask was mixed with 8 ml of deionized water and acid-washed sand in an ice-cold mortar. The homogenate was centrifuged at 2000 g for 5 min. The supernatant was assayed for a-amylase activity within 12 h of preparation. Amylase (a-1,4-glucan- 4-glucanohydrolase, EC ) was assayed with the substrate 1% soluble starch in 20 mmol l )1 phosphate buffer (ph 6Æ9). A mixture of 0Æ5 ml of extract and 0Æ5 ml of 20 mmol l )1 phosphate buffer (ph 6Æ9) containing 1% soluble starch was incubated at 25 C for 3 min. The reducing sugars released therein were then determined by the method of Bernfeld (1955) with reference to a standard curve of maltose. One a-amylase unit (U) was defined as the amount of enzyme producing 1 lmol of maltose per min at 25 C on soluble starch.

3 912 J.R. HAN ET AL. Main nutritional components assay The crude protein content of the samples was calculated from the nitrogen content (as N 6Æ25) as determined by the micro-kjedahl method. The crude fat content was estimated gravimetrically after continuous ether extraction of the dried samples in a Soxhlet apparatus. The reducing sugar content was determined by the Folin capacity method (Huang 1989). The starch content was determined with the multi-starch plant sample assay method described by Huang (1989). The starch degradation rate was calculated as follows: RESULTS Effect of fermentation time The a-amylase activity, starch content and ergosterol yield of every sample were determined after 10, 15, 20 and 25 days. The results (Table 1) showed that the a-amylase activity of G. lucidum reached its maximum value of 267 U g )1 of culture on day 20 after inoculation. Prolongation of fermentation time from 10 to 25 days increased significantly the degradation rate of starch (P <0Æ01). When plotting the degradation rate of starch vs the fermentation Starch degradation rate (%Þ ¼ Starch content of fermented sample starch content of the control 100: Starch content of control Measure of ergosterol Ergosterol is membrane component of most fungi and ergosterol levels are commonly used to estimate fungal biomass on various substrates (Charcosset and Chauvet 2001). In present study, the ergosterol content in fermented products was measured by the method of Matcham et al. (1985) with some modifications. The fermented samples were extracted by mixing with ethanol in the proportion of 2 ml ethanol to 1 g of sample. The mixture was steeped for 1 h, then the supernatant was decanted and the residue washed with two further aliquots of ethanol. Following clarification by centrifugation for 10 min at 2000 g, the three washings were combined. The extracts were evaporated to dryness under oxygen-free nitrogen prior to saponification in a solution of 1 mol l )1 KOH in 95% (v/v) ethanol for 1 h at 70 C. After cooling, the mixtures were diluted with two volumes of water and the nonsaponifiable fraction containing ergosterol extracted with three washings of petroleum ether. The washings were combined and dried over sodium sulfate prior to assay. The ergosterol extracts were analysed on a Hitachi HPLC (Hitachi, Tokyo, Japan). The samples were eluted with 97 : 3 methanol/water (v/v) with a flow rate of 0Æ5 ml min )1 and monitored at 282 nm in a Hitachi L-4000 UV detector. Fifty microlitres of ergosterol was injected into the HPLC system. The analysis was calibrated against a standard curve obtained from ergosterol solutions of 0 60 lg ml )1. Experimentation and analysis All experiments were replicated in three flasks and the data are presented as the mean and standard error of three independent experiments. Duncan s multiple range test (Du 1985) was used to determine the significant differences among mean values at the 1% level of confidence. time, a positive linear relation was found. The longer the fermentation time was, the higher the degradation rate, so on day 25 after inoculation, this fungus gave the highest degradation rate of starch (50Æ2%). Considering that the degradation rate of starch was affected by both amylase activity and mycelial biomass, we tested the effect of fermentation time on ergosterol yield as an indicator of mycelial biomass in every sample (Table 1). The results showed that the ergosterol yield per flask had also a positive correlation with the fermentation time. So on day 25 after inoculation, G. lucidum gave the highest ergosterol yield (690 lg/flask). Effect of supplementary nitrogen sources Due to the nitrogen deficiency in cornmeal, addition of nitrogen to the basal medium was required for increasing the mycelial biomass and enhancing the degradation rate of starch. We examined the effect of supplementary nitrogen Table 1 Effect of fermentation time on starch degradation and mycelial biomass of Ganoderma lucidum Fermentation time (days) a-amylase activity (U g )1 of culture) Degradation rate of starch (%) Ergosterol yield* (lg flask )1 ) ± 9A 41Æ7 ± 3Æ3A 540 ± 36A ± 11B 43Æ6 ± 3Æ1A 570 ± 37A ± 13C 47Æ9 ± 3Æ7B 650 ± 44B ± 10B 50Æ2 ± 3Æ9B 690 ± 45B Data are mean values from three independent experiments ± standard errors. Mean values in the same column followed by different letters are significantly different at the P < 0Æ01 level according to Duncan s multiple range test. *The ergosterol yield was used to estimate the mycelial biomass of G. lucidum during solid-state fermentation.

