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Production and characterization of exopolysaccharides from submerged culture of Phellinus linteus KCTC 6190
Enzyme and Microbial Technology 33 (2003) 309–319
Production and characterization of exopolysaccharides from submerged culture of Phellinus linteus KCTC 6190 Hye-Jin Hwang a , Sang-Woo Kim a , Jang-Won Choi b , Jong-Won Yun a,∗ b
a Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, South Korea Department of Natural Resources, Daegu University, Kyungsan, Kyungbuk 712-714, South Korea
Received 10 February 2003; accepted 10 May 2003
Abstract The optimal temperature and initial pH for both mycelial growth and exopolysaccharide (EPS) production in shake flask cultures of Phellinus linteus KCTC 6190 were found to be 30 ◦ C and pH 4.0, respectively. Optimal medium composition was determined to be sucrose 50 g/l, corn steep powder 3 g/l, KH2 PO4 0.68 g/l, and CaCl2 0.55 g/l. Under optimal culture conditions, the maximum mycelial biomass and EPS achieved in a 5-l stirred-tank fermenter indicated 11 and 3.3 g/l, respectively. To the best of our knowledge, this is the highest polysaccharide yield amongst liquid cultures of P. linteus reported in the literature. The three groups of EPSs (designated as Fr-I, -II, and -III) were obtained from the culture filtrates by a gel filtration chromatography on Sepharose CL-4B and the different fractions were analyzed directly using the size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis. It was revealed that all fractions of EPS were polysaccharides consisted of mainly mannose, galactose, and glucose. The molecular weights of Fr-I, -II, and -III of EPS were determined to be 433,400 (±7801), 31,470 (±283), and 12,950 (±90) g/mol, respectively. The SEC/MALLS analysis revealed that molecular dimension of the Fr-I was a spherical form, whereas the Fr-II was a random coil in an aqueous solution. © 2003 Elsevier Inc. All rights reserved. Keywords: Exopolysaccharide; MALLS; Mushroom; Phellinus linteus; Submerged culture
1. Introduction During the past decades, much interest has been generated in the polysaccharides produced by numerous microorganisms specially mushrooms because of their various biological and pharmacological activities including anti-tumor, immuno-stimulating, and hypoglycemic activities, etc. [1–5]. In order to obtain polysaccharides from mushrooms, most investigators have exerted their efforts to cultivate edible and medicinal mushrooms on solid artificial media (for fruit body production) rather than submerged culture (for mycelial extract and/or exopolysaccharide (EPS) production) [6–8]. Submerged culture obviously gives rise to potential advantages of higher mycelial production in a compact space and shorter time without significant problem of contamination [9–12]. Although enormous efforts have been made to obtain optimal submerged culture conditions for bioactive polysaccharide production from several mushrooms, the currently ∗
Corresponding author. Tel.: +82-53-850-6556; fax: +82-53-850-6559. E-mail address: [email protected] (J.-W. Yun).
0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0141-0229(03)00131-5
available reports for nutritional requirements and environmental conditions in submerged cultures are quite limited to a few kinds of mushrooms [13–15]. Phellinus linteus, a basidiomycete belonging to the family Polyporaceae, has been well known as a medicinally potent mushroom due to its high anti-tumor activity. The anti-tumor activity of the polysaccharides from the fruit body of this mushroom was first reported in 1968 [16], thereafter a wide variety of further reports have been documented by many investigators [17–22]. Many kinds of Phellinus mushrooms (e.g. P. igniarius, P. hartigii, P. gilvus, P. pini, etc.) are known and they have different medicinal effects such as anti-tumor and immuno-stimulating activities [23–27]. In the present study, submerged culture conditions of P. linteus were optimized for the production of both mycelial biomass and EPS, both of which are known to have many pharmacological activities as mentioned above. Furthermore, the pure polysaccharides were isolated by a gel filtration chromatography and their molecular features were characterized by a multi-angle laser-light scattering (MALLS) system.
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2. Materials and methods
2.5. Estimation of mycelial and EPS concentrations
2.1. Microorganism
Samples collected at various intervals from shake flask were centrifuged at 10,000 × g for 20 min, and the resulting supernatant was filtered through a Whatman filter paper No. 2 (Whatman International Ltd., Maidstone, England). The resulting culture filtrate was mixed with four times its volume of absolute ethanol, stirred vigorously and left overnight at 4 ◦ C. The precipitated EPS was centrifuged at 10,000 × g for 20 min discarding the supernatant. The precipitate of crude EPS was lyophilized and the weight was estimated. Dry weight of mycelium was measured after repeated washing of the mycelial pellet with distilled water and drying at 90 ◦ C for overnight to a constant weight (Fig. 1). The filtrate from a membrane filtration was analyzed by HPLC (Shimadzu Co., Osaka, Japan) using an Aminex HPX-42C column equipped with a refractive index detector for quantitative analysis of residual sugar concentration.
P. linteus KCTC 6190 was kindly obtained from Rural Development Administration, Korea. The stock culture was maintained on potato dextrose agar (PDA) slant and subcultured every 1 month, and the slants were incubated at 28 ◦ C for 6 days and then stored at 4 ◦ C. 2.2. Inoculum preparation P. linteus was initially grown on PDA medium (2.4% potato dextrose broth and 2% agar) in a petri dish, and then transferred to the seed culture medium by punching out 5 mm of the agar plate culture with a sterilized self-designed cutter. The seed culture was grown in a 250 ml flask containing 50 ml of PMP medium (2.4% potato dextrose broth, 1% malt extract, and 0.1% peptone) at 28 ◦ C on a rotary shaker incubator at 150 rpm for 4 days. 2.3. Culture conditions The flask culture experiments were performed in a 250 ml flask containing 50 ml of the media inoculated with 5% (v/v) of the seed culture. To find the optimal culture temperature and pH in the optimal medium, the following factors were studied: (1) Temperature: The inoculated flasks were incubated at 20, 25, 28, 30, and 35 ◦ C. (2) Initial pH: The medium was adjusted to different levels of pH with 3N HCl or NaOH. The final pHs before sterilization ranged from 3 to 9. The data presented in the results section represents the mean of three independent experiments. The fermentation medium was inoculated with 5% (v/v) of the seed culture and then cultivated at 30 ◦ C in a 5-l stirred-tank fermenter (KoBioTech Co., Seoul, Korea). Unless otherwise specified, fermentations were performed under the following conditions: temperature, 30 ◦ C; aeration rate, 2 vvm; agitation speed, 50 rpm; initial pH, 4.0; working volume, 3-l. 2.4. Purification of the EPS The ethanol precipitates of the EPS components were dissolved in 0.2 M NaCl buffer to a concentration of 10 g/l, and loaded onto a Sepharose CL-4B column (2.4 cm × 100 cm, Sigma Chemical Co., St. Louis, MO). The column was eluted with the same buffer at a flow rate of 0.6 ml/min. The total sugar content of EPS produced from P. linteus was determined by phenol sulfuric acid method [28] using glucose as the standard. Total protein was determined by the Lowry method [29] with bovine serum albumin as the standard. The protein moiety in the EPS was monitored by absorbance at 280 nm, whilst the carbohydrate moiety was monitored at 480 nm. The active fractions of EPS were pooled and lyophilized until further analysis.
