Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1226-1
ORIGINAL PAPER
Production of bioactive polysaccharides by Inonotus obliquus under submerged fermentation supplemented with lignocellulosic biomass and their antioxidant activity Xiangqun Xu • Yan Hu • Lili Quan
Received: 11 March 2014 / Accepted: 17 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract The effect of lignocellulose degradation in wheat straw, rice straw, and sugarcane bagasse on the accumulation and antioxidant activity of extra- (EPS) and intracellular polysaccharides (IPS) of Inonotus obliquus under submerged fermentation were first evaluated. The wheat straw, rice straw, and sugarcane bagasse increased the EPS accumulation by 91.4, 78.6, and 74.3 % compared with control, respectively. The EPS and IPS extracts from the three lignocellulose media had significantly higher hydroxyl radical- and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the control medium. Of the three materials, wheat straw was the most effective lignocellulose in enhancing the mycelia growth, accumulation and antioxidant activity of I. obliquus polysaccharides (PS). The carbohydrate and protein content, as well as the monosaccharide compositions of the EPS and IPS extracts, were correlated with sugar compositions and dynamic contents during fermentation of individual lignocellulosic materials. The enhanced accumulation of bioactive PS of cultured I. obliquus supplemented with rice straw, wheat straw, and bagasse was evident. Keywords Biodegradation Inonotus obliquus Lignocellulosic biomass Polysaccharides Submerged fermentation
X. Xu (&) Y. Hu L. Quan Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China e-mail: [email protected]; [email protected]
Introduction In recent years, edible and medicinal higher fungi and their secondary metabolites have been widely studied due to their health-promoting properties and relatively low toxicity [1]. Inonotus obliquus (I. obliquus) is one of the most efficient wood-degrading white-rot fungus that belongs to the family Hymenochaetaceae of Basidiomycetes and causes the simultaneous decay of lignin, cellulose, and hemicellulose of birch. This mushroom is well known as one of the most popular medicinal species due to its therapeutic effects. Many biological activities have been attributed to I. obliquus [2]. Polysaccharides (PS), one of the main active components of I. obliquus, were reported to exhibit many biological activities such as antioxidant [3], antitumor [4–6], and immune-stimulating [7, 8] effects. As demonstrated by our group before, the PS extracts from both the wild sclerotia and cultured mycelia including extracellular (EPS) and intracellular (IPS) extracts under submerged fermentation were effective in scavenging hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and in inhibiting lipid peroxidation [9]. Both the PS extracts from the mycelia of I. obliquus with higher polysaccharide contents showed a stronger antioxidant activity than those from the wild sclerotia [9]. Recently, we developed a method to enhance the accumulation of I. obliquus polyphenols under submerged fermentation using the fungus’s ability in lignocellulose degradation [10, 11]. We first demonstrated that I. obliquus in submerged fermentation could effectively decompose the lignocellulose in corn stover, rice straw, wheat straw, and sugarcane bagasse added in a liquid medium [11] as in nature where I. obliquus degraded wood polymers. Correspondingly, the accumulation and
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antioxidant activity of both the extracellular and intracellular polyphenols by I. obliquus were significantly enhanced in the four lignocellulosic biomass-containing culture with lignocellulose degradation [10, 11]. We also found that corn stover as a lignocellulose resource could increase the accumulation and antioxidant activity of the EPS and IPS from the cultures of I. obliquus [12, 13]. However, the mechanism of the enhancement, i.e., whether and how the derivation and bioconversion of cellulose and hemicellulose in the lignocellulosic biomass to the EPS and IPS of I. obliquus under submerged culture, is not clear. In addition, the use of lignocellulosic biomass varies from region to region and depends on their calorific values, nutritive value, lignin content, density, palatability. The right selection of the substrate is of great importance for an efficient and economical production of the compounds of interest. Along this line, in this study, we first evaluated the derivation and bioconversion effectiveness and efficiency of the lignocellulose in sugarcane bagasse, rice straw, and wheat straw into EPS and IPS by I. obliquus. The dynamic changes of monosaccharide compositions in the culture broth and production of EPS and IPS in the different lignocellulosic materials-containing media during fermentation were investigated and correlated to the degradation of cellulose, hemicellulose, and lignin. The chemical contents and monosaccharide compositions of all the EPS and IPS were analyzed. The antioxidant activity of all the EPS and IPS extracts was comparatively studied.
Methods Lignocellulosic materials After washed and dried at 50 °C, rice straw, wheat straw, and sugarcane bagasse were milled using an herbal grinder and passed through a 60-mesh sieve and trapped in a 100-mesh screen. The particles with sizes between 60- and 100-mesh were collected and added in the liquid culture for better lignocellulose degradation [11].
Liquid fermentation I. obliquus (CBS314.39) was purchased from the Centraal Bureau voor Schimmelcultuur, Utrecht, the Netherlands. Malt extract agar with mycelia was cultured in the medium (g L-1: glucose 20, peptone 3, yeast extract 2, KH2PO4 1, MgSO4 1.5, and CaCl2 0.1 for 4–5 days on a rotary shaker at 28 °C with a speed of 150 rpm. The seed culture was added into the control medium or lignocellulose-containing
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medium and incubated at a rate of 8 % (v/v) and at 28 °C in a rotary shaker at 150 rpm. The control medium (g L-1) contained corn flour 53, peptone 3, KH2PO4 1, ZnSO42H2O 0.01, K2HPO4 0.4, FeSO47H2O 0.05, MgSO47H2O 0.5, CuSO45H2O 0.02, CoCl2 0.01, MnSO4H2O 0.08, pH = 6.0 optimized by the response surface methodology (RSM) in our previous work that demonstrated corn flour and peptone were the best carbon source and nitrogen source [12]. The lignocellulose-containing medium (g L-1) contained corn flour 35, lignocellulosic material 30, and all of the other components were the same as the control medium. The corn flour contained in the medium was drastically hydrolyzed by a-amylase and glucoamylase into glucose to avoid the significant interference in the quantitative analysis of the EPS from early fermentation of I. obliquus before use [12].
Extraction of EPS and IPS The culture broth separated from the mycelia was concentrated to one quarter of the original volume with a rotary evaporator under reduced pressure at 60 °C. The concentrate was mixed with four-times volume of chilled 95 % (v/v) ethanol, then stirred vigorously and left at 4 °C overnight. The supernatant was collected for monosaccharide compositions analysis in Sect. 2.4. The ethanol precipitate was collected, then centrifuged (6,5009g for 10 min) and lyophilized. The lyophilized samples were termed as CT-EPS, RS-EPS, WS-EPS, and SB-EPS from the control, rice straw, wheat straw, and sugarcane bagassecontaining medium, respectively. As EPS could be produced by the degraded lignocellulosic biomass and fungi itself, RS-EPS, WS-EPS, and SB-EPS were the mixtures containing EPS produced from fungi itself and EPS from degraded lignocellulosic biomass. The data of carbohydrate composition and antioxidant activity measured below were obtained from the mixtures. The Sevage method was employed to remove protein after neutrase treatment with some modification [14]. The supernatant was concentrated and the aqueous solution was dialyzed against distilled water for 48 h then lyophilized. The deproteinated extracts from the control, rice straw, wheat straw, and sugarcane bagasse-containing media were named as CT-DEPS, RS-DEPS, WS-DEPS, and SB-DEPS, respectively. The mycelial masses suspended in distilled water were first heated for 1.5 h and then heated for 3 h in an autoclave at 121 °C to extract heat-stable mycelial product, followed by filtration [9]. The filtrate was then put through ethanol precipitation, dialysis, and lyophilization. The lyophilized samples were termed as CT-IPS, RS-IPS, WSIPS, and SB-IPS from the control, rice straw, wheat straw, and sugarcane bagasse-containing medium, respectively.
Bioprocess Biosyst Eng
The mycelia were filtered, washed through a 60-mesh sieve, and dried at 45 °C for 48 h for the determination of the biomass dry weight. The EPS production was calculated by the deduction of reducing sugar of total carbohydrate in the culture broth during fermentation. The reducing sugar concentration of the culture broth was measured by the DNS method [15]. The total carbohydrate content of the extracts was determined by phenol–sulfuric acid method [16]. The saccharide compositions of culture broth after precipitation of EPS were measured by gas chromatography after converting them into acetylated derivatives [17] with rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), and glucose (Glu) as the standards. The supernatant collected, in Sect. 2.3, was evaporated to dryness under a steam of nitrogen and then hydrolyzed and acetylated [12, 13]. The alditol acetates were analyzed by a gas chromatograph Varian CP-3800 (Varian Inc., Palo ALto, CA) equipped with a Varian CP-Sil 5 CB capillary chromatography column (25 m 9 0.53 mm, 0.25-lm film thickness) and a flameionization detector. The monosaccharide components were identified by matching the GC retention time with the six standard compounds [13]. Chemical analysis of EPS and IPS extracts The total carbohydrate and protein content of the extracts was determined by phenol–sulfuric acid method [16] and the method of Bradford with bovine serum albumin as a standard [18]. The total phenolic content was determined using Folin–Ciocalteu reagent [19]. The monosaccharide compositions of the extracts were analyzed using the same method in Sect. 2.4. Assay for hydroxyl and DPPH radical-scavenging activity The lyophilized samples obtained after 12 days of cultivation were dissolved in water in a concentration gradient (0.5–5 mg mL-1). Hydroxyl radical-scavenging activity of all the EPS and IPS extracts was determined according to the salicylic method with some modifications [12]. The DPPH free radical-scavenging activity was measured according to the method described by Yang et al. [20]. The IC50 that is the concentration of an inhibitor where the response is reduced by half was calculated by using median-effect analysis.
