Optimization of Hydroxyl Radical Scavenging Activity of Exopolysaccharides from Inonotus Obliquus in Submerged Fermentation Using Response Surface Methodology

J. Microbiol. Biotechnol. (2010), 20(4), 835–843 doi: 10.4014/jmb.0909.09017 First published online 2 February 2010 Optimization of Hydroxyl Radical Scavenging Activity of Exopolysaccharides from Inonotus obliquus in Submerged Fermentation Using Response Surface Methodology Chen, Hui1, Xiangqun Xu1*, and Yang Zhu2 1School of Science, Zhejiang Sci-Tech University, Hangzhou 310018, China 2TNO Quality of Life, Department of Bioscience, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received: September 15, 2009 / Revised: November 28, 2009 / Accepted: December 18, 2009 The objectives of this study were to investigate the effect Basidiomycetes [22, 29], and has been a folk remedy for a of fermentation medium on the hydroxyl radical scavenging long time in Russia and northern latitudes [11, 24, 29]. activity of exopolysaccharides from Inonotus obliquus by Many fungal triterpenoids, steroids, and phenolic compounds response surface methodology (RSM). A two-level fractional from I. obliquus have various biological activities [11, 18, factorial design was used to evaluate the effect of different 39]. In particular, polysaccharides from I. obliquus exhibit components of the medium. Corn flour, peptone, and strong immunomodulating, antitumor, and antioxidant KH2PO4 were important factors significantly affecting activities [22, 26, 31, 34]. hydroxyl radical scavenging activity. These selected variables The limited natural resource and difficult artificial were subsequently optimized using path of steepest ascent cultivation of I. obliquus to obtain fruit body make it (descent), a central composite design, and response surface impossible to obtain a large quantity of polysaccharides. analysis. The optimal medium composition was (% w/v): Submerged cultures offer a promising alternative. Many corn flour 5.30, peptone 0.32, KH2PO4 0.26, MgSO4 0.02, medicinal mushroom polysaccharides are being commercially and CaCl2 0.01. Under the optimal condition, the hydroxyl produced by submerged culture [6, 14, 16] because of a radical scavenging rate (49.4%) was much higher than high productivity compared with production from fruit that using either basal fermentation medium (10.2%) and bodies [20, 23]. In addition, submerged culture is fast, single variable optimization of fermentation medium cost-effective, easy to control, and without heavy metal (35.5%). The main monosaccharides components of the contamination. In submerged fermentation, production of RSM optimized polysaccharides are rhamnose, arabinose, polysaccharides is sensitive to medium components and xylose, mannose, glucose, and galactose with molar proportion fermentation process parameters. The optimization of at 1.45%, 3.63%, 2.17%, 15.94%, 50.00%, and 26.81%. medium and fermentation process parameters has focused Keywords: Inonotus obliquus, exopolysaccharides, hydroxyl on getting the maximum biomass or yield of polysaccharides radical scavenging activity, medium optimization, response for most medicinal mushrooms in previous studies [8, 13, surface methodology, monosaccharides component 14, 16, 21, 35]. Little is known about the qualitative effect of fermentation condition on the bioactivity of polysaccharides from submerged culture. In submerged fermentation, the biological activities of polysaccharides are associated with Edible and medicinal mushrooms have an established nutritional condition [3]. Carbon source, nitrogen source, history of use in the human diet and traditional therapies and inorganic salts affect both the yield and bioactivity of [11, 24, 29]. Many mushroom-derived preparations are polysaccharides. Although previous works have been done widely applied in modern clinical practice in China, Japan, on the accumulation of polysaccharides from the culture of Korea, and other Asian countries, either for preventive or many different mushrooms [13, 14, 16, 21, 35], the factors curative purposes. The medicinal mushroom Inonotus I. obliquus obliquus I. obliquus affecting the activity of polysaccharides of have ( ) belongs to the family Hymenochaetaceae, not yet been reported. In order to obtain exopolysaccharides (EPSs) with the maximum activities, we optimized the *Corresponding author Phone: +86-571-86843228; Fax: +86-571-87702220; medium composition to maximize the antioxidant activity E-mail: [email protected] of EPSs. It is well-known that reactive oxygen species 836 Chen et al. (ROS), such as hydroxyl radicals, superoxide anion, and Shake-Flask Culture hydrogen peroxide, are related to the pathogenesis of The harvested seed culture was added into 250-ml Erlenmeyer various diseases [1, 4]. Hydroxyl radical is the most reactive flasks containing 100 ml of liquid fermentation medium to be optimized in