2.6. Analysis of carbohydrates and amino acids The total sugar content of EPS produced from P. linteus was determined by phenol sulfuric acid method [28] using glucose as the standard. Sugar composition was analyzed by gas chromatography (Varian Co., Model: Star 3600CX, Lexington, MA) with a fused silica capillary column (Na form, 300 mm × 0.25 mm, Supelco Inc., Bellefonte, PA) and a flame ionization detector. Total protein was determined by the Lowry method [29] with bovine serum albumin as the standard. The composition of amino acid was analyzed by amino acid analyzer (Amersham Pharmacia Biotech Ltd., Model: Pharmacia Biochrom 20, Cambridge, UK) with a high performance ion exchange column (No. 3906, 200 mm × 4.6 mm). 2.7. SEC/MALLS analysis The molecular weights of the EPS were estimated by size exclusion chromatography (SEC) coupled with MALLS system (Wyatt Technology, Santa Babara, CA). The polysaccharide samples were dissolved in a phosphate/chloride buffer (ionic strength = 0.1, pH 6.8) containing 0.04% diaminotetraacetic acid-disodium salt (Na2 EDTA) and 0.01% sodium azide and filtered through 0.22 m filter membranes (Millex HV type, Millipore Co., Bedford, MA) prior to injection into the SEC/MALLS system [30]. The chromatographic system consisted of a degasser (Degasys, DG-1200, uniflow, HPLC Technology, Macclesfield, UK), a high performance pump (Model 590 Programmable Solvent Delivery Module, Waters, Millipore, Watford, UK), an injection valve (Rheodyne Inc., Cotati, CA) fitted with a 100-l loop, and three SEC columns (Shodex PROTEIN KW-802.5, 803, 804, Showa Denko K.K., Tokyo, Japan) were connected in series. The flow rate was 0.8 ml/min and the injection volume was 100 l. During the calculation of molecular weights of each EPS, the value of dn/dc, so-called
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Fig. 1. Recovery process of polysaccharide produced by submerged culture of P. linteus KCTC 6190.
“specific refractive index increment” was used from the data in literature [30] after modification by reflecting the ratio of carbohydrate (100%) and protein contents (0%) in EPS, in which the estimated dn/dc was 0.14.
not shown). Of all nitrogen sources tested, the best mycelial growth and EPS production were observed in the medium containing corn steep powder (Table 2) and its maximum concentration for both mycelial growth and EPS production was determined to be 3 g/l (data not shown).
3. Results 3.1. Effect of carbon and nitrogen sources To find a suitable carbon source for the EPS production, P. linteus was cultivated in media containing various carbon sources at a concentration of 1% (w/v) for 8 days. Among nine carbon sources examined, fructose, mannitol, sorbitol, and sucrose were favorable to mycelial growth and EPS production (Table 1). Sorbitol gave rise to the highest mycelial growth and EPS production. Although fructose, maltose, and lactose also gave good mycelial growth, they led to low EPS yields. It is noteworthy that this fermentation system can have a significant impact on the industrial application because of low cost of carbon sources. Therefore, the optimal concentration of carbon source for both mycelial growth and EPS production was determined to be sucrose 50 g/l (data
Table 1 Effect of carbon sources on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in flask culturesa ,b Carbon sources
Dry cell weight (g/l)
Controlc Cellobiose Fructose Glucose Lactose Maltose Mannitol Sorbitol Sucrose Xylose
2.20 2.21 3.39 2.00 3.10 3.00 2.54 3.83 2.78 1.22
a
± ± ± ± ± ± ± ± ± ±
0.42 0.13 0.40 0.13 0.48 0.04 0.38 0.35 0.08 0.05
Exopolysaccharides (g/l) 1.36 1.38 1.51 1.27 1.29 1.14 1.61 1.61 1.60 0.59
± ± ± ± ± ± ± ± ± ±
0.06 0.02 0.01 0.35 0.01 0.26 0.07 0.13 0.03 0.17
Final pH 3.96 3.67 3.46 3.66 3.42 3.77 3.63 3.21 3.55 3.81
The flask culture experiments were carried out for 8 days. Values are mean ± S.D. of triple determinations. c Control means PMP medium. b
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Table 2 Effect of nitrogen sources on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in flask culturesa ,b Nitrogen sources
Dry cell weight (g/l)
Controlc Corn steep powder Malt extract Meat peptone Polypeptone Soy peptone Tryptone Yeast extract NH4 Cl NH4 NO3 KNO3 NaNO3
2.73 5.72 3.05 0.50 2.57 1.30 2.09 1.47 0.61 0.82 1.60 2.09
± ± ± ± ± ± ± ± ± ± ± ±
0.15 0.24 0.63 0.04 0.11 0.60 0.53 0.55 0.01 0.02 0.16 0.05
Exopolysaccharides (g/l) 1.85 1.97 1.78 0.39 1.57 1.45 1.57 1.59 0.63 0.88 1.05 0.90
± ± ± ± ± ± ± ± ± ± ± ±
0.06 0.11 0.20 0.16 0.09 0.08 0.14 0.08 0.02 0.02 0.05 0.17
Final pH 4.33 4.58 4.27 4.38 4.32 4.41 4.36 4.41 4.50 4.53 4.35 4.31
a
The flask culture experiments were carried out for 8 days. Values are mean ± S.D. of triple determinations. c Control means the medium containing 5% sucrose without addition of nitrogen source. b
3.2. Effect of bioelement In several fungal fermentations, mineral ions are usually recognized as favorable bioelements for mycelial growth and production of secondary metabolites like polysaccharides [14]. In this work, the most effective bioelement for mycelial growth and EPS production among six different kinds of inorganic salts tested was CaCl2 (Table 3). 3.3. Effect of temperature and initial pH To find optimal temperature for the mycelial growth and EPS production, P. linteus was cultivated in shake flask culture at various temperatures (20–35 ◦ C), where the optimum temperature was found to be 30 ◦ C (Fig. 2A). This temperature optimum is quite similar to the results reported by Kang et al. [17] and Chi et al. [18] from other liquid cultures of different species of P. linteus. It is comparable that many Table 3 Effect of mineral sources on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in flask culturesa ,b Mineral sources (5 mM)
Dry cell weight (g/l)
Controlc CaCl2 FeSO4 KH2 PO4 K2 HPO4 MgSO4 MnSO4
2.75 3.33 1.91 2.88 2.92 2.85 3.16
a
± ± ± ± ± ± ±
0.01 0.05 0.03 0.08 0.20 0.01 0.42
Exopolysaccharides (g/l) 1.36 2.03 0.96 1.44 1.43 1.51 1.49
± ± ± ± ± ± ±
0.02 0.01 0.02 0.06 0.02 0.01 0.11
Final pH 3.59 2.98 3.45 3.37 3.33 3.31 3.34
The flask culture experiments were carried out for 8 days. b Values are mean ± S.D. of triple determinations. c Control means the medium containing 5% sucrose, 0.3% corn steep powder without addition of mineral source.