Statistical analysis The results were expressed as mean ± standard deviation (SD) of three independent experiments performed. Tests of significant differences were determined by Duncan’s multiple range tests at p = 0.05 or independent sample t test (p = 0.05) by one-way analysis of variance (ANOVA) of the data.
Results Time courses of mycelial biomass Figure 1 shows that the mycelial biomass from all the four media gradually increased during day 2–9. From day 7–9, the wheat straw medium was the most suitable medium for the cell growth of I. obliquus compared with the other three media. The addition of sugarcane bagasse and rice straw did not result in a better growth of I. obliquus than the control. The maximal mycelial biomass from the control, wheat straw, rice straw, and sugarcane bagasse-containing media was 10.8, 12.4, 9.5, and 8.0 g L-1 on day 9, 9, 10, and 9, respectively. As illustrated in Fig. 2, the reducing sugars from the hydrolyzed corn flour starch at the beginning stage was gradually consumed and decreased to a low level of 2 mg L-1 in the control and individual lignocellulosecontaining medium on day 5, 4, 5, and 6, respectively. During day 2–5, the medium supplemented with straw and
wheat straw medium rice straw medium sugarcane bagasse medium control medium
16 14
Mycelial biomass (g/L)
Determination of mycelia biomass, EPS and IPS production, and saccharide compositions of culture broth during fermentation
12 10 8 6 4 2 0 2
4
6
8
10
12
Time (day) Fig. 1 Time profiles of mycelial growth in submerged fermentation of I. obliquus in the control (open circle), wheat straw (filed square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
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wheat straw medium rice straw medium sugarcane bagasse medium control medium
16
A 1.8 1.6
EPS production (g/L)
Reducing sugar (mg/mL)
20
12 8 4
wheat straw medium rice straw medium sugarcane bagasse medium control medium
1.4 1.2 1.0 0.8 0.6 0.4 0.2
0 2
4
6
8
10
12
0.0 2
Time (day)
4
6
8
10
12
Time (day) Fig. 2 Sugar consumption during submerged fermentation of I. obliquus in the control (open circle), wheat straw (filed square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
wheat straw medium rice straw medium sugarcane bagasse medium control medium
B 1.4 1.2
IPS production (g/L)
bagasse (during day 2–3) had lower reducing sugar contents because the lignocellulosic material-containing media had a lower starting concentration (35 g L-1) of corn flour than the control medium (50 g L-1). During day 4–7, the bagasse medium had higher reducing sugar contents than the control medium. These reducing sugars might come from the cellulose and hemicellulose degradation. On the other hand, during the time, cell growth in the bagasse medium was so poor that sugars would be consumed slowly (Fig. 1).
1.0 0.8 0.6 0.4 0.2 0.0 2
EPS and IPS production during fermentation Figure 3 shows the time course of EPS production (Fig. 3a) and IPS production (Fig. 3b) of I. obliquus in the control and lignocellulose-containing medium. There were significant differences in the EPS production between the four media. In the control group, EPS production maximized at 0.7 g L-1 after 9 days of fermentation. The maximum EPS production in the wheat straw, rice straw, and bagasse medium reached 1.34, 1.25, and 1.22 g L-1 after 11, 11, and 10 days, respectively (Fig. 3a). As shown in Fig. 3b, compared with the control medium, only wheat straw promoted the IPS accumulation. The IPS production increased to the maximum level of 0.94, 0.99, 0.48, and 0.55 g L-1 in the control, wheat straw, rice straw, and bagasse-containing medium on day 9, 9, 10, and 9, respectively, implying a strong relationship with the cell growth (Fig. 1).
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4
6
8
10
12
Time (day) Fig. 3 Time course of EPS production (a) and IPS production (b) of I. obliquus in the control (open circle), wheat straw (filled square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
Chemical contents and monosaccharide compositions of EPS and IPS Table 1 is a summary of sugar and protein contents of four crude EPS extracts, four crude IPS extracts, and four deproteinated EPS extracts from the control and lignocellulose-containing media obtained after 12 days of cultivation. The sugar content of the crude IPS extracts (CT-IPS, WS-IPS, RS-IPS, and SB-IPS) was significantly higher than that of the counterpart crude EPS extracts (CTEPS, WS-EPS, RS-EPS, and SB-EPS) from the control, wheat straw, rice straw, and bagasse-containing medium.
Bioprocess Biosyst Eng Table 1 Major chemical content of the PS extracts Sugar content (wt%)
Protein content (wt%)
WS-EPS
22.39 ± 1.03b
21.95 ± 1.71b
RS-EPS
20.36 ± 2.62ab
27.51 ± 3.36c
SB-EPS
41.98 ± 2.61c
17.29 ± 3.56ab
CT-EPS
17.69 ± 0.40a
14.87 ± 1.74a
WS-DEPS
47.95 ± 2.10e
36.94 ± 1.96g
RS-DEPS
53.63 ± 0.96fg
31.35 ± 3.36f
SB-DEPS
55.29 ± 2.30g
10.70 ± 0.81e
CT-DEPS
50.66 ± 1.62ef
15.14 ± 2.09e
WS-IPS
51.67 ± 1.85 I
15.14 ± 0.12 I
RS-IPS
48.29 ± 2.43 I
15.27 ± 0.26 I
SB-IPS CT-IPS
68.14 ± 2.38 II 47.43 ± 1.70 I
13.05 ± 0.13 I 25.06 ± 5.94 II
CT-, RS-, WS-, and SB- represent extracts from the control, rice straw, wheat straw, and sugarcane bagasse-containing media. EPS, IPS, and DEPS are extracts from the culture broth, mycelia, and deproteinated EPS * Data with different letters in the same group are significantly different at p \ 0.05
The order of sugar content was SB-EPS (-IPS) [ WS-EPS (-IPS) [ RS-EPS (IPS) [ CT-EPS (IPS). The protein content of the EPS extracts was higher than that of the respective IPS counterparts except for those from the control medium with the order being RS-EPS [ WSEPS [ SB-EPS [ CT-EPS, but CT-IPS [ WS-IPS * RSIPS * SB-IPS. The results indicated that lignocellulose degradation could significantly increase the sugar content of the EPS and IPS and protein content of the EPS but decrease the protein content of the IPS extracts. Of the
Table 2 Monosaccharide compositions of the PS extracts
Sugar component (mol%) Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
WS-EPS
10.5 ± 0.4g
9.1 ± 0.4h
14.5 ± 0.5g
27.6 ± 0.5g
27.0 ± 0.5a
11.3 ± 0.2e
RS-EPS
6.5 ± 0.2e
7.8 ± 0.5g
8.6 ± 0.5f
26.8 ± 0.4f
35.9 ± 0.6d
14.4 ± 0.3 g
SB-EPS
7.2 ± 0.2f
7.3 ± 0.3efg
6.6 ± 0.2e
22.6 ± 0.3e
41.2 ± 0.5f
15.1 ± 0.5 h
CT-EPS
11.4 ± 0.5h
7.4 ± 0.4fg
8.7 ± 0.4f
19.0 ± 0.2d
40.0 ± 0.6e
13.5 ± 0.1f
2.7 ± 0.1c
6.5 ± 0.3cd
4.8 ± 0.3d
26.7 ± 0.6f
48.9 ± 0.6g
10.4 ± 0.3d
WS-DEPS CT-, RS-, WS-, and SBrepresent the extracts from the control, rice straw, wheat straw, and sugarcane bagassecontaining media. EPS, IPS, and DEPS are the extracts from culture broth, mycelia, and deproteinated EPS
three materials, bagasse was the most effective in enhancing sugar content, and rice straw was the most effective in protein content of EPS. The deproteinization treatment of the crude EPS extracts resulted in a significant increase in the sugar content with the order SBDEPS [ RS-DEPS [ CT-DEPS [ WS-DEPS. However, only the protein content of SB-DEPS decreased and of CTDEPS did no change after deproteinization. WS-DEPS and RS-DEPS had higher protein content than WS-EPS and RS-EPS. The results may be explained that the EPS extracts from the wheat straw and rice straw-containing medium contained other compounds such as small molecular sugar and non-saccharide components except exopolysaccharide and protein. The deproteinated process and the following dialysis process removed not only free protein but also small molecular impurities. The results suggested that all the EPS and IPS from the four media were polysaccharide-protein conjugates. Table 2 is a summary of the monosaccharide compositions of the EPS, DEPS, and IPS extracts from the four media obtained after 12 days of cultivation. All the PS extracts were composed of Rha, Ara, Xyl, Man, Glu, and Gal, but the molar proportions were different. Man and Glu were the dominant monosaccharides in the EPS extracts, and Glu was the major component in the IPS extracts, which significantly higher than in the EPS extracts. The Man content of WS-EPS, RS-EPS, and SB-EPS were higher than that of CT-EPS. The EPS from the wheat straw medium had the highest content of Man, Rha, Ara, and Xyl among all the extracts. After deproteinization process, the molar proportions of Rha, Ara, and Xyl of the EPS extracts significantly decreased.
