Title

雞腿菇和花松茸之液態培養及其生理活性

Translated Titles

Submerged cultivation of Coprinus comatus and Sparassis crispa and their physiological activities

DOI

10.6845/NCHU.2006.00081

Authors

蔡淑瑤

Key Words

雞腿菇 ; 花松茸 ; 液態培養 ; 品質評估 ; 抗氧化性質 ; 多醣 ; 癌細胞毒性 ; Coprinus comatus ; Sparassis crispa ; submerged culture ; quality evaluation ; antioxidant properties ; polysaccharides ; tumor cytotoxicity

PublicationName

中興大學食品暨應用生物科技學系所學位論文

Volume or Term/Year and Month of Publication

2006年

Academic Degree Category

博士

Advisor

毛正倫

Content Language

繁體中文

Chinese Abstract

本研究探討雞腿菇及花松茸之液態深層培養的條件,冀能提高雞腿菇和花松茸菌絲體及胞外多醣產量,並分析培養所得菌絲體及發酵液之品質特性、抗氧化性質及對腫瘤細胞之毒性,且評估以熱水及鹼所萃取之多醣的理化性質及對癌細胞毒性之研究。 雞腿菇(Coprinus comatus (Mull.: Fr.) S. F. Gray)因其形狀像雞腿而得名,亦稱毛頭鬼傘,是一種食藥兼用的真菌類。經研究發現雞腿菇具有降血糖、提高免疫活性、抗腫瘤、抑菌、抑制病毒和抗致突變性等功效。花松茸(Sparassis crispa(Wulf.)Fr.),別名為繡球菌、山珊瑚和甘藍蘑菇等。目前經研究則發現花松茸所分離出β-葡聚醣,具有抗腫瘤能力及免疫調節作用。 一般人工栽培由接種到生成子實體需耗費很長的時間,應用發酵技術生產菌絲體及多醣,具有發酵時間短及品質穩定。本研究乃在探討雞腿菇和花松茸之液態培養,以獲得生產菌絲體及胞外多醣之最適培養基組成。在雞腿菇液態培養部分,經探討後,在振盪培養轉速 100 rpm 及 6 天的培養時間,發現使用碳源和氮源分別為 2% 果糖和 0.5% 酵母萃取物時,並將酸鹼值調成 pH 5.0 和 20°C的培養溫度下,可獲得最高產量的菌絲體(7.64 g/L)和胞外多醣(0.58 g/L)。在花松茸液態培養部分,經探討後,在振盪培養轉速 100 rpm 及 6 天的培養時間,發現使用碳源和氮源分別為 2% 葡萄糖和 0.5% 酵母萃取物時,並將酸鹼值調成 pH 4.0 和 20°C的培養溫度下,可獲得最高產量菌絲體(8.73 g/L)和胞外多醣(0.45 g/L)。 本研究進一步針對雞腿菇及花松茸兩種食藥用菇之菌絲體及發酵液的非揮發性呈味成分及生理活性物質予以探討。結果發現,這兩種菇類的菌絲體和發酵液都含有多量的碳水化合物。雞腿菇菌絲體、雞腿菇發酵液、花松茸菌絲體及花松茸發酵液之總可溶性糖和糖醇含量分別為111.84、523.61、61.37和556.73 mg/g,其中在雞腿菇和花松茸之發酵液中以葡萄糖含量最高,分別為445.44和494.75 mg/g。而雞腿菇菌絲體、雞腿菇發酵液、花松茸菌絲體及花松茸發酵液之總游離胺基酸的含量分別為18.03、91.13、24.41和81.78 mg/g。