kinds of mushrooms have relatively low temperature optima ranging from 20 to 25 ◦ C in their submerged cultures [6,14]. In order to investigate the effect of initial pH on mycelial growth and EPS production, P. linteus was cultivated in the optimized medium under different initial pHs (3.0–9.0) in shake flask culture. Fig. 2B shows the effect of initial pH on the mycelial growth and EPS production of P. linteus. The highest mycelial concentration was obtained at pH 5, whereas the maximum EPS production was achieved at pH 4. 3.4. Fermentation results Fig. 3 shows the typical time profiles of substrate consumption, mycelial growth, and EPS production in a 5-l stirred-tank fermenter under optimized culture conditions. The mycelial growth was continuously increased towards the end of fermentation and its final mycelial concentration indicated 11 g/l at day 15, whereas the maximum EPS production (3.3 g/l) was achieved at day 14. The initial pH of the fermentation broth slowly decreased from 4.0 to 3.3. So as to enhance the EPS production, the effects of operating parameters (e.g. effects of agitation intensity and aeration rate) were further studied in a stirred-tank fermenter. However, the maximum concentrations of mycelial growth and EPS were not increased to a notable level (data not shown). 3.5. Separation of EPS by Sepharose CL-4B chromatography The crude EPS from submerged culture of P. linteus was purified by gel filtration in Sepharose CL-4B column, where three polysaccharide peaks were eluted (Fig. 4). It was revealed that all fractions of EPS were polysaccharides without protein moiety. 3.6. Chemical composition of EPS The detailed compositions of carbohydrate in EPS from P. linteus are illustrated in Table 4. The compositional analysis revealed that all three fractions are polysaccharides without protein moiety, consisted of mainly mannose, galactose, and glucose. Unfortunately, it is difficult to directly compare the chemical composition in the present study with those of other reports, since most of previous data in the literature are obtained from extracts of fruit bodies or mycelia not of EPS. 3.7. Purification and characterization of EPS with SEC/MALLS system The elution profiles of EPS observed for the determination of molecular mass in SEC/MALLS system was in good agreement with that of Sepharose CL-4B chromatography (Fig. 5A). Three principle peaks appeared between 16–20
Fig. 2. Effect of temperature (A) and initial pH (B) on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in the flask culture. (䊉) mycelial dry weight, (䊊) EPS. All experimental data were mean ± S.D. of triple determinations.
Fig. 3. Typical time profiles for the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in a 5-l stirred-tank fermenter. (䊉) mycelial dry weight, (䊊) EPS, (䉱) pH, () residual sugar.
Fig. 4. Elution profiles on Sepharose CL-4B gel filtration chromatography for the isolation of EPSs from the culture filtrates of P. linteus KCTC 6190. For detailed chromatographic conditions, see Section 2.
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Table 4 Carbohydrate compositions of EPSs (Fr-I, -II, and -III) produced from submerged culture of P. linteus KCTC 6190a Carbohydrate
Fucose Ribose Arabinose Xylose Mannose Galactose Glucose a b
Composition (%) Fr-I
Fr-II
Fr-III
ndb
4.03 nd 2.54 2.98 15.87 31.15 43.43
nd 8.89 nd nd 10.76 6.67 73.68
14.64 nd nd 41.55 25.19 18.62
For detailed analysis conditions, see Section 2. nd: not detected.
and 25–32 ml elution volume. It also showed that the intensity radii of the MALLS and RI signals on the top of the three peaks were different. The molecular mass values for three eluted fractions were calculated for the portions of peaks, which lying within the peak ranges. These ranges were defined by a common detection limit for the MALLS and RI chromatograms in the peak regions. Fig. 5B shows the logarithmic plots of molecular weight of three EPS as a function of elution volume. The primary retention factor in SEC is the fraction of the pore volume of the
column packing, which is accessible by the molecule. This is determined by the size of the molecule: a small molecule is able to enter the pores and thus elutes more slowly than a larger molecule or a complex of molecules. The molecular weight of EPS separated by the SEC system decreased over the elution volume for larger molecules of Fr-I. On the contrary, the amount of scatter in molecular weight data slightly increased in the later ranges of elution for Fr-II and -III. This phenomenon is presumably due to the combination for small sized molecules, decrease in Rayleigh scatter with particle size, and low quantity of material present, making the system too dilute to be measured accurately [33]. In actuality, the weight average molar mass (Mw ) of Fr-I, -II, and -III were determined to be 433,400 (±7801), 31,470 (±283), and 12,950 (±90) g/mol, respectively (Table 5). For each of these moments of the distribution, the root mean square (RMS) radius was calculated. These data provided a measure of EPS molecular size in terms of the RMS distance from the molecular center of gravity to its edge (i.e. for a spherical molecule this is equivalent to the radius). The significant difference between three RMS radii (Rn , Rw , and Rz ) of Fr-I is presumably resulted from its aggregative conformation. In contrast to Fr-I, the RMS radii for the rest of two fractions ranged from 16 to 34 nm show no clear trends (Table 5). The calculated differential molecular weight distributions for each of the three fractions were shown in Fig. 6A. Each
Fig. 5. (A) Elution profiles of EPSs for the determination of molecular mass in SEC/MALLS system. For detailed analysis conditions, see Section 2. (- - - -) Elution profiles from MALLS detector, (—) elution profiles from refractive index detector. The dotted curves at elution volumes over 31.5 ml represent baseline noise generated by buffer solution. (B) Logarithmic plots of molecular weight of three groups of EPS produced from submerged culture of P. linteus KCTC 6190 as a function of elution volume. The differential refractive index signal, in arbitrary unit, is also shown as solid line.
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Table 5 Relevant molecular parameters of three groups of EPS produced from submerged culture of P. linteus KCTC 6190 in MALLS analysis Parametersa
Fr-I (error %)
Fr-II (error %)
Fr-III (error %)
Mn (g/mol) Mw (g/mol) Mz (g/mol) Mw /Mn Rn (nm) Rw (nm) Rz (nm)
3.649 × (2.1%) 4.334 × 105 (1.8%) 5.079 × 105 (3.0%) 1.19 28.9 (1.0%) 31.1 (0.9%) 33.4 (0.7%)
3.113 × (0.9%) 3.147 × 104 (0.9%) 3.181 × 104 (1.9%) 1.01 16.3 (3.0%) 16.3 (3.0%) 16.4 (2.9%)
1.240 × 104 (0.8%) 1.295 × 104 (0.7%) 1.353 × 104 (1.6%) 1.04 33.9 (2.4%) 32.6 (2.4%) 31.2 (2.5%)
105
104
Mw /Mn means polydispersity ratio. Rn , Rw , and Rz refer number, weight, z-average square mean radius of gyration, respectively. a M , M , and M refer number, weight, z-average molecular weight, respectively. n w z
step of this distribution plot indicates a fraction of a sample, i.e. the position of the vertical part of the step shows the amount of the material in this fraction. The three EPSs showed almost Gaussian distribution and the peak molecular weights for the fractions of I, II, and III were 467,600, 31,830, and 11,520 g/mol, respectively. The Fr-I exhibited a wide range of distribution, whereas Fr-II showed the narrowest distribution with a steep slope, Fr-III being similar. One can conjecture from these results that Fr-I is polydisperse, whilst the rest of fractions are mostly monodisperse. Fig. 6B displays the cumulative molar mass distribution plots of these EPSs. The cumulative molar mass distribu-
tion shows the dependence of the weight fraction versus the molecular mass. The utility of this plot is suggested by the dashed lines in this figure, which indicate that 50% of the sample mass is below (and conversely, above) 397,500, 31,360, and 12,500 g/mol in molecular weight, respectively (corresponding to Fr-I, -II, and -III). The combination of the plots of molecular weight and its cumulative distribution as a function of elution volume provide a complete quantitative characterization of the polysaccharide samples. Therefore, this plot is particularly useful in determining what molecular weights are contained in the high and low molecular tails of the polysaccharide samples.
Fig. 6. (A) Differential molecular weight distributions of three groups of EPS produced from submerged culture of P. linteus KCTC 6190. (B) Cumulative distribution of molar mass of three groups of EPS produced from submerged culture of P. linteus KCTC 6190.
