Study On Submerged Fermentation Of Shiraia Polysaccharide And Its Antioxidant Activity And Structure

Shiraia bambusicola is a parasitic fungus on bamboo branches,its peduncle has high medicinal value as a traditional Chinese medicine,and can be used to treat various diseases,such as rheumatoid arthritis,sciatica,injury,chronic bronchitis,etc.The stilbene quinones and polysaccharides are the most important active ingredients in their stroma extracts.Current research shows that Shiraia polysaccharides have protective effects on liver injury in mice,and have certain antioxidant and anti-inflammatory activities.At present,the supply of Shiraia stroma is limited,and submerged fermentation is an effective and rapid method for large-scale production of medicinal fungi.In this project,based on two kinds of Sh.bambusicola isolated from the fruiting bodies of Shiraia from two different habitats,two kinds of Shiraia mycelium polysaccharides were obtained by submerged fermentation.The biological productivity,antioxidant capacity and immunoregulatory activity of the two kinds of mycelium polysaccharides were studied and compared with the corresponding stroma.The effects of medium components and fermentation conditions on polysaccharide content and antioxidant activity during submerged fermentation were further explored,and a submerged fermentation Shiraia polysaccharide culture system was constructed.Finally,the crude polysaccharide was separated and purified,and the structure of the obtained single polysaccharide component was identified and analyzed.The main results of the paper are as follows:（1）Determination of biological productivity and biological activity of stroma polysaccharides and mycelium polysaccharides:Crude polysaccharides were extracted from the Shiraia stroma and corresponding mycelia of the two places by water extraction and alcohol precipitation,determination of the biological productivity,antioxidant properties and immunomodulatory activity of polysaccharides.The results showed that in 100 g of lyophilized biomass of fermented mycelium,9.12±0.36 g of Zhejiang Shiraia mycelium polysaccharide（ZSIP）was produced,and the Zhejiang Shiraia stroma polysaccharide（ZSSP）yield was 3.94±0.92 g/100 g of biomass（DM）.At the same time,the polysaccharide content of Yunnan Shiraia mycelium polysaccharide（YSIP）and Yunnan Shiraia stroma polysaccharide（YSSP）yield was 5.44±0.28 g/100 g DM and 2.33±1.14 g/100 g DM,respectively.In addition,at a concentration of 5 mg/m L,the hydroxyl radical scavenging activities of ZSIP and ZSSP were 89.29%±1.36%and 47.54%±1.12%,respectively,5 mg/m L YSIP had a higher hydroxyl radical scavenging activity of 70.02%±1.02%than YSSP（56.10%±0.89%）.And the ZSIP and YSIP have 100%2,2′-diaza-bis（3-ethyl-benzothiazole-6-sulfonic acid）ammonium salt（ABTS）radical scavenging activity at a concentration of 3 mg/m L,which is equivalent to0.025 mg/m L of ascorbic acid.The immunoregulation results showed that ZSIP enhanced the production of IL-6 in RAW264.7 macrophages and increased the production of inducible nitric oxide synthase（i NOS）than ZSSP.In summary,it can be seen that the biological productivity and biological activity of Shiraia polysaccharides are significantly improved by submerged fermentation.（2）Construction and optimization of submerged fermentation Shiraia polysaccharide culture system:The effects of culture medium and fermentation conditions on the polysaccharide content and antioxidant activity of Sh.bambusicola after submerged fermentation were investigated.The basic components of the submerged fermentation medium were determined by single factor test（g/L）:30 g mannitol,24 g potato glucose,2.25g fish peptone,1 g K₂HPO₄·3H₂O and 0.50 g Mg SO₄·7H₂O,p H natural.The orthogonal experiment confirmed that the fermentation conditions were:inoculation amount 2%（v/v）,fermentation time 60 h,initial p H 7,under these conditions,polysaccharide yield reached11.85±0.42 g/100 g DM.（3）Isolation,purification and structure-activity relationship analysis of Shiraia polysaccharides:Sevag method and Sephadex G-150 column chromatography were used to prepare two pure polysaccharides,named SIP-1 and SIP-2.High-performance gel permeation chromatography（HPGPC）was used to detect the molecular weights of SIP-1 and SIP-2 of5.71×10³k Da and 0.10×10³k Da,respectively.The high-performance liquid chromatography（HPLC）determined that the monosaccharide components of SIP-1 and SIP-2 were mainly composed of mannose,glucose and galactose,and the molar ratios were 1.00:7.26:1.09 and1.00:1.52:1.05,respectively.Further structural studies of two pure polysaccharides were performed using infrared spectroscopy and nuclear magnetic resonance scanning,the results show that SIP-1 is anα-pyranoglucose mainly containingα-D-glucopyranose andβ-D-galactopyranosyl;and SIP-2 is aβ-pyranose glucan containingβ-D-glucopyranose,β-D-mannopyranoside andβ-D-galactopyranosyl.The in vitro antioxidant activity of pure polysaccharides was measured.At 2 mg/m L,the hydroxyl radical scavenging rates of SIP-1and SIP-2 were 39.76%±0.57%and 91.22%±2.97%.At the same time,at 4 mg/m L,compared with the ABTS⁺radical scavenging ability of SIP-1（67.40%±3.61%）,SIP-2 has a stronger scavenging ability（99.07%±0.99%）.Different structures may be responsible for their different antioxidant activities.This research is of great significance to the fermentation of polysaccharides from Sh.bambusicola,and provides new ideas for the development of other high-bioactive medicinal fungal products,which has theoretical significance and application value.