阿魏侧耳深层发酵条件及其胞外多糖理化性质的研究
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摘要
本研究主要包括阿魏侧耳液体培养基的筛选、发酵条件的优化和胞外多糖理化性质的分析三方面的内容,其结果如下:
1、通过单因子试验和正交试验,确定适合菌丝生长的优化培养基配方为:VBl0.002%,葡萄糖3%,蛋白胨0.15%,(NH4)_2SO_40.05%,酵母膏0.3,MgSO_40.05%,KH_2PO_40.1%,ZnSO_40.02%,NaCl0.03%。适合产生胞外多糖的优化培养基配方为:VBl0.002%,葡萄糖1%,蛋白胨0.15%,(NH4)_2SO_40.05%,酵母膏0.3,MgSO_40.05%,KH_2PO_40.05%,ZnSO_40.01%,NaCl0.03%。
2、确定摇瓶(250mL)发酵条件为:装液量100mL,接种量10%,起始pH5.0,培养温度25℃,摇床转速150r/min,接种菌龄5d,发酵10d。50L发酵罐发酵条件为装液量30L,接种菌龄5d,接种量10%,温度25℃,起始pH5.0,转速150r/min,通气量1.2m~3/h,罐压0.04Mpa,培养108h。
3、DEAE-纤维素柱层析结果表明:胞外水溶性多糖含有一个中性多糖组分和一个糖蛋白组分。
4、气相色谱分析结果表明:胞外水不溶性多糖由鼠李糖、葡萄糖和4种未知单糖组成,鼠李糖和葡萄糖的摩尔比为:鼠李糖:葡萄糖=4.08:5.55
This paper included mainly three aspects: screening of liquid culture medium of Pleurotus ferulae Lanzi, optimization of fermentation conditions, analyses of physical and chemical properties of EPS, the results of which were as follows:1. By monofactorial and orthogonal test, the optimal composition of liquid culture medium for the mycelia's growth and the producing of EPS were respectively determined. The former was composed by 0.002% VB_1, 3% Glucose, 0. 15% Peptone, 0. 05% (NH4)_2SO_4, 0.3% extract of yeast, 0.05% MgSO_4, 0. 1%KH_2PO_4,0.02%ZnSO_4 , 0. 03%NaCl. The later was composed by 0.002% VB_1, 1% Glucose, 0. 15% Peptone, 0.05% (NH4)_2SO_4, 0.3% extract of yeast, 0.05% MgSO_4, 0. 05%KH_2PO_4, 0. 01%ZnSO_4, 0. 03%NaCl.2. The optimal flask (250ml) fermentation conditions were as follows: culture volume was 100mL, inoculum quantity was 10%, initial pH value was 5.0, culture temperature was 25℃, rotation speed was 150rpm, strain age of spawn was 5 days, culture time was 10 days.The optimal jar (50L) fermentation conditions were as follows: culture volume was 30L, strain age of spawn was 5 days, inoculum quantity was 10%, initial pH value was 5.0, culture temperature was 25 ℃, rotation speed was 150rpm, ventilation quantity was 1. 2m~3/h , pressure inside the jar was 0. 04Mpa , culture time was 108 hours.3. The result of DEAE-cellulose elution indicated that there was one neutral polysaccharide and one glycoprotein in the water-soluble EPS.4. Analysed with gas chromatography the monosaccharide composition and molecular ratio of water-fast EPS was gained: rha:glu = 4. 08:5. 55, and 4 unknownmonosaccharide.
