裂褶菌胞外多糖的研究
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摘要
本文对裂褶菌胞外多糖的发酵条件，提取工艺及结构进行了研究，确定了裂褶菌产糖的最佳培养基、培养条件、提取工艺条件及多糖的结构。研究的主要内容和结论如下：
1．在裂褶菌胞外多糖的发酵研究中通过单因子试验和正交试验确定了裂褶菌产糖的最优培养基配方为：葡萄糖45g/L，酵母膏3g/L，KH_2PO_4 0.5g/L，MgSO_4·7H_2O 0.5g/L；最适发酵条件为：接种龄66h，接种量10％，培养温度27℃，装液量20％，初始pH自然。得到摇瓶条件下菌体最大得率20.75g/L，胞外多糖最大得率2.70g/L。经7L发酵罐的分批发酵得到菌丝干重产率为8.81g/L，胞外多糖产率为0.84g/L。多糖与菌体比为1:10。补水稀释发酵试验结果为菌丝干重得率19.98g/L，裂褶菌胞外多糖得率1.89g/L，分别为分批发酵的2.27倍和2.25倍，而多糖菌体比未变。
2．对裂褶菌胞外多糖的提取工艺进行了初步研究。确定了最佳的脱色方法为活性碳吸附法，最适加量为0.5％。最佳脱蛋白方法为等电点法与Sevag法2次相结合的方法。工艺路线为：发酵液→除去菌体→活性碳脱色→浓缩→脱蛋白→乙醇沉淀→复溶→沉淀→纯裂褶菌胞外多糖
3．进行了裂褶菌胞外多糖的结构分析。由凝胶柱层析，聚丙烯酰胺凝胶电泳，高效液相色谱分析可知裂褶菌胞外多糖为均一组分，分子量4×10~4u。通过完全水解，纸层析，气相色谱分析单糖组分，红外光谱，酶解反应，高碘酸氧化和固体核磁共振分析结构，证明了裂褶菌胞外多糖是以葡萄糖为单一组分的β-(1-3)-D葡聚糖，其中每三个葡萄糖分子上连有一个β-(1-6)的分支。
The fermentation conditions, the extraction methods and the structure of schizophyllan were studied in this thesis. The main contents and results were as follows:
Fermentation schizophyllan by Schizophyllum commune was optimized. The results of single factor and orthogonal experiment showed that the optimized medium composition contained: glucose 45g/L,yeast extract 3g/L,KH2PO4 0.5g/L,MgSO4.7H2O 0.5g/L. The optimized conditions were inoculum age 66h,inoculum size 10%,temperature 27 C,volume of medium 20% and pH 6.Maxium yield of schizophyllan and mycelium of flask were 2.70g/L and 20.75g/L respectively .But the yields of schizophyllan and mycelium were 0.84g/L and 8.81g/L respectively in batch culture. The result of water-fed experiment showed that the yields of mycelium and schizophyllan were greatly improved and them were 19.98g/L and 1.89g/L respectively.
The extraction method of exo-polysoccharide from schizophyllum commune by submerged cultivation was studied. The extraction technology was improved and the best methods of removing color and Proteins were achieved.
The structure of schizophyllan from schizophyllum commune by submerged cultivation was also researched. The purified schizophyllan was proved to be homogeneous with molecular weight 4 + 104u by sephadex G-200 column chromatography, PAGE and HPLC. Its monomer was determined by hydrolysis, PC, GC and its structure was analyzed by IR, enzymolysis, periodate oxidation, CP/MAS13C NMR, the results showed that schizophyllan was only composed of glucose and it consisted of a backbone chain of 1,3-B-D-glucopyranose units linked with single 1,6-B-D-glucopyranoses at about every third glucose molecule in the basic chain.
