丝状真菌抗线虫活性物质的研究
|
摘要
本论文对总状共头霉Sr．菌(SyncephaJastrum racemosum)产生抗线虫活性物质的发酵条件进行了优化，并分析了发酵滤液的理化性质，对活性物质分离进行了探讨。
通过培养基组分的单因子试验、方差和显著性水平分析，确定了最佳发酵培养基配方为：去皮马铃薯200g／L煮汁，蔗糖20g／L，pH自然；最适发酵条件为500ml三角瓶装量100ml，接种量2％，170r／min，26℃，培养4d。发酵滤液1／2浓度的杀线虫活性达到85％以上。5L发酵罐放大培养条件为一级种子静置培养24h，按接种量4％接入装有3.5L发酵培养基的5L发酵罐中，起始通气量1：0.5，搅拌转速300～400r／min，发酵54h。发酵滤液1／2浓度的杀线虫活性达到95％以上。
本论文研究了菌体形态与发酵滤液抗线虫活性之间的关系，发现摇瓶条件下形成直径2～4mm的乳白色菌球，发酵滤液抗线活性较高，菌球形态较为稳定。又通过扫描电镜观察不同发酵条件下形成的菌丝球内部、外部形态，发现当菌球过大时，外部菌丝纤细致密，内部形成空腔，菌丝干瘪，养分传输受阻致使发酵滤液活性低下；当菌球过小时，菌丝质地坚密，不易伸展，影响传质，发酵滤液活性也较低；只有形成直径2mm～4mm的大小适中的菌球，外部菌丝大多饱满，内部菌丝松软，有利于外部养分和氧的传递，发酵滤液抗线虫活性较高。通过研究菌体形态与发酵滤液抗线虫活性之间的关系，为发酵结果提供表观依据。
对发酵滤液的理化性质进行了研究，结果表明抗线虫活性物质是一种水溶性物质，不易溶于有机溶剂，具有热稳定性，121℃处理30min，仍保持较高的活性。
对发酵滤液中的活性物质进行分离，发现Sr．菌产生的抗线虫活性物质包括有机酸类物质和非有机酸类活性组分。经过HPLC、GC-MS分析，得知有机酸类含有草酸、柠檬酸，琥珀酸和一个未知组分，还含有长链饱和脂肪酸和不饱和脂肪酸的可能性；通过凝胶柱G25、薄层TLC对非有机酸类活性成分进行分离，结合红外光谱分析初步得出其为含有氨基、糖环的多组分化合物，有待进一步纯化。
The fermentation process of the nematicidal bio-active substances from Syncephalastrum racemosum, primary separation and the properties of the bio-active substances were studied.
By single factor and statistical analysis, the optimum medium, which is composed of sucrose 20g/L and peeled potato 200g/L with natural pH, and the optimal condition were determined. The 2×106 spores of fungus were incubated in 500ml conical flask with 100ml liquid cultural medium under 170r/min for 4 days at 26 ℃. The nematicidal rate reached above 85% in the one time diluted fermentation solution. During fermentation process on fermenter the nematicidal rate of one time diluted fermentation solution reached above 95% from 44h to 52h.
The relationship between the morpha of Sr. and the nematicidal activity of the bio-active substances was studied. It was found that the pellets with diameter from 2mm to 4mm were formed under the optimal conditions. On the basis of observing The morpha of fungus under different condition was observed by the Scan Electronic Microscope (SEM). The result showed that when the pellet was too big, the filamentous were compact on the surface of pellets and hollow was formed inside because of the nutrient prohibition; when pellet was too small, the filamentous were hard and the nematicidal activity of the bio-active substances was low. Only when the pellets with diameter from 2mm to 4mm were formed, the filamentous were plump and the nematicidal activity was high. The gist of fermentation was provided by the study of the relationship between the morpha of Sr. and the nematicidal activity of the bio-active substances.
Some properties of the fermentation liquid were studied. The results showed that nematicidal bio-active substances were water-solubility and steady with heat. It kept high bio-activity after being dealed with 30min in 121 ℃.
The primary separation of bio-active sunstances was studied, too. It was concluded that nematicidal bio-active substances was composed of organic acids and non-acids. It was found that oxalic acid, citric acid and some acids else were metabolized by Sr. through HPLC, GC-MS. In addition, it was probably saturated and unsaturated fatty acids were metabolized, too. Primary separated by TLC and Sephadex G25, the bio-active substances were composed of compounds with amido and gluco- excepting organic acids. The purification of bio-active
substances was necessary in the future.
