Effect of plant oil and surfactant on the production of mycelial biomass and polysaccharides in submerged culture of Grifola frondosa

Biochemical Engineering Journal 38 (2008) 198–205

Effect of plant oil and surfactant on the production of mycelial biomass and polysaccharides in submerged culture of Grifola frondosa Chienyan Hsieh a,∗ , Hui-Liang Wang a , Chien-Cheng Chen a , Tai-Hao Hsu b , Mei-Hua Tseng b a

Department of Biotechnology, National Kaohsiung Normal University, 62 Shenjhong Rd., Yanchao Township, Kao-Hsiung County 824, Taiwan b Department of Bioindustry Technology, Da Yeh University, Chang-Hua, Taiwan Received 29 October 2006; received in revised form 11 June 2007; accepted 1 July 2007

Abstract Effects of various plant oil addition and surfactant addition at different stages of cell growth phase on the cell growth and production of bioactive metabolites, such as exopolysaccharide (EPS) and intracellular polysaccharide (IPS) in the submerged culture of Grifola frondosa were studied with 2% glucose medium. Olive, safflower seed, soy and sunflower oil were favorable plant oil sources to the mycelial growth of G. frondosa. The highest cell growth (∼12.64 ± 0.47 g/l cell dry weight) can be obtained on day 13 of cultivation in the medium containing 1% all the plant oil sources. EPS production was slightly enhanced by olive oil but significantly inhibited by safflower seed oil and sunflower oil after 13 days of cultivation. With 4% glucose, 0.5% plant oils were selected to add on 0-, 3-, 7-, and 9-day of cultivation to exam the effect on the cell growth at different stages. Amongst four plant oil sources examined, cell growth yielded relatively high mycelial biomass (11.22 ± 1.14 g/l) and that was achieved in 4% glucose medium with 0.5% soybean oil. The higher EPS production and slightly lower cell growth were found in 4% glucose media; the maximum EPS production was 2.248 ± 0.107 g/l found in 4% glucose media with olive oil addition. Tween 80 and Span 80 addition had shown to increase cell growth and the maximal cell concentration of 9.10 ± 0.80 g/l was obtained with 1% Span 80 addition. Both EPS and IPS production were found to decrease with all the tested concentrations of Tween 80 and Span 80 addition. Span 80 added at the vegetative growth phase in 4% glucose media yielded the highest mycelial biomass of G. frondosa (8.95 ± 0.57 g/l); meanwhile Tween 80 added at the beginning cultivation had resulted in the highest EPS production (1.451 ± 0.098 g/l). Tween 20 and Span 20 addition were shown to have serious inhibition on cell growth of G. frondosa and also on polysaccharides production. The results obtained were useful in better understanding the regulation and optimization of G. frondosa culture for efficient production of cell mass and polysaccharides in the submerged culture. © 2007 Elsevier B.V. All rights reserved. Keywords: Grifola frondosa; Plant oil; Tween 80; Span 80; Polysaccharides

1. Introduction Grifola frondosa is a Basidiomycete fungus belonging to the order Aphyllopherales, and family Polyporaceae [1]. G. frondosa (Maitake) is also called the king of mushrooms and the hen of the woods [1]. Maitake is used as a Chinese medicine called ‘keisho’ “Shen nong ben cao jing” and has been frequently used as remedy for spleen, stomach, nerve tense, and hemorrhoids. In Japan G. frondosa has become a relatively popular food in recent years because of its bioactive ingredients. It contains a polysaccharide compound beta-glucan that was not found in other mushrooms; beta-glucan is also reported to help

∗

Corresponding author at: Tel.: +886 7 7172930x7317; fax: +886 7 6051353. E-mail address: [email protected] (C. Hsieh).

