墨汁鬼伞液体发酵培养条件及生物活性研究
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摘要
墨汁鬼伞(Coprinus atramentarius)又名柳树蘑,柳树钻,是鬼伞属中最有代表性的一种,在真菌界被认定为一种不可食用品种,但其同时具有多种医学保健功能。当前关于墨汁鬼伞的研究报道主要是固体栽培及生物学特性研究,未见关于墨汁鬼伞液体发酵培养的报道,也没有针对其生物活性物质的深入研究。
本文对墨汁鬼伞进行液体发酵深层培养,对影响液体发酵的培养基和发酵条件进行全面研究,确定其初步培养条件,然后以菌体生长和胞外多糖为指标对培养基和发酵条件进行了优化。通过全因子中心组合实验设计对发酵培养基进行优化表明:当葡萄糖、玉米粉、麸皮、KH_2PO_4和MgSO_4·7H_2O分别为13.51、17.07、7.62、2.29和2.00 g/L时,胞外多糖最大预测值为293.73 mg/L;当上述变量为10.07、18.02、11.63、1.58和4.00 g/L时,菌丝体最大生长量为6.24 g/L。通过Box-Behnken实验设计对发酵条件进行了优化,得到当温度、转速、培养时间、装液量分别为28.4℃、134.5 r/min、119.28 h、180.5 mL/500 mL时,胞外多糖最大预测值为415.58 mg/L;当上述变量为20.45℃、129 r/min、75.12 h、194.5 mL/500 mL时,菌丝体最大生长量为7.26 g/L。
对墨汁鬼伞发酵液进行初步分离,得到六个粗组分,为胞内外粗蛋白、粗多糖,及胞内外去除蛋白和多糖的小分子组分。
体外试验表明,胞外蛋白和胞外多糖的抗氧化活性最强,非酶糖基化反应抑制率最高。剂量0.1 mg/mL时,胞外蛋白羟自由基清除率为73.57 %(同浓度Vc为66.74 %),超氧自由基清除率为79.36 %(同浓度Vc为65.74 %);胞外多糖羟自由基清除率为59.10 %,超氧自由基清除率为52.15 %。剂量0.1 mg/mL时,胞外蛋白和多糖非酶糖基化反应抑制率分别为65.70 %、42.36 %。活性验证试验表明,墨汁鬼伞胞外多糖和胞外蛋白具有良好的抗氧化活性和降血糖功效。
为更好地发挥墨汁鬼伞液体发酵成分的活性功能,需对活性粗组分进行分离纯化,精制成高纯度的具有生物活性的制剂。本文对胞外蛋白和胞外多糖进行了初步分离纯化。采用?KTA explorer 100及与其相配套的标准DEAE-纤维素柱和标准凝胶柱对墨汁鬼伞胞外多糖进行了初步分离纯化。采用TOYOPEARL CM-650 M、TOYOPEARL SP-650 M离子交换柱对墨汁鬼伞胞外蛋白进行分离,检测各个组分的体外抗氧化活性,通过SDS-PAGE对活性组分的分子量进行初步探明。
Coprinus atramentarius, a typical species belonging to the genus Agaricus, has been designated as a kind of pharmaceutical fungi with favorable health care function. Solid state fermentation of C. atramentarius has been reported, and most research on this macrofungus at present has been concentrated on the aspect of basic biological characteristics investigation. Few reports regarding its biological active components and other culture method have been obtained.
In this thesis, liquid fermentation has been acquired and researches on the substance with biological activities have been carried out. At the stage of liquid fermentation study, biomass and the yield of extracellular polysaccharide have been taken as the index. Extensive one-factor experiments revealed the key factors from various culture medium components and fermentation conditions. RSM (response surface methodology) was taken advantage of to detect the relationship between the key factors. CCD (central composite design) and Box-Behnken experiment design were adopted for culture medium and fermentation conditions optimization, respectively. By solving the quadratic regression model equation using appropriate statistic methods, the optimum concentrations for the submerged fermentation medium were determined: glucose, corn powder, wheat bran, KH_2PO_4 and MgSO_4·7H_2O were 13.51, 17.07, 7.62, 2.29 and 2 g/L, respectively, then the predicted yield of the extracellular polysaccharide was 293.73mg/L. When the above values were 10.07, 18.02, 11.63, 1.58 and 4 g/L, the maximum biomass was 6.24 g/L. And the second RSM analysis disclosed the optimum fermentation conditions for the submerged fermentation were: temperature28.4℃, shaking rate134.5 r/min, culture time 119.28 h, volume of medium 186.5 mL/500 mL, then the predicted yield of the extracellular polysaccharide was 415.58 mg/L. When the above values were 20.45℃、129 r/min、75.12 h、194.5 mL/500 mL, the maximum biomass was 7.26 g/L.