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Fig. 7. The double logarithmic plots of RMS radius vs. molecular mass for three groups of EPS (Fr-I, -II, and -III) from culture filtrates of P. linteus KCTC 6190. (A) Fr-I, (B) Fr-II, and (C) Fr-III.
The overall slopes for each EPS in the double logarithmic plots of RMS radius versus molecular mass were shown in Fig. 7. The slope of 0.33 obtained from the plot for the Fr-I indicates that this molecule exists nearly as a spherical form in an aqueous solution (Fig. 7A), whereas the Fr-II was almost random coils with the slope of 0.67 (Fig. 7B). However, no definitive conclusions for the molecular dimension of Fr-III was made because no clear trend was obvious as shown in Fig. 7C. For all fractions of EPS, the values of RMS radii at the same molecular weight were different. This result indicates that these samples have different branched structures, and thus in EPS there could be a number of different types of aggregates and complex, which might have very different conformations depending on their Mw . 4. Discussion 4.1. Fermentation for EPS production It was revealed that the nutritional requirement for EPS production in P. linteus was not always consistent with that of mycelial growth. Moreover, feature of EPS produced by
submerged culture of P. linteus differs by strains and their culture conditions. In case of carbon source, taking into account that most sugar alcohols are expensive ingredients, we selected sucrose as a suitable carbon source and its optimum concentration was found to be 50 g/l. Contrary to our findings, Lee et al. [32] reported that the maximum polysaccharide was produced for the media containing mannose or arabinose, whereas polysaccharide production was not detected in sucrose and sorbitol medium. According to the results from other investigators, a combined medium of glucose and sucrose was proved to give higher yield of mycelial growth of P. linteus (by 30% increase) than those containing only glucose or sucrose [17,18]. Lee et al. [32] pointed out that different carbon source altered slightly the carbohydrate compositions in polysaccharides. Thus, each mushroom requires different carbon source for maximum mycelial growth or EPS production. In comparison with the results reported by other investigators, the requirement of nitrogen source in liquid culture of P. linteus was also different for the strain, i.e. the mycelial growth and EPS production were frequently favorable in peptone medium in most of P. linteus fermentation [32]. Chi et al. [18] reported an interesting finding that
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several amino acids were more favorable nitrogen sources than complex nitrogen sources for mycelial growth of P. linteus. However, in the present study, corn steep powder yielded the highest mycelial growth and EPS production, whilst three types of peptones examined were not efficient nitrogen sources. The supplementation of several kinds of bioelements has been proved to be important in fungal fermentations. Jonathan and Fasidi [36] suggested that several mineral ions (Mg, K, and Ca) promoted mycelial growth of Lentinus subnudus (Berk) and Schizophyllum commune. Kang et al. [17] have reported that KH2 PO4 and CaCl2 were the most effective mineral sources for mycelial growth of Phellinus sp. In the present study, supplementation with calcium ions led to increase in both mycelial growth and EPS production and other mineral ions examined had negligible effects except ferrous ion (Table 3). The optimum environmental conditions for mycelial growth and EPS production of Phellinus mushrooms in liquid cultures are dependent on strains. Lee et al. [32] reported that the mycelial growth of P. linteus L13202 was optimum at pH 7.0, whilst EPS production was maximal at pH 5.0. It has been reported that a wide variety of mushrooms have also acidic pH optima for mycelial growth and EPS production [13,14] and particularly several P. linteus have pH optima at pH 5–7 [37,38]. Lee et al. [32] found an important result that the culture pH affected the monosaccharide composition in polysaccharide, i.e. the glucose amount was decreased from 90 to 83% by changing culture pH from 5.0 to 9.0. To the best of our knowledge, the EPS production obtained in this work is the highest yield amongst liquid cultures of P. linteus reported in the literature. Several investigators have made efforts to improve the mycelial biomass and EPS in P. linteus by either fed-batch culture in a stirred-tank fermenter or airlift fermenter. Kim et al. [38] increased the mycelial concentration by 28% by intermittent feeding of glucose. Choi and Lee [37] insisted that the use of an airlift fermenter was more efficient than stirred-tank fermenter, achieving higher mycelial yield. 4.2. Molecular characterization of EPS Although there are many reports on polysaccharides extracted from mycelia of P. linteus, any useful data about EPS obtained from culture filtrates of P. linteus are scarcely available. One of the most important differences between EPS and mycelia-extracted polysaccharides from P. linteus is the molecular size and chemical composition. Lee et al. [39] reported that four groups of polysaccharides from mycelia had molecular weights ranging from 9400 to 15,000 Da. More recently, Kim et al. [40] isolated an acidic proteoglycan from fruit body of P. linteus with molecular mass of 150,000 Da. In contrast, one of the EPS in the present investigation (Fr-I) had markedly high molecular weight (433,400 g/mol). Lee et al. [31] reported that all fractions of EPS produced
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from submerged culture of P. linteus IY001 contained a large amount of aspartic acid, glycine, glutamic acid, and alanine. Lee et al. [32] reported that the EPS produced by submerged culture of P. linteus L13202 was composed of mannose, galactose, and glucose, these composition ranges were 3–12, 2–10, and 80–95% depending on carbon sources, respectively. Collectively, the molecular weight and chemical composition of polysaccharides produced from fruit body of P. linteus or its submerged culture product is strongly dependent on strain, extraction method, and culture conditions [41]. Therefore, a comparative study of medicinal activities between the EPS and mycelia-extracted polysaccharides deserves further investigation. The advantages of SEC are known over other techniques of molecular weight determination, especially in terms of simplicity of operation and the ability to determine molecular weight averages. By combining SEC and MALLS detection, weight, number and z-average values for both mass and size may be obtained for most samples. Moreover, chromatographic analysis permits access to the molar mass distribution and polydispersity of the samples [34]. In the course of MALLS analysis for microbial polysaccharides, the linear relations between logarithmic RMS radius and molecular mass can be considered as estimations of the molecular conformations of each EPS [34]. However, it should be noted that some of the RMS radius data could be close to the lower limit of resolution for MALLS instrument so that the RMS radius data should be used carefully. This is presumably resulted from the fact that a number of different behaviors present at different parts of the molecular weight distribution spectrum. The RMS radius plot for the Fr-III in this work may be a good example for this presumption (Fig. 7C). The molecular conformations of each EPS could be identified from the double logarithmic plot of RMS radius versus molecular mass according to the following equation: log ri = k + a log Mi ; where, ri is a square mean radius of EPS molecule, k is the intercept on the Y axis, and a is a slope for determining the molecular conformation of the EPS. For polymers of sphere-like structure, random coils, or rigid rods, their corresponding values of slope are known as 1/3, 1/2, and 1, respectively, according to the following dimensional relationships: for spheres, ri3 ∝ Mi ; for random coils, ri2 ∝ Mi ; for rigid rods, ri ∝ Mi [34,35]. These polydispersities (Mw /Mn ) of EPSs evaluated from the ratio of the weight average molar mass to the number average molar mass are summarized in Table 5. Eventually, the polydispersity ratio of Fr-I was higher than those of Fr-II and -III. The low values of polydispersity ratio (Mw /Mn ) for EPSs mean that these EPS molecules exist in much less dispersed in aqueous solution without forming large aggregates. Rew et al. [25] reported that the molecular size, degree of branching and building components were different depending on the growth medium. In addition, the structure and molecular mass of polysaccharides have been found to play
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an important role in their biological activities [24,42]. In this regard, it is important to mention here that the characterization data for polysaccharides from submerged culture of mushrooms should be obtained for each culture condition.