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.07.001

strengthen the body’s natural immune system and improve general health [2,3]. Except health benefits, Maitake mushrooms with firm, meaty texture contains a distinctive rich, woody flavor. Usually, dried fruiting body of G. frondosa is used to lengthen the shelf-life. Moreover, dried fruiting body of G. frondosa is also used to produce health foods, including G. frondosa tea, whole G. frondosa powder, powder of G. frondosa extracts, G. frondosa granules, and G. frondosa drinks [4]. The major anti-tumor substances, which have been obtained from extracts of the fruit body and liquid-cultured mycelium, were attributed to polysaccharides [4,5]. These polysaccharides have been identified as many types of glucans in that more than 20 anti-tumor polysaccharides have been isolated and purified from G. frondosa; each active polysaccharide has a basic structure of a (1–6)-␤-branched (1–3)-␤-d-glucan and heteroglycan or heteroglycan–protein complex as the major component [5–8].

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The D-fraction from the fruiting body powder of G. frondosa was observed to induce angiogenesis in vivo but enhance proliferation and migration in human vascular endothelial cell in vitro. The D-fraction also increased plasma vascular endothelial growth factor concentration [8]. Although bioactive polysaccharides were obtained from both fruiting body and liquid-cultured mycelium of G. frondosa [5–9], most previous studies’ efforts have been focusing on cultivating this mushroom in solid artificial media (for fruiting body production) rather than in submerged cultures (for mycelial extract and/or exopolysaccharide (EPS) production). However, because the solid culture of G. frondosa needs to take long time to complete a fruiting body, investigators have recently exerted their efforts to prepare this mushroom from submerged culture in the form of mycelium for use in the formulation of nutraceuticals and functional foods [10]. Submerged culture gives rise to potential advantages of higher mycelial production in a compact space and shorter time with fewer chances of contamination [11–13]. And, it ensures standardized quality and year around production [14]. According to previous studies, various plant oils had been used to accelerate mycelial growth in some mushroom species and proved to have a stimulatory effect [15]. Also, many researchers had reported that fatty acid, oil and surfactant promoted the production of fungal metabolites [16,17]. Banchio and Gramajo had also reported that the uptake of long-chain length fatty acids with both mediated transport and simple diffusion was superior to medium-chain length fatty acids with simple diffusion only in Streptomyces coelicolor [18]. They also found the uptake of fatty acid was stimulated by the presence of glucose. So far, the role of G. frondosa, the effects of fatty acids and surfactants on cell growth, and polysaccharide production by G. frondosa have not been reported yet. This study attempts to understand more of plant oil addition and surfactant addition in submerged culture for mycelial growth and polysaccharides production by G. frondosa. This study investigates the effect of carbon source concentration in the submerged culture on cell growth by using four kinds of plant oil additions and surfactant additions.

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containing 100 ml of the medium with 4 units of a cutter square of activated mycelia. Mycelia agar squares (5 mm × 5 mm) were obtained by a self-designed cutter to use as inoculums in a shake-flask culture. The media consisted of the following components: 4 g/l yeast extract, 10 g/l malt extract, 2 g/l glucose, 1 g/l molasses, and 10 ml mineral salt solution. And the mineral salt solution contained 120 g/l MgSO4 ·7H2 O, 6 g/l NaCl, 20 g/l KH2 PO4 , 20 g/l CaCl2 , 10 g/l FeSO4 ·7H2 O, and 1.8 g/l ZnCl2 . The flasks were incubated on a New Brunswick rotary shaker (Model G24) at 25 ◦ C, 150 rpm for 7 days. Then, the mycelium was homogenized by a sterilized blender for 30 s to be the inoculums in the following experiments [20]. 2.3. Flask culture conditions The flask culture experiments were performed in 250 ml flasks containing 100 ml of medium, which were the same compositions used for inoculum preparation. After inoculating with 10% (v/v) of the seed culture, the culture was incubated at 25 ◦ C on a rotary shaker incubator at 100 rpm, and samples were collected at various intervals from the shake flasks for analyzing biomass dry weight, exopolysaccharides (EPS), and intracellular polysaccharides (IPS). The effects of factors affecting cell growth and the production of components such as EPS and IPS by G. frondosa were studied using shake-flask culture on rotary incubator shaker (Wisdom 721 SR-Incubator-Shaker) as described above. Effects of plant oil additions on G. frondosa culture were studied by substituting various plant oils such as olive, safflower seed, soy, and sunflower oil for fermentation medium in a one at a time fashion. The concentrations of plant oils used were from 0.1% to 1%. Effects of surfactants on G. frondosa culture were also studied using shake-flask culture. Surfactants such as Tween 80, Tween 20, Span 80, and Span 20 were supplemented, all at volume fractions of 0.5%, in liquid media and the culture was cultivated at 25 ◦ C on a rotary shaker incubator at 150 rpm. 2.4. Analytical methods