4 SOLID-STATE FERMENTATION OF CORNMEAL BY GANODERMA LUCIDUM 913 Table 2 Effect of supplementary nitrogen sources on mycelial biomass and starch degradation by Ganoderma lucidum. Control means without supplementation of nitrogen sources to basal medium Table 3 Effect of supplementary saccharide on mycelial biomass and starch degradation by Ganoderma lucidum. Control means without supplementation of saccharide to basal medium Adjuvant Degradation rate of starch (%) Ergosterol yield* (lg flask )1 ) Adjuvant Degradation rate of starch (%) Ergosterol yield*(lg flask )1 ) Yeast extract 65Æ2 ± 3Æ9A 905 ± 50A Casamino acid 58Æ7 ± 3Æ6C 795 ± 41C Peptone 61Æ9 ± 3Æ8B 835 ± 49B (NH 4 ) 2 SO 4 53Æ3 ±3Æ3D 720 ± 39D NH 4 NO 3 51Æ4 ±3Æ1D 695 ± 40E Control 50Æ2 ± 3Æ1E 690 ± 36E Data are mean values from three independent experiments ± standard errors. Mean values in the same column followed by different letters are significantly different at the P < 0Æ01 level according to Duncan s multiple range test. *The ergosterol yield was used to estimate the mycelial biomass of G. lucidum during solid-state fermentation. Glucose 60Æ5 ± 3Æ8A 795 ± 43A Fructose 49Æ8 ± 3Æ0B 710 ± 39B Sucrose 59Æ5 ± 3Æ7A 775 ± 44A Maltose 58Æ3 ± 3Æ7A 780 ± 44A Lactose 50Æ6 ± 3Æ1B 715 ± 40B Control 50Æ2 ± 3Æ1B 690 ± 40B Data are mean values from three independent experiments ± standard errors. Mean values in the same column followed by different letters are significantly different at the P < 0Æ01 level according to Duncan s multiple range test. *The ergosterol yield was used to estimate the mycelial biomass of G. lucidum during solid-state fermentation. sources on degradation rate of starch and ergosterol yield (Table 2). In this experiment, the basal medium served as control. The supplementation of nitrogen sources (2 g l )1 ) was conducted through salt solution. Among five kinds of nitrogen sources, yeast extract gave the highest degradation rate of starch and ergosterol yield respectively, followed by peptone. Three kinds of organic nitrogen sources (i.e. yeast extract, casamino acid and peptone) were more effective than two kinds of inorganic nitrogen sources [i.e. (NH 4 ) 2 SO 4 and NH 4 NO 3 ]. Effect of supplementary saccharides In cornmeal as amylaceous substrate, starch is the main carbon source that can be utilized by the strain. Generally, compared with polysaccharides, glucose and maltose are easily utilized by various fungi. We examined the effect of selected saccharides on degradation rate of starch and ergosterol yield (Table 3). The supplementation of saccharides were conducted through salt solution and remained the concentration of 5 g l )1 of salt solution. The results showed that supplementation of three types of saccharides (i.e. glucose, sucrose and maltose) to the basal medium caused a significant increase in both the degradation rate of starch and the ergosterol yield as compared with control (P < 0Æ01). Glucose gave the highest degradation rate of starch and ergosterol yield respectively. Identification of optimum SSF condition Through orthogonal experiments (Table 4), the theoretical optimum culture medium for SSF of G. lucidum was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented Table 4 Result of orthogonal experiment to assess optimal conditions for solid-state fermentation by Ganoderma lucidum Experimental no. Yeast extract (g l )1 ) Glucose (g l )1 ) Particle size of cornmeal (mesh) Degradation rate of starch (%) Æ2 ±4Æ Æ Æ4 ±4Æ Æ5 ±4Æ Æ8 ±4Æ Æ Æ5 ±4Æ Æ4 ±4Æ Æ6 ±3Æ Æ Æ5 ±4Æ Æ7 ±4Æ0 Table 5 Variance analysis of orthogonal experiment Variation source d.f. Sum of squares F value F 