CN105648001A - 对橙色粘球菌jch-04次级代谢产物分离纯化提取抗霉枯草菌素的方法 - Google Patents
对橙色粘球菌jch-04次级代谢产物分离纯化提取抗霉枯草菌素的方法 Download PDFInfo
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- RCIPRGNHNAEGHR-ZLHAWHIKSA-N 3-[(3s,6s,13s,16r,19r,22r,25r,28s)-6,13,19,22-tetrakis(2-amino-2-oxoethyl)-16-(hydroxymethyl)-25-[(4-hydroxyphenyl)methyl]-10-(11-methyltridecyl)-2,5,8,12,15,18,21,24,27-nonaoxo-1,4,7,11,14,17,20,23,26-nonazabicyclo[26.3.0]hentriacontan-3-yl]propanamide Chemical compound C([C@H]1NC(=O)[C@@H]2CCCN2C(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CC(NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](CO)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@@H](CC(N)=O)NC1=O)CCCCCCCCCCC(C)CC)C1=CC=C(O)C=C1 RCIPRGNHNAEGHR-ZLHAWHIKSA-N 0.000 title claims abstract description 22
- 108700030603 mycosubtiline Proteins 0.000 title claims abstract description 22
- 238000000746 purification Methods 0.000 title claims abstract description 17
- 229930000044 secondary metabolite Natural products 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000000926 separation method Methods 0.000 title claims abstract description 10
- 241000863423 Myxococcus fulvus Species 0.000 title abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 57
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 230000004151 fermentation Effects 0.000 claims abstract description 29
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000011347 resin Substances 0.000 claims abstract description 24
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 238000005227 gel permeation chromatography Methods 0.000 claims description 11
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 abstract 1
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- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
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- LGZXYFMMLRYXLK-UHFFFAOYSA-N mercury(2+);sulfide Chemical compound [S-2].[Hg+2] LGZXYFMMLRYXLK-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了对橙色粘球菌JCH-04次级代谢产物分离纯化提取抗霉枯草菌素的方法,该橙色粘球菌JCH-04(Myxococcus?fulvus?JCH-04)在中国微生物菌种保藏管理委员会普通微生物中心保藏;取菌种接种于种子培养基的摇瓶中培养得种子液;将种子液接种于发酵培养基的三角瓶中培养得发酵液;将发酵液过滤,收集树脂装入层析柱中,洗脱液浓缩得粗提物;粗提物中加入水和乙酸乙酯,混匀后离心,吸取水相,干燥得冻干粉;冻干粉甲醇溶解,离心,取上清液浓缩,通过Sephadex?LH-20凝胶层析分离,收集140~180min馏分;馏分浓缩,通过半制备色谱分离,收集保留时间39.159?min和42.919?min的组分,分别为C14?Mycosubtilin和C15?Mycosubtilin。本发明方法简单,由橙色粘球菌次级代谢产物分离纯化提取抗霉枯草菌素,扩大了抗菌脂肽的提取途径。