1．在裂褶菌胞外多糖的发酵研究中通过单因子试验和正交试验确定了裂褶菌产糖的最优培养基配方为：葡萄糖45g/L，酵母膏3g/L，KH_2PO_4 0.5g/L，MgSO_4·7H_2O 0.5g/L；最适发酵条件为：接种龄66h，接种量10％，培养温度27℃，装液量20％，初始pH自然。得到摇瓶条件下菌体最大得率20.75g/L，胞外多糖最大得率2.70g/L。经7L发酵罐的分批发酵得到菌丝干重产率为8.81g/L，胞外多糖产率为0.84g/L。多糖与菌体比为1:10。补水稀释发酵试验结果为菌丝干重得率19.98g/L，裂褶菌胞外多糖得率1.89g/L，分别为分批发酵的2.27倍和2.25倍，而多糖菌体比未变。
2．对裂褶菌胞外多糖的提取工艺进行了初步研究。确定了最佳的脱色方法为活性碳吸附法，最适加量为0.5％。最佳脱蛋白方法为等电点法与Sevag法2次相结合的方法。工艺路线为：发酵液→除去菌体→活性碳脱色→浓缩→脱蛋白→乙醇沉淀→复溶→沉淀→纯裂褶菌胞外多糖
3．进行了裂褶菌胞外多糖的结构分析。由凝胶柱层析，聚丙烯酰胺凝胶电泳，高效液相色谱分析可知裂褶菌胞外多糖为均一组分，分子量4×10~4u。通过完全水解，纸层析，气相色谱分析单糖组分，红外光谱，酶解反应，高碘酸氧化和固体核磁共振分析结构，证明了裂褶菌胞外多糖是以葡萄糖为单一组分的β-(1-3)-D葡聚糖，其中每三个葡萄糖分子上连有一个β-(1-6)的分支。
The fermentation conditions, the extraction methods and the structure of schizophyllan were studied in this thesis. The main contents and results were as follows:
Fermentation schizophyllan by Schizophyllum commune was optimized. The results of single factor and orthogonal experiment showed that the optimized medium composition contained: glucose 45g/L,yeast extract 3g/L,KH2PO4 0.5g/L,MgSO4.7H2O 0.5g/L. The optimized conditions were inoculum age 66h,inoculum size 10%,temperature 27 C,volume of medium 20% and pH 6.Maxium yield of schizophyllan and mycelium of flask were 2.70g/L and 20.75g/L respectively .But the yields of schizophyllan and mycelium were 0.84g/L and 8.81g/L respectively in batch culture. The result of water-fed experiment showed that the yields of mycelium and schizophyllan were greatly improved and them were 19.98g/L and 1.89g/L respectively.
The extraction method of exo-polysoccharide from schizophyllum commune by submerged cultivation was studied. The extraction technology was improved and the best methods of removing color and Proteins were achieved.
The structure of schizophyllan from schizophyllum commune by submerged cultivation was also researched. The purified schizophyllan was proved to be homogeneous with molecular weight 4 + 104u by sephadex G-200 column chromatography, PAGE and HPLC. Its monomer was determined by hydrolysis, PC, GC and its structure was analyzed by IR, enzymolysis, periodate oxidation, CP/MAS13C NMR, the results showed that schizophyllan was only composed of glucose and it consisted of a backbone chain of 1,3-B-D-glucopyranose units linked with single 1,6-B-D-glucopyranoses at about every third glucose molecule in the basic chain.
引文
[1] J．韦伯斯特着，张素轩译．真菌导论[M]，北京，中国林业出版社，1982，11：230．
[2] 裘维蕃主编．菌物学大全[M]，北京，科学出版社，1998，3：156．
[3] 洪震等．食用药用真菌技术及发酵生产，北京，中国农业科技出版社，1992．
[4] 戴芳澜．中国真菌总汇[M]，北京，科学出版社，1979，9：714．
[5] Shinichi Kitamura, et al.Biopolymers, 1996, 39, 407～416.
[6] 李兆兰．裂褶菌多糖的结构研究[J]，南京大学学报，1994，30(3)：482～487．
[7] Udo Rau. Production of schizophyllan[J],Methods in Biotechnology,1999,Vol 10(Carbohydrate Biotechnology Protocols):43～55.
[8] Norisuye. T,et al.J.Polym. Sci.Polym. Phys. Ed, 1980,18:547～558.
[9] S.Kitamura,et al. Biopolymers, 1989,28:639-654.
[10] T. Yanaki,et al.Carbohydr. Polym, 1985,5:275-283.1997, 16 (2) : 90～94.
[11] 王淼，丁霄霖．葡聚糖生物活性与结构关系[J]，无锡轻工大学学报，1997，16(2)：90～94．
[12] N.Komatsu. Gann, 1969,60:137～144.
[13] Motoji Suzuki,et al. Jap. J.Antiobiotics, 1977,30(6):356～361.
[14] Harukaza Mashiba,et al. Jap. J.Exp. Med, 1983,53(4):195～198.
[15] John A,Bohn,et al. Carbohydrat.polymers, 1995,28:3～14.
[16] Noboru,Nagumo.Jap Jantiobiotics, 1974,27(2): 101～111.
[17] 井出透．癌的化学疗法，1981，8(9)：1457．
[18] 夏冬，林志彬等．裂褶菌胞内多糖和胞外多糖对小鼠免疫功能的影响[J]，药学学报，1990，25(3)：161～166．
[19] Moo-SungKim,et al.Allured' s Cosmetics and Toiletrie Agazine,2000,115(7):79～86.
[20] Schulz. D,et al. Carbohydr. Polym, 1992,18:295～299.