通过培养基组分的单因子试验、方差和显著性水平分析，确定了最佳发酵培养基配方为：去皮马铃薯200g／L煮汁，蔗糖20g／L，pH自然；最适发酵条件为500ml三角瓶装量100ml，接种量2％，170r／min，26℃，培养4d。发酵滤液1／2浓度的杀线虫活性达到85％以上。5L发酵罐放大培养条件为一级种子静置培养24h，按接种量4％接入装有3.5L发酵培养基的5L发酵罐中，起始通气量1：0.5，搅拌转速300～400r／min，发酵54h。发酵滤液1／2浓度的杀线虫活性达到95％以上。
本论文研究了菌体形态与发酵滤液抗线虫活性之间的关系，发现摇瓶条件下形成直径2～4mm的乳白色菌球，发酵滤液抗线活性较高，菌球形态较为稳定。又通过扫描电镜观察不同发酵条件下形成的菌丝球内部、外部形态，发现当菌球过大时，外部菌丝纤细致密，内部形成空腔，菌丝干瘪，养分传输受阻致使发酵滤液活性低下；当菌球过小时，菌丝质地坚密，不易伸展，影响传质，发酵滤液活性也较低；只有形成直径2mm～4mm的大小适中的菌球，外部菌丝大多饱满，内部菌丝松软，有利于外部养分和氧的传递，发酵滤液抗线虫活性较高。通过研究菌体形态与发酵滤液抗线虫活性之间的关系，为发酵结果提供表观依据。
对发酵滤液的理化性质进行了研究，结果表明抗线虫活性物质是一种水溶性物质，不易溶于有机溶剂，具有热稳定性，121℃处理30min，仍保持较高的活性。
对发酵滤液中的活性物质进行分离，发现Sr．菌产生的抗线虫活性物质包括有机酸类物质和非有机酸类活性组分。经过HPLC、GC-MS分析，得知有机酸类含有草酸、柠檬酸，琥珀酸和一个未知组分，还含有长链饱和脂肪酸和不饱和脂肪酸的可能性；通过凝胶柱G25、薄层TLC对非有机酸类活性成分进行分离，结合红外光谱分析初步得出其为含有氨基、糖环的多组分化合物，有待进一步纯化。
The fermentation process of the nematicidal bio-active substances from Syncephalastrum racemosum, primary separation and the properties of the bio-active substances were studied.
By single factor and statistical analysis, the optimum medium, which is composed of sucrose 20g/L and peeled potato 200g/L with natural pH, and the optimal condition were determined. The 2×106 spores of fungus were incubated in 500ml conical flask with 100ml liquid cultural medium under 170r/min for 4 days at 26 ℃. The nematicidal rate reached above 85% in the one time diluted fermentation solution. During fermentation process on fermenter the nematicidal rate of one time diluted fermentation solution reached above 95% from 44h to 52h.
The relationship between the morpha of Sr. and the nematicidal activity of the bio-active substances was studied. It was found that the pellets with diameter from 2mm to 4mm were formed under the optimal conditions. On the basis of observing The morpha of fungus under different condition was observed by the Scan Electronic Microscope (SEM). The result showed that when the pellet was too big, the filamentous were compact on the surface of pellets and hollow was formed inside because of the nutrient prohibition; when pellet was too small, the filamentous were hard and the nematicidal activity of the bio-active substances was low. Only when the pellets with diameter from 2mm to 4mm were formed, the filamentous were plump and the nematicidal activity was high. The gist of fermentation was provided by the study of the relationship between the morpha of Sr. and the nematicidal activity of the bio-active substances.
Some properties of the fermentation liquid were studied. The results showed that nematicidal bio-active substances were water-solubility and steady with heat. It kept high bio-activity after being dealed with 30min in 121 ℃.
The primary separation of bio-active sunstances was studied, too. It was concluded that nematicidal bio-active substances was composed of organic acids and non-acids. It was found that oxalic acid, citric acid and some acids else were metabolized by Sr. through HPLC, GC-MS. In addition, it was probably saturated and unsaturated fatty acids were metabolized, too. Primary separated by TLC and Sephadex G25, the bio-active substances were composed of compounds with amido and gluco- excepting organic acids. The purification of bio-active
substances was necessary in the future.