2. Materials and methods 2.1. Organism G. frondosa (TARI 619908) used in this study was obtained from Taiwan Agricultural Research Institute, Wufeng, Taiwan [19,20]. The cultures were maintained on potato–agar–dextrose slopes. The slopes were inoculated and incubated at 25 ◦ C for 14 days, and then stored at 4 ◦ C [19]. 2.2. Inoculum preparation A modified agar plate (4 g/l yeast extract, 10 g/l malt extract, 2 g/l glucose, 1.0 g/l molasses, 10 ml/l mineral salt solution, and 15 g/l agar) for mycelium activation culture was prepared; the pH was initially adjusted to 5, followed by autoclaving at 121 ◦ C for 15 min. The mycelium was grown at 25 ◦ C for 7 days. The experimental inoculums were prepared in 250 ml Erlenmeyer flasks

2.4.1. Cell concentration The cell concentration was termed as the dry weight per unit volume. Ten millilitres fermentation broth was obtained and subjected to centrifuge at 4185 × g (6000 rpm) for 15 min (HERMLE, model Z200A). Then, the sediment produced at 4185 × g was washed, resuspended, and centrifuged twice with 10 ml distilled water. Next, the sediment was dried to a constant weight and then frozen. 2.4.2. Determination of polysaccharides In order to determine the extracellular polysaccharides, 4 volumes of 95% ethanol was added in the fermentation broth filtrate and then left overnight at 4 ◦ C to precipitate the crude polysaccharides. Next, the precipitated polysaccharides were collected by centrifugation (HERMLE, model Z160M) at 10,000 rpm for 10 min, and they were dried to remove the residual ethanol with a freezing dryer. According to the method developed by Dubois et

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al., the total polysaccharide content was determined by a phenolsulfuric acid assay [21]. And the filtered mycelia were washed twice with the same volume of distilled water, and then collected for determining intracellular polysaccharides and cell concentration. The procedure for IPS concentration in this study was the same as that mentioned above but prior to ethanol precipitation, and 100 mg of freeze–dried mycelia was ground into powder and extracted with 10 ml of 121 ◦ C distilled water for 30 min [22].

caused by the uptake of plant oils as the carbon sources supplies the cell growth continuously. This result is also consistent with a previous study that various carbon sources were suitable for the cell growth of Cordyceps sinensis and the cell could grow better with lower final pH of fermentation broth [26]. On the production of EPS and IPS by G. frondosa the oil addition were shown that the increased concentration of olive oil and soybean oil led to a higher EPS production, but lower EPS production was found in safflower seed oil and sunflower oil addition. This result showed that the EPS production was significantly inhibited by safflower seed oil and sunflower oil. The lowest EPS production (0.412 ± 0.004 g/l), half less than that in the control one (0.907 ± 0.015 g/l), was obtained when 1% safflower oil was supplemented. On the other hand, the EPS production increased to 0.923 ± 0.012 g/l and 1.151 ± 0.018 g/l when the concentration increased up to 1% of soybean oil and olive oil addition, respectively. It is noteworthy that oleic acid, presented in the safflower seed oil around 10–20% and sunflower oil around 21.3% instead of linoleic acid, presented in the safflower seed oil around 70–80% and sunflower oil around 66.2%, seemed to play a critical role in inhibiting the production of polysaccharides. A previous study reported that linoleic acid drastically suppressed polysaccharide formation during submerged culture of Ganoderma lucidum [23]. Furthermore, Stasinopoulos and Seviour [27] also demonstrated that linoleic acid had a strong inhibitory effect on the polysaccharide production from Acremonium persicinum. The amount of oleic acid in olive oil, safflower seed oil, soybean oil and sunflower oil are 84%, 10–20%, 30–60%, and 21.3%, respectively and that of linoleic acid, 4%, 70–80%, 45–75%, and 66.2%, respectively. For the specific IPS production, the oil addition led to significantly inhibiting IPS

