Fermentation Process Optimization Of Phellinus Igniarius And Metabolic Regulation Of Its Polysaccharides

Phellinus igniarius is a precious medicinal mushroom belonging to the subphylum Basidomycotina, class Hymenochaetacae, order Polyporales, family Hymenochaetaceae, genus Phellinus. It contains many kinds of compounds such as polysaccharides, flavonoids, triterpenoids, larch mushroom acids and so on. These compounds are able to prevent and cure many diseases including anti-cancer, anti-liver fibrosis, anti-pneumonia, anti-angiogenesis and enhancing immunity. Because of the physiological complexity, particularity and high requirement of growing environment, it is very difficult to cultivate fruit body with high quantity of bioactive compounds, In addition, the amounts of natural P. igniarius were decreased sharply due to the mass collection in recent years. It is a promising approach by artificial cultivation and fungi fermentation to address the contradiction between supply and demand. Using this method, polysaccharides from P. igniarius can be produced in large scale and used in medical care causes.In order to illuminate the mechanism of mycelia growth and biosynthesis of extracellular polysaccharides (EPS) by P. igniarius, the optimization of fermentation process was carried out. The seed culture with a large amount of loose pellets and a chemical defined media for mycelial growth and EPS synthesis were obtained. Fermentation kinetics, mycelial morphology, contents of amino acids and the characterization of EPS were used to analyze the metabolic regulation of EPS biosynthesis. Response surface methodology was used for optimizing extraction conditions of mycelia and the preliminary characterization of EPS was analyzed. The anti-hyperlipemia activity of intracellular polysaccharides (IPS) and EPS was analyzed by adult rats. The main results were as follows: 1. The optimization of fermentation process including seed culture and a chemical defined media were carried out. Glass beads and seed age affected the amount of pellets formation. The optimal seed medium was including 3% glucose,1.5% yeast source,0.1% MgSO₄ and 0.1% KH₂PO₄. Inoculation volume and inoculation time were 10% and 96 h, respectively. During the seed culture, a glass bead was used to increase the amount of pellets. After optimization of seed culture, the amounts of pellets were increased 62.5%. The diameter of pellets and core pellets decreased 39.1% and 30.3%, compared with control. A chemically defined medium for mycelial growth and EPS production by submerged culture of P. igniarius was investigated. The mainly defined medium compositions were optimized by orthogonal matrix method. The optimal defined medium (per liter) included 40.0 g glucose,4.0 g glutamic acid,4.0 g (NH₄)₂SO₄,1.0 g MgSO₄ 1.0 g KH₂PO₄ and initial pH value was 6.0. Under the optimal medium, the maximal mycelial biomass and EPS production were 12.33±0.89 g. L"1 and 1.21±0.08 g. L^-1 at 192 hour in shake flask.2. Effects of different kinds of carbon sources and phytohormones on EPS biosynthesis were studied. Our results showed that lactose was able to significantly increase the production of mycelia and EPS. Plant oil was benefit to mycelia growth. Especially, rapeseed oil significantly increased the production of mycelia, whereas it inhibited EPS synthesis. Adding 3% lactose in initial medium was benefical for EPS biosynthesis, and the maximal production of dry cell weight (DCW) and EPS were 17.436±2.227 g. L^-1 and 0.963±0.391 g. L^-1, respectively, while adding 1% rapeseed oil in initial medium was helpful for mycelia growth, the maximal production of DCW and EPS were 34.633±1.6 g. L^-1 and 0.73±0.063 g. L^-respectively. Morphology analysis revealed that some of mycelia cultured in lactose were hollow, but mycelia cultured in rapeseed oil were robust. Effect of phytohormones including indole-3-acetic acid (IAA), indoIe-3-butyric acid (IBA) and 1-naphthalentacetic acid (NAA) on mycelial growth of medicinal mushroom Phellinus linteus were investigated. The production of DCW and EPS with addition of5.0mg. L^-1 NAA was 6.24±0.18 g. L^-1 at 168 h and 0.86±0.01 g. L^-1 at 192 h, which were enhanced by 15.98% and 56.36% compared to the control, respectively. However, the molecular weight and infrared spectrum of EPS were coincident with the control. Results indicated that NAA at the proper concentration was beneficial in stimulating mycelial growth and EPS biosynthesis, whereas it could not alter the molecular structure of EPS. The fermentation dynamic analysis showed thet specific growth rate was accord with specific production rate. These results indicated that the positive effect of NAA on EPS biosynthesis maybe cause by enhancing the mycelial growth.3. The methods of high performance liquid chromatography, infrared spectrometry and gas chromatography were used to analysis the preliminary character of EPS. The results showed that it contained 49.7% sugar,18.0% protein. It contained four parts of polysaccharide, molecular weight (g. mol^-1) were 6.4×10⁶,3.3xl0⁵,2.7×10⁵ and 2.9xlO³, respectively. The structure of monosaccharide was mainly galactose according to gas chromatogphy. The structure of polysaccharide could be composed ofβ-galactan.4. Response surface methodology was applied to optimize the extraction conditions of IPS of P. igniarius. The optimal conditions were extraction temperature 70℃, extraction time 1.5 h and the ratio of mycelia to water 1:6.2. Under the optimal condition, the maximal yield of crude IPS from mycelia was 50.39±0.41 mg. g^-1. Compared with the extraction of dry power mycelia, the optimal extraction process was favor to save extraction time and decrease extraction temperature.5. The EPS and IPS extracted from mycelia can obviously reduce the level of the blood cholesterol (TC), serum triglyceride (TG), and serum low-density lipoprotein (LDL), and increase the level of high-density lipoprotein (HDL) of the hyperlipemia rats. Polysaccharide could decrease the level of TC, TG and LDL remarkably with decreasing rate of 19.7%,28.3% and 32.9%. Polysaccharide could increase the levels of HDL with the highest rates of 12.6%. Polysaccharides showed a significant effect of antihyperlipidaemia activity. The results indicated that polysaccharides from submerged culture of medicinal fungus have favorable potency to develop anti-hyperlipermia drugs.