引文
1．杨新玲，张利兰编译．植物寄生线虫防治的新策略[J]．world pesticides Vol．23 No．5(2001)
2．董锦艳等．杀线虫菌物毒素的研究进展(Ⅰ)(Ⅱ)[J]．中国生物防治2001，5月，8月
3．王明祖．中国植物线虫研究[M]．武汉：湖北科学技术出版社，1998．168-289
4. Robin N H, Susan L F M. Novel Management Strategies for Plant Parasitic Nematodes [M]. Plenum Press(New York): Biological Control of Plant Disease, 1992, 273-276
5．[美]V．H．Dropkin，植物线虫学导论[J]．厦门大学出版社，1991，198～203
6．向红琼，冯志新．粗皮侧耳粗毒素的提取及其杀线虫活性的研究[J]．云南大学学报，1999，14(增刊)：82—87
7．李国红，董锦艳等．侧耳及其相关菌对线虫的作用[J]．中国生物防治，2001，17(1)：26-29
8．喻子牛主编 微生物农药及其产业化[M]．科学出版社，2000，276～283
9．刘维志主编 植物病原线虫学[M]．中国农业出版社，1998，360～365
10．陶黎明编译，作物寄生线虫的防治[J]．世界农药，2000年第二十二卷第五期
11．齐淑华等，爱福丁防治番茄根结线虫病[J]．植物保护，2000，26(5)：46-47
12．刘志波，刘志俊等，溴甲烷的禁用及其替代品的开发[J]．农药，1999，38(1)：38-40
13．高仁恒，刘杏忠．捕食线虫真菌[J]．生物学通报，1995年第30卷第1期
14．向红琼，冯志新等．食线虫担子菌的研究现状与展望[J]．植物病理学报2002，vol．32：8～15
15．向红琼等：Pleurotus ostreatus对线虫作用机理的研究[J]．植物病理学报2000，vol．30：35～7363
16．向红琼等：杀线虫真菌——粗皮侧耳初报[J]．山地农业生物学报1998，17(2)：87～91
17．张克勤，何世川等，真菌和细菌在线虫生防中的作用及其研究进展[J]．杀虫微生物，1991(3)：55—61
18．高学彪，邓穗儿等，淡紫拟青霉MCWA18菌株对南方根结线虫的寄生和防治作用[J]．中国生物防治，1998，14(4)：163-166
19．张新德等，食线虫真菌防治大豆孢囊线虫的初步研究[J]．北京农业大学学报，17：87—91
20．王昌家，宋超英等，淡紫拟青霉菌料防治大豆孢囊线虫的后效研究[J]．中国生物防治，1997，13：26-28
21．丘文杰，潘沧桑淡紫拟青霉防治植物线虫及其几丁质霉作用的研究进展
22．刘杏忠 等，淡紫拟青霉发酵液生理活性的初步鉴定[J]．北京农业大学学报，1991，12(11)：45-46
23. Stifling G R. Bilogical control of Plant Parasitic Nematodes Progress, Problems and Prospects. C.A.B International,1991,微生物农药及其产业化[M]．
24．童建华，庄占兴，阿维菌素生产与应用现状[J]．农药，1999，38(9)：38-39
25．施跃峰，微生物杀虫剂研究进展[J]．植物保护，2000年26卷第5期
26．高菊芳编译，生物农药的作用、应用与功效[J]．世界农药，2001年二十三卷第一期
27．董锦艳等：真菌杀线虫代谢物的研究进展[J]．菌物系统2001，20(2)：285～296
28. O. STERNER, W. ETZEL et al, Omphalotin, a new peptide with potent nematicidal activity from Omphaloyus olearius (Ⅰ) (Ⅱ), [J]. Natural Product Letters, Volume 10, pp. 25-32; 33-38
29．张业江，徐汉虹，非洲山毛豆的主要杀虫活性成分研究[J]．天然产物研究与开发，2000，Vol．12 No．6
30．邢来君，李明春编著．普通真菌学[M]．高教出版社，1991，319-320
31．孙建华，土壤真菌培养物对松材线虫生长和繁殖抑制作用的研究[J]．南开大学学报自然科学版，1997，Vol．30，82～87
32. Job S.K. Charles, A.V. Vijayalakshmi et al, A new report on the biological suppression of cyst nematode, Heterodera oryzicola infecting banana in Kerala, Indian[J].Journal of Tropical Agiculture 38 (2000): 102—103
33. R. V.S. Amorim, E.S. Melo et al, Chitosan from Syncephalastrum racemosum, used as a film support for lipase immobilization[J]. Bioresourse Technology 89 (2003) 35-39