RS-DEPS
2.3 ± 0.2bc
5.8 ± 0.3ab
3.2 ± 0.1b
18.7 ± 0.3d
30.6 ± 0.4b
39.4 ± 0.5 k
SB-DEPS
3.5 ± 0.4d
5.5 ± 0.4a
2.1 ± 0.2a
22.7 ± 0.4e
34.9 ± 0.5c
31.3 ± 0.4i
CT-DEPS
2.5 ± 0.2bc
5.9 ± 0.3abc
4.5 ± 0.2cd
26.3 ± 0.3f
26.6 ± 0.3a
34.2 ± 0.5j
WB-IPS
3.2 ± 0.2d
6.8 ± 0.4def
4.2 ± 0.1c
11.0 ± 0.3a
71.0 ± 0.5j
3.8 ± 0.2a
RS-IPS
2.3 ± 0.1bc
5.4 ± 0.3a
2.9 ± 0.2b
13.2 ± 0.1b
72.1 ± 0.4k
4.1 ± 0.1a
SB-IPS CT-IPS
1.8 ± 0.2a 2.1 ± 0.2ab
6.7 ± 0.1de 6.2 ± 0.2bcd
1.8 ± 0.2a 5.0 ± 0.4d
18.6 ± 0.2d 18.0 ± 0.2c
62.8 ± 0.6i 59.7 ± 0.5h
8.3 ± 0.2b 9.0 ± 0.4c
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A 40 35 30 25 20 15 10 5 0
2
4
6
8
wheat straw medium rice straw medium sugarcane bagasse medium control medium
B
wheat straw medium rice straw medium sugarcane bagasse medium control medium
Arabinose concentration (mg/L)
Rhamnose concentration (mg/L)
Fig. 4 Time course of contents of rhamnose (a), arabinose (b), xylose (c), mannose (d), glucose (e), and galactose (f) of the culture broth after precipitation of PS during fermentation in the control (open circle), wheat straw (filed square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
10
70 60 50 40 30 20 10 0 2
4
Time (day)
Xylose concentration (mg/L)
90 75 60 45 30 15 2
4
6
8
35 30
20 15 10 5 0 2
10
4
16 12 8 4 0
Time (day)
Saccharide compositions and contents of culture broth during fermentation In order to better understand whether the saccharides hydrolyzed from lignocellulose degradation were employed by I. obliquus to biosynthesize the EPS except for supplying follow-up carbon resource, the sugar compositions of the fermentation broths after removal of the EPS were analyzed and illustrated in Fig. 4. Glu was identified as the primary sugar during the period of
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8
10
Galactose concentration (mg/L)
Glucose concentration (g/L)
wheat straw medium ricestraw medium sugarcane bagasse medium control medium
6
8
10
wheat straw medium rice straw medium sugarcane bagasse medium control medium
F
4
6
Time (day)
E
2
10
25
Time (day)
20
8
wheat straw medium rice straw medium sugarcane bagasse medium control medium
D Mannose concentration (mg/L)
wheat straw medium rice straw medium sugarcane bagasse medium control medium
C
6
Time (day)
60 50 40 30 20 10 0 2
4
6
8
10
Time (day)
fermentation, particularly in the initial stage when overwhelming majority of sugars existed in the form of Glu. Then, Glu from the hydrolyzed corn flour starch at the beginning stage was rapidly consumed in the control and lignocellulose-containing medium. During day 2–5, the medium supplemented with the wheat straw, rice straw, and bagasse (during day 2–4) had lower Glu contents partly because the lignocellulose-containing media had a lower starting concentration (35 g L-1) of corn flour than the control medium (50 g L-1), but also because more Glu
Bioprocess Biosyst Eng
WS-EPS RS-EPS SB-EPS CT-EPS Mannitol
Hydroxyl radical-scavenging activity (%)
A
100
Hydroxyl radical-scavenging activity (%)
WS-EPS (filed square), RS-EPS (filed circle), and SB-EPS (open square) (a); CT-DEPS (open circle), WS-DEPS (filed square), RSDEPS (filed circle), and SB-DEPS (open square) (b); CT-IPS (open circle), WS-IPS (filed square), RS-IPS (filed circle), and SB-IPS (open square) (c). (Filed left inverted triangle) represent mannitol. Each point is the mean ± SD of triplicates
80 60 40 20 0
1
2
3
4
5
Concentration (mg/mL) WS-DEPS RS-DEPS SB-DEPS CT-DEPS Mannitol
B
might be employed to produce more EPS in the lignocellulose-containing media (Fig. 3A). Contrary to the decreased Glu content, Xyl and Gal in the three lignocellulose media increased accordingly with time. Rha and Man increased continuously in the bagasse medium, but increased till the 6th day in the wheat straw medium and 8th day in the rice straw medium and then decreased. Ara increased till the 6th day in the wheat straw medium and 4th day in the rice straw and bagasse-containing medium and then decreased. The broth of the control medium had a much lower level of the five sugars than that of the lignocellulose media and the content of the sugars changed little during the entire fermentation. Scavenging effect on hydroxyl and DPPH radicals
60 50 40 30 20 0
1
2
3
4
5
Concentration (mg/mL) WS-IPS RS-IPS SB-IPS CT-IPS Mannitol
C Hydroxyl radical-scavenging activity (%)
b Fig. 5 Hydroxyl radical-scavenging rates of CT-EPS (open circle),
90 75 60 45 30 15 0
1
2
3
4
Concentration (mg/mL)
5
Figure 5 showed the hydroxyl radical-scavenging effect of the crude EPS (Fig. 5a), DEPS (Fig. 5b), and IPS extracts (Fig. 5c). All extracts exhibited a hydroxyl radical-scavenging activity in a dose-dependent manner (0.5–5.0 mg mL-1). The scavenging activity of EPS and IPS from the lignocellulose media was significantly stronger than that from the control medium and mannitol which was in accordance with our previous work using corn stover, which might be because lignocellulose decomposition by I. obliquus stimulated the mycelia to produce more PS with higher antioxidant effect to protect themselves [12]. According to the effective concentrations corresponding to IC50 values (Table 3), the order of hydroxyl radical-scavenging activity was RS-EPS * WSEPS [ SB-EPS [ CT-EPS, and WS-IPS [ RS-IPS [ SBIPS [ CT-IPS. The crude EPS extracts had a higher activity than the respective IPS and DEPS counterparts, respectively. The DPPH free radical is a stable free radical, which has been widely adopted as a tool for estimating the free radical-scavenging activities of antioxidants. As can be seen in Fig. 6, moderate DPPH radical-scavenging activity of the EPS (Fig. 6a), DEPS (Fig. 6b), and IPS (Fig. 6c) extracts from the four media was evident in a dose-dependent manner at the concentrations from 0.5 to 3 mg L-1. Again, the PS extracts except for SB-IPS from the three lignocellulose media were more effective than those respective counterparts from the control medium, respectively, according to the IC50 values (Table 3). Wheat straw was
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2.20 ± 0.04c
0.65 ± 0.04b
WS
EPS
2.29 ± 0.04d
0.60 ± 0.03b
BS
3.60 ± 0.05f
0.67 ± 0.03b
SB
WS 2.15 ± 0.04e 1.88 ± 0.03b
CT 1.62 ± 0.02d 5.95 ± 0.05h
DEPS
3.55 ± 0.05 g 3.31 ± 0.04e
2.90 ± 0.05f
SB
0.88 ± 0.02a
US
8.54 ± 0.06j
7.84 ± 0.04 h
CT
4.97 ± 0.05g
0.62 ± 0.01b
WS
IPS
6.19 ± 0.06i
0.47 ± 0.01a
RS
–
1.32 ± 0.04c
SB
DPPH radical-scavenging activity (%)
DPPH radical-scavenging activity (%)
* IC50 value is the effective concentration at which hydroxyl radicals, DPPH radicals, and lipid peroxidation was inhibited by 50 %
14.49 ± 0.08k
8.83 ± 0.07i
CT
DPPH radical-scavenging activity (%)
EPS, IPS, and DEPS are the extracts from culture broth, mycelia, and deproteinated EPS. CT, WS, RS, and SB represent extracts from the control, wheat straw, rice straw, and sugarcane bagasse-containing media
DPPH radicals
Hydroxyl radicals
IC50(mB/mL)
Table 3 IC50 values of the PS extracts in antioxidant property
Bioprocess Biosyst Eng
A
B
70
60
50
40
30
20
WS-EPS RS-EPS SB-EPS CT-EPS
0
0
0
1
Concentration (mg/mL)
1
1
2
2
2
3
3
3
4
30
4
20
10
4
5
60
50
40
WS-DEPS RS-DEPS SB-DEPS CT-DEPS
20 5
Concentration (mg/mL)
C
50
40
30
WS-IPS RS-IPS SB-IPS CT-IPS
0
5
Concentration (mg/mL)
Fig. 6 DPPH radical-scavenging rates of CT-EPS (open circle), WSEPS (filed square), RS-EPS (filed circle), and SB-EPS (open square) (a); CT-DEPS (open circle), WS-DEPS (filed square), RS-DEPS (filed circle), and SB-DEPS (open square) (b); and CT-IPS (open circle), WS-IPS (filed square), RS-IPS (filed circle), and SB-IPS (open square) (c). Each point is the mean ± SD of triplicates
Bioprocess Biosyst Eng Table 4 Monosaccharide compositions of hemicellulose in lignocellulosic materials
Sugar component (mol%) Rha
Ara
Xyl
References Man
Wheat straw
–
9.2–16.5
68.5–79.5
1.7–2.0
Rice straw
0.7–0.9
11.2–16.9
26.9–53.4
–
Sugarcane bagasse
3.0–6.5
9.3–11.7
78.0–82.2
1.0–1.4
the best material in enhancing the DPPH-scavenging activity of EPS and IPS. Different from the precede results on hydroxyl radical-scavenging, DEPS exhibited a higher DPPH radical-scavenging activity than EPS.