3. Results and discussion 3.1. Effects of plant oil on cell growth and the production of polysaccharides Plant oil, which has the function of antifoam agent in fermentation, has been reported to be favorable to the mycelial growth in several medicinal mushrooms, and to increase the production of bioactive metabolites [23–25]. In this research effects of the additives of olive, safflower seed, soy and sunflower oil on G. frondosa in the submerged fermentation were studied, all at volume fractions of 0.1–1% with 2% glucose (Table 1). The mycelial growth of G. frondosa was found increased when all of the additives oil concentration increased. The highest cell growth (∼12.61 ± 1.22 g/l cell dry weight) was obtained on day 13 in the medium, containing 1% soybean oil. The pH of broth was found to decline with increasing oil addition. The lowest pH (2.7 ± 0.1) was found in 1% of olive oil and safflower oil addition. The stimulation of cell growth by oil in this study might be caused by the partial incorporation of lipids in the cell membrane, thereby facilitating the uptake of nutrients from the medium [23]. The lower pH in oil addition media might be

Table 1 Effects of plant oils on the cell growth and the production of EPS and IPS in a 2% glucose media pH Control Olive-0.1%a Olive-0.25%b Olive-0.5%c Olive-1.0%d Safflower-0.1%a Safflower-0.25%b Safflower-0.5%c Safflower-1.0%d Soybean-0.1%a Soybean-0.25%b Soybean-0.5%c Soybean-1.0%d Sunflower-0.1% a Sunflower-0.25%b Sunflower-0.5%c Sunflower-1.0%d

4.3 4.2 3.6 3.3 2.8 3.9 3.7 3.6 2.7 4.3 3.7 3.3 3.7 4.1 3.6 2.8 3.0

CDW (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.3 0.1 0.1 0.6 0.1 0.1 0.3 0.1 0.2 0.2 0.4 0.2 0.1 0.3

3.46 4.55 6.08 8.28 11.71 4.04 5.70 7.88 8.49 3.83 5.97 8.55 12.61 4.70 6.22 8.48 11.64

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.40 0.40 0.22 0.74 0.17 0.74 0.30 0.01 0.05 0.15 0.29 1.22 1.00 0.60 0.55 0.47

EPS (g/l) 0.907 0.889 0.957 1.018 1.151 0.467 0.438 0.465 0.412 0.533 0.741 0.743 0.923 0.725 0.651 0.463 0.629

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

IPS (mg/g) 0.015 0.060 0.080 0.082 0.018 0.021 0.067 0.047 0.004 0.009 0.027 0.036 0.012 0.047 0.049 0.016 0.056

28.5 9.5 11.9 8.3 14.0 5.5 11.7 13.9 18.3 6.6 17.0 10.9 13.0 10.4 15.0 12.6 20.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.3 1.4 0.1 0.2 2.5 0.9 0.3 0.5 0.3 0.4 0.6 0.8 0.6 1.7 0.5 0.1 1.0

Abbreviations: EPS, exopolysaccharide; IPS, intracellular polysaccharide; CDW, dry cell weight. Control without addition. All experiments were carried out in triplicate and data were expressed as means ± S.D. a 0.1% addition. b 0.25% addition. c 0.5% addition. d 1.0% addition.