34. Heng-Chien HO, Ling-Yun Chen et al, Identification of a fungal protein of Syncephalastrum racemosum as Aspartic Proteinase[J]. ARCHIEVES OF BIOCHENISTRY AND BIOPHYSICS Vol. 334, No. 1, October 1, pp. 97-103,1996
35. Heng-Chien HO, Pyng-Fang Shiau, and Shih-Lu Wu, Single-Column
Purification of Syncephapepsin—An Aspartic Proteinase from Syncephalastrum racemosum[J].PROTEIN EXPRESSION AND PURIFICATIN 12, 399—402, (1998)
36．孙建华，宇克莉等，应用Sr真菌代谢物防治植物线虫病害的初步研究[J]．云南农业大学学报，1999，14(增刊)：122-123
37．孙建华等：Sr18真菌代谢物防治番茄根结线虫病研究[J]．华北农学报2002，17(1)：119～123
38．马承铸，顾真容等，杀虫抗生素的线虫生测法及其应用[J]．上海农业学报1995，11(1)：78～82
39．万树青，杀线虫剂生物活性测定[J]．农药，1994年33卷5期
40．王成树，李农昌等，真菌杀虫剂标准化问题的探讨[J]．中国生物防治，1998，14(3)134—138
41．赵博光，苦豆碱对松材线虫的杀线活性[J]．林业科学，Vol．32，No．3
42．天津轻工业学院，等工业发酵分析[M]．中国轻工业出版社
43．大连轻工业学院，等食品分析[M]．中国轻工业出版社，1995
44. M. Pazouki, T. Panda, Understanding the morphology of fungi [J]. Bioprocess Engineering 22(2000) 127—143
45．白冬梅，赵学明等，应用HPLC-反相色谱法测定米根霉乳酸发酵液中的有机酸[J]．工业微生物，2001，Vol．31 No．1
46．杨毅，李崎等，反相高效液相色谱法(RP-HPLC)测定啤酒中有机酸[J]．食品与发酵工业，2003，Vol．29 No．8
47．王明慈主编，概率论与数理统计[M]．高等教育出版社，1998
48. C.DJIAN,M.PONCHET, AND J.C.CAYROL Nematocidal Properties of Carboxylic Acids and Derivatives [J]. PESTICIDE BIOCHEMISTRY AND PHYSIOLIGY 50, 229-239 (1994)
49．赵永芳编著，生物化学技术原理及其应用[M．武汉大学出版社，1994
50．中草药有效成分提取与分离[M]．中国科学院上海药物研究所编著，上海科学技术出版社，1983
2．董锦艳等．杀线虫菌物毒素的研究进展(Ⅰ)(Ⅱ)[J]．中国生物防治2001，5月，8月
3．王明祖．中国植物线虫研究[M]．武汉：湖北科学技术出版社，1998．168-289
4. Robin N H, Susan L F M. Novel Management Strategies for Plant Parasitic Nematodes [M]. Plenum Press(New York): Biological Control of Plant Disease, 1992, 273-276
5．[美]V．H．Dropkin，植物线虫学导论[J]．厦门大学出版社，1991，198～203
6．向红琼，冯志新．粗皮侧耳粗毒素的提取及其杀线虫活性的研究[J]．云南大学学报，1999，14(增刊)：82—87
7．李国红，董锦艳等．侧耳及其相关菌对线虫的作用[J]．中国生物防治，2001，17(1)：26-29
8．喻子牛主编 微生物农药及其产业化[M]．科学出版社，2000，276～283
9．刘维志主编 植物病原线虫学[M]．中国农业出版社，1998，360～365
10．陶黎明编译，作物寄生线虫的防治[J]．世界农药，2000年第二十二卷第五期
11．齐淑华等，爱福丁防治番茄根结线虫病[J]．植物保护，2000，26(5)：46-47
12．刘志波，刘志俊等，溴甲烷的禁用及其替代品的开发[J]．农药，1999，38(1)：38-40
13．高仁恒，刘杏忠．捕食线虫真菌[J]．生物学通报，1995年第30卷第1期
14．向红琼，冯志新等．食线虫担子菌的研究现状与展望[J]．植物病理学报2002，vol．32：8～15
15．向红琼等：Pleurotus ostreatus对线虫作用机理的研究[J]．植物病理学报2000，vol．30：35～7363
16．向红琼等：杀线虫真菌——粗皮侧耳初报[J]．山地农业生物学报1998，17(2)：87～91
17．张克勤，何世川等，真菌和细菌在线虫生防中的作用及其研究进展[J]．杀虫微生物，1991(3)：55—61
18．高学彪，邓穗儿等，淡紫拟青霉MCWA18菌株对南方根结线虫的寄生和防治作用[J]．中国生物防治，1998，14(4)：163-166
19．张新德等，食线虫真菌防治大豆孢囊线虫的初步研究[J]．北京农业大学学报，17：87—91
20．王昌家，宋超英等，淡紫拟青霉菌料防治大豆孢囊线虫的后效研究[J]．中国生物防治，1997，13：26-28
21．丘文杰，潘沧桑淡紫拟青霉防治植物线虫及其几丁质霉作用的研究进展
22．刘杏忠 等，淡紫拟青霉发酵液生理活性的初步鉴定[J]．北京农业大学学报，1991，12(11)：45-46