Discussion White-rot fungi hydrolyzed cellulose and/or hemicellulose to support its mycelial growth and metabolism, and degraded lignin while lignin could not be used as carbon or energy to support fungi survival [21, 22]. Cellulose was the major component in the rice straw (39.6 %) and sugarcane bagasse (48.8 %) but hemicellulose was the major component in the wheat straw (38.0 %) [11]. Our previous work demonstrated that cellulose in the rice straw and sugarcane bagasse degraded faster than hemicellulose but hemicellulose was degraded faster than cellulose in the wheat straw and significantly degraded at the initial stage of fermentation by I. obliquus under submerged fermentation [11]. The biomass of mycelia in the wheat straw medium was higher than that in the rice straw and sugarcane bagasse media. The analysis of hydrolases and polysaccharide synthetases activity is under way for better explanation. Although the wheat straw medium showed the highest values in growth yield and EPS production, good mycelia growth does not seem to be determining factor for a high production yield of EPS because the mycelia yield from the control medium was higher than that from the rice straw and sugarcane bagasse media, but the EPS production was significantly lower (Fig. 3a). Therefore, the effect of mycelial growth on EPS production was complicated when the culture was under the lignocellulose degradation. The results demonstrated that EPS might be partly produced by the degraded lignocellulosic biomass. Nevertheless, the lignocellulosic materials of wheat straw, rice straw, and bagasse could increase EPS production and antioxidant activity, which is in agreement with our previous research on corn stover [12]. The different lignocellulose degradation rate [11] and the monosaccharide compositions in hemicellulose of the three materials generated different sugars in the culture broth. The hemicellulose in wheat straw had higher content of Xyl than that in rice straw and higher content of Gal than that in bagasse (Table 4) [23–25]. It was previously reported that
Glu
Gal
1.8–6.2
3.4–14.4
[23]
24.6–53.1
4.7–7.8
[24]
2.2–4.1
0.3–0.7
[25]
Xyl had significant effects on EPS production in other higher fungi including Grifola frondosa [26] and Cordyceps taii [27]. Hemicellulose is mainly composed of Xyl, Ara, and Gal in wheat straw. Xyl, Ara, and Glu are major hemicellulose components in rice straw. Xyl and Ara are the major sugars in sugarcane bagasse (Table 4). Compared with the rice straw- and bagasse-containing medium, there were more Rha, Ara, Xyl, Man, and Gal in the wheat straw medium before the 6th day. This might be because the hemicellulose was degraded faster than cellulose in the wheat straw and significantly degraded at the initial stage of fermentation, while cellulose was the major component and degraded faster than hemicellulose in the rice straw and sugarcane bagasse found in our previous work [11]. It should be noted that there were big differences in amount between glucose and other sugars because glucose came from the corn flour except for cellulose and hemicellulose. The contradictory results on hydroxyl radical- and DPPH radical-scavenging activity of the PS (Figs. 5, 6) extracts might be attributed to the different solubility in the two reaction systems, i.e., the reaction mixture for hydroxyl radical assay was aqueous solution but for DPPH radical assay was methanol solution [13]. The results indicated that the antioxidant activity of EPS, IPS, and DEPS was not proportional with the sugar content (Table 1). Higher protein content (Table 1), higher Man, Rha, and Xyl ratio (Table 2) may result in the higher antioxidant activity [7, 12, 28]. Particularly, the EPS extract from the wheat straw medium that contained 22 % protein, and 27.6 % Man, 10.5 % Rha, 14.5 % Xyl was the most effective antioxidant. In conclusion, the lignocellulose degradation in the wheat straw, rice straw, and sugarcane bagasse simultaneously enhanced the accumulation and antioxidant activity of I. obliquus PS. Of the three materials, the wheat straw was the best lignocellulosic biomass. The production and activity of PS were likely related to the lignocellulose compositions and structure. Studies on the activity of lignocellulose hydrolases and PS synthetases during fermentation are ongoing for better explanation of the difference of individual lignocellulosic biomass. Acknowledgments This research was supported by the research grant from the Science and Technology Department of Zhejiang Province, China (2012C23075).
