白舞菇多醣最適化搖瓶培養條件及其高壓粗萃物之抗發炎效果

Flask culture of White Grifola frondosa for the optimization condition of production polysaccharides and its anti-inflammatory effect by high pressure extraction

陳英維(Ying-Wei Chen)

指導教授 ： 徐源泰

國立臺灣大學/生物資源暨農學院/園藝暨景觀學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01361

摘要

白舞菇 ( Grifola frondosa ) 為擔子菌綱多孔菌屬之食藥用真菌，是黑舞菇的變異種，因外觀呈白色而有較佳的商品價值。近年來，多數相關研究指出代謝產物-多醣為主要活性成分之一，這些自子實體及菌絲體萃取所得之多醣體具有抗腫瘤、保肝及增強免疫力等多種藥理功效。本研究欲增加白舞菇菌絲體多醣產量以利後續進行機能性分析，故利用搖瓶培養白舞菇菌絲體，藉由改變碳氮源、最適碳氮源濃度及初始pH值，探討其對菌絲生長及多醣產量之影響。並進一步將前述試驗結果所得之菌絲體，以熱水及不同條件之高壓進行萃取，分析菌絲體多醣粗萃物對小鼠巨噬細胞株RAW 264.7之抗發炎活性。
實驗結果發現，葡萄糖、果糖、蔗糖及玉米澱粉等四種碳源中，以玉米澱粉可得最高菌絲濃度9.24±0.12 g/L，而以果糖可得高多胞內多醣產量0.12±0.004 g/g DCW。在20、30、40及50 g/L果醣濃度中，以30 g/L果醣可得最高菌體濃度，而以20 g/L果糖可得最高胞內多醣產量。酵母萃取物 (Yeast Extract, YE) 及麥芽萃取物等兩種氮源中，以YE可得最高菌絲及多醣產量。培養基初始pH 4、5、6、7中，以pH 6.0可得最高菌絲及多醣產量，pH 5.0次之。在萃取率方面，以熱水萃取可得最高菌絲體粗多醣萃取率，高壓組中，冷凍乾燥菌絲體粉末以400 MPa可得最高菌絲體粗多醣萃取率，其餘則無顯著差異，而經500 MPa熱風乾燥菌絲體又比經400 MPa冷凍乾燥菌絲體增加20%萃取率。雖然高壓萃取無法顯著提升粗多醣萃取率，但具有可改進萃取之效用。另一方面，將熱水及高壓萃取所得菌絲體多醣粗萃物，與小鼠巨噬細胞株RAW 264.7進行處理，以脂多醣 (LPS) 刺激細胞使其發炎後，藉由三種模式 (預發炎、修復發炎及共培養模式)，偵測前發炎因子NO、ROS含量，評估菌絲體多醣粗萃物之抗發炎效果。MTS結果顯示，菌絲體多醣粗萃物對小鼠巨噬細胞株RAW 264.7不具毒性，細胞存活率皆達90%以上。各處理於預發炎及修復發炎模式下，皆可顯著抑制或減少NO生成之效果，且具濃度效應，其中又以預發炎模式效果最佳。而在共培養模式下，各處理均無法減少NO生成。
綜合上述研究結果，不同培養條件會影響白舞菇菌絲體的生長及代謝產物多醣的產量，因此可根據目標產物，選擇最適化的培養條件，作為未來以發酵槽放大生產的參考依據。在抗發炎活性方面，熱水萃取及高壓萃取所得之菌絲體粗多醣萃取物，具有可預防NO生成之效果，故具有抗發炎之潛力，未來可將白舞菇多醣應用在健康食品上，增進其產業利用性及附加價值。

關鍵字

白舞菇 ； 多醣 ； 高壓萃取 ； 小鼠巨噬細胞株 ； 抗發炎活性

並列摘要

The edible mushroom White Grifola frondosa is a Basidiomycete fungus belongs to the family Polyporaceae. For recent years, most studies related to Grifola frondosa are focusing on pharmacological and biological activities of polysaccharide. Polysaccharide extracted from the fruiting body and mycelium have been reported to have medical effects such as antitumor, hepato-protective activities and also can strengthen the immune system. In this study, White Grifola frondosa was cultivated in shaking flask under different media compositions and conditions in order to get the higher production of mycelial biomass and polysaccharide. Moreover, compared with the impact of mycelial polysaccharide by hot water and high hydrostatic pressure extraction and evaluated its anti-inflammatory activity in RAW 264.7 cell line.
Among four kinds of carbon sources-Glucose, Fructose, Sucrose and Corn Starch, the result showed that Corn Starch was the most suitable carbon source for both mycelial biomass production. While Fructose can got more production of intracellular polysaccharide which was 20 grams per liter of medium. The use of Yeast Extract was the optimal nitrogen source than Malt Extract for both mycelial biomass and polysaccharide production. In addition, among four tested initial pH in cultural media, pH 6.0 was the optimal condition for the production of mycelial biomass and polysaccharide, followed by pH5. In terms of extraction, the crude extract of vacuum freeze drying mycelial polysaccharide obtained the higher extraction yield by hot water extraction than high hydrostatic pressure extraction, followed by 400 MPa but no significant difference between 50-200 MPa. Besides, the extraction of hot air drying mycelial by 500 MPa improved 20% than vacuum freeze drying by 400 MPa. Although there is no obvious enhancement on the crude extract of mycelial polysaccharide by High Hydrostatics Pressure extraction, it can improve the extraction. On the other hand, the crude extracts of mycelial polysaccharide (CEMP) by hot water and high hydrostatic pressure on the production of pro-inflammatory factors (NO, ROS) in the three lipopolysaccharide-stimulated inflammation models (preventive inflammation model, repairing inflammation model and co-treatment model) with RAW 264.7 cell line were evaluated. Effect of CEMP on the production of NO were measured by three models (preventive inflammation model, repairing inflammation model and co-treatment inflammation model). Cell viability results showed that crude extract of mycelial polysaccharide had no significant cytotoxicity in RAW 264.7 cell line. All of CEMP significantly inhibited or decreased the production of NO with dose-dependent manner on preventive and repairing inflammation models. Especially, the preventive inflammation model had the best effect on inhibition of NO production. However, all of CEMO on co-treatment inflammation model had no effect on inhibition of NO production.
In conclusion, all of the above results demonstrated that different nutritions and environmental factors influenced mycelial growth and polysaccharide production. The finding may provide a useful information in the future for production of White Grifola frondosa on a large scale. Moreover, all of CEMP by hot water and high hydrostatic pressure extraction had better anti-inflammatory activity in preventive inflammation models… Therefore, CEMP can be applied to the development of functional food to increase the industrial utilities and to promote the additional values of White Grifola frondosa.

並列關鍵字

White Grifola frondosa ； polysaccharide ； high hydrostatic pressure extraction ； RAW 264.7 cell line ； anti-inflammation activity

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