23. Stifling G R. Bilogical control of Plant Parasitic Nematodes Progress, Problems and Prospects. C.A.B International,1991,微生物农药及其产业化[M]．
24．童建华，庄占兴，阿维菌素生产与应用现状[J]．农药，1999，38(9)：38-39
25．施跃峰，微生物杀虫剂研究进展[J]．植物保护，2000年26卷第5期
26．高菊芳编译，生物农药的作用、应用与功效[J]．世界农药，2001年二十三卷第一期
27．董锦艳等：真菌杀线虫代谢物的研究进展[J]．菌物系统2001，20(2)：285～296
28. O. STERNER, W. ETZEL et al, Omphalotin, a new peptide with potent nematicidal activity from Omphaloyus olearius (Ⅰ) (Ⅱ), [J]. Natural Product Letters, Volume 10, pp. 25-32; 33-38
29．张业江，徐汉虹，非洲山毛豆的主要杀虫活性成分研究[J]．天然产物研究与开发，2000，Vol．12 No．6
30．邢来君，李明春编著．普通真菌学[M]．高教出版社，1991，319-320
31．孙建华，土壤真菌培养物对松材线虫生长和繁殖抑制作用的研究[J]．南开大学学报自然科学版，1997，Vol．30，82～87
32. Job S.K. Charles, A.V. Vijayalakshmi et al, A new report on the biological suppression of cyst nematode, Heterodera oryzicola infecting banana in Kerala, Indian[J].Journal of Tropical Agiculture 38 (2000): 102—103
33. R. V.S. Amorim, E.S. Melo et al, Chitosan from Syncephalastrum racemosum, used as a film support for lipase immobilization[J]. Bioresourse Technology 89 (2003) 35-39
34. Heng-Chien HO, Ling-Yun Chen et al, Identification of a fungal protein of Syncephalastrum racemosum as Aspartic Proteinase[J]. ARCHIEVES OF BIOCHENISTRY AND BIOPHYSICS Vol. 334, No. 1, October 1, pp. 97-103,1996
35. Heng-Chien HO, Pyng-Fang Shiau, and Shih-Lu Wu, Single-Column
Purification of Syncephapepsin—An Aspartic Proteinase from Syncephalastrum racemosum[J].PROTEIN EXPRESSION AND PURIFICATIN 12, 399—402, (1998)
36．孙建华，宇克莉等，应用Sr真菌代谢物防治植物线虫病害的初步研究[J]．云南农业大学学报，1999，14(增刊)：122-123
37．孙建华等：Sr18真菌代谢物防治番茄根结线虫病研究[J]．华北农学报2002，17(1)：119～123
38．马承铸，顾真容等，杀虫抗生素的线虫生测法及其应用[J]．上海农业学报1995，11(1)：78～82
39．万树青，杀线虫剂生物活性测定[J]．农药，1994年33卷5期
40．王成树，李农昌等，真菌杀虫剂标准化问题的探讨[J]．中国生物防治，1998，14(3)134—138
41．赵博光，苦豆碱对松材线虫的杀线活性[J]．林业科学，Vol．32，No．3
42．天津轻工业学院，等工业发酵分析[M]．中国轻工业出版社
43．大连轻工业学院，等食品分析[M]．中国轻工业出版社，1995
44. M. Pazouki, T. Panda, Understanding the morphology of fungi [J]. Bioprocess Engineering 22(2000) 127—143
45．白冬梅，赵学明等，应用HPLC-反相色谱法测定米根霉乳酸发酵液中的有机酸[J]．工业微生物，2001，Vol．31 No．1
46．杨毅，李崎等，反相高效液相色谱法(RP-HPLC)测定啤酒中有机酸[J]．食品与发酵工业，2003，Vol．29 No．8
47．王明慈主编，概率论与数理统计[M]．高等教育出版社，1998
48. C.DJIAN,M.PONCHET, AND J.C.CAYROL Nematocidal Properties of Carboxylic Acids and Derivatives [J]. PESTICIDE BIOCHEMISTRY AND PHYSIOLIGY 50, 229-239 (1994)
49．赵永芳编著，生物化学技术原理及其应用[M．武汉大学出版社，1994
50．中草药有效成分提取与分离[M]．中国科学院上海药物研究所编著，上海科学技术出版社，1983