另外,類味精(MSG-like)成分的含量在兩種發酵液中分別為15.81及17.73 mg/g,多於兩種菌絲體所含有的3.44及3.91 mg/g;而呈味5'-核苷酸的含量在兩種發酵液中為2.71及3.20 mg/g,多於兩種菌絲體所含有的1.72及2.20 mg/g。以等價鮮味值來看,雞腿菇菌絲體、雞腿菇發酵液、花松茸菌絲體及花松茸發酵液分別相當於52、415、70 和315 g MSG/100 g。整體而言,雞腿菇和花松茸之發酵液具有非常強的鮮味。 雞腿菇和花松茸之菌絲體和發酵液,針對其乙醇和熱水萃取物之抗氧化性質及所含抗氧化成分加以探討。從所得結果可了解,雞腿菇菌絲體熱水萃比乙醇萃有較佳的抗氧化性質,包括抗氧化力、還原力、清除DPPH和羥自由基能力;而雞腿菇發酵液熱水萃比乙醇萃有較佳的抗氧化性質,包括抗氧化力、還原力、清除羥自由基能力和螯合亞鐵離子之能力,其中以雞腿菇菌絲體熱水萃之整體抗氧化性質最佳。花松茸菌絲體乙醇萃比熱水萃有較佳的抗氧化性質,包括抗氧化力、還原力、清除DPPH自由基能力,而花松茸發酵液熱水萃比乙醇萃有較佳的抗氧化性質,包括抗氧化力、清除羥自由基能力和螯合亞鐵離子之能力,其中以花松茸菌絲體乙醇萃之整體抗氧化性質最佳。整體而言,雞腿菇和花松茸之菌絲體具有較佳的抗氧化性質。 另外,以鹼溶液比熱水能萃取出更多之雞腿菇及花松茸多醣,而菌絲體不管是熱水萃或鹼萃,其多醣萃取率都是最高的。分析雞腿菇及花松茸多醣組成分可知,發酵液不管以熱水萃取或是鹼萃取,其多醣含量都是最高的。雞腿菇及花松茸鹼萃多醣之微量蛋白含量高於熱水萃多醣。由元素分析發現雞腿菇及花松茸鹼萃多醣含N、C及H之量皆高於雞腿菇及花松茸熱水萃多醣所含。由中性單糖組成分析來看,發現雞腿菇及花松茸之熱水萃及鹼萃多醣主要是由甘露糖和葡萄糖所組成。以膠體層析法得知雞腿菇與花松茸熱水萃多醣的分子量較其鹼萃多醣大。 以MTT分析法評估雞腿菇及花松茸之乙醇萃及熱水萃對腫瘤細胞生長的抑制情形,並以鹼萃及熱水萃所純化的雞腿菇及花松茸多醣對腫瘤細胞生長情況,進行評估及比較。另外,藉由對A549和SVEC細胞株之細胞週期分析結果,以確定其毒殺腫瘤細胞之可能途徑。結果發現,雞腿菇之子實體乙醇萃、菌絲體乙醇萃、子實體鹼萃多醣和菌絲體鹼萃多醣對癌細胞之生長抑制率IC50,在A549分別為0.69、1.69、0.24和2.58 mg/mL;在HCT116分別為1.72、1.53、1.14和1.58 mg/mL;在HL-60分別為2.22、0.83、nd和2.29 mg/mL、在MCF-7分別為0.94、1.80、1.11和3.22 mg/mL、在SK-Hep-1分別為1.25、1.98、2.56和8.46 mg/mL;以及在SVEC分別為0.88、1.95、3.25和5.53 mg/mL。而花松茸菌絲體乙醇萃對癌細胞之生長抑制率IC50分別為A549之1.22 mg/mL、HCT116之3.00 mg/mL、HL-60之1.66 mg/mL、MCF-7之2.14 mg/mL、SK-Hep-1之2.85 mg/mL和SVEC之1.70 mg/mL。對於A549和SVEC細胞株之細胞週期,除了雞腿菇菌絲體乙醇萃取物,明顯在G0/G1期有累積作用,可使細胞週期停滯於G0/G1期,而其它萃取物則對細胞週期比率無顯著的影響。 綜合上述得知雞腿菇和花松茸之菌絲體及發酵液皆含有豐富的營養成分及必需胺基酸和鮮味物質,且在抗氧化效果、抑制癌細胞生長之能力及多醣生理活性物質逐一被證實的情況下,不僅成為高蛋白質、低脂質及無膽固醇的健康食品,更提供食品調味的新選擇。