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References 1. Zhong JJ, Xiao JH (2009) Secondary metabolites from higher fungi: discovery, bioactivity, and bioproduction. Adv Biochem Eng Biot 113:79–150 2. Zheng W, Miao K, Liu Y, Zhao Y, Zhang M, Pan S, Dai Y (2010) Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production. Appl Microbiol Biotechnol 87:1237–1254 3. Ju HK, Chung HW, Hong SS, Park JH, Lee J, Kwon SW (2010) Effect of steam treatment on soluble phenolic content and antioxidant activity of the Chaga mushroom (Inonotus obliquus). Food Chem 119:619–625 4. Kim YO, Park HW, Kim JH, Lee JY, Moon SH, Shin CS (2006) Anti-cancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus. Life Sci 79:72–80 5. Zhang LX, Fan C, Liu SC, Zang ZF, Jiao LL, Zhang LP (2011) Chemical composition and antitumor activity of polysaccharide from Inonotus obliquus. J Med Plants Res 5:1251–1260 6. Fan LP, Ding SD, Ai LZ, Deng KQ (2012) Antitumor and immunomodulatory activity of water-soluble polysaccharide from Inonotus obliquus. Carbohyd Polym 90:870–874 7. Kim YO, Han SB, Lee HW, Ahn HJ, Yoon YD, Jung JK, Kim HM, Shin CS (2005) Immunostimulating effect of the endopolysaccharide produced by submerged culture of Inonotus obliquus. Life Sci 77:2438–2456 8. Won DP, Lee JS, Kwon DS, Lee KE, Shin WC, Hong EK (2011) Immunostimulating activity by polysaccharides isolated from fruiting body of Inonotus obliquus. Mol Cells 31:165–173 9. Xu XQ, Wu YD, Chen H (2011) Comparative antioxidative characteristics of polysaccharide-enriched extracts from natural sclerotia and cultured mycelia in submerged fermentation of Inonotus obliquus. Food Chem 127:74–79 10. Xu XQ, Zhu JW (2011) Enhanced phenolic antioxidants production in submerged cultures of Inonotus obliquus in a ground corn stover medium. Biochem Eng J 58–59:103–109 11. Zhu LH, Xu XQ (2013) Stimulatory effect of different lignocellulosic materials for phenolic compound production and antioxidant activity from Inonotus obliquus in submerged fermentation. Appl Biochem Biotechnol 169:2138–2152 12. Chen H, Yan MC, Zhu JW, Xu XQ (2011) Enhancement of exopolysaccharide production and antioxidant activity in submerged cultures of Inonotus obliquus by lignocellulose decomposition. J Ind Microbiol Biotechnol 38:291–298 13. Xiang YL, Xu XQ, Li J (2012) Chemical properties and antioxidant activity of exopolysaccharides fractions from mycelial culture of Inonotus obliquus in a ground corn stover medium. Food Chem 134:1899–1905
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14. Yan Q, Han L, Jiang ZQ (2004) Removal of protein from the crude astragalus polysaccharides by proteases. Food Sci Technol 6:23–26 15. Miller G (1959) Use of dinitrosalicylic reagent for the determination of reducing sugar. Anal Chem 31:426–428 16. Dubois M, Gilles KA, Hamilton J, Rebers PA, Smith F (1956) Colorimetric method for determination of sugar and relative substances. Anal Chem 28:350–366 17. Chaplin MF, Kennedy JF (1994) Carbohydrate analysis: a practical approach. Oxford University Press, Oxford 18. Bradford MM (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 19. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic phosphotunstic acid reagents. Am J Enol Vitic 16:144–158 20. Yang ZY, Tu YY, Xia HL (2007) Suppression of free-radicals and protection against H2O2-induced oxidative damage in HPF-1 cell by oxidized phenolic compounds present in black tea. Food Chem 105:1349–1356 21. Bak JS, Ko JK, Choi IG, Park YC, Seo JH, Kim KH (2009) Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw. Biotechnol Bioeng 104:471–482 22. Sun FH, Li J, Yuan YX, Yan ZY, Liu XF (2011) Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover. Int Biodeterior Biodegradation 65:931–938 23. Sun RC, Lawther JM, Banks WB (1996) Fractional and structural characterization of wheat straw hemicelluloses. Carbohyd Polym 29:325–331 24. Sun RC, Tomkinson J, Ma PL, Liang SF (2000) Comparative study of hemicelluloses from rice straw by alkali and hydrogen peroxide treatments. Carbohyd Polym 42:111–122 25. Xu F, Sun JX, Liu CF, Sun RC (2006) Comparative study of alkali- and acidic organic solvent-soluble hemicellulosic polysaccharides from sugarcane bagasse. Carbohyd Res 341:253–261 26. Shih IL, Tsai KL, Hsieh C (2007) Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. Biochem Eng J 33:193–201 27. Xiao JH, Xiao DM, Xiong Q, Liang ZQ, Zhong JJ (2010) Nutritional requirements for the hyperproduction of bioactive exopolysaccharides by submerged fermentation of the edible medicinal fungus Cordyceps taii. Biochem Eng J 49:241–249 28. Chen H, Zhang M, Qu Z, Xie B (2008) Antioxidant activities of different fractions of polysaccharide conjugates from green tea (Camellia sinensis). Food Chem 106:559–563
ORIGINAL PAPER
Production of bioactive polysaccharides by Inonotus obliquus under submerged fermentation supplemented with lignocellulosic biomass and their antioxidant activity Xiangqun Xu • Yan Hu • Lili Quan
Received: 11 March 2014 / Accepted: 17 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract The effect of lignocellulose degradation in wheat straw, rice straw, and sugarcane bagasse on the accumulation and antioxidant activity of extra- (EPS) and intracellular polysaccharides (IPS) of Inonotus obliquus under submerged fermentation were first evaluated. The wheat straw, rice straw, and sugarcane bagasse increased the EPS accumulation by 91.4, 78.6, and 74.3 % compared with control, respectively. The EPS and IPS extracts from the three lignocellulose media had significantly higher hydroxyl radical- and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the control medium. Of the three materials, wheat straw was the most effective lignocellulose in enhancing the mycelia growth, accumulation and antioxidant activity of I. obliquus polysaccharides (PS). The carbohydrate and protein content, as well as the monosaccharide compositions of the EPS and IPS extracts, were correlated with sugar compositions and dynamic contents during fermentation of individual lignocellulosic materials. The enhanced accumulation of bioactive PS of cultured I. obliquus supplemented with rice straw, wheat straw, and bagasse was evident. Keywords Biodegradation Inonotus obliquus Lignocellulosic biomass Polysaccharides Submerged fermentation
X. Xu (&) Y. Hu L. Quan Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China e-mail: [email protected]; [email protected]
Introduction In recent years, edible and medicinal higher fungi and their secondary metabolites have been widely studied due to their health-promoting properties and relatively low toxicity [1]. Inonotus obliquus (I. obliquus) is one of the most efficient wood-degrading white-rot fungus that belongs to the family Hymenochaetaceae of Basidiomycetes and causes the simultaneous decay of lignin, cellulose, and hemicellulose of birch. This mushroom is well known as one of the most popular medicinal species due to its therapeutic effects. Many biological activities have been attributed to I. obliquus [2]. Polysaccharides (PS), one of the main active components of I. obliquus, were reported to exhibit many biological activities such as antioxidant [3], antitumor [4–6], and immune-stimulating [7, 8] effects. As demonstrated by our group before, the PS extracts from both the wild sclerotia and cultured mycelia including extracellular (EPS) and intracellular (IPS) extracts under submerged fermentation were effective in scavenging hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and in inhibiting lipid peroxidation [9]. Both the PS extracts from the mycelia of I. obliquus with higher polysaccharide contents showed a stronger antioxidant activity than those from the wild sclerotia [9]. Recently, we developed a method to enhance the accumulation of I. obliquus polyphenols under submerged fermentation using the fungus’s ability in lignocellulose degradation [10, 11]. We first demonstrated that I. obliquus in submerged fermentation could effectively decompose the lignocellulose in corn stover, rice straw, wheat straw, and sugarcane bagasse added in a liquid medium [11] as in nature where I. obliquus degraded wood polymers. Correspondingly, the accumulation and
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antioxidant activity of both the extracellular and intracellular polyphenols by I. obliquus were significantly enhanced in the four lignocellulosic biomass-containing culture with lignocellulose degradation [10, 11]. We also found that corn stover as a lignocellulose resource could increase the accumulation and antioxidant activity of the EPS and IPS from the cultures of I. obliquus [12, 13]. However, the mechanism of the enhancement, i.e., whether and how the derivation and bioconversion of cellulose and hemicellulose in the lignocellulosic biomass to the EPS and IPS of I. obliquus under submerged culture, is not clear. In addition, the use of lignocellulosic biomass varies from region to region and depends on their calorific values, nutritive value, lignin content, density, palatability. The right selection of the substrate is of great importance for an efficient and economical production of the compounds of interest. Along this line, in this study, we first evaluated the derivation and bioconversion effectiveness and efficiency of the lignocellulose in sugarcane bagasse, rice straw, and wheat straw into EPS and IPS by I. obliquus. The dynamic changes of monosaccharide compositions in the culture broth and production of EPS and IPS in the different lignocellulosic materials-containing media during fermentation were investigated and correlated to the degradation of cellulose, hemicellulose, and lignin. The chemical contents and monosaccharide compositions of all the EPS and IPS were analyzed. The antioxidant activity of all the EPS and IPS extracts was comparatively studied.
Methods Lignocellulosic materials After washed and dried at 50 °C, rice straw, wheat straw, and sugarcane bagasse were milled using an herbal grinder and passed through a 60-mesh sieve and trapped in a 100-mesh screen. The particles with sizes between 60- and 100-mesh were collected and added in the liquid culture for better lignocellulose degradation [11].
Liquid fermentation I. obliquus (CBS314.39) was purchased from the Centraal Bureau voor Schimmelcultuur, Utrecht, the Netherlands. Malt extract agar with mycelia was cultured in the medium (g L-1: glucose 20, peptone 3, yeast extract 2, KH2PO4 1, MgSO4 1.5, and CaCl2 0.1 for 4–5 days on a rotary shaker at 28 °C with a speed of 150 rpm. The seed culture was added into the control medium or lignocellulose-containing
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medium and incubated at a rate of 8 % (v/v) and at 28 °C in a rotary shaker at 150 rpm. The control medium (g L-1) contained corn flour 53, peptone 3, KH2PO4 1, ZnSO42H2O 0.01, K2HPO4 0.4, FeSO47H2O 0.05, MgSO47H2O 0.5, CuSO45H2O 0.02, CoCl2 0.01, MnSO4H2O 0.08, pH = 6.0 optimized by the response surface methodology (RSM) in our previous work that demonstrated corn flour and peptone were the best carbon source and nitrogen source [12]. The lignocellulose-containing medium (g L-1) contained corn flour 35, lignocellulosic material 30, and all of the other components were the same as the control medium. The corn flour contained in the medium was drastically hydrolyzed by a-amylase and glucoamylase into glucose to avoid the significant interference in the quantitative analysis of the EPS from early fermentation of I. obliquus before use [12].
