Anti-complementary Activities of Exo- and Endo-biopolymer Produced by Submerged Mycelial Culture of Eight Different Mushrooms

Abstract

The Elfvingia applanata (EA), Hericium erinaceum (HE),Grifola frondosa (GF), Pholiota nameko (PN), Pleurotus eryngii (PE), Trametes suaveolens (TS), Fomes fomentarius (FF), and Inonotus obliquus (IO) could produce the endo- (EN) and exo-biopolymer (EX) in submerged culture. The highest anti-complementary activity of the EN was exhibited by PN (49.1%), followed by HE (38.6%), TS (37.0%),and FF (33.0%),whereas the high activity of the EX was found with GF (59.8%),followed by HE (36.3%),TS (30.8%),and IO (28.8%). The EN of P. nameko (EN-PN) and EX of G. frondosa (EX-GF) were found to contain 78.6% and 41.2% carbohydrates, while 21.4% and 58.8% protein, respectively. The sugar and amino acid compositions of EN-PN and EX-GF were also analyzed in detail.

Among various immune systems, the complement system consisting of a series of enzymes in blood serum which plays an important role in host resistance as a primary humoral mediated antigen-antibody reaction. The complement system is major effectors of the humoral and innate immunity involved in host defense. A number of substances from chemicals, plant or microbial origins have been reported to modulate the complement cascade (Hildebert and Jordan, 1988). The function of the complement system in innate and specific humoral immunity is to promote phagocytosis of microbes on which complement is activated, to stimulate inflammation, and to induce the lysis of these microbes. The biological importance of complement is emphasized by severe symptoms that cause complement deficiencies. The regulation of complement activity affects an important in controlling immune responses (Walport, 1993).

Polysaccharides are essential constituents for all living organisms as they are associated with a variety of vital functions. The polysaccharides of natural origin have emerged as an important class of bioactive materials. Polysaccharides possessing anti-complementary activity have been reported from bacteria, fungi and higher plants. Mushrooms have been proved as an interesting source for new secondary metabolites with a variety of different biological activities. Active substances of various mushrooms can act as biological response modifiers (Maeda and Chihara, 1971) and also can have many pharmacological effects, i.e. immuno-modulating, hypoglycemic, hypolipidemic, and anti-tumor actions. In addition to above biological activities, some anti-complementary activities have also been reported from Ganoderma lucidum (Lee et al., 1994), Lentinus edodes (Song et al., 1998), and Pleurotus ostreatus (Kweon et al., 1990). Recently, immuno-active polysaccharide-protein has been isolated from many kinds of mushroom species. The activation of the complement system by fungal biopolymers is found to be closely related with anti-tumor effect and anti-complementary activity was also proportionate to anti-tumor actions (Lee et al., 1994). Wang et al. (1996) investigated immuno-modulating activity of lectins from Tricholoma mongolicum. The immuno-modulating activity of β-glucan obtained from liquid-culture of G. frondosa has also been documented by Suzuki et al. (1989).

In this study, the anti-complementary activities of EN and EX produced from submerged culture of EA, HE, GF, PN, PE, TS, FF, and IO were investigated. Chemical compositions of EN and EX produced from chosen strain were also analyzed.

Materials and Methods

Organism and culture medium

The cultures of EA (KACC 50174), HE (KACC 42140), GF (KACC 50027), PN (KACC 50453), PE (KACC 50037), TS (KCTC 26205), FF (KCTC 6363), and IO (KACC 50069) were obtained from the Korean Agricultural Culture Collection (KACC) and Korean Collection of Type Cultures (KCTC). The seed cultures were grown in 250-ml flask, containing 100 ml of potato dextrose broth (pH 5.0) and incubated on a rotary shaker (120 rpm) at 25℃ for approximately 7 d. One hundred ml of the medium with mycelial pellets was homogenized aseptically in a Sorvall omni-mixer for 3 min in an ice bath and inoculated in the liquid media at the rate of 2% (v/v) for submerged cultivation. The mushroom complete medium (MCM) with the following composition (g/l): glucose 20, yeast extract 2, peptone 2, KH₂PO₄ 0.46, K₂HPO₄ 1.0, and MgSO₄·7H₂O 0.5, with pH 5.0 was used to perform submerged mycelial culture for the production of biopolymers. The submerged mycelial cultures were carried out in 500-ml flask, containing 200 ml of the medium at 120 rpm/25℃/7~15 d.