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production in all the four oil sources examined (Table 1). The lowest IPS content (5.5 mg/g-DW) was found in 0.1% safflower seed oil medium, one-fifth less than the control one (without oil addition). From the above results, when 2% glucose was used as a carbon source, that plant oils addition could stimulate the mycelial growth of G. frondosa but slightly increase the EPS production. This result was not consistent with the previous reports [23–25,28]. However, it should be mentioned that the carbon source used in those previous studies was from 3% to 5%. And the following experiments were performed with 4% glucose and the cell growth at different stages with plant oil addition. 3.2. High glucose concentration with plant oil addition at different stages of cell growth A comparison of 4% and 2% glucose media with 0.5% (v/v) plant oils addition for cell growth and polysaccharides production was shown in Fig. 1. The higher EPS production and slightly lower cell growth were found in 4% glucose media; the maximum EPS production was 2.248 ± 0.107 g/l found in 4% glucose media with olive oil addition, while the maximum cell concentration was 8.55 ± 0.29 g/l found in 2% glucose media with soybean oil addition (Fig. 1). Under the supplement of 0.5% plant oil the higher glucose media seem not to favor cell growth but EPS production. It is noteworthy that higher glucose resulted in less cell growth in this study (Fig. 1). This result was similar to the previous report of Babchio and Gramajo, which indicated that the uptake of long-chain fatty acid (>16 C) was stimulated in the presence of glucose [18]. However, on the other hand, the carbon catabolite repression was found in high glucose concentration media and it resulted in the preferential utilization of glucose from mixture of carbon sources due to the repression of the expression of the gene encoding enzymes

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Table 2 Effects of 0.5% (v/v) plant oils addition at different stage of cell growth in a 4% glucose media pH Controla Olive-3b Olive-7c Olive-9d Safflower-3b Safflower-7c Safflower-9d Soybean-3b Soybean-7c Soybean-9d Sunflower-3b Sunflower-7c Sunflower-9d

3.7 3.3 3.1 4.1 3.3 4.2 4.4 3.2 3.5 3.7 3.6 4.2 4.4

CDW (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.4 0.1 0.1 0.1

4.27 9.85 8.33 6.59 8.85 7.32 5.63 11.22 8.05 7.73 9.29 7.32 5.63

± ± ± ± ± ± ± ± ± ± ± ± ±

0.31 0.25 0.33 0.30 0.13 0.25 0.27 1.14 0.35 0.50 0.32 0.25 0.27

EPS (g/l) 1.813 2.031 2.520 2.201 0.814 1.219 1.028 1.087 1.850 1.343 1.036 1.419 1.228

± ± ± ± ± ± ± ± ± ± ± ± ±

IPS (mg/g) 0.074 0.827 0.134 0.087 0.082 0.104 0.067 0.096 0.068 0.030 0.134 0.104 0.067

22.1 12.5 11.5 11.0 5.4 6.7 6.3 22.2 6.7 4.5 4.2 15.3 14.8

± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 0.9 0.4 0.7 0.5 0.7 0.2 3.8 0.4 0.2 0.5 1.6 3.8

Abbreviations: EPS, exopolysaccharide; IPS, intracellular polysaccharide; CDW, dry cell weight. All experiments were carried out in triplicate and data were expressed as means ± S.D. a Control without oil addition. b Addition at day 3 of cultivation. c Addition at day 7 of cultivation. d Addition at day 9 of cultivation.

required for the utilization of less favored carbon source [30]. To better understand the effect of plant oils on the cell growth and polysaccharides production, 0.5% plant oils were selected to add in 0-, 3-, 7-, and 9-day of cultivation, which were termed the initial stage of cell growth, the vegetative growth phase, the stationary phase, and the post-stationary stage, to exam the effect on cell growth and polysaccharides production. The results showed that the four plant oils adding at initial stage of cell growth were able to stimulate the mycelial growth of G. frondosa in 2% glucose media as well as in 4% glucose media (Table 2). Amongst

Fig. 1. Comparison of 4% and 2% glucose media with 0.5% (v/v) plant oils addition for cell growth and polysaccharides production.