1、通过单因子试验和正交试验,确定适合菌丝生长的优化培养基配方为:VBl0.002%,葡萄糖3%,蛋白胨0.15%,(NH4)_2SO_40.05%,酵母膏0.3,MgSO_40.05%,KH_2PO_40.1%,ZnSO_40.02%,NaCl0.03%。适合产生胞外多糖的优化培养基配方为:VBl0.002%,葡萄糖1%,蛋白胨0.15%,(NH4)_2SO_40.05%,酵母膏0.3,MgSO_40.05%,KH_2PO_40.05%,ZnSO_40.01%,NaCl0.03%。
2、确定摇瓶(250mL)发酵条件为:装液量100mL,接种量10%,起始pH5.0,培养温度25℃,摇床转速150r/min,接种菌龄5d,发酵10d。50L发酵罐发酵条件为装液量30L,接种菌龄5d,接种量10%,温度25℃,起始pH5.0,转速150r/min,通气量1.2m~3/h,罐压0.04Mpa,培养108h。
3、DEAE-纤维素柱层析结果表明:胞外水溶性多糖含有一个中性多糖组分和一个糖蛋白组分。
4、气相色谱分析结果表明:胞外水不溶性多糖由鼠李糖、葡萄糖和4种未知单糖组成,鼠李糖和葡萄糖的摩尔比为:鼠李糖:葡萄糖=4.08:5.55
This paper included mainly three aspects: screening of liquid culture medium of Pleurotus ferulae Lanzi, optimization of fermentation conditions, analyses of physical and chemical properties of EPS, the results of which were as follows:1. By monofactorial and orthogonal test, the optimal composition of liquid culture medium for the mycelia's growth and the producing of EPS were respectively determined. The former was composed by 0.002% VB_1, 3% Glucose, 0. 15% Peptone, 0. 05% (NH4)_2SO_4, 0.3% extract of yeast, 0.05% MgSO_4, 0. 1%KH_2PO_4,0.02%ZnSO_4 , 0. 03%NaCl. The later was composed by 0.002% VB_1, 1% Glucose, 0. 15% Peptone, 0.05% (NH4)_2SO_4, 0.3% extract of yeast, 0.05% MgSO_4, 0. 05%KH_2PO_4, 0. 01%ZnSO_4, 0. 03%NaCl.2. The optimal flask (250ml) fermentation conditions were as follows: culture volume was 100mL, inoculum quantity was 10%, initial pH value was 5.0, culture temperature was 25℃, rotation speed was 150rpm, strain age of spawn was 5 days, culture time was 10 days.The optimal jar (50L) fermentation conditions were as follows: culture volume was 30L, strain age of spawn was 5 days, inoculum quantity was 10%, initial pH value was 5.0, culture temperature was 25 ℃, rotation speed was 150rpm, ventilation quantity was 1. 2m~3/h , pressure inside the jar was 0. 04Mpa , culture time was 108 hours.3. The result of DEAE-cellulose elution indicated that there was one neutral polysaccharide and one glycoprotein in the water-soluble EPS.4. Analysed with gas chromatography the monosaccharide composition and molecular ratio of water-fast EPS was gained: rha:glu = 4. 08:5. 55, and 4 unknownmonosaccharide.
引文
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2.陈坚,堵国成.发酵工程实验技术[M].北京:化学工业出版社,2003:139-148.
3.丁保金,金丽琴,吕建新.多糖的生物活性研究进展[J].中国药学杂志,2004,39(8):561-564.
4.董洪新,吕作舟.阿魏侧耳多糖的分离纯化与抗肿瘤活性的研究[J].微生物学通报,2003,30(2):16-19.
5.董晓燕.生物化学实验[M].北京:化学工业出版社,2003:126-132.
6.杜巍,李元瑞,袁静.灰树花深层培养中影响因素的研究[J].食品工业科技,2003,24(3):44-46.
7.杜宇野.香菇多糖的研究进展[J].中国食用菌,1994,14(4):11.
8.樊锦艳,王秋颖.食药用真菌多糖的研究及开发利用[J].食品工业科技,2003,24(12):106-107.
9.甘勇,吕作舟.阿魏蘑多糖理化性质及免疫活性研究[J].菌物系统,2001,20(2):228-232
10.宫志远,于淑芳,曲玲.阿魏蘑菌丝生长的营养需求及环境条件研究[J].中国食用菌,2002,21(4):38-41.