Description
技术领域
本发明涉及橙色粘球菌JCH-04,具体涉及一种对橙色粘球菌JCH-04次级代谢产物分离纯化提取抗霉枯草菌素的方法。
背景技术
抗菌脂肽具有区别于常规抗生素的全新抗菌机制,不仅不易产生耐药性,而且对传统药物已具有耐药性的菌株仍然有良好的杀菌和抑菌功效;同时,其抗菌谱之广、抗菌效果佳、不残留,对G—、G+菌、真菌和支原体等均具有良好的抑制和杀灭作用,随着达托霉素、棘白霉素等新一代脂肽类抗生素的上市,越来越多的科研工作者将目光转移至对该类抗菌物质的研发上,使其成为绿色安全、新型高效的抗菌药物的研发方向。
抗菌脂肽常见于解淀粉芽孢杆菌和苏云金芽孢杆菌的次级代谢产物中,而粘细菌作为天然活性物质的生产者,是一类重要的药源菌,可产生异常丰富的次级代谢产物,包括芳香族,杂环类,醌类,大环类,聚醚类,多烯类,肽类化合物等,却未见报道其可产抗菌脂肽类物质。
发明内容
本发明的目的在于:提供一种对橙色粘球菌JCH-04次级代谢产物分离纯化提取抗霉枯草菌素的方法,对橙色粘球菌JCH-04进行发酵培养,由其次级代谢产物分离纯化提取抗霉枯草菌素(Mycosubtilin),它是抗菌脂肽的一种,扩大抗菌脂肽的提取途径。
本发明的技术解决方案是:对橙色粘球菌JCH-04的次级代谢产物进行分离纯化提取抗霉枯草菌素(Mycosubtilin),该橙色粘球菌JCH-04(MyxococcusfulvusJCH-04)在中国微生物菌种保藏管理委员会普通微生物中心保藏,地址:北京市朝阴区大屯路中国科学院微生物研究所,保藏日期2009年1月22日,保藏编号为:CGMCCNo.2893;取一环菌种接种于装有25mL种子培养基的250mL摇瓶中,30℃180rpm培养2天得种子液;将种子液以体积比1:10接种于含有100mL发酵培养基的500mL三角瓶中,30℃180rpm培养6天,得发酵液;将1000mL发酵液用60目筛过滤,收集树脂,装入层析柱中,用质量浓度20%甲醇冲洗,再用纯甲醇洗脱,收集洗脱液,50℃旋转蒸发,浓缩得粗提物;粗提物1.9631g中分别加入50mL的水和乙酸乙酯,混匀后离心,吸取水相,冷冻干燥得冻干粉;冻干粉563.5mg用3mL纯甲醇溶解,离心,取上清液,通过SephadexLH-20凝胶层析分离,收集140~180min的馏分;将馏分于50℃旋蒸浓缩至2mL,通过半制备色谱分离,收集保留时间为39.159min和42.919min的组分,分别为C14Mycosubtilin和C15Mycosubtilin。
其中,对橙色粘球菌JCH-04的次级代谢产物进行分离纯化提取抗霉枯草菌素(Mycosubtilin)的具体步骤是:
(1)种子液培养:取上一环保藏编号为CGMCCNo.2893的橙色粘球菌JCH-04的菌种接种于装有25mL种子培养基的250mL摇瓶中,30℃180rpm培养2天,得种子液;其中,使用的液体种子培养基配方g/L计为:土豆淀粉5.0~15,葡萄糖2~5,酵母粉0.5~10,脱脂奶粉2.0~10,CaCl20.5~2.0,MgSO4·7H2O0.5~2.0,Fe3+-Na-EDTA0.005~0.02,去离子水定容至1000mL,pH7.2;
(2)发酵培养:将上述种子液以体积比1:10接种于含有100mL发酵培养基的500mL三角瓶中,30℃180rpm培养6天,得发酵液;其中,使用的液体发酵培养基配方g/L计为:土豆淀粉5.0~15,葡萄糖2~5,酵母粉0.5~10,脱脂奶粉2.0~10,CaCl20.5~2.0,MgSO4·7H2O0.5~2.0,Fe3+-Na-EDTA0.005~0.02,甘油5.0~15,XAD-16大孔吸附树脂15~30,去离子水定容至1000mL,pH7.2;
(3)提取粗产物:将上述1000mL发酵液用60目筛过滤,收集树脂,先用水洗去树脂表面残留的发酵液,然后将树脂装入玻璃层析柱中,层析柱规格2×50cm,用100mL质量浓度20%甲醇以1mL/min的流速流过树脂柱清洗树脂,再用纯甲醇以1mL/min的流速洗脱树脂,收集甲醇洗脱液200mL,将200mL甲醇洗脱液于50℃旋转蒸发,浓缩得油脂状粗提物,称重为1.9631g;
(4)萃取:粗提物1.9631g中分别加入50mL乙酸乙酯和50mL水,溶解粗提物,10000rpm离心3min,乙酸乙酯相和水相分层,用移液器吸取水相,将水相冷冻干燥得冻干粉,称重563.5mg;
(5)SephadexLH-20凝胶层析:用3mL纯甲醇溶解冻干粉563.5mg,离心取上清液,通过SephadexLH-20凝胶层析分离,凝胶层析柱规格为2×50cm,SephadexLH-20凝胶量为20g,洗脱剂为甲醇,流速0.6mL/min,收集140~180min的馏分;