0Æ05 Yeast extract 2 70Æ3 15Æ98 19 Glucose 2 10Æ9 2Æ48 Particle size of cornmeal 2 3Æ1 0Æ70 Error 2 4Æ4 Sum 8 88Æ7 with 3 g yeast extract and 7Æ5 g glucose per litre. Under the optimum culture condition, the degradation rate of starch by G. lucidum reached its maximum values of 70Æ4%. The result of orthogonal experiment also showed that, though the single factor effect of either yeast extract or glucose on the degradation rate of starch was not significant (P < 0Æ05) (Table 5), the interaction of several factors achieved a better degree of starch degradation than any individual factor.

5 914 J.R. HAN ET AL. Table 6 Main nutritional components of fermented product of Ganoderma lucidum and a nonfermented control on a dry basis Component (%) Nonfermented Fermented P (t-test) Protein 11Æ0 ±0Æ5 16Æ5 ±0Æ7 <0Æ01 Crude fat 10Æ3 ± 0Æ6 8Æ5 ± 0Æ3 <0Æ01 Starch 64Æ5 ±1Æ5 25Æ3 ±0Æ8 <0Æ001 Reducing sugar 4Æ2 ± 0Æ2 20Æ6 ± 0Æ8 <0Æ001 Effect of SSF on the main nutritional components content Under the condition of orthogonal experiment 2, the main nutritional components content of the fermented product and the control were summarized in Table 6. The starch content of the nonfermented control reached 64Æ5%; the reducing sugar content was only 4Æ2%. However, the starch content of the fermented product decreased significantly (P < 0Æ001) from 64Æ5 to25æ3%; while the reducing sugar content increased significantly (P <0Æ001) from 4Æ2 to 20Æ6%. SSF also produced a significant (P < 0Æ01) increase from 11Æ0 to16æ5% in protein content. After SSF, the crude fat content decreased significantly (P < 0Æ01). DISCUSSION Fermentation has historically been one of the most common ways to enhance the nutritional properties and palatability of agricultural and dairy products. After SSF of G. lucidum, the starch content of cornmeal was reduced greatly, and quite a proportion of the starch was converted into dextrin and reducing sugar. The digesting and absorbing ratio of cornmeal was strikingly increased. This also demonstrated that G. lucidum was able to produce high-active a-amylase during SSF on cornmeal. High a-amylase activity was attributed to the close contact of hyphae of G. lucidum with the substrate in SSF, which resulted in a high degradation rate of starch. Yang et al. (1992) had discussed the importance of a-amylase in SLF of Candida tropicalis. However, in their research, the a-amylase was not produced by C. tropicalis during SLF, but commercial amylase was added into the medium at the same time as inoculation with the yeast. A large and diverse spectrum of chemical compounds with a pharmacological activity has been isolated from the mycelium, fruiting bodies and sclerotia of Ganoderma mushrooms: triterpenoids, polysaccharides, proteins, amino acids, nucleotides, alkaloids, steroids, lactones, fatty acids and enzymes (Hirose et al. 1988). The quality and content of these physiologically active substances vary from strain to strain and also depend on culture conditions (Habijanič et al. 2001). In this experiment, the ergosterol in the fermented product was not only as an indicator of mycelial biomass but also a kind of physiologically active substances of G. lucidum (Lin et al. 1991). Although, in our research, no attempt was made to determine other physiologically active substances, it is likely that the fermented products contain more kinds of these physiologically active substances besides ergosterol. This implied that cornmeal could be processed into many kinds of special functional foods containing some bioactive substances after SSF by G. lucidum. Functional foods as a marketing term was