Acknowledgment This work was supported by the Research Grant, Daegu University 2002.
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Production and characterization of exopolysaccharides from submerged culture of Phellinus linteus KCTC 6190 Hye-Jin Hwang a , Sang-Woo Kim a , Jang-Won Choi b , Jong-Won Yun a,∗ b
a Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, South Korea Department of Natural Resources, Daegu University, Kyungsan, Kyungbuk 712-714, South Korea
Received 10 February 2003; accepted 10 May 2003
Abstract The optimal temperature and initial pH for both mycelial growth and exopolysaccharide (EPS) production in shake flask cultures of Phellinus linteus KCTC 6190 were found to be 30 ◦ C and pH 4.0, respectively. Optimal medium composition was determined to be sucrose 50 g/l, corn steep powder 3 g/l, KH2 PO4 0.68 g/l, and CaCl2 0.55 g/l. Under optimal culture conditions, the maximum mycelial biomass and EPS achieved in a 5-l stirred-tank fermenter indicated 11 and 3.3 g/l, respectively. To the best of our knowledge, this is the highest polysaccharide yield amongst liquid cultures of P. linteus reported in the literature. The three groups of EPSs (designated as Fr-I, -II, and -III) were obtained from the culture filtrates by a gel filtration chromatography on Sepharose CL-4B and the different fractions were analyzed directly using the size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis. It was revealed that all fractions of EPS were polysaccharides consisted of mainly mannose, galactose, and glucose. The molecular weights of Fr-I, -II, and -III of EPS were determined to be 433,400 (±7801), 31,470 (±283), and 12,950 (±90) g/mol, respectively. The SEC/MALLS analysis revealed that molecular dimension of the Fr-I was a spherical form, whereas the Fr-II was a random coil in an aqueous solution. © 2003 Elsevier Inc. All rights reserved. Keywords: Exopolysaccharide; MALLS; Mushroom; Phellinus linteus; Submerged culture
1. Introduction During the past decades, much interest has been generated in the polysaccharides produced by numerous microorganisms specially mushrooms because of their various biological and pharmacological activities including anti-tumor, immuno-stimulating, and hypoglycemic activities, etc. [1–5]. In order to obtain polysaccharides from mushrooms, most investigators have exerted their efforts to cultivate edible and medicinal mushrooms on solid artificial media (for fruit body production) rather than submerged culture (for mycelial extract and/or exopolysaccharide (EPS) production) [6–8]. Submerged culture obviously gives rise to potential advantages of higher mycelial production in a compact space and shorter time without significant problem of contamination [9–12]. Although enormous efforts have been made to obtain optimal submerged culture conditions for bioactive polysaccharide production from several mushrooms, the currently ∗
Corresponding author. Tel.: +82-53-850-6556; fax: +82-53-850-6559. E-mail address: [email protected] (J.-W. Yun).
0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0141-0229(03)00131-5
available reports for nutritional requirements and environmental conditions in submerged cultures are quite limited to a few kinds of mushrooms [13–15]. Phellinus linteus, a basidiomycete belonging to the family Polyporaceae, has been well known as a medicinally potent mushroom due to its high anti-tumor activity. The anti-tumor activity of the polysaccharides from the fruit body of this mushroom was first reported in 1968 [16], thereafter a wide variety of further reports have been documented by many investigators [17–22]. Many kinds of Phellinus mushrooms (e.g. P. igniarius, P. hartigii, P. gilvus, P. pini, etc.) are known and they have different medicinal effects such as anti-tumor and immuno-stimulating activities [23–27]. In the present study, submerged culture conditions of P. linteus were optimized for the production of both mycelial biomass and EPS, both of which are known to have many pharmacological activities as mentioned above. Furthermore, the pure polysaccharides were isolated by a gel filtration chromatography and their molecular features were characterized by a multi-angle laser-light scattering (MALLS) system.
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2. Materials and methods
2.5. Estimation of mycelial and EPS concentrations
2.1. Microorganism
Samples collected at various intervals from shake flask were centrifuged at 10,000 × g for 20 min, and the resulting supernatant was filtered through a Whatman filter paper No. 2 (Whatman International Ltd., Maidstone, England). The resulting culture filtrate was mixed with four times its volume of absolute ethanol, stirred vigorously and left overnight at 4 ◦ C. The precipitated EPS was centrifuged at 10,000 × g for 20 min discarding the supernatant. The precipitate of crude EPS was lyophilized and the weight was estimated. Dry weight of mycelium was measured after repeated washing of the mycelial pellet with distilled water and drying at 90 ◦ C for overnight to a constant weight (Fig. 1). The filtrate from a membrane filtration was analyzed by HPLC (Shimadzu Co., Osaka, Japan) using an Aminex HPX-42C column equipped with a refractive index detector for quantitative analysis of residual sugar concentration.
P. linteus KCTC 6190 was kindly obtained from Rural Development Administration, Korea. The stock culture was maintained on potato dextrose agar (PDA) slant and subcultured every 1 month, and the slants were incubated at 28 ◦ C for 6 days and then stored at 4 ◦ C. 2.2. Inoculum preparation P. linteus was initially grown on PDA medium (2.4% potato dextrose broth and 2% agar) in a petri dish, and then transferred to the seed culture medium by punching out 5 mm of the agar plate culture with a sterilized self-designed cutter. The seed culture was grown in a 250 ml flask containing 50 ml of PMP medium (2.4% potato dextrose broth, 1% malt extract, and 0.1% peptone) at 28 ◦ C on a rotary shaker incubator at 150 rpm for 4 days. 2.3. Culture conditions The flask culture experiments were performed in a 250 ml flask containing 50 ml of the media inoculated with 5% (v/v) of the seed culture. To find the optimal culture temperature and pH in the optimal medium, the following factors were studied: (1) Temperature: The inoculated flasks were incubated at 20, 25, 28, 30, and 35 ◦ C. (2) Initial pH: The medium was adjusted to different levels of pH with 3N HCl or NaOH. The final pHs before sterilization ranged from 3 to 9. The data presented in the results section represents the mean of three independent experiments. The fermentation medium was inoculated with 5% (v/v) of the seed culture and then cultivated at 30 ◦ C in a 5-l stirred-tank fermenter (KoBioTech Co., Seoul, Korea). Unless otherwise specified, fermentations were performed under the following conditions: temperature, 30 ◦ C; aeration rate, 2 vvm; agitation speed, 50 rpm; initial pH, 4.0; working volume, 3-l. 2.4. Purification of the EPS The ethanol precipitates of the EPS components were dissolved in 0.2 M NaCl buffer to a concentration of 10 g/l, and loaded onto a Sepharose CL-4B column (2.4 cm × 100 cm, Sigma Chemical Co., St. Louis, MO). The column was eluted with the same buffer at a flow rate of 0.6 ml/min. The total sugar content of EPS produced from P. linteus was determined by phenol sulfuric acid method [28] using glucose as the standard. Total protein was determined by the Lowry method [29] with bovine serum albumin as the standard. The protein moiety in the EPS was monitored by absorbance at 280 nm, whilst the carbohydrate moiety was monitored at 480 nm. The active fractions of EPS were pooled and lyophilized until further analysis.