[21] Kojima E,et al.Agric.biol.chem,1986,50:231～232.
[22] U.Rau,et al. Oxygen controlled batch cultivations of schizophyllum commune for enhance production of branched β-1,3-glucans[J], Bioprocess Engineering, 1994,11:161～165.
[23] U.Rau,et al.Ehanced production of branched β-1,3-glucans with schizophyllum commune by oxygen limitation[J],GIT Fachz. Lab., 1992, 36(4):331～337.
[24] U.Rau,et al. Process integrated downstream processing of branched β-1,3-glucans (schizophyllan)[J], GIT Fachz. Lab., 1992,36(12): 1233 1238.
[25] Weuskens, U.Rau,et al. Continuous production of an Exocellular polysaccharide by schizophyllum commune[J], Dechema. Biotechnol. conf., 1990,4:725～728.
[26] K.Cordes,U.Rau,et al. Influence of process parameters and dowmstream processing on viscous data on native glucan solutions[J],Dechema Biotechnol.conf., 1989,3:1067～1070.
[27] 李兆兰．裂褶菌胞内多糖的分离纯化鉴定及其性质[J]，真菌学报，1994，13(4)：267～271．
[28] 李兆兰．裂褶菌胞内和胞外多糖的及其几种理化性质的分析[J]，南京大学学报，1991，27(1)：143～152．
[29] 周义发．裂褶菌多糖的构象研究[J]，生物化学与生物物理进展，1995，22(1)：53～55．
[30] 夏鉴清．不同碳氮源对裂褶菌多糖产量影响的方差分析法[J]，上海师范大学学报，1989，18(4)．
[31] 钱秀萍．几种营养条件对裂褶菌生长及产L-苹果酸的影响[J]，食用菌学报，1996，3(3)：18～24．
[32] 王凤仙．三十烷醇提高裂褶菌深层培养菌丝球产量的研究[J]，中国食用菌，1990，9(6)：17．
[33] 江学斌．裂褶菌深层培养的研究，硕士论文，华南理工大学，1996，3：1～50．
[34] 胡德群，胡鸣．裂褶菌多糖的研究[J]，四川中草药研究，1994，7：21～23．
[35] 张光绮．猴头菌深层培养物化学成分的初步研究[J]，食用菌，1984(2) ．
[36] 方积年．多糖的分离纯化及其纯度鉴定与分子量测定[J]，药学通报，1984，19(10)：46～49．
[37] 肇玉明．香菇多糖提取和初步分析，硕士论文，天津轻工业学院，1998， 1：1～45．
[38] 张翼伸．多糖的结构测定[J]，生物化学和生物物理进展，1983，5：18～23．
[39] 方一韦．具有药理活性多糖的研究现况[J]，分析化学，1994，22(9)：955～960．
[40] 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．
[41] Hohmaji.J Acta Chem. Scand, 1972,26:661.
[42] Akio HTRATA,et al.Biol Pharm Bull, 1994,17(11 ): 1437～1440.
[43] 范秀容编．微生物学实验[M]，北京，高等教育出版社，1998，3．
[44] 天津轻工业学院，等．工业发酵分析[M]，北京，中国轻工业出版社．
[45] 天津轻工业学院，等．食品分析[M]，北京，中国轻工业出版社，1983．
[46] 赵永芳编．生物化学技术原理及其应用[M]，武汉，武汉大学出版社，2000，10．
[47] 威廉氏斯．BL等编，实用生物化学原理和技术[M]，北京，科学出版社，1979．
[48] 李春喜，王文林．生物统计学[M]，北京，科学出版社，1997．
[49] 张惟杰．复合多糖的生化技术研究[M]，浙江，浙江大学出版社，1994．
[2] 裘维蕃主编．菌物学大全[M]，北京，科学出版社，1998，3：156．
[3] 洪震等．食用药用真菌技术及发酵生产，北京，中国农业科技出版社，1992．
[4] 戴芳澜．中国真菌总汇[M]，北京，科学出版社，1979，9：714．
[5] Shinichi Kitamura, et al.Biopolymers, 1996, 39, 407～416.
[6] 李兆兰．裂褶菌多糖的结构研究[J]，南京大学学报，1994，30(3)：482～487．
[7] Udo Rau. Production of schizophyllan[J],Methods in Biotechnology,1999,Vol 10(Carbohydrate Biotechnology Protocols):43～55.
[8] Norisuye. T,et al.J.Polym. Sci.Polym. Phys. Ed, 1980,18:547～558.
[9] S.Kitamura,et al. Biopolymers, 1989,28:639-654.
[10] T. Yanaki,et al.Carbohydr. Polym, 1985,5:275-283.1997, 16 (2) : 90～94.