Extraction of EPS and IPS The culture broth separated from the mycelia was concentrated to one quarter of the original volume with a rotary evaporator under reduced pressure at 60 °C. The concentrate was mixed with four-times volume of chilled 95 % (v/v) ethanol, then stirred vigorously and left at 4 °C overnight. The supernatant was collected for monosaccharide compositions analysis in Sect. 2.4. The ethanol precipitate was collected, then centrifuged (6,5009g for 10 min) and lyophilized. The lyophilized samples were termed as CT-EPS, RS-EPS, WS-EPS, and SB-EPS from the control, rice straw, wheat straw, and sugarcane bagassecontaining medium, respectively. As EPS could be produced by the degraded lignocellulosic biomass and fungi itself, RS-EPS, WS-EPS, and SB-EPS were the mixtures containing EPS produced from fungi itself and EPS from degraded lignocellulosic biomass. The data of carbohydrate composition and antioxidant activity measured below were obtained from the mixtures. The Sevage method was employed to remove protein after neutrase treatment with some modification [14]. The supernatant was concentrated and the aqueous solution was dialyzed against distilled water for 48 h then lyophilized. The deproteinated extracts from the control, rice straw, wheat straw, and sugarcane bagasse-containing media were named as CT-DEPS, RS-DEPS, WS-DEPS, and SB-DEPS, respectively. The mycelial masses suspended in distilled water were first heated for 1.5 h and then heated for 3 h in an autoclave at 121 °C to extract heat-stable mycelial product, followed by filtration [9]. The filtrate was then put through ethanol precipitation, dialysis, and lyophilization. The lyophilized samples were termed as CT-IPS, RS-IPS, WSIPS, and SB-IPS from the control, rice straw, wheat straw, and sugarcane bagasse-containing medium, respectively.
Bioprocess Biosyst Eng
The mycelia were filtered, washed through a 60-mesh sieve, and dried at 45 °C for 48 h for the determination of the biomass dry weight. The EPS production was calculated by the deduction of reducing sugar of total carbohydrate in the culture broth during fermentation. The reducing sugar concentration of the culture broth was measured by the DNS method [15]. The total carbohydrate content of the extracts was determined by phenol–sulfuric acid method [16]. The saccharide compositions of culture broth after precipitation of EPS were measured by gas chromatography after converting them into acetylated derivatives [17] with rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), and glucose (Glu) as the standards. The supernatant collected, in Sect. 2.3, was evaporated to dryness under a steam of nitrogen and then hydrolyzed and acetylated [12, 13]. The alditol acetates were analyzed by a gas chromatograph Varian CP-3800 (Varian Inc., Palo ALto, CA) equipped with a Varian CP-Sil 5 CB capillary chromatography column (25 m 9 0.53 mm, 0.25-lm film thickness) and a flameionization detector. The monosaccharide components were identified by matching the GC retention time with the six standard compounds [13]. Chemical analysis of EPS and IPS extracts The total carbohydrate and protein content of the extracts was determined by phenol–sulfuric acid method [16] and the method of Bradford with bovine serum albumin as a standard [18]. The total phenolic content was determined using Folin–Ciocalteu reagent [19]. The monosaccharide compositions of the extracts were analyzed using the same method in Sect. 2.4. Assay for hydroxyl and DPPH radical-scavenging activity The lyophilized samples obtained after 12 days of cultivation were dissolved in water in a concentration gradient (0.5–5 mg mL-1). Hydroxyl radical-scavenging activity of all the EPS and IPS extracts was determined according to the salicylic method with some modifications [12]. The DPPH free radical-scavenging activity was measured according to the method described by Yang et al. [20]. The IC50 that is the concentration of an inhibitor where the response is reduced by half was calculated by using median-effect analysis.
Statistical analysis The results were expressed as mean ± standard deviation (SD) of three independent experiments performed. Tests of significant differences were determined by Duncan’s multiple range tests at p = 0.05 or independent sample t test (p = 0.05) by one-way analysis of variance (ANOVA) of the data.
Results Time courses of mycelial biomass Figure 1 shows that the mycelial biomass from all the four media gradually increased during day 2–9. From day 7–9, the wheat straw medium was the most suitable medium for the cell growth of I. obliquus compared with the other three media. The addition of sugarcane bagasse and rice straw did not result in a better growth of I. obliquus than the control. The maximal mycelial biomass from the control, wheat straw, rice straw, and sugarcane bagasse-containing media was 10.8, 12.4, 9.5, and 8.0 g L-1 on day 9, 9, 10, and 9, respectively. As illustrated in Fig. 2, the reducing sugars from the hydrolyzed corn flour starch at the beginning stage was gradually consumed and decreased to a low level of 2 mg L-1 in the control and individual lignocellulosecontaining medium on day 5, 4, 5, and 6, respectively. During day 2–5, the medium supplemented with straw and
wheat straw medium rice straw medium sugarcane bagasse medium control medium
16 14
Mycelial biomass (g/L)
Determination of mycelia biomass, EPS and IPS production, and saccharide compositions of culture broth during fermentation
12 10 8 6 4 2 0 2
4
6
8
10
12
Time (day) Fig. 1 Time profiles of mycelial growth in submerged fermentation of I. obliquus in the control (open circle), wheat straw (filed square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
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wheat straw medium rice straw medium sugarcane bagasse medium control medium
16
A 1.8 1.6
EPS production (g/L)
Reducing sugar (mg/mL)
20
12 8 4
wheat straw medium rice straw medium sugarcane bagasse medium control medium
1.4 1.2 1.0 0.8 0.6 0.4 0.2
0 2
4
6
8
10
12
0.0 2
Time (day)
4
6
8
10
12
Time (day) Fig. 2 Sugar consumption during submerged fermentation of I. obliquus in the control (open circle), wheat straw (filed square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
wheat straw medium rice straw medium sugarcane bagasse medium control medium
B 1.4 1.2
IPS production (g/L)
bagasse (during day 2–3) had lower reducing sugar contents because the lignocellulosic material-containing media had a lower starting concentration (35 g L-1) of corn flour than the control medium (50 g L-1). During day 4–7, the bagasse medium had higher reducing sugar contents than the control medium. These reducing sugars might come from the cellulose and hemicellulose degradation. On the other hand, during the time, cell growth in the bagasse medium was so poor that sugars would be consumed slowly (Fig. 1).
1.0 0.8 0.6 0.4 0.2 0.0 2
EPS and IPS production during fermentation Figure 3 shows the time course of EPS production (Fig. 3a) and IPS production (Fig. 3b) of I. obliquus in the control and lignocellulose-containing medium. There were significant differences in the EPS production between the four media. In the control group, EPS production maximized at 0.7 g L-1 after 9 days of fermentation. The maximum EPS production in the wheat straw, rice straw, and bagasse medium reached 1.34, 1.25, and 1.22 g L-1 after 11, 11, and 10 days, respectively (Fig. 3a). As shown in Fig. 3b, compared with the control medium, only wheat straw promoted the IPS accumulation. The IPS production increased to the maximum level of 0.94, 0.99, 0.48, and 0.55 g L-1 in the control, wheat straw, rice straw, and bagasse-containing medium on day 9, 9, 10, and 9, respectively, implying a strong relationship with the cell growth (Fig. 1).
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4
6
8
10
12
Time (day) Fig. 3 Time course of EPS production (a) and IPS production (b) of I. obliquus in the control (open circle), wheat straw (filled square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
Chemical contents and monosaccharide compositions of EPS and IPS Table 1 is a summary of sugar and protein contents of four crude EPS extracts, four crude IPS extracts, and four deproteinated EPS extracts from the control and lignocellulose-containing media obtained after 12 days of cultivation. The sugar content of the crude IPS extracts (CT-IPS, WS-IPS, RS-IPS, and SB-IPS) was significantly higher than that of the counterpart crude EPS extracts (CTEPS, WS-EPS, RS-EPS, and SB-EPS) from the control, wheat straw, rice straw, and bagasse-containing medium.