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the four plant oils addition in 4% glucose media, the highest cell concentration (11.22 ± 1.14 g/l) was obtained on day 13 cultivation of the medium, which 0.5% soybean oil was added at the vegetative growth phase. However, the cell concentration with oil addition at the post-stationary stage was less than that at the other stages. It is worth to mention that the cell growth obtained from olive oil was less than that from soybean oil. The fact that the cell yields were higher in the presence of fatty acids with the mixture oleic/linoleic (1:1) > linoleic > oleic reported by Butler and Huzel [29]. In this study, the EPS production was significantly increased with the plant oils addition in 4% glucose media, but the result was quite different from in 2% glucose media. The maximal EPS production (2.520 ± 0.134 g/l) was obtained in olive oil addition at the stationary phase. The finding showed that the oil addition at the vegetative growth phase had shown higher cell growth but less EPS production. And the oil-uptake might be inhibited by the carbon catabolite repression of glucose, because glucose might be used as a main carbon source for cell synthesis and polysaccharide production under this circumstance. On the other hand, the oil addition at the stationary phase had less glucose presented in cell broth. The study found that once the effect of carbon catabolite repression was decreased, the uptake of fatty acids became active and the oil-uptake might be mainly used for polysaccharides production [30]. The utilization of carbon source seemed to shift the balance between cell production and their metabolic product. Once the energy source flew to cell production, their metabolic product (polysaccharides) might obtain less energy for synthesis. On the other hand, high-polysaccharide production did not result in high-concentration cell production.

3.3. Effects of surfactants on cell growth and the production of polysaccharides Volume fractions of 0.1–1% of two hydrophilic Tween series and two hydrophobic Span series surfactants were studied in the submerged culture of G. frondosa in 2% glucose media. The results of cell growth and polysaccharides production were shown in Table 3 and Fig. 2. When the addition of Tween 80 (polyoxyethylene sorbitan monooleate) and Span 80 (sorbitan monooleate) were used in the study, the finding had shown that the cell growth increased and the maximal cell concentration (9.10 ± 0.80 g/l) was obtained from the 1% Span 80 addition after 13 days of cultivation. And both EPS and IPS production were found to be decreased with all concentrations of Tween 80 and Span 80 addition tested. Tween 80 and Span 80, having an 18 C side chain, had been reported that they could be hydrolyzed by microbial enzymes, such as lipase, to release oleic acid [31]. That is, the activity of lipase was induced by the presence of Tween 80 or Span 80 surfactants; oleic acids releasing from the hydrolyzates of Tween 80 and Span 80 were able to enhance the cell growth. However, the increase of cell growth from the media with Tween 80 was less than that from the media with Span 80 and was far less than that from the media with plant oil. In fact, Tween 80 showed a slower hydrolysis rate than olive oil or Span 80, because lipases are known to have higher activity at oil–water interface than the aqueous solution, a phenomenon known as interfacial activation [32]. And Tween 80 provided oleic acid for cell growth through a mode of controlled release [33]. The finding showed that the less polysaccharides production was similar to that from plant oil addition in 2% glucose media and it might be because Tween 80 would increase cell growth instead of polysaccharides production.

Table 3 Effects of surfactants on the cell growth and the production of EPS and IPS in a 2% glucose media pH Control T80-0.1%a T80-0.25%b T80-0.5%c T80-1.0%d T20-0.1%a T20-0.25%b T20-0.5%c T20-1.0%d S80-0.1%a S80-0.25%b S80-0.5%c S80-1.0%d S20-0.1%a S20-0.25%b S20-0.5%c S20-1.0%d

4.3 3.6 3.5 3.5 3.6 3.9 3.7 3.9 3.8 3.6 3.3 3.2 3.1 4.3 4.3 4.4 4.7

CDW (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.2 0.3 0.1 0.2 0.3 0.3 0.1 0.2 0.2 0.1