Recovery of EX and EN

The supernatant and the hot water extract of mycelia were treated with ethanol. The ethanol precipitate was dissolved in water, dialyzed, and lyophilized to obtain an EN and EX. The recovery procedure for EX and EN from submerged mycelial culture of mushrooms is shown in Fig. 1.

Fig. 1.

Schematic diagram depicting recovery process of exo- and endo-biopolymer from submerged mycelial culture of mushrooms.

Assay of anti-complementary activity

Anti-complementary activity was measured by the complement fixation test based on complement consumption and the degree of red blood cell lysis by the residual complement. Fifty microliter of water solution of biopolymers was mixed with equal volumes of normal human serum (NHS) and GVB⁺⁺ (gelatin veronal buffered saline, pH 7.4) containing Mg⁺⁺ (500 µg) and Ca⁺⁺ (150 µg). The mixtures were incubated at 37℃ for 30 min and the residual total complement hemolysis (TCH₅₀) was determined by using IgM hemolysin sensitized sheep erythrocytes at 1 × 10⁸ cell/ml. At the same time, the NHS was incubated with deionized water (DIW) and GVB⁺⁺ (GVB containing 500 µg Mg⁺⁺ and 150 µg Ca⁺⁺) to provide a control. The anti-complementary activity of biopolymers was expressed as the percentage inhibition of the TCH₅₀ of control.

Chemical analysis of EX and EN

Total protein content of the EN and EX was determined with bovine serum albumin (BSA) as a standard (Lowry et al., 1951). The amino acid composition was analyzed by a Biochrom 20 (Pharmacia Biotech. Ltd., U.S.A.) amino acid autoanalyzer with a Na-form column after hydrolysis of the protein. The total sugar content was determined by the phenol sulfuric acid method, using a mannose and galactose mixture (1 : 1) as the standard (Dubios et al., 1964). The sugar composition was analyzed by a GC 3600 gas chromatography (Varian Co., U.S.A.) based on the hydrolysis and acethylation method (Jones and Albersheim, 1972).

Results and Discussion

Production of EX and EN

Eight different mushrooms were cultivated for production of EX and EN. Favourable growth was achieved with EA, GF, PN, and PE while high yield of EX and EN were obtained from GF and PN, respectively (Table 1).

Table 1.

Mycelial growth, exo- and endo-biopolymer yields by submerged mycelial culture of eight different mushrooms

^aCulture time at which the maximum mycelial mass was reached. All the values are given as the mean based on results of triplicate experiments.

Anti-complementary activities of EX and EN

The water soluble EXs and ENs obtained through the submerged mycelial cultures of eight kinds of mushrooms were analyzed for anti-complementary activity (Figs. 2 and 3) by total hemolytic complement assay (TCH₅₀). Their activities were compared at the concentration of 1000 µg/ml. The highest activity (49.1%) was recorded with EN-PN followed by EN-HE (38.6%) and EN-TS (37.0%).

Fig. 2.

The anti-complementary activities of the endo-biopolymers obtained from the submerged mycelial cultures of eight different types of mushrooms. LPS: Lipopolysaccharide was used for the positive control. The concentration of each sample and LPS was 1000 µg/ml. Each value is the mean ± S.D. of triplicate. EN: endo-biopolymer, EA: Elfvingia applanata, FF: Fomes fomentarius, GF: Grifola frondosa, HE: Hericium erinaceum, IO: Inonotus obliquus, PN: Pholiota nameko, PE: Pleurotus eryngii, TS: Trametes suaveolens.

Fig. 3.

The anti-complementary activities of exo-biopolymers obtained from the submerged mycelial cultures of eight different types of mushrooms. LPS: Lipopolysaccharide was used for the positive control. The concentration of each sample and LPS was 1000 µg/ml. Each value is the mean ± S.D. of triplicate. EX: exo-biopolymer, EA: Elfvingia applanata, FF: Fomes fomentarius, GF: Grifola frondosa, HE: Hericium erinaceum, IO: Inonotus obliquus, PN: Pholiota nameko, PE: Pleurotus eryngii, TS: Trametes suaveolens.