Fractional alcohol precipitation was made use of to divide the C. atramentarius broth into six intra- and extra-cellular components. Free radicals scavenging efficiency is one reliable index meaning to the antioxidant activities as well as the inhibition rate of non-enzymatic glycation reaction to the hypoglycemic effect. Hydroxyl radicals and super-oxide anion scavenging efficiency were measured, and the non-enzymatic glycation reaction inhibition rate of the six components was detected.
Biological activities determination proved the extra-cellular protein and the extra-cellular polysaccharide to be the components with most satisfactory antioxidant activity and hypoglycemic effect.
Further separation and purification were essential to acquire more information about the active substances, as well as to make use of them. The extra-cellular polysaccharide was purified by DEAE and Superdex 200, and TOYOPEARL SP-650M was utilize to purify the extra-cellular protein. From the extra-cellular protein, one protein named EPP was determined to possess both antioxidant activity and hypoglycemic effect.
本文对墨汁鬼伞进行液体发酵深层培养,对影响液体发酵的培养基和发酵条件进行全面研究,确定其初步培养条件,然后以菌体生长和胞外多糖为指标对培养基和发酵条件进行了优化。通过全因子中心组合实验设计对发酵培养基进行优化表明:当葡萄糖、玉米粉、麸皮、KH_2PO_4和MgSO_4·7H_2O分别为13.51、17.07、7.62、2.29和2.00 g/L时,胞外多糖最大预测值为293.73 mg/L;当上述变量为10.07、18.02、11.63、1.58和4.00 g/L时,菌丝体最大生长量为6.24 g/L。通过Box-Behnken实验设计对发酵条件进行了优化,得到当温度、转速、培养时间、装液量分别为28.4℃、134.5 r/min、119.28 h、180.5 mL/500 mL时,胞外多糖最大预测值为415.58 mg/L;当上述变量为20.45℃、129 r/min、75.12 h、194.5 mL/500 mL时,菌丝体最大生长量为7.26 g/L。
对墨汁鬼伞发酵液进行初步分离,得到六个粗组分,为胞内外粗蛋白、粗多糖,及胞内外去除蛋白和多糖的小分子组分。
体外试验表明,胞外蛋白和胞外多糖的抗氧化活性最强,非酶糖基化反应抑制率最高。剂量0.1 mg/mL时,胞外蛋白羟自由基清除率为73.57 %(同浓度Vc为66.74 %),超氧自由基清除率为79.36 %(同浓度Vc为65.74 %);胞外多糖羟自由基清除率为59.10 %,超氧自由基清除率为52.15 %。剂量0.1 mg/mL时,胞外蛋白和多糖非酶糖基化反应抑制率分别为65.70 %、42.36 %。活性验证试验表明,墨汁鬼伞胞外多糖和胞外蛋白具有良好的抗氧化活性和降血糖功效。
为更好地发挥墨汁鬼伞液体发酵成分的活性功能,需对活性粗组分进行分离纯化,精制成高纯度的具有生物活性的制剂。本文对胞外蛋白和胞外多糖进行了初步分离纯化。采用?KTA explorer 100及与其相配套的标准DEAE-纤维素柱和标准凝胶柱对墨汁鬼伞胞外多糖进行了初步分离纯化。采用TOYOPEARL CM-650 M、TOYOPEARL SP-650 M离子交换柱对墨汁鬼伞胞外蛋白进行分离,检测各个组分的体外抗氧化活性,通过SDS-PAGE对活性组分的分子量进行初步探明。
Coprinus atramentarius, a typical species belonging to the genus Agaricus, has been designated as a kind of pharmaceutical fungi with favorable health care function. Solid state fermentation of C. atramentarius has been reported, and most research on this macrofungus at present has been concentrated on the aspect of basic biological characteristics investigation. Few reports regarding its biological active components and other culture method have been obtained.
In this thesis, liquid fermentation has been acquired and researches on the substance with biological activities have been carried out. At the stage of liquid fermentation study, biomass and the yield of extracellular polysaccharide have been taken as the index. Extensive one-factor experiments revealed the key factors from various culture medium components and fermentation conditions. RSM (response surface methodology) was taken advantage of to detect the relationship between the key factors. CCD (central composite design) and Box-Behnken experiment design were adopted for culture medium and fermentation conditions optimization, respectively. By solving the quadratic regression model equation using appropriate statistic methods, the optimum concentrations for the submerged fermentation medium were determined: glucose, corn powder, wheat bran, KH_2PO_4 and MgSO_4·7H_2O were 13.51, 17.07, 7.62, 2.29 and 2 g/L, respectively, then the predicted yield of the extracellular polysaccharide was 293.73mg/L. When the above values were 10.07, 18.02, 11.63, 1.58 and 4 g/L, the maximum biomass was 6.24 g/L. And the second RSM analysis disclosed the optimum fermentation conditions for the submerged fermentation were: temperature28.4℃, shaking rate134.5 r/min, culture time 119.28 h, volume of medium 186.5 mL/500 mL, then the predicted yield of the extracellular polysaccharide was 415.58 mg/L. When the above values were 20.45℃、129 r/min、75.12 h、194.5 mL/500 mL, the maximum biomass was 7.26 g/L.