3.46 4.19 3.95 5.17 3.67 2.96 3.25 3.02 2.33 5.91 5.46 7.61 9.10 2.74 2.69 2.20 1.73

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.20 0.62 0.06 0.02 0.21 0.29 0.30 0.46 0.17 1.17 1.18 0.80 0.12 0.09 0.18 0.05

EPS (g/l) 1.907 0.589 0.599 0.626 0.743 0.589 0.716 0.911 0.773 0.606 0.676 0.662 0.828 0.740 0.805 0.884 0.938

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

IPS (mg/g) 0.015 0.018 0.027 0.039 0.001 0.022 0.063 0.071 0.189 0.033 0.015 0.038 0.041 0.048 0.071 0.051 0.033

28.5 9.7 10.3 10.8 18.5 18.5 22.6 30.4 11.3 14.8 14.3 9.0 13.9 16.7 12.2 6.3 2.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.3 0.1 0.1 0. 3 0.9 0.7 7.9 0.8 0.5 2.3 2.6 2.5 2.4 5.7 0.7 0.6 0.2

Abbreviations: EPS, exopolysaccharide; IPS, intracellular polysaccharide; CDW, dry cell weight; T80, Tween 80; T20, Tween 20; S80, Span 80; S20, Span 20. Control without addition. All experiments were carried out in triplicate and data were expressed as means ± S.D. a 0.1% addition. b 0.25% addition. c 0.5% addition. d 1.0% addition.

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Fig. 2. Comparison of 4% and 2% glucose media with 0.5% (v/v) surfactants addition for cell growth and polysaccharides production.

With 12 C side chain, surfactants of Tween 20 (polyoxyethylene sorbitan monolaurate) and Span 20 (sorbitan monolaurate) addition were shown to have significant inhibition on the cell growth of G. frondosa (Table 3). The less carbon chain of surfactants exhibited higher diffusion rate in cell wall of G. frondosa. The high concentration of surfactant might damage the cell membrane or interact with other bio-compounds in cell and then resulted in low cell growth. However, Tween 80 and Span 80, with longer side chain of surfactants, might also be able to diffuse through the cell wall with less concentration of surfactants. To accommodate the surfactants media the composition of fatty acid in cell was altered. This explanation is consistent with a

previous study which had shown that the presence of Tween 80, the containing fatty acids a decrease in the amount of n-palmitate and an increase of octadecenoate were observed [34]. With higher concentration of glucose, the effects of surfactants on cell growth at different growth stages and on EPS and IPS production were shown in Table 4. The comparison of 4% glucose and 2% glucose was shown in Fig. 2. A higher cell growth and EPS production were obtained with Tween 80 and Span 80 in 4% glucose media. Span 80 added at the vegetative growth phase in 4% glucose media yielded the highest mycelial biomass of G. frondosa (8.95 ± 0.57 g/l); meanwhile Tween 80 added at the beginning of cultivation had resulted in

Table 4 Effects of 0.5% (v/v) surfactants addition at different stage of cell growth in a 4% glucose media pH Controla T80-3b T80-7c T80-9d T20-3b T20-7c T20-9d S80-3b S80-7c S80-9d S20-3b S20-7c S20-9d

4.1 3.6 3.5 4.2 4.1 4.7 4.8 3.2 3.2 3.9 4.3 4.5 4.5

CDW (g/l) ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.2 0.3 0.5 0.2 0.2 0.1 0.2 0.2 0.1 0.1 0.1

3.83 4.86 4.56 4.18 1.96 0.65 0.89 8.95 6.62 6.55 3.52 3.53 2.89

± ± ± ± ± ± ± ± ± ± ± ± ±

0.31 0.85 0.68 0.65 0.18 0.04 0.07 0.57 0.10 0.40 0.16 0.11 0.02

EPS (g/l) 1.312 1.276 1.047 0.963 1.209 1.081 1.157 1.006 0.806 0.782 1.111 1.134 1.081