[11] 王淼，丁霄霖．葡聚糖生物活性与结构关系[J]，无锡轻工大学学报，1997，16(2)：90～94．
[12] N.Komatsu. Gann, 1969,60:137～144.
[13] Motoji Suzuki,et al. Jap. J.Antiobiotics, 1977,30(6):356～361.
[14] Harukaza Mashiba,et al. Jap. J.Exp. Med, 1983,53(4):195～198.
[15] John A,Bohn,et al. Carbohydrat.polymers, 1995,28:3～14.
[16] Noboru,Nagumo.Jap Jantiobiotics, 1974,27(2): 101～111.
[17] 井出透．癌的化学疗法，1981，8(9)：1457．
[18] 夏冬，林志彬等．裂褶菌胞内多糖和胞外多糖对小鼠免疫功能的影响[J]，药学学报，1990，25(3)：161～166．
[19] Moo-SungKim,et al.Allured' s Cosmetics and Toiletrie Agazine,2000,115(7):79～86.
[20] Schulz. D,et al. Carbohydr. Polym, 1992,18:295～299.
[21] Kojima E,et al.Agric.biol.chem,1986,50:231～232.
[22] U.Rau,et al. Oxygen controlled batch cultivations of schizophyllum commune for enhance production of branched β-1,3-glucans[J], Bioprocess Engineering, 1994,11:161～165.
[23] U.Rau,et al.Ehanced production of branched β-1,3-glucans with schizophyllum commune by oxygen limitation[J],GIT Fachz. Lab., 1992, 36(4):331～337.
[24] U.Rau,et al. Process integrated downstream processing of branched β-1,3-glucans (schizophyllan)[J], GIT Fachz. Lab., 1992,36(12): 1233 1238.
[25] Weuskens, U.Rau,et al. Continuous production of an Exocellular polysaccharide by schizophyllum commune[J], Dechema. Biotechnol. conf., 1990,4:725～728.
[26] K.Cordes,U.Rau,et al. Influence of process parameters and dowmstream processing on viscous data on native glucan solutions[J],Dechema Biotechnol.conf., 1989,3:1067～1070.
[27] 李兆兰．裂褶菌胞内多糖的分离纯化鉴定及其性质[J]，真菌学报，1994，13(4)：267～271．
[28] 李兆兰．裂褶菌胞内和胞外多糖的及其几种理化性质的分析[J]，南京大学学报，1991，27(1)：143～152．
[29] 周义发．裂褶菌多糖的构象研究[J]，生物化学与生物物理进展，1995，22(1)：53～55．
[30] 夏鉴清．不同碳氮源对裂褶菌多糖产量影响的方差分析法[J]，上海师范大学学报，1989，18(4)．
[31] 钱秀萍．几种营养条件对裂褶菌生长及产L-苹果酸的影响[J]，食用菌学报，1996，3(3)：18～24．
[32] 王凤仙．三十烷醇提高裂褶菌深层培养菌丝球产量的研究[J]，中国食用菌，1990，9(6)：17．
[33] 江学斌．裂褶菌深层培养的研究，硕士论文，华南理工大学，1996，3：1～50．
[34] 胡德群，胡鸣．裂褶菌多糖的研究[J]，四川中草药研究，1994，7：21～23．
[35] 张光绮．猴头菌深层培养物化学成分的初步研究[J]，食用菌，1984(2) ．
[36] 方积年．多糖的分离纯化及其纯度鉴定与分子量测定[J]，药学通报，1984，19(10)：46～49．
[37] 肇玉明．香菇多糖提取和初步分析，硕士论文，天津轻工业学院，1998， 1：1～45．
[38] 张翼伸．多糖的结构测定[J]，生物化学和生物物理进展，1983，5：18～23．
[39] 方一韦．具有药理活性多糖的研究现况[J]，分析化学，1994，22(9)：955～960．
[40] 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．
[41] Hohmaji.J Acta Chem. Scand, 1972,26:661.
[42] Akio HTRATA,et al.Biol Pharm Bull, 1994,17(11 ): 1437～1440.
[43] 范秀容编．微生物学实验[M]，北京，高等教育出版社，1998，3．
[44] 天津轻工业学院，等．工业发酵分析[M]，北京，中国轻工业出版社．
[45] 天津轻工业学院，等．食品分析[M]，北京，中国轻工业出版社，1983．
[46] 赵永芳编．生物化学技术原理及其应用[M]，武汉，武汉大学出版社，2000，10．
[47] 威廉氏斯．BL等编，实用生物化学原理和技术[M]，北京，科学出版社，1979．
[48] 李春喜，王文林．生物统计学[M]，北京，科学出版社，1997．
[49] 张惟杰．复合多糖的生化技术研究[M]，浙江，浙江大学出版社，1994．