Bioprocess Biosyst Eng Table 1 Major chemical content of the PS extracts Sugar content (wt%)
Protein content (wt%)
WS-EPS
22.39 ± 1.03b
21.95 ± 1.71b
RS-EPS
20.36 ± 2.62ab
27.51 ± 3.36c
SB-EPS
41.98 ± 2.61c
17.29 ± 3.56ab
CT-EPS
17.69 ± 0.40a
14.87 ± 1.74a
WS-DEPS
47.95 ± 2.10e
36.94 ± 1.96g
RS-DEPS
53.63 ± 0.96fg
31.35 ± 3.36f
SB-DEPS
55.29 ± 2.30g
10.70 ± 0.81e
CT-DEPS
50.66 ± 1.62ef
15.14 ± 2.09e
WS-IPS
51.67 ± 1.85 I
15.14 ± 0.12 I
RS-IPS
48.29 ± 2.43 I
15.27 ± 0.26 I
SB-IPS CT-IPS
68.14 ± 2.38 II 47.43 ± 1.70 I
13.05 ± 0.13 I 25.06 ± 5.94 II
CT-, RS-, WS-, and SB- represent extracts from the control, rice straw, wheat straw, and sugarcane bagasse-containing media. EPS, IPS, and DEPS are extracts from the culture broth, mycelia, and deproteinated EPS * Data with different letters in the same group are significantly different at p \ 0.05
The order of sugar content was SB-EPS (-IPS) [ WS-EPS (-IPS) [ RS-EPS (IPS) [ CT-EPS (IPS). The protein content of the EPS extracts was higher than that of the respective IPS counterparts except for those from the control medium with the order being RS-EPS [ WSEPS [ SB-EPS [ CT-EPS, but CT-IPS [ WS-IPS * RSIPS * SB-IPS. The results indicated that lignocellulose degradation could significantly increase the sugar content of the EPS and IPS and protein content of the EPS but decrease the protein content of the IPS extracts. Of the
Table 2 Monosaccharide compositions of the PS extracts
Sugar component (mol%) Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
WS-EPS
10.5 ± 0.4g
9.1 ± 0.4h
14.5 ± 0.5g
27.6 ± 0.5g
27.0 ± 0.5a
11.3 ± 0.2e
RS-EPS
6.5 ± 0.2e
7.8 ± 0.5g
8.6 ± 0.5f
26.8 ± 0.4f
35.9 ± 0.6d
14.4 ± 0.3 g
SB-EPS
7.2 ± 0.2f
7.3 ± 0.3efg
6.6 ± 0.2e
22.6 ± 0.3e
41.2 ± 0.5f
15.1 ± 0.5 h
CT-EPS
11.4 ± 0.5h
7.4 ± 0.4fg
8.7 ± 0.4f
19.0 ± 0.2d
40.0 ± 0.6e
13.5 ± 0.1f
2.7 ± 0.1c
6.5 ± 0.3cd
4.8 ± 0.3d
26.7 ± 0.6f
48.9 ± 0.6g
10.4 ± 0.3d
WS-DEPS CT-, RS-, WS-, and SBrepresent the extracts from the control, rice straw, wheat straw, and sugarcane bagassecontaining media. EPS, IPS, and DEPS are the extracts from culture broth, mycelia, and deproteinated EPS
three materials, bagasse was the most effective in enhancing sugar content, and rice straw was the most effective in protein content of EPS. The deproteinization treatment of the crude EPS extracts resulted in a significant increase in the sugar content with the order SBDEPS [ RS-DEPS [ CT-DEPS [ WS-DEPS. However, only the protein content of SB-DEPS decreased and of CTDEPS did no change after deproteinization. WS-DEPS and RS-DEPS had higher protein content than WS-EPS and RS-EPS. The results may be explained that the EPS extracts from the wheat straw and rice straw-containing medium contained other compounds such as small molecular sugar and non-saccharide components except exopolysaccharide and protein. The deproteinated process and the following dialysis process removed not only free protein but also small molecular impurities. The results suggested that all the EPS and IPS from the four media were polysaccharide-protein conjugates. Table 2 is a summary of the monosaccharide compositions of the EPS, DEPS, and IPS extracts from the four media obtained after 12 days of cultivation. All the PS extracts were composed of Rha, Ara, Xyl, Man, Glu, and Gal, but the molar proportions were different. Man and Glu were the dominant monosaccharides in the EPS extracts, and Glu was the major component in the IPS extracts, which significantly higher than in the EPS extracts. The Man content of WS-EPS, RS-EPS, and SB-EPS were higher than that of CT-EPS. The EPS from the wheat straw medium had the highest content of Man, Rha, Ara, and Xyl among all the extracts. After deproteinization process, the molar proportions of Rha, Ara, and Xyl of the EPS extracts significantly decreased.
RS-DEPS
2.3 ± 0.2bc
5.8 ± 0.3ab
3.2 ± 0.1b
18.7 ± 0.3d
30.6 ± 0.4b
39.4 ± 0.5 k
SB-DEPS
3.5 ± 0.4d
5.5 ± 0.4a
2.1 ± 0.2a
22.7 ± 0.4e
34.9 ± 0.5c
31.3 ± 0.4i
CT-DEPS
2.5 ± 0.2bc
5.9 ± 0.3abc
4.5 ± 0.2cd
26.3 ± 0.3f
26.6 ± 0.3a
34.2 ± 0.5j
WB-IPS
3.2 ± 0.2d
6.8 ± 0.4def
4.2 ± 0.1c
11.0 ± 0.3a
71.0 ± 0.5j
3.8 ± 0.2a
RS-IPS
2.3 ± 0.1bc
5.4 ± 0.3a
2.9 ± 0.2b
13.2 ± 0.1b
72.1 ± 0.4k
4.1 ± 0.1a
SB-IPS CT-IPS
1.8 ± 0.2a 2.1 ± 0.2ab
6.7 ± 0.1de 6.2 ± 0.2bcd
1.8 ± 0.2a 5.0 ± 0.4d
18.6 ± 0.2d 18.0 ± 0.2c
62.8 ± 0.6i 59.7 ± 0.5h
8.3 ± 0.2b 9.0 ± 0.4c
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A 40 35 30 25 20 15 10 5 0
2
4
6
8
wheat straw medium rice straw medium sugarcane bagasse medium control medium
B
wheat straw medium rice straw medium sugarcane bagasse medium control medium
Arabinose concentration (mg/L)
Rhamnose concentration (mg/L)
Fig. 4 Time course of contents of rhamnose (a), arabinose (b), xylose (c), mannose (d), glucose (e), and galactose (f) of the culture broth after precipitation of PS during fermentation in the control (open circle), wheat straw (filed square), rice straw (filed circle), sugarcane bagasse (open square)-containing medium
10
70 60 50 40 30 20 10 0 2
4
Time (day)
Xylose concentration (mg/L)
90 75 60 45 30 15 2
4
6
8
35 30
20 15 10 5 0 2
10
4
16 12 8 4 0
Time (day)
Saccharide compositions and contents of culture broth during fermentation In order to better understand whether the saccharides hydrolyzed from lignocellulose degradation were employed by I. obliquus to biosynthesize the EPS except for supplying follow-up carbon resource, the sugar compositions of the fermentation broths after removal of the EPS were analyzed and illustrated in Fig. 4. Glu was identified as the primary sugar during the period of
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8
10
Galactose concentration (mg/L)
Glucose concentration (g/L)
wheat straw medium ricestraw medium sugarcane bagasse medium control medium
6
8
10
wheat straw medium rice straw medium sugarcane bagasse medium control medium
F
4
6
Time (day)
E
2
10
25
Time (day)
20
8
wheat straw medium rice straw medium sugarcane bagasse medium control medium
D Mannose concentration (mg/L)
wheat straw medium rice straw medium sugarcane bagasse medium control medium
C
6
Time (day)
60 50 40 30 20 10 0 2
4
6
8
10
Time (day)
fermentation, particularly in the initial stage when overwhelming majority of sugars existed in the form of Glu. Then, Glu from the hydrolyzed corn flour starch at the beginning stage was rapidly consumed in the control and lignocellulose-containing medium. During day 2–5, the medium supplemented with the wheat straw, rice straw, and bagasse (during day 2–4) had lower Glu contents partly because the lignocellulose-containing media had a lower starting concentration (35 g L-1) of corn flour than the control medium (50 g L-1), but also because more Glu
Bioprocess Biosyst Eng
WS-EPS RS-EPS SB-EPS CT-EPS Mannitol
Hydroxyl radical-scavenging activity (%)
A
100
Hydroxyl radical-scavenging activity (%)
WS-EPS (filed square), RS-EPS (filed circle), and SB-EPS (open square) (a); CT-DEPS (open circle), WS-DEPS (filed square), RSDEPS (filed circle), and SB-DEPS (open square) (b); CT-IPS (open circle), WS-IPS (filed square), RS-IPS (filed circle), and SB-IPS (open square) (c). (Filed left inverted triangle) represent mannitol. Each point is the mean ± SD of triplicates
80 60 40 20 0
1
2
3
4
5
Concentration (mg/mL) WS-DEPS RS-DEPS SB-DEPS CT-DEPS Mannitol
B
might be employed to produce more EPS in the lignocellulose-containing media (Fig. 3A). Contrary to the decreased Glu content, Xyl and Gal in the three lignocellulose media increased accordingly with time. Rha and Man increased continuously in the bagasse medium, but increased till the 6th day in the wheat straw medium and 8th day in the rice straw medium and then decreased. Ara increased till the 6th day in the wheat straw medium and 4th day in the rice straw and bagasse-containing medium and then decreased. The broth of the control medium had a much lower level of the five sugars than that of the lignocellulose media and the content of the sugars changed little during the entire fermentation. Scavenging effect on hydroxyl and DPPH radicals
60 50 40 30 20 0
1
2
3
4
5
Concentration (mg/mL) WS-IPS RS-IPS SB-IPS CT-IPS Mannitol
C Hydroxyl radical-scavenging activity (%)
b Fig. 5 Hydroxyl radical-scavenging rates of CT-EPS (open circle),
90 75 60 45 30 15 0
1
2
3
4
Concentration (mg/mL)