the following procedures (described below). The inoculate among the oxygen radicals and induces severe damage to o the adjacent biomolecules [5]. In this paper, the scavenging rate was 10% (v/v). The culture was incubated at 28 C for 9 days on a rotary shaker maintained at 150 rpm to produce mycelia and rate of hydroxyl radical was chosen as an indicator to exopolysaccharides. evaluate the antioxidant activity of EPS from I. obliquus. We used response surface methodology (RSM) including Extraction and Purification of Exopolysaccharides the fractional factorial design, the steepest ascent (descent) Harvested fermentation broth was filtered in a vacuum to separate method, and the central composite design (CCD) in the mycelia from broth. The filtrate was concentrated under vacuum experimental design [9, 12, 17]. Response surface methodology into one quarter of the original volume. The concentrate was mixed is a well-known method applied in the optimization of with four times volume of absolute ethanol, stirred vigorously, and o medium constituents and other critical variables responsible left overnight at 4 C. The precipitated exopolysaccharides were for the production of biomolecules. Statistical experimental repeatedly washed with 95% ethanol to remove adherent sugar g designs can be employed at various phases of the residue and other small molecules, and then centrifuged (6,500 × optimization process, such as for screening experiments for 10 min) and lyophilized. The EPS extract was obtained [16]. The Sevage method was employed to remove protein. The EPS and for finding optimum conditions for a desired response. extract water solution was mixed with Sevage reagent [chloroform: Single variable optimization methods are tedious and can butanol=5:1 (v/v)] and oscillated intensively. The crude EPS was also lead to misinterpretation of results because they obtained after the treated water solution had been concentrated, overlook the interaction between different factors involved precipitated, and lyophilized as stated above. [15]. This method had been successfully applied in the optimization of medium compositions [10, 21, 25, 33] and Assay of Crude EPS Hydroxyl Radical Scavenging Activity fermentation processes [32, 36, 38] to improve the production The hydroxyl radical (·OH) scavenging activity of crude EPS was of polysaccharides. However, to find the most important measured with the salicylic acid method as described by Smirnoff factors among related factors is the most important thing and Cumbes [28] with some modification. The reaction mixture (4 ml) firstly considered. The optimization process involves three contained 1 ml of H2O2 (8.8 mmol/l), 1 ml of FeSO4 (9 mmol/l), major steps: performing the statistically designed experiments, 1 ml of salicylic and (9 mmol/l), and 1 ml of crude EPS solution (1 mg/ml). The H2O2 was added into the mixture to start up the estimating the coefficients in a mathematical model, and o reaction. The reaction mixture was incubated at 37 C for 60 min, predicting the response followed by checking the adequacy and then centrifuged at 15,000 ×g for 6 min. The absorbance (A) of of the model [7, 19, 21, 27]. Finally, gas chromatography the reaction solutions at 510 nm was measured. The scavenging rate (GC) was employed to investigate the monosaccharide was calculated according to the equation: components of exopolysaccharides from different media. A0 –()Ax –Ax0 Scavening rate (%)=--------------------------------× 1 0 0 % (1) A0 MATERIALS AND METHODS where A0 is the absorbance for control (double-distilled water), Ax is the absorbance for the reaction mixture with EPS sample solution, Materials and Ax0 is the absorbance for background (i.e., the reaction mixture Corn flour and soybean powder were purchased from a local market. without H2O2). Yeast extract, peptone, and malt extract were purchased from Hangzhou Microbiology Institute (Hangzhou, Zhejiang, China). Glucose Selection of Carbon Source and Nitrogen Source and other chemical reagents were of analytical grade. The basal medium designed by Kim et al. [22] with some modification was employed. The basal medium contained (% w/v) glucose 3, Microorganism peptone 0.4, MgSO4 0.05, KH2PO4 0.15, and CaCl2 0.01. For I. obliquus (CBS314.39) was from the Centraal Bureau voor screening carbon source, glucose was replaced by sucrose, fructose, Schimmelcultuur, Utrecht, The Netherlands. It was maintained on maltose, corn flour, or soluble starch while keeping other components malt extract agar slants containing (% w/v) malt extract 3, peptone in the medium constant. For screening the nitrogen source, soybean o 0.3, and agar 1.5 at pH 5.6±0.2. The slants were cultivated at 25 C powder, yeast extract, KNO3, or (NH4)2SO4 were used while keeping for about 2 weeks. When the mycelia overgrew the slants,

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