initiated in the 1980s and is used to describe foods fortified with ingredients capable of producing health benefits. This concept is becoming increasingly popular with consumers because of a heightened awareness of the link between health, nutrition and diet. Food manufacturers are enthusiastic about developing such products because the added ingredients give increased value to food (Hilliam 1998). In this case, all bioactive components present in the fermented product should be assayed. The importance of further studies on SSF of G. lucidum in cornmeal or other cereals can readily be seen. ACKNOWLEDGEMENT Support for this research by the Shanxi Province Science Foundation (No ) is gratefully acknowledged. REFERENCES Adaskaveg, J.E., Gilbertson, R.L. and Blanchette, R.A. (1990) Comparative studies of delignification caused by Ganoderma species. Appl Environ Microbiol 56, Bernfeld, P. (1955) Alpha-amylase. In Methods in Enzymology, Vol. 1 ed. Colowick, S. and Kaplan, N.O. pp New York: Academic Press. Charcosset, J.Y. and Chauvet, E. (2001) Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes. Appl Environ Microbiol 67, Do, J.H. and Kim, S.D. (1988) Properties of amylase produced from higher fungi Ganoderma lucidum. Korean J Appl Microbiol Bioeng 8, Du, R.J. (1985) Biological Statistics. Beijing: Higher Education Press. Habijanič, J., Berovič, M., Wraber, B., Hodžar, D. and Boh, B. (2001) Immunostimulatory effects of fungal polysaccharides from Ganoderma lucidum submerged biomass cultivation. Food Technol Biotechnol 39, Han, J.R. (2003) Solid-state fermentation of cornmeal with the basidiomycete Hericium erinaceum for degrading starch and upgrading nutritional value. Int J Food Microbiol 80, Hilliam, M. (1998) The market for functional foods. Int Dairy J 8, Hirose, K., Muto, S., Nimura, K., Ohara, M., Oguchi, Y., Matsunaga, K., Kadochi, J., Sugita, N. et al. (1988) Antiviral Agent. Japan Patent No Kureha Chemical Industry Co., Tokyo, Japan. Hsieh, C. and Yang, F.C. (2004) Reusing soy residue for the solid-state fermentation of Ganoderma lucidum. Biores Technol 91,

6 SOLID-STATE FERMENTATION OF CORNMEAL BY GANODERMA LUCIDUM 915 Huang, W.K. (1989) Examination and Analysis of Food. Beijing: China Light Industry Press. Jong, S.C. and Birmingham, J.M. (1992) Medicinal benefits of the mushroom Ganoderma. In Advances in Applied Microbiology, Vol. 37 ed. Neidleman, S.L. and Laskin, A.I. pp New York: Academic Press. Lin, C.N., Tome, W.P. and Won, S.J. (1991) Novel cytotoxic principles of Formosan Ganoderma lucidum. J Nat Prod 54, Matcham, S.E., Jordan, B.R. and Wood, D.A. (1985) Estimation of fungal biomass in a solid substrate by three independent methods. Appl Microbiol Biotechnol 21, Mizuno, T., Wang, G., Zhang, J., Kawagishi, H., Nishitoba, T. and Li, J. (1995) Ganoderma lucidum and Ganoderma tsugae: bioactive substances and medicinal effects. Food Rev Int 11, Pandey, A., Soccol, C.R. and Mitchell, D. (2000) New developments in solid-state fermentation. I. Bioprocesses and products. Process Biochem 35, Perumal, K. and Kalaichelvan, P.T. (1996) Production of extracellular lignin peroxidase and laccase by Ganoderma lucidum PTK-3 on sugarcane bagasse lignin. Indian J Exp Biol 34, Wang, Y.W. and Wang, Y. (1990) Study on nutrient physiology of some species of Ganoderma. Edible Fungi of China 9, Yang, J.X., Tao, S.X. and Song, T.S. (1992) Improvement of nutritional value in enzymatically hydrolyzed corn meal by Candida tropicalis. Acta Nutrimenta Sinica 14, Yoon, S., Kim, M.K., Hong, J.S. and Kim, M.S. (1994) Production of polygalacturonase from Ganoderma lucidum. Korean J Mycol 22,