5
Figure 5 showed the hydroxyl radical-scavenging effect of the crude EPS (Fig. 5a), DEPS (Fig. 5b), and IPS extracts (Fig. 5c). All extracts exhibited a hydroxyl radical-scavenging activity in a dose-dependent manner (0.5–5.0 mg mL-1). The scavenging activity of EPS and IPS from the lignocellulose media was significantly stronger than that from the control medium and mannitol which was in accordance with our previous work using corn stover, which might be because lignocellulose decomposition by I. obliquus stimulated the mycelia to produce more PS with higher antioxidant effect to protect themselves [12]. According to the effective concentrations corresponding to IC50 values (Table 3), the order of hydroxyl radical-scavenging activity was RS-EPS * WSEPS [ SB-EPS [ CT-EPS, and WS-IPS [ RS-IPS [ SBIPS [ CT-IPS. The crude EPS extracts had a higher activity than the respective IPS and DEPS counterparts, respectively. The DPPH free radical is a stable free radical, which has been widely adopted as a tool for estimating the free radical-scavenging activities of antioxidants. As can be seen in Fig. 6, moderate DPPH radical-scavenging activity of the EPS (Fig. 6a), DEPS (Fig. 6b), and IPS (Fig. 6c) extracts from the four media was evident in a dose-dependent manner at the concentrations from 0.5 to 3 mg L-1. Again, the PS extracts except for SB-IPS from the three lignocellulose media were more effective than those respective counterparts from the control medium, respectively, according to the IC50 values (Table 3). Wheat straw was
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123
2.20 ± 0.04c
0.65 ± 0.04b
WS
EPS
2.29 ± 0.04d
0.60 ± 0.03b
BS
3.60 ± 0.05f
0.67 ± 0.03b
SB
WS 2.15 ± 0.04e 1.88 ± 0.03b
CT 1.62 ± 0.02d 5.95 ± 0.05h
DEPS
3.55 ± 0.05 g 3.31 ± 0.04e
2.90 ± 0.05f
SB
0.88 ± 0.02a
US
8.54 ± 0.06j
7.84 ± 0.04 h
CT
4.97 ± 0.05g
0.62 ± 0.01b
WS
IPS
6.19 ± 0.06i
0.47 ± 0.01a
RS
–
1.32 ± 0.04c
SB
DPPH radical-scavenging activity (%)
DPPH radical-scavenging activity (%)
* IC50 value is the effective concentration at which hydroxyl radicals, DPPH radicals, and lipid peroxidation was inhibited by 50 %
14.49 ± 0.08k
8.83 ± 0.07i
CT
DPPH radical-scavenging activity (%)
EPS, IPS, and DEPS are the extracts from culture broth, mycelia, and deproteinated EPS. CT, WS, RS, and SB represent extracts from the control, wheat straw, rice straw, and sugarcane bagasse-containing media
DPPH radicals
Hydroxyl radicals
IC50(mB/mL)
Table 3 IC50 values of the PS extracts in antioxidant property
Bioprocess Biosyst Eng
A
B
70
60
50
40
30
20
WS-EPS RS-EPS SB-EPS CT-EPS
0
0
0
1
Concentration (mg/mL)
1
1
2
2
2
3
3
3
4
30
4
20
10
4
5
60
50
40
WS-DEPS RS-DEPS SB-DEPS CT-DEPS
20 5
Concentration (mg/mL)
C
50
40
30
WS-IPS RS-IPS SB-IPS CT-IPS
0
5
Concentration (mg/mL)
Fig. 6 DPPH radical-scavenging rates of CT-EPS (open circle), WSEPS (filed square), RS-EPS (filed circle), and SB-EPS (open square) (a); CT-DEPS (open circle), WS-DEPS (filed square), RS-DEPS (filed circle), and SB-DEPS (open square) (b); and CT-IPS (open circle), WS-IPS (filed square), RS-IPS (filed circle), and SB-IPS (open square) (c). Each point is the mean ± SD of triplicates
Bioprocess Biosyst Eng Table 4 Monosaccharide compositions of hemicellulose in lignocellulosic materials
Sugar component (mol%) Rha
Ara
Xyl
References Man
Wheat straw
–
9.2–16.5
68.5–79.5
1.7–2.0
Rice straw
0.7–0.9
11.2–16.9
26.9–53.4
–
Sugarcane bagasse
3.0–6.5
9.3–11.7
78.0–82.2
1.0–1.4
the best material in enhancing the DPPH-scavenging activity of EPS and IPS. Different from the precede results on hydroxyl radical-scavenging, DEPS exhibited a higher DPPH radical-scavenging activity than EPS.
Discussion White-rot fungi hydrolyzed cellulose and/or hemicellulose to support its mycelial growth and metabolism, and degraded lignin while lignin could not be used as carbon or energy to support fungi survival [21, 22]. Cellulose was the major component in the rice straw (39.6 %) and sugarcane bagasse (48.8 %) but hemicellulose was the major component in the wheat straw (38.0 %) [11]. Our previous work demonstrated that cellulose in the rice straw and sugarcane bagasse degraded faster than hemicellulose but hemicellulose was degraded faster than cellulose in the wheat straw and significantly degraded at the initial stage of fermentation by I. obliquus under submerged fermentation [11]. The biomass of mycelia in the wheat straw medium was higher than that in the rice straw and sugarcane bagasse media. The analysis of hydrolases and polysaccharide synthetases activity is under way for better explanation. Although the wheat straw medium showed the highest values in growth yield and EPS production, good mycelia growth does not seem to be determining factor for a high production yield of EPS because the mycelia yield from the control medium was higher than that from the rice straw and sugarcane bagasse media, but the EPS production was significantly lower (Fig. 3a). Therefore, the effect of mycelial growth on EPS production was complicated when the culture was under the lignocellulose degradation. The results demonstrated that EPS might be partly produced by the degraded lignocellulosic biomass. Nevertheless, the lignocellulosic materials of wheat straw, rice straw, and bagasse could increase EPS production and antioxidant activity, which is in agreement with our previous research on corn stover [12]. The different lignocellulose degradation rate [11] and the monosaccharide compositions in hemicellulose of the three materials generated different sugars in the culture broth. The hemicellulose in wheat straw had higher content of Xyl than that in rice straw and higher content of Gal than that in bagasse (Table 4) [23–25]. It was previously reported that
Glu
Gal
1.8–6.2
3.4–14.4
[23]
24.6–53.1
4.7–7.8
[24]
2.2–4.1
0.3–0.7
[25]
Xyl had significant effects on EPS production in other higher fungi including Grifola frondosa [26] and Cordyceps taii [27]. Hemicellulose is mainly composed of Xyl, Ara, and Gal in wheat straw. Xyl, Ara, and Glu are major hemicellulose components in rice straw. Xyl and Ara are the major sugars in sugarcane bagasse (Table 4). Compared with the rice straw- and bagasse-containing medium, there were more Rha, Ara, Xyl, Man, and Gal in the wheat straw medium before the 6th day. This might be because the hemicellulose was degraded faster than cellulose in the wheat straw and significantly degraded at the initial stage of fermentation, while cellulose was the major component and degraded faster than hemicellulose in the rice straw and sugarcane bagasse found in our previous work [11]. It should be noted that there were big differences in amount between glucose and other sugars because glucose came from the corn flour except for cellulose and hemicellulose. The contradictory results on hydroxyl radical- and DPPH radical-scavenging activity of the PS (Figs. 5, 6) extracts might be attributed to the different solubility in the two reaction systems, i.e., the reaction mixture for hydroxyl radical assay was aqueous solution but for DPPH radical assay was methanol solution [13]. The results indicated that the antioxidant activity of EPS, IPS, and DEPS was not proportional with the sugar content (Table 1). Higher protein content (Table 1), higher Man, Rha, and Xyl ratio (Table 2) may result in the higher antioxidant activity [7, 12, 28]. Particularly, the EPS extract from the wheat straw medium that contained 22 % protein, and 27.6 % Man, 10.5 % Rha, 14.5 % Xyl was the most effective antioxidant. In conclusion, the lignocellulose degradation in the wheat straw, rice straw, and sugarcane bagasse simultaneously enhanced the accumulation and antioxidant activity of I. obliquus PS. Of the three materials, the wheat straw was the best lignocellulosic biomass. The production and activity of PS were likely related to the lignocellulose compositions and structure. Studies on the activity of lignocellulose hydrolases and PS synthetases during fermentation are ongoing for better explanation of the difference of individual lignocellulosic biomass. Acknowledgments This research was supported by the research grant from the Science and Technology Department of Zhejiang Province, China (2012C23075).
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