(6)半制备色谱分离:将上述馏分于50℃旋转蒸发浓缩至2mL,再采用半制备色谱分离纯化,制备柱为C18柱(HederaODS-2,5μm,10.0×250mm),检测波长215nm,进样量2mL,收集保留时间为39.159min和42.919min的组分,于50℃旋转蒸发除去乙腈后,冷冻干燥,分别得C14Mycosubtilin31.4mg和C15Mycosubtilin28.6mg。
其中,C14Mycosubtilin分子结构式为:
,
C15Mycosubtilin为其同系物,在脂肪酸直碳链上多一个亚甲基(-CH2)。
本发明的优点是:
1、从橙色粘球菌的次级代谢产物中发现了抗霉枯草菌素的两个同系类化合物,扩大抗菌脂肽的提取途径,并提供了发酵制备、分离纯化方法,实现有效分离。
2、利用本发明的方法,提高了得率,从1L的发酵液中提取到C14Mycosubtilin31.4mg和C15Mycosubtilin28.6mg,收率远高于酸沉淀法,经HPLC分析,纯度达到了98%以上。
3、本发明涉及到抗霉枯草菌素(Mycosubtilin)同系物对G—、G+菌、真菌和支原体等均具有良好的抑制和杀灭作用,不易产生耐药性,对于开发绿色安全、新型高效的抗菌药物具有重大意义。
附图说明
图1为G1红外光谱图。
图2为G2红外光谱图。
图3为茚三酮实验结果。
图4为G1质谱分析结果。
图5为G2质谱分析结果。
图6为G1氨基酸分析结果。
图7为G2氨基酸分析结果。
图8为G1二级质谱分析结果。
图9为C14MycosubtilinHPLC图。
图10为C15MycosubtilinHPLC图。
具体实施方式
下面结合具体实施例进一步说明本发明的技术解决方案,实施例不能理解为是对技术方案的限制。
实施例:依以下步骤对橙色粘球菌JCH-04的次级代谢产物进行分离纯化提取得抗霉枯草菌素(Mycosubtilin)
(1)种子液培养:取上一环保藏编号为CGMCCNo.2893的橙色粘球菌JCH-04的菌种接种于装有25mL种子培养基的250mL摇瓶中,30℃180rpm培养2天,得种子液;其中,使用的液体种子培养基配方g/L计为:土豆淀粉5.0~15,葡萄糖2~5,酵母粉0.5~10,脱脂奶粉2.0~10,CaCl20.5~2.0,MgSO4·7H2O0.5~2.0,Fe3+-Na-EDTA0.005~0.02,去离子水定容至1000mL,pH7.2;
(2)发酵培养:将上述种子液以体积比1:10接种于含有100mL发酵培养基的500mL三角瓶中,30℃180rpm培养6天,得发酵液;其中,使用的液体发酵培养基配方g/L计为:土豆淀粉5.0~15,葡萄糖2~5,酵母粉0.5~10,脱脂奶粉2.0~10,CaCl20.5~2.0,MgSO4·7H2O0.5~2.0,Fe3+-Na-EDTA0.005~0.02,甘油5.0~15,XAD-16大孔吸附树脂15~30,去离子水定容至1000mL,pH7.2;
(3)提取粗产物:将上述1000mL发酵液用60目筛过滤,收集树脂,先用水洗去树脂表面残留的发酵液,然后将树脂装入玻璃层析柱中,层析柱规格2×50cm,用100mL质量浓度20%甲醇以1mL/min的流速流过树脂柱清洗树脂,再用纯甲醇以1mL/min的流速洗脱树脂,收集甲醇洗脱液200mL,将200mL甲醇洗脱液于50℃旋转蒸发,浓缩得油脂状粗提物,称重1.9631g;
(4)萃取:粗提物1.9631g中分别加入50mL乙酸乙酯和50mL水,溶解粗提物,10000rpm离心3min,乙酸乙酯相和水相分层,用移液器吸取水相,将水相冷冻干燥得冻干粉,称重563.5mg;
(5)SephadexLH-20凝胶层析:用3mL甲醇溶解冻干粉563.5mg,离心取上清液,50℃旋转蒸发浓缩至2mL,用0.45μm滤膜过滤后,上样,通过SephadexLH-20凝胶层析分离,凝胶层析柱规格为2×50cm,SephadexLH-20凝胶量为20g,洗脱剂为甲醇,流速0.6mL/min,收集140~180min的馏分;
(6)半制备色谱分离:将上述馏分于50℃旋转蒸发浓缩至2mL,再采用半制备色谱分离纯化,制备柱为C18柱(HederaODS-2,5μm,10.0×250mm),检测波长215nm,进样量2mL,收集保留时间为39.159min和42.919min的组分,于50℃旋转蒸发除去乙腈后,冷冻干燥,分别得G1和G2冻干粉31.4mg和28.6mg。
上述半制备色谱梯度洗脱程序见表1,收集保留时间为39.159min和42.919min的组分,分别命名为G1、G2。
表1半制备色谱梯度洗脱程序
注:A,乙腈;B,水
结构鉴定:对G1、G2分别进行红外光谱分析,发现二者红外光谱相似,基本可以重合,且与蛋白质类的红外光谱图相似,见图1、图2。
茚三酮实验:通过茚三酮实验,G1和G2与茚三酮反应不显色,在110℃经6mol/L盐酸水解2h后,与茚三酮反应显橘红色,见图3,说明G1和G2为脂肽类化合物。
质谱分析:通过质谱分析,获得G1、G2的[M+Na]+的m/z分别为1065.5、1079.5,得到G1的相对分子质量为1042.5,G2的相对分子质量为1056.5,见图4、图5。