2.6. Analysis of carbohydrates and amino acids The total sugar content of EPS produced from P. linteus was determined by phenol sulfuric acid method [28] using glucose as the standard. Sugar composition was analyzed by gas chromatography (Varian Co., Model: Star 3600CX, Lexington, MA) with a fused silica capillary column (Na form, 300 mm × 0.25 mm, Supelco Inc., Bellefonte, PA) and a flame ionization detector. Total protein was determined by the Lowry method [29] with bovine serum albumin as the standard. The composition of amino acid was analyzed by amino acid analyzer (Amersham Pharmacia Biotech Ltd., Model: Pharmacia Biochrom 20, Cambridge, UK) with a high performance ion exchange column (No. 3906, 200 mm × 4.6 mm). 2.7. SEC/MALLS analysis The molecular weights of the EPS were estimated by size exclusion chromatography (SEC) coupled with MALLS system (Wyatt Technology, Santa Babara, CA). The polysaccharide samples were dissolved in a phosphate/chloride buffer (ionic strength = 0.1, pH 6.8) containing 0.04% diaminotetraacetic acid-disodium salt (Na2 EDTA) and 0.01% sodium azide and filtered through 0.22 m filter membranes (Millex HV type, Millipore Co., Bedford, MA) prior to injection into the SEC/MALLS system [30]. The chromatographic system consisted of a degasser (Degasys, DG-1200, uniflow, HPLC Technology, Macclesfield, UK), a high performance pump (Model 590 Programmable Solvent Delivery Module, Waters, Millipore, Watford, UK), an injection valve (Rheodyne Inc., Cotati, CA) fitted with a 100-l loop, and three SEC columns (Shodex PROTEIN KW-802.5, 803, 804, Showa Denko K.K., Tokyo, Japan) were connected in series. The flow rate was 0.8 ml/min and the injection volume was 100 l. During the calculation of molecular weights of each EPS, the value of dn/dc, so-called
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Fig. 1. Recovery process of polysaccharide produced by submerged culture of P. linteus KCTC 6190.
“specific refractive index increment” was used from the data in literature [30] after modification by reflecting the ratio of carbohydrate (100%) and protein contents (0%) in EPS, in which the estimated dn/dc was 0.14.
not shown). Of all nitrogen sources tested, the best mycelial growth and EPS production were observed in the medium containing corn steep powder (Table 2) and its maximum concentration for both mycelial growth and EPS production was determined to be 3 g/l (data not shown).
3. Results 3.1. Effect of carbon and nitrogen sources To find a suitable carbon source for the EPS production, P. linteus was cultivated in media containing various carbon sources at a concentration of 1% (w/v) for 8 days. Among nine carbon sources examined, fructose, mannitol, sorbitol, and sucrose were favorable to mycelial growth and EPS production (Table 1). Sorbitol gave rise to the highest mycelial growth and EPS production. Although fructose, maltose, and lactose also gave good mycelial growth, they led to low EPS yields. It is noteworthy that this fermentation system can have a significant impact on the industrial application because of low cost of carbon sources. Therefore, the optimal concentration of carbon source for both mycelial growth and EPS production was determined to be sucrose 50 g/l (data
Table 1 Effect of carbon sources on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in flask culturesa ,b Carbon sources
Dry cell weight (g/l)
Controlc Cellobiose Fructose Glucose Lactose Maltose Mannitol Sorbitol Sucrose Xylose
2.20 2.21 3.39 2.00 3.10 3.00 2.54 3.83 2.78 1.22
a
± ± ± ± ± ± ± ± ± ±
0.42 0.13 0.40 0.13 0.48 0.04 0.38 0.35 0.08 0.05
Exopolysaccharides (g/l) 1.36 1.38 1.51 1.27 1.29 1.14 1.61 1.61 1.60 0.59
± ± ± ± ± ± ± ± ± ±
0.06 0.02 0.01 0.35 0.01 0.26 0.07 0.13 0.03 0.17
Final pH 3.96 3.67 3.46 3.66 3.42 3.77 3.63 3.21 3.55 3.81
The flask culture experiments were carried out for 8 days. Values are mean ± S.D. of triple determinations. c Control means PMP medium. b
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Table 2 Effect of nitrogen sources on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in flask culturesa ,b Nitrogen sources
Dry cell weight (g/l)
Controlc Corn steep powder Malt extract Meat peptone Polypeptone Soy peptone Tryptone Yeast extract NH4 Cl NH4 NO3 KNO3 NaNO3
2.73 5.72 3.05 0.50 2.57 1.30 2.09 1.47 0.61 0.82 1.60 2.09
± ± ± ± ± ± ± ± ± ± ± ±
0.15 0.24 0.63 0.04 0.11 0.60 0.53 0.55 0.01 0.02 0.16 0.05
Exopolysaccharides (g/l) 1.85 1.97 1.78 0.39 1.57 1.45 1.57 1.59 0.63 0.88 1.05 0.90
± ± ± ± ± ± ± ± ± ± ± ±
0.06 0.11 0.20 0.16 0.09 0.08 0.14 0.08 0.02 0.02 0.05 0.17
Final pH 4.33 4.58 4.27 4.38 4.32 4.41 4.36 4.41 4.50 4.53 4.35 4.31
a
The flask culture experiments were carried out for 8 days. Values are mean ± S.D. of triple determinations. c Control means the medium containing 5% sucrose without addition of nitrogen source. b
3.2. Effect of bioelement In several fungal fermentations, mineral ions are usually recognized as favorable bioelements for mycelial growth and production of secondary metabolites like polysaccharides [14]. In this work, the most effective bioelement for mycelial growth and EPS production among six different kinds of inorganic salts tested was CaCl2 (Table 3). 3.3. Effect of temperature and initial pH To find optimal temperature for the mycelial growth and EPS production, P. linteus was cultivated in shake flask culture at various temperatures (20–35 ◦ C), where the optimum temperature was found to be 30 ◦ C (Fig. 2A). This temperature optimum is quite similar to the results reported by Kang et al. [17] and Chi et al. [18] from other liquid cultures of different species of P. linteus. It is comparable that many Table 3 Effect of mineral sources on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in flask culturesa ,b Mineral sources (5 mM)
Dry cell weight (g/l)
Controlc CaCl2 FeSO4 KH2 PO4 K2 HPO4 MgSO4 MnSO4
2.75 3.33 1.91 2.88 2.92 2.85 3.16
a
± ± ± ± ± ± ±
0.01 0.05 0.03 0.08 0.20 0.01 0.42
Exopolysaccharides (g/l) 1.36 2.03 0.96 1.44 1.43 1.51 1.49
± ± ± ± ± ± ±
0.02 0.01 0.02 0.06 0.02 0.01 0.11
Final pH 3.59 2.98 3.45 3.37 3.33 3.31 3.34
The flask culture experiments were carried out for 8 days. b Values are mean ± S.D. of triple determinations. c Control means the medium containing 5% sucrose, 0.3% corn steep powder without addition of mineral source.