English Abstract

The research reported herein is designed to study the submerged culture of Coprinus comatus and Sparassis crispa to produce high contents dry biomass and polysaccharide of mycelia. Therefore, the evaluation of quality properties, antioxidant properties, and tumor cytotoxicity of mycelia and fermentation filtrate. Furthermore, the evaluation physicochemical properties of hot water and alkali extracts polysaccharides, and tumor cytotoxicity. Coprinus comatus (Muller: Fries) S. F. Gray (Coprinaceae), the shaggy mane or chicken drumstick mushroom and also known as the lawyer’s wig mushroom, is a newly cultivated edible and medicinal mushroom. C. comatus have been reported to lower blood glucose, immunomodulating, antitumor activity, antibacterial activity, antivirus activity, antimutagenic effect. Sparassis crispa (Wulf: Fries), also called cauliflower fungus, is a newly cultivated mushroom and looks like a cauliflower or coral. Recent research indicated β-(1,3)-D-glucan could be extracted from its fruiting bodies, which exhibited the effects of anti-tumor and immunoregulation. Growing mushroom mycelium in liquid culture on a defined nutrient broth has been a simple and fast alternative method to produce fungal biomass. The research reported herein was to study the optimal conditions for submerged culture of C. comatus and S. crispa, to evaluate the dry biomass and polysaccharides of their mycelia and fermentation filtrate. With regard to C. comatus submerged culture, the optimal conditions were pH 5.0, 20°C, 6 days and 100 rpm with carbon and nitrogen sources being 2% fructose and 0.5% yeast extract. Contents of dry biomass and polysaccharides from C. comatus were 7.64 and 0.58 g/L. With regard to S. crispa submerged culture, the optimal conditions were pH 4.0, 20°C, 6 days and 100 rpm with carbon and nitrogen sources being 2% glucose and 0.5% yeast extract. Contents of dry biomass and polysaccharides from S. crispa were 8.73 and 0.45 g/L. Furthermore, the non-volatile components and physiological activity in the two forms of C. comatus and S. crispa mycelia and filtrate were studied. Both mycelia and filtrate of C. comatus and S. crispa were high in contents of carbohydrate. Content of total sugars and polyols were 111.84, 523.61, 61.37 and 556.73 mg/g for C. comatus mycelia, C. comatus filtrate, S. crispa mycelia and S. crispa filtrate, respectively. Glucose contents were the highest in both C. comatus and S. crispa filtrate were 445.44 and 494.75 mg/g, respectively. Contents of total free amino acids were 18.03, 91.13, 24.41 and 81.78 mg/g for C. comatus mycelia, C. comatus filtrate, S. crispa mycelia and S. crispa filtrate, respectively. The contents of monosodium glutamate (MSG)-like components from C. comatus and S. crispa in filtrate (15.81 and 17.73 mg/g) were higher than that in mycelia (3.44 and 3.91 mg/g). The contents of flavor 5'-nucleotides from C. comatus and S. crispa in filtrate (2.71 and 3.20 mg/g) were higher than that in mycelia (1.72 and 2.20 mg/g). EUC values were 52, 415, 70 and 315 g MSG/100 g for C. comatus mycelia, C. comatus filtrate, S. crispa mycelia and S. crispa filtrate, respectively. Overall, filtrate of C. comatus and S. crispa possessed highly intense umami taste. In addition, to investigate the antioxidant properties of ethanolic and hot water extracts of mycelia and filtrate from C. comatus and S. crispa mycelia and filtrate, including antioxidant activity, reducing power, scavenging abilities on radicals and chelating abilities on metal ions. The contents of potential antioxidant components in these extracts were also determined. Hot water extracts were more effective than ethanolic extracts from C. comatus mycelia in