11.郝利民,邓桂芳,李政等.5种因子对裂褶菌菌丝生长及胞外多糖产生的影响[J].食品与发酵工业,2004,30(8):75-78.
12.洪霞.真菌药物研制的十年进展简述[J].中国食用菌,1994,14(2):4-7.
13.胡群宝,郭宝江.螺旋藻多糖和糖蛋白的提纯及其理化特性研究[J].海洋科学,2001,25(7):41-44.
14.贾士儒.生物工艺与工程实验技术[M].北京:中国轻工业出版社,2002:140-166.
15.孔乐生.灵芝多糖的分离纯化及理化性质研究[D].上海交通大学硕士学位论文,2001.
16.雷德柱,于淑娟,曹珍年.灰树花胞外多糖的结构分析[J].菌物系统,2002,21(2):280-282.
17.李平作,徐柔,章克昌.灵芝胞外多糖深层发酵培养基的优化[J].无锡轻工大学学报,1998,17(4):26-30.
18.李平作,章克昌.灵芝胞外多糖的分离纯化及生物活性[J].微生物学报,2000,40(2):217-221.
19.李绍平.冬虫夏草的质量评价及多糖成分的研究[D].中国药科大学博士学位论文,2000.
20.李小定,荣建华,吴谋成.真菌多糖生物活性研究进展[J].食用菌学报,2002,9(4):50-58.
21.李小定,吴谋成,曾晓波等.灰树花多糖的分离、纯化与理化性质[J].华中农业大学学报,2002,21(2):186-188.
22.李信,许雷.蛹虫草菌胞外多糖发酵工艺优化[J].化工冶金,1998,9(3):254-259.
23.刘国诠.生物工程下游技术(第二版)[M].北京:化学工业出版社,2003:162-172.
24.刘三第.真菌的药用价值[J].食用菌学报,1997,4(1):55.
25.刘祖同,罗信昌.食用蕈菌生物技术及应用[M].北京:清华大学出版社,2002.
26.罗长才.冬虫夏草深层发酵技术及其生物活性多糖的研究[D].江南大学硕士学位论文,2001.
27.史锋.生物化学实验[M].杭州:浙江大学出版社,2002,20-26.
28.宋频然,常继东.灵芝真菌发酵胞外多糖反馈抑制的初步研究[J].华东理工大学学报,2003,29(3):311-314.
29.宋旭红,于红,张月明等.新疆阿魏蘑菇提取物抗肿瘤实验研究[J].营养学报,2002,24(2):139-143.
30.孙东平,潘锋,史小丽等.灵芝菌发酵培养基的优化及灵芝胞外多糖的分离纯化[J].中草药,2000,31(12):941-943.
31.孙克,许正宏,敖宗华等.灰树花发酵条件的研究[J].药物生物技术,2002,9(2):91-93.
32.田庚元,王晨,冯宇澄.枸杞子糖蛋白的分离纯化、物化性质及糖肽键特征[J].生物化学与生物物理学报,1995,27(2):201-206.
33.汪维云,吴守一,朱金华.灰树花液体深层培养工艺研究[J].生物工程学报,1999,15(3):378-382.
34.汪维云,朱金华.食用菌液体深层发酵技术研究进展及展望[J].食用菌,2001,17(2):11-12.
35.王翎,程显好,陈冠军等.虫花菌胞外糖蛋白的分离、纯化及其性质研究[J].真菌学报,1996,15(1):48-52.
36.王允祥,陆兆新,吕凤霞.翅鳞伞深层发酵胞外多糖优化研究[J].生物工程学报,2004,20(3):414-422.
37.肖彩霞,王玉红,章克昌.黑木耳深层发酵工艺条件的研究[J].生物技术,2004,14(5):70-72.
38.徐建平.我国药用真菌研究概况[J].江苏食用菌,1990,6:1-3.
39.杨新美.食用菌研究法[M].北京:中国农业出版社,1998:86-103.
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