Among the EXs (Fig. 3), a highest activity (59.8%) was observed EX-GF, followed by EX-HE (36.3%), EX-TS (30.8%), and EX-IO (28.8%).

In earlier studies, the anti-complementary activity has been reported from fruiting bodies (Kweon et al., 1990), mycelia (Song et al., 1998), and culture broth (Jeong et al., 2004) of various mushrooms. An activation of the complement system by some mushroom polysaccharides is reported to enhance macrophage activation, cytolysis, and anti-tumor activity (Suzuki et al., 1989; Wang et al., 1996). The anti-complementary activity of lentinan (Chihara et al., 1970) isolated from Lentinus edodes and Schizophyllan (Tabata et al., 1981) from Schzophyllum commune has been demonstrated earlier and these polysaccharides, which found responsible for it, are now in clinical use as anti-cancer drugs. Similarly, many workers (Lee et al., 1994; Suzuki et al., 1989) have shown a correlation between the activation of the complement system and anti-tumor effect of polysaccharides. Therefore, the results obtained from this study can suggest that EN-PN and EX-GF may have a potential as an anti-tumor substance.

Amino acid and sugar composition of EN-PN and EX-GF

The EN-PN was found to contain 21.4% and 78.6% of total sugar and protein contents, respectively. Out of seventeen different kinds of amino acids which constitute the protein moiety, the major amino acids were appeared to be glutamic acid (12.6%), glysine (12.2%), lysine (11.9%), and aspartic acid (10.9%), while glucose (81.1%) appeared to be the major carbohydrate of the sugar moiety (Table 2).

Table 2.

Sugar and amino acid compositions of the endo-biopolymers obtained from the mycelia of Pholiota nameko

^*Calculated on the basis of total sugar or protein.

The EX-GF seems to be a glycoprotein, which contains 62.2% carbohydrate and 37.8% protein (Table 3). Serine (12.3%), threonine (11.6%), aspartic acid (11.6%), glycine (9.8%), and glutamic acid (9.4%) were found as the major amino acids in the EX-GF, while mannose (49.0%), glucose (15.7%), and fucose (18.4%) were the major sugars present in the carbohydrate moiety.

Table 3.

Sugar and amino acid compositions of the exo-biopolymers obtained from the submerged mycelial culture of Grifola frondosa

^*Calculated on the basis of total sugar and protein.

Yang et al. (1987) reported that the polysaccharide-peptide isolated from deep-layer cultured mycelia of Coriolus versicolor is composed of glucose (74.6%) with the remainder being galactose, mannose, xylose and fucose. Kim et al. (1994) also noticed that the polymer from ascocarp of Cordyceps militaris was mainly composed of glucose (78.6%) as observed in the present studies (endopolymer). Most anti-complementary polysaccharides generally obtained from mushrooms are composed of glycopeptide or proteoglycan (Moon et al., 2002), and contain arabinose and galactose as a component sugar together with a significant amount of glucose (Yamada et al., 1990). Our results are complementary with the findings of Yamada et al. (1985), who also showed the involvement of the carbohydrate moiety in executing anti-complementary activity.

Song et al. (1998) while working with endo-polymers of various mushrooms reported that the polymer could be of glycopeptides nature, consisting of sugar and amino acids. Many mushroom proteins have been reported to activate lymphocytes and to stimulate cell proliferation and cytokine secretion in vitro (Wang et al., 1998), while some mushroom proteins are known to activate immune cells and inhibit the growth of implanted tumor cell in vivo (Wang et al., 1996). Song et al. (1998) reported a polysaccharide from the mycelia of L edodes, which stimulated the polyclonal antibody production in vitro. The protein-bound polysaccharide produced from mycelia of C. versicolor is characterized as an agent capable of modifying the host biological response by stimulating the immune system and thereby augmenting various therapeutic effects (Yang et al., 1987). Accordingly, mushroom proteins may affect the host's immune system, and are therefore thought to have potential in anti-complementary activity. Therefore, it can be suggested that carbohydrate and protein moieties in polymer contribute to the expression of anti-complementary activity. In this context, anti-complementary biopolymers from PN and GF are required further purification and a more detailed study on the structure and its property.