antioxidant activity, reducing power, scavenging ability on DPPH and hydroxyl radicals as evidenced by lower EC50 values. Hot water extracts were more effective than ethanolic extracts from C. comatus filtrate in antioxidant activity, reducing power, scavenging ability on hydroxyl radicals and chelating ability on ferrous ions as evidenced by lower EC50 values. Overall, for both extracts, hot water extracts from C. comatus mycelia were more effective among antioxidant properties assayed. Ethanolic extracts were more effective than hot water extracts from S. cirpsa mycelia in antioxidant activity, reducing power, and scavenging ability on DPPH as evidenced by lower EC50 values. Hot water extracts were more effective than ethanolic extracts from S. crispa filtrate in antioxidant activity, scavenging ability on hydroxyl radicals and chelating ability on ferrous ions as evidenced by lower EC50 values. Overall, for both extracts, ethanolic extracts from S. crispa mycelia were more effective among antioxidant properties assayed. With regard to the hot water or alkali extraction from C. comatus and S. crispa fruit body, mycelia and filtrate, the yield was the highest in both mushrooms for mycelia polysaccharides. However, the highest total sugar contents were found in hot water and alkali extracts polysaccharides from filtrate. The microprotein contents of alkali extracts from C. comatus and S. crispa were higher than that of their hot water extracts polysaccharides. In elemental analysis, nitrogen, carbon and hydrogen contents of alkali extracts polysaccharides from C. comatus and S. crispa were higher than that of their hot water extracts polysaccharides. Neutral sugars were mannose and glucose for all polysaccharides isolated. Using gel filtration, the molecular weights of hot water extracts from C. comatus and S. crispa were higher than that of their alkali extracts. Besides, the studies of the effect of ethanolic and hot water extracts of C. comatus and S. crispa fruit body and mycelia and polysaccharide therefrom inhabited cancer cell viability were studied using MTT test. Furthermore, evaluation possibility mechanism of induced cytotoxicity in A549 and SVEC cell line by cell cycle analysis. IC50 values in A549 cell line were 0.69, 1.69, 0.24 and 2.58 mg/mL; in HCT116 cell line were 1.72, 1.53, 1.14 and 1.58 mg/mL; in HL-60 cell line were 2.22, 0.83, not detected and 2.29 mg/mL; in MCF-7 cell line were 0.94, 1.80, 1.11 and 3.22 mg/mL; in SK-Hep-1 cell line were 1.25, 1.98, 2.56 and 8.46 mg/mL; in SVEC cell line were 0.88, 1.95, 3.25 and 5.53 mg/mL for fruit body in ethanolic extracts, mycelia in ethanolic extracts, fruit body in alkali extracts polysaccharide, and mycelia in alkali extracts polysaccharide from C. comatus, respectively. IC50 values in A549 cell line was 1.22 mg/mL; in HCT116 cell line was 3.00 mg/mL; in HL-60 cell line was 1.66 mg/mL; in MCF-7 cell line was 2.14 mg/mL; in SK-Hep-1 cell line was 2.85 mg/mL; in SVEC cell line was 1.70 mg/mL for mycelia in ethanolic extracts from S. crispa, respectively. Cell cycle analysis revealed that ethanolic extracts of mycelia from C. comatus induced apoptosis on A549 via G0/G1 cell cycle arrest. Overall, mycelia and filtrate of C. comatus and S. crispa contained abundant nutritional components and essential amino acids and umami components, and bioactive polysaccharides, possessed antioxidant properties and tumor cytotoxicity. Based on the results, it is a high protein, low fat and no cholesterol health food, and is an alternative to food flavoring.

Topic Category 農業暨自然資源學院 > 食品暨應用生物科技學系所
生物農學 > 生物科學
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Times Cited
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