kinds of mushrooms have relatively low temperature optima ranging from 20 to 25 ◦ C in their submerged cultures [6,14]. In order to investigate the effect of initial pH on mycelial growth and EPS production, P. linteus was cultivated in the optimized medium under different initial pHs (3.0–9.0) in shake flask culture. Fig. 2B shows the effect of initial pH on the mycelial growth and EPS production of P. linteus. The highest mycelial concentration was obtained at pH 5, whereas the maximum EPS production was achieved at pH 4. 3.4. Fermentation results Fig. 3 shows the typical time profiles of substrate consumption, mycelial growth, and EPS production in a 5-l stirred-tank fermenter under optimized culture conditions. The mycelial growth was continuously increased towards the end of fermentation and its final mycelial concentration indicated 11 g/l at day 15, whereas the maximum EPS production (3.3 g/l) was achieved at day 14. The initial pH of the fermentation broth slowly decreased from 4.0 to 3.3. So as to enhance the EPS production, the effects of operating parameters (e.g. effects of agitation intensity and aeration rate) were further studied in a stirred-tank fermenter. However, the maximum concentrations of mycelial growth and EPS were not increased to a notable level (data not shown). 3.5. Separation of EPS by Sepharose CL-4B chromatography The crude EPS from submerged culture of P. linteus was purified by gel filtration in Sepharose CL-4B column, where three polysaccharide peaks were eluted (Fig. 4). It was revealed that all fractions of EPS were polysaccharides without protein moiety. 3.6. Chemical composition of EPS The detailed compositions of carbohydrate in EPS from P. linteus are illustrated in Table 4. The compositional analysis revealed that all three fractions are polysaccharides without protein moiety, consisted of mainly mannose, galactose, and glucose. Unfortunately, it is difficult to directly compare the chemical composition in the present study with those of other reports, since most of previous data in the literature are obtained from extracts of fruit bodies or mycelia not of EPS. 3.7. Purification and characterization of EPS with SEC/MALLS system The elution profiles of EPS observed for the determination of molecular mass in SEC/MALLS system was in good agreement with that of Sepharose CL-4B chromatography (Fig. 5A). Three principle peaks appeared between 16–20
Fig. 2. Effect of temperature (A) and initial pH (B) on the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in the flask culture. (䊉) mycelial dry weight, (䊊) EPS. All experimental data were mean ± S.D. of triple determinations.
Fig. 3. Typical time profiles for the mycelial growth and EPS production by submerged culture of P. linteus KCTC 6190 in a 5-l stirred-tank fermenter. (䊉) mycelial dry weight, (䊊) EPS, (䉱) pH, () residual sugar.
Fig. 4. Elution profiles on Sepharose CL-4B gel filtration chromatography for the isolation of EPSs from the culture filtrates of P. linteus KCTC 6190. For detailed chromatographic conditions, see Section 2.
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Table 4 Carbohydrate compositions of EPSs (Fr-I, -II, and -III) produced from submerged culture of P. linteus KCTC 6190a Carbohydrate
Fucose Ribose Arabinose Xylose Mannose Galactose Glucose a b
Composition (%) Fr-I
Fr-II
Fr-III
ndb
4.03 nd 2.54 2.98 15.87 31.15 43.43
nd 8.89 nd nd 10.76 6.67 73.68
14.64 nd nd 41.55 25.19 18.62
For detailed analysis conditions, see Section 2. nd: not detected.
and 25–32 ml elution volume. It also showed that the intensity radii of the MALLS and RI signals on the top of the three peaks were different. The molecular mass values for three eluted fractions were calculated for the portions of peaks, which lying within the peak ranges. These ranges were defined by a common detection limit for the MALLS and RI chromatograms in the peak regions. Fig. 5B shows the logarithmic plots of molecular weight of three EPS as a function of elution volume. The primary retention factor in SEC is the fraction of the pore volume of the
column packing, which is accessible by the molecule. This is determined by the size of the molecule: a small molecule is able to enter the pores and thus elutes more slowly than a larger molecule or a complex of molecules. The molecular weight of EPS separated by the SEC system decreased over the elution volume for larger molecules of Fr-I. On the contrary, the amount of scatter in molecular weight data slightly increased in the later ranges of elution for Fr-II and -III. This phenomenon is presumably due to the combination for small sized molecules, decrease in Rayleigh scatter with particle size, and low quantity of material present, making the system too dilute to be measured accurately [33]. In actuality, the weight average molar mass (Mw ) of Fr-I, -II, and -III were determined to be 433,400 (±7801), 31,470 (±283), and 12,950 (±90) g/mol, respectively (Table 5). For each of these moments of the distribution, the root mean square (RMS) radius was calculated. These data provided a measure of EPS molecular size in terms of the RMS distance from the molecular center of gravity to its edge (i.e. for a spherical molecule this is equivalent to the radius). The significant difference between three RMS radii (Rn , Rw , and Rz ) of Fr-I is presumably resulted from its aggregative conformation. In contrast to Fr-I, the RMS radii for the rest of two fractions ranged from 16 to 34 nm show no clear trends (Table 5). The calculated differential molecular weight distributions for each of the three fractions were shown in Fig. 6A. Each
Fig. 5. (A) Elution profiles of EPSs for the determination of molecular mass in SEC/MALLS system. For detailed analysis conditions, see Section 2. (- - - -) Elution profiles from MALLS detector, (—) elution profiles from refractive index detector. The dotted curves at elution volumes over 31.5 ml represent baseline noise generated by buffer solution. (B) Logarithmic plots of molecular weight of three groups of EPS produced from submerged culture of P. linteus KCTC 6190 as a function of elution volume. The differential refractive index signal, in arbitrary unit, is also shown as solid line.
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Table 5 Relevant molecular parameters of three groups of EPS produced from submerged culture of P. linteus KCTC 6190 in MALLS analysis Parametersa
Fr-I (error %)
Fr-II (error %)
Fr-III (error %)
Mn (g/mol) Mw (g/mol) Mz (g/mol) Mw /Mn Rn (nm) Rw (nm) Rz (nm)
3.649 × (2.1%) 4.334 × 105 (1.8%) 5.079 × 105 (3.0%) 1.19 28.9 (1.0%) 31.1 (0.9%) 33.4 (0.7%)
3.113 × (0.9%) 3.147 × 104 (0.9%) 3.181 × 104 (1.9%) 1.01 16.3 (3.0%) 16.3 (3.0%) 16.4 (2.9%)
1.240 × 104 (0.8%) 1.295 × 104 (0.7%) 1.353 × 104 (1.6%) 1.04 33.9 (2.4%) 32.6 (2.4%) 31.2 (2.5%)
105
104
Mw /Mn means polydispersity ratio. Rn , Rw , and Rz refer number, weight, z-average square mean radius of gyration, respectively. a M , M , and M refer number, weight, z-average molecular weight, respectively. n w z
step of this distribution plot indicates a fraction of a sample, i.e. the position of the vertical part of the step shows the amount of the material in this fraction. The three EPSs showed almost Gaussian distribution and the peak molecular weights for the fractions of I, II, and III were 467,600, 31,830, and 11,520 g/mol, respectively. The Fr-I exhibited a wide range of distribution, whereas Fr-II showed the narrowest distribution with a steep slope, Fr-III being similar. One can conjecture from these results that Fr-I is polydisperse, whilst the rest of fractions are mostly monodisperse. Fig. 6B displays the cumulative molar mass distribution plots of these EPSs. The cumulative molar mass distribu-
tion shows the dependence of the weight fraction versus the molecular mass. The utility of this plot is suggested by the dashed lines in this figure, which indicate that 50% of the sample mass is below (and conversely, above) 397,500, 31,360, and 12,500 g/mol in molecular weight, respectively (corresponding to Fr-I, -II, and -III). The combination of the plots of molecular weight and its cumulative distribution as a function of elution volume provide a complete quantitative characterization of the polysaccharide samples. Therefore, this plot is particularly useful in determining what molecular weights are contained in the high and low molecular tails of the polysaccharide samples.
Fig. 6. (A) Differential molecular weight distributions of three groups of EPS produced from submerged culture of P. linteus KCTC 6190. (B) Cumulative distribution of molar mass of three groups of EPS produced from submerged culture of P. linteus KCTC 6190.