氨基酸分析:经氨基酸分析仪分析,得到G1和G2的氨基酸组成,见图6、表2、图7、表3。
表2G1氨基酸组成分析结果
表3G2氨基酸组成分析结果
二级质谱分析:经二级质谱分析,得到氨基酸的连接顺序,见图8,与已知化合物进行比较,结果表明G1为C14Mycosubtilin,G2为C15Mycosubtilin。
根据离子峰之间的差值推测氨基酸的连接顺序,,G1中存在肽的片段chain1:Gln—Pro—Ser—Asn,根据,G1中存在肽的片段chain2:Gln—Asn—Tyr—Asn。综合chain1和chain2两个氨基酸片段,G1中氨基酸的连接顺序应为Asn—Tyr—Asn—Gln—Pro—Ser—Asn,分子中包含3个Asn,1个Gln,1个Ser,1个Tyr,1个Pro,共7个氨基酸,和氨基酸分析仪分析的结果相符合。
HPLC分析检测:色谱柱为C18柱,粒径5μm,规格4.6×250mm,采用二极管PDA进行检测,检测波长:220nm,梯度洗脱程序见表4,C14Mycosubtilin保留时间为7.4min,C15Mycosubtilin保留时间为8.2min,见图9、图10,纯度在98%以上。
表4.HPLC检测流动相洗脱程序
注:A,乙腈;B,水。
Claims (3)
1.对橙色粘球菌JCH-04的次级代谢产物进行分离纯化提取抗霉枯草菌素的方法,该橙色粘球菌JCH-04(MyxococcusfulvusJCH-04)在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为:CGMCCNo.2893;其特征是:取一环菌种接种于装有25mL种子培养基的250mL摇瓶中,30℃180rpm培养2天得种子液;将种子液以体积比1:10接种于含有100mL发酵培养基的500mL三角瓶中,30℃180rpm培养6天,得发酵液;将1000mL发酵液用60目筛过滤,收集树脂,装入层析柱中,用质量浓度20%甲醇冲洗,再用纯甲醇洗脱,收集洗脱液,50℃旋转蒸发,浓缩得粗提物1.9631g;粗提物中分别加入50mL的水和乙酸乙酯,混匀后离心,吸取水相,冷冻干燥得冻干粉563.5mg;冻干粉用3mL纯甲醇溶解,离心,取上清液通过SephadexLH-20凝胶层析分离,流速0.6mL/min,收集140~180min的馏分;将馏分于50℃旋蒸浓缩至2mL,通过半制备色谱分离,收集保留时间为39.159min和42.919min的组分,分别为C14Mycosubtilin和C15Mycosubtilin。
2.根据权利要求1所述的对橙色粘球菌JCH-04的次级代谢产物进行分离纯化提取抗霉枯草菌素的方法,其特征是该方法具体步骤是:
(1)种子液培养:取上一环保藏编号为CGMCCNo.2893的橙色粘球菌JCH-04的菌种接种于装有种子培养基的摇瓶中,30℃180rpm培养2天,得种子液;其中,1000mL种子培养基配方为:10g土豆淀粉,2g葡萄糖,2g安琪酵母,4g脱脂奶粉,1g氯化钙,1g硫酸镁,8mg乙二胺四乙酸铁钠盐,用去离子水定容至1000mL,pH为7.2;
(2)发酵培养:将上述种子液以体积比1:10接种于含有100mL发酵培养基的500mL三角瓶中,30℃180rpm培养6天,得发酵液;其中1000mL发酵培养基配方为:10g土豆淀粉,2g葡萄糖,2g安琪酵母,4g脱脂奶粉,1g氯化钙,1g硫酸镁,8mg乙二胺四乙酸铁钠盐,5g甘油,20gXAD-16大孔吸附树脂,用去离子水定容至1000mL,pH为7.2;
(3)提取粗产物:将上述1000mL发酵液用60目筛过滤,收集树脂,先用水洗去树脂表面残留的发酵液,然后将树脂装入玻璃层析柱中,层析柱规格2×50cm,用100mL质量浓度20%甲醇以1mL/min的流速流过树脂柱清洗树脂,再用纯甲醇以1mL/min的流速洗脱树脂,收集甲醇洗脱液200mL,将200mL甲醇洗脱液于50℃旋转蒸发,浓缩得油脂状粗提物,称重1.9631g;
(4)萃取:粗提物1.9631g中分别加入50mL乙酸乙酯和50mL水,溶解粗提物,10000rpm离心3min,乙酸乙酯相和水相分层,用移液器吸取水相,将水相冷冻干燥得冻干粉,称重563.5mg;
(5)SephadexLH-20凝胶层析:用3mL纯甲醇溶解冻干粉563.5mg,离心取上清液,通过SephadexLH-20凝胶层析分离,凝胶层析柱规格为2×50cm,SephadexLH-20凝胶量为20g,洗脱剂为纯甲醇,流速0.6mL/min,收集140~180min的馏分;
(6)半制备色谱分离:将上述馏分于50℃旋转蒸发浓缩至2mL,再采用半制备色谱分离纯化,制备柱为C18柱(HederaODS-2,5μm,10.0×250mm),检测波长215nm,进样量2mL,收集保留时间为39.159min和42.919min的组分,于50℃旋转蒸发除去乙腈后,冷冻干燥,分别得C14Mycosubtilin31.4mg和C15Mycosubtilin28.6mg。