Fractional alcohol precipitation was made use of to divide the C. atramentarius broth into six intra- and extra-cellular components. Free radicals scavenging efficiency is one reliable index meaning to the antioxidant activities as well as the inhibition rate of non-enzymatic glycation reaction to the hypoglycemic effect. Hydroxyl radicals and super-oxide anion scavenging efficiency were measured, and the non-enzymatic glycation reaction inhibition rate of the six components was detected.
Biological activities determination proved the extra-cellular protein and the extra-cellular polysaccharide to be the components with most satisfactory antioxidant activity and hypoglycemic effect.
Further separation and purification were essential to acquire more information about the active substances, as well as to make use of them. The extra-cellular polysaccharide was purified by DEAE and Superdex 200, and TOYOPEARL SP-650M was utilize to purify the extra-cellular protein. From the extra-cellular protein, one protein named EPP was determined to possess both antioxidant activity and hypoglycemic effect.
引文
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77 Wang Y X, Lu Z X . Optimization of processing parameters for the mycelial growth and extracellular polysaccharide production by Boletus spp. ACCC 50328[J]. Process Biochemistry, 2005, 40: 1043-1051.
78 Han C C, Yuan J H, Wang Y Z, et al. Hypoglycemic activity of fermented mushroom of Coprinus comatus rich in vanadium[J]. Journal of Trace Elements in Medicine and Biology, 2006(20): 191-196.
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81 Francis F, Sabu A, Madhavan K N, et al. Use of response surface methodology for optimizing process parameters for the production ofα-amylase by Aspergillus oryzae[J]. Biochemical Engineering Journal, 2003, 15: 107-115.
82 Murat E. Response surface methodological approach for inclusion of perfluorocarbon in actinorhodin fermentation medium[J]. Process Biochemistry, 2002, 38: 667-673.
83 Adinarayana K, Ellaiah P, Srinivasulu B, et al. Response surface methodological approach to optimize the nutritional parameters for neomycin production by Streptomyces marinensis under solid-state fermentation[J]. Process Biochemistry, 2003, 38: 1565-1572.
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85张惟杰主编.糖复合物生化研究技术(第二版)[M].杭州,浙江大学出版社, 1999.
86 Fhernanda R S, Elaine R. Structural characterization of a polysaccharide and aβ-glucan isolated from the edible mushroom Flammulina velutipes[J]. Phytochemistry, 2006, 67: 2189-2196.
87 Hisiao Y M, Huang Y L, Tang S C, et al. Effect of a fungal immunomodulatory protein from Ganoderma tsugae on cell cycle and interferon-gamma production through phosphatidylinositol 3-kinase signal pathway[J]. Process Biochemistry, 2008, 43: 423-430.
88 Wang H X, Ng T B. Isolation of a novel ubiquitin-like protein from Pleurotus astreatus mushroom with anti-human immunodeficiency virus, translation-inhibitiory and ribonuclease activies. Biochem Biophys Res Commun, 2000, 276(2): 587-593.
89 Yu L G, Fernig D G, White M R, et al. Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibts epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear import. J Biol Chem, 1999, 274(8): 4890-4899.
90 Lam S K, Ng T B. Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem Biophys Res Commun, 2001, 285(4): 1071-1075.
91 Cui Y, Kma D S, Park K C. Antioxidant effects of Inonotus obliquus[J]. J Ethnopharmacol, 2005, 96(1-2): 79-85.
92 Michal Z, Andrej S, Anna S. Antioxidant and radical-scavenging activities of Slovak honeys– An electron paramagnetic resonance study[J]. Food chemistry, 2008, 110: 512-521.
93 Barbara R, Lopes R, Paula B A, et al. Comparative study of phytochemicals andantioxidant potential of wild edible mushroom caps and stipes[J]. Food chemistry, 2008, 110: 47-56.
94丁重阳.鸡腿蘑(Coprinus comatus)防治糖尿病生物活性物质结构鉴定及液体发酵的研究[D].南京,南京农业大学, 2007.
95 Mustafa K, Tepe B. Chemical composition, antioxidant and antimicrobial properties of the essential oils of three Salvia species from Turkish flora[J]. Bioresource technology, 2008, 99:4096-4104.
96李建武,肖能赓,余瑞元等.生物化学实验原理和方法[M].北京:北京大学出版社, 1994: 125.
97蔡武城,袁厚积.生物物质常用化学分析法[M].北京:科学出版社, 1982: 95.