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Fig. 7. The double logarithmic plots of RMS radius vs. molecular mass for three groups of EPS (Fr-I, -II, and -III) from culture filtrates of P. linteus KCTC 6190. (A) Fr-I, (B) Fr-II, and (C) Fr-III.
The overall slopes for each EPS in the double logarithmic plots of RMS radius versus molecular mass were shown in Fig. 7. The slope of 0.33 obtained from the plot for the Fr-I indicates that this molecule exists nearly as a spherical form in an aqueous solution (Fig. 7A), whereas the Fr-II was almost random coils with the slope of 0.67 (Fig. 7B). However, no definitive conclusions for the molecular dimension of Fr-III was made because no clear trend was obvious as shown in Fig. 7C. For all fractions of EPS, the values of RMS radii at the same molecular weight were different. This result indicates that these samples have different branched structures, and thus in EPS there could be a number of different types of aggregates and complex, which might have very different conformations depending on their Mw . 4. Discussion 4.1. Fermentation for EPS production It was revealed that the nutritional requirement for EPS production in P. linteus was not always consistent with that of mycelial growth. Moreover, feature of EPS produced by
submerged culture of P. linteus differs by strains and their culture conditions. In case of carbon source, taking into account that most sugar alcohols are expensive ingredients, we selected sucrose as a suitable carbon source and its optimum concentration was found to be 50 g/l. Contrary to our findings, Lee et al. [32] reported that the maximum polysaccharide was produced for the media containing mannose or arabinose, whereas polysaccharide production was not detected in sucrose and sorbitol medium. According to the results from other investigators, a combined medium of glucose and sucrose was proved to give higher yield of mycelial growth of P. linteus (by 30% increase) than those containing only glucose or sucrose [17,18]. Lee et al. [32] pointed out that different carbon source altered slightly the carbohydrate compositions in polysaccharides. Thus, each mushroom requires different carbon source for maximum mycelial growth or EPS production. In comparison with the results reported by other investigators, the requirement of nitrogen source in liquid culture of P. linteus was also different for the strain, i.e. the mycelial growth and EPS production were frequently favorable in peptone medium in most of P. linteus fermentation [32]. Chi et al. [18] reported an interesting finding that
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several amino acids were more favorable nitrogen sources than complex nitrogen sources for mycelial growth of P. linteus. However, in the present study, corn steep powder yielded the highest mycelial growth and EPS production, whilst three types of peptones examined were not efficient nitrogen sources. The supplementation of several kinds of bioelements has been proved to be important in fungal fermentations. Jonathan and Fasidi [36] suggested that several mineral ions (Mg, K, and Ca) promoted mycelial growth of Lentinus subnudus (Berk) and Schizophyllum commune. Kang et al. [17] have reported that KH2 PO4 and CaCl2 were the most effective mineral sources for mycelial growth of Phellinus sp. In the present study, supplementation with calcium ions led to increase in both mycelial growth and EPS production and other mineral ions examined had negligible effects except ferrous ion (Table 3). The optimum environmental conditions for mycelial growth and EPS production of Phellinus mushrooms in liquid cultures are dependent on strains. Lee et al. [32] reported that the mycelial growth of P. linteus L13202 was optimum at pH 7.0, whilst EPS production was maximal at pH 5.0. It has been reported that a wide variety of mushrooms have also acidic pH optima for mycelial growth and EPS production [13,14] and particularly several P. linteus have pH optima at pH 5–7 [37,38]. Lee et al. [32] found an important result that the culture pH affected the monosaccharide composition in polysaccharide, i.e. the glucose amount was decreased from 90 to 83% by changing culture pH from 5.0 to 9.0. To the best of our knowledge, the EPS production obtained in this work is the highest yield amongst liquid cultures of P. linteus reported in the literature. Several investigators have made efforts to improve the mycelial biomass and EPS in P. linteus by either fed-batch culture in a stirred-tank fermenter or airlift fermenter. Kim et al. [38] increased the mycelial concentration by 28% by intermittent feeding of glucose. Choi and Lee [37] insisted that the use of an airlift fermenter was more efficient than stirred-tank fermenter, achieving higher mycelial yield. 4.2. Molecular characterization of EPS Although there are many reports on polysaccharides extracted from mycelia of P. linteus, any useful data about EPS obtained from culture filtrates of P. linteus are scarcely available. One of the most important differences between EPS and mycelia-extracted polysaccharides from P. linteus is the molecular size and chemical composition. Lee et al. [39] reported that four groups of polysaccharides from mycelia had molecular weights ranging from 9400 to 15,000 Da. More recently, Kim et al. [40] isolated an acidic proteoglycan from fruit body of P. linteus with molecular mass of 150,000 Da. In contrast, one of the EPS in the present investigation (Fr-I) had markedly high molecular weight (433,400 g/mol). Lee et al. [31] reported that all fractions of EPS produced
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from submerged culture of P. linteus IY001 contained a large amount of aspartic acid, glycine, glutamic acid, and alanine. Lee et al. [32] reported that the EPS produced by submerged culture of P. linteus L13202 was composed of mannose, galactose, and glucose, these composition ranges were 3–12, 2–10, and 80–95% depending on carbon sources, respectively. Collectively, the molecular weight and chemical composition of polysaccharides produced from fruit body of P. linteus or its submerged culture product is strongly dependent on strain, extraction method, and culture conditions [41]. Therefore, a comparative study of medicinal activities between the EPS and mycelia-extracted polysaccharides deserves further investigation. The advantages of SEC are known over other techniques of molecular weight determination, especially in terms of simplicity of operation and the ability to determine molecular weight averages. By combining SEC and MALLS detection, weight, number and z-average values for both mass and size may be obtained for most samples. Moreover, chromatographic analysis permits access to the molar mass distribution and polydispersity of the samples [34]. In the course of MALLS analysis for microbial polysaccharides, the linear relations between logarithmic RMS radius and molecular mass can be considered as estimations of the molecular conformations of each EPS [34]. However, it should be noted that some of the RMS radius data could be close to the lower limit of resolution for MALLS instrument so that the RMS radius data should be used carefully. This is presumably resulted from the fact that a number of different behaviors present at different parts of the molecular weight distribution spectrum. The RMS radius plot for the Fr-III in this work may be a good example for this presumption (Fig. 7C). The molecular conformations of each EPS could be identified from the double logarithmic plot of RMS radius versus molecular mass according to the following equation: log ri = k + a log Mi ; where, ri is a square mean radius of EPS molecule, k is the intercept on the Y axis, and a is a slope for determining the molecular conformation of the EPS. For polymers of sphere-like structure, random coils, or rigid rods, their corresponding values of slope are known as 1/3, 1/2, and 1, respectively, according to the following dimensional relationships: for spheres, ri3 ∝ Mi ; for random coils, ri2 ∝ Mi ; for rigid rods, ri ∝ Mi [34,35]. These polydispersities (Mw /Mn ) of EPSs evaluated from the ratio of the weight average molar mass to the number average molar mass are summarized in Table 5. Eventually, the polydispersity ratio of Fr-I was higher than those of Fr-II and -III. The low values of polydispersity ratio (Mw /Mn ) for EPSs mean that these EPS molecules exist in much less dispersed in aqueous solution without forming large aggregates. Rew et al. [25] reported that the molecular size, degree of branching and building components were different depending on the growth medium. In addition, the structure and molecular mass of polysaccharides have been found to play
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an important role in their biological activities [24,42]. In this regard, it is important to mention here that the characterization data for polysaccharides from submerged culture of mushrooms should be obtained for each culture condition.
Acknowledgment This work was supported by the Research Grant, Daegu University 2002.
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