± ± ± ± ± ± ± ± ± ± ± ± ±

IPS (mg/g) 0.074 0.141 0.093 0.091 0.132 0.168 0.067 0.203 0.116 0.182 0.176 0.131 0.072

15.8 9.6 21.1 15.3 11.7 9.4 11.7 17.0 11.3 9.3 6.6 4.7 4.4

± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 0.6 4.2 3.7 0.7 0.9 2.7 8.4 1.1 0.9 6.3 0.8 0.2

Abbreviations: EPS, exopolysaccharide; IPS, intracellular polysaccharide; CDW, dry cell weight; T80, Tween 80; T20, Tween 20; S80, Span 80; S20, Span 20. All experiments were carried out in triplicate and data were expressed as means ± S.D. a Control without addition. b Addition at day 3 of cultivation. c Addition at day 7 of cultivation. d Addition at day 9 of cultivation.

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the highest EPS production (1.451 ± 0.098 g/l). These results were consistent with those in previous studies which showed the effect of Tween 80 on stimulatory enzyme secretion by Candida lipolytica in submerged cultures [35]. It was reported that the addition of Tween 80 to a C. lipolytica culture medium would increase cellular viability. The maximum stimulation of enzyme production was found when Tween 80 was added at the beginning of cultivation, and the highest enzyme production was obtained in the medium with 1% Tween 80 [35]. In this study the finding showed that Tween 20 and Span 20 addition had inhibition on cell growth of G. frondosa at all stages (Table 4). Especially, adding Tween 20 on day 7, the lowest cell concentration was found to be as low as 0.65 ± 0.04 g/l and the results were close to those in the surfactant addition in 2% glucose media. 4. Conclusion The feasibility of using plant oils and surfactants for the mycelial growth and polysaccharides production of G. frondosa was investigated in this study. It can be concluded that plant oils such as olive, safflower seed, soybean, and sunflower oil and surfactants such as Tween 80 and Span 80 could be favorable used as an additive for the mycelial growth of G. frondosa. However, the application on the production of polysaccharides in 2% glucose was not as good as in 4% glucose. A higher cell growth and EPS production were obtained with Tween 80 and Span 80 in 4% glucose media (Fig. 2). Span 80 added at the vegetative growth phase in 4% glucose media yielded the highest mycelial biomass of G. frondosa; meanwhile Tween 80 added at the beginning of cultivation had resulted in the highest EPS production. Acknowledgement The authors wish to thank the National Science Council of the R.O.C. for financial supports (NSC91-2214-E-006). References [1] P. Stamets, Growing Gourmet and Medicinal Mushrooms, Ten Speed Press, Berkeley, CA, 1993. [2] K. Adachi, H. Nanba, H. Kuroda, Potentiation of host-mediated antitumor activity in mice by beta-GLUCAN obtained from Grifola frondosa (Maitake), Chem. Pharm. Bull. 35 (1987) 262–270. [3] H. Nanba, Antitumor activity of orally administered D-fraction from Maitake mushroom (Grifola frondosa), J. Naturopath. Med. 1 (1993) 10–15. [4] T. Mizuno, T. Sakai, G. Chihara, Health foods and medicinal usages of mushrooms, Food Rev. Int. 11 (1995) 69–81. [5] T. Mizuno, K. Ohsawa, N. Hagiwara, R. Kuboyama, Fractionation and characterization of antitumor polysaccharides from Maitake, Grifola frondosa, Agric. Biol. Chem. 50 (1986) 1679–1688. [6] N. Ohno, Y. Adachi, I. Suzuki, K. Sato, S. Oikawa, T. Yadomae, Characterization of the antitumor glucan obtained from liquid-cultured Grifola frondosa, Chem. Pharm. Bull. 34 (1986) 1709–1715. [7] N. Ohno, Y. Adachi, I. Suzuki, S. Oikawa, K. Sato, K.Y. Suzuki, et al., Two different conformations of antitumor glucans obtained from Grifola frondosa, Chem. Pharm. Bull. 34 (1986) 2555–2560.

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