3.根据权利要求2所述的对橙色粘球菌JCH-04的次级代谢产物进行分离纯化提取抗霉枯草菌素的方法,其特征是:C14Mycosubtilin的分子式为C48H74O14N12,C15Mycosubtilin的分子式为C49H76O14N12;C14Mycosubtilin分子结构式为:
,
C15Mycosubtilin为其同系物,在脂肪酸直碳链上多一个亚甲基(-CH2)。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110452940A (zh) * | 2019-09-04 | 2019-11-15 | 台州职业技术学院 | 一种链霉菌的次级代谢产物的分离提取方法 |
CN112316111A (zh) * | 2020-11-05 | 2021-02-05 | 新疆师范大学 | 一种抗霉枯草菌素在制备抗肿瘤药物中的应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101638625A (zh) * | 2009-02-26 | 2010-02-03 | 淮阴工学院 | 橙色粘球菌jch-04及抗菌代谢产物的培养方法 |
CN102132788A (zh) * | 2011-03-22 | 2011-07-27 | 淮阴工学院 | 水产养殖用橙色粘球菌微生态制剂的制备及应用方法 |
CN104004693A (zh) * | 2014-06-17 | 2014-08-27 | 江南大学 | 一种短小芽孢杆菌及其在控制白酒中土臭味的应用 |
US20150344905A1 (en) * | 2014-05-28 | 2015-12-03 | Bayer Cropscience Lp | Compositions and methods for controlling fungal and bacterial diseases in plants |
-
2016
- 2016-01-28 CN CN201610057944.1A patent/CN105648001A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101638625A (zh) * | 2009-02-26 | 2010-02-03 | 淮阴工学院 | 橙色粘球菌jch-04及抗菌代谢产物的培养方法 |
CN102132788A (zh) * | 2011-03-22 | 2011-07-27 | 淮阴工学院 | 水产养殖用橙色粘球菌微生态制剂的制备及应用方法 |
US20150344905A1 (en) * | 2014-05-28 | 2015-12-03 | Bayer Cropscience Lp | Compositions and methods for controlling fungal and bacterial diseases in plants |
CN104004693A (zh) * | 2014-06-17 | 2014-08-27 | 江南大学 | 一种短小芽孢杆菌及其在控制白酒中土臭味的应用 |
Non-Patent Citations (4)
Title |
---|
IMEN BEN SLIMENE 等: "Putative use of a Bacillus subtilis L194 strain for biocontrol of Phoma medicaginis in Medicago truncatula seedlings", 《RESEARCH IN MICROBIOLOGY》 * |
NOPPORN THASANA 等: "Bacillus subtilis SSE4 produces subtulene A, a new lipopeptide antibiotic possessing an unusual C15 unsaturated b-amino acid", 《FEBS LETTERS》 * |
姜莉莉 等: "枯草芽孢杆菌在防治植物病害上的应用及研究进展", 《安徽农学通报》 * |
张华: "生防菌B10-26抑菌活性物质研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110452940A (zh) * | 2019-09-04 | 2019-11-15 | 台州职业技术学院 | 一种链霉菌的次级代谢产物的分离提取方法 |
CN110452940B (zh) * | 2019-09-04 | 2021-03-30 | 台州职业技术学院 | 一种链霉菌的次级代谢产物的分离提取方法 |
CN112316111A (zh) * | 2020-11-05 | 2021-02-05 | 新疆师范大学 | 一种抗霉枯草菌素在制备抗肿瘤药物中的应用 |
CN112316111B (zh) * | 2020-11-05 | 2024-03-29 | 新疆师范大学 | 一种抗霉枯草菌素在制备抗肿瘤药物中的应用 |
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