CN104630119A - 抑藻活性物质紫色杆菌素及其制备方法 - Google Patents
抑藻活性物质紫色杆菌素及其制备方法 Download PDFInfo
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- XAPNKXIRQFHCHN-QGOAFFKASA-N violacein Chemical compound O=C\1NC2=CC=CC=C2C/1=C(C(=O)N1)/C=C1C1=CNC2=CC=C(O)C=C21 XAPNKXIRQFHCHN-QGOAFFKASA-N 0.000 title claims abstract description 27
- LEJQUNAZZRYZKJ-UHFFFAOYSA-N violacein Natural products Oc1ccc2NCC(C3=CC(=C4/C(=O)Nc5ccccc45)C(=O)N3)c2c1 LEJQUNAZZRYZKJ-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 15
- 239000013543 active substance Substances 0.000 title claims abstract description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 241000699340 Rugamonas sp. A3 Species 0.000 claims abstract description 12
- 239000000287 crude extract Substances 0.000 claims abstract description 10
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- 241000195493 Cryptophyta Species 0.000 claims description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 230000001580 bacterial effect Effects 0.000 claims description 22
- 239000000463 material Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 241000206757 Heterosigma akashiwo Species 0.000 claims description 18
- 239000012153 distilled water Substances 0.000 claims description 13
- 235000015097 nutrients Nutrition 0.000 claims description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 11
- 229960001701 chloroform Drugs 0.000 claims description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 238000005286 illumination Methods 0.000 claims description 8
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- 229920005654 Sephadex Polymers 0.000 claims description 6
- 239000012507 Sephadex™ Substances 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 5
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 4
- RJRZRMYKFWYPNX-UHFFFAOYSA-N [NH4+].[NH4+].CCO.[O-]S([O-])(=O)=O Chemical compound [NH4+].[NH4+].CCO.[O-]S([O-])(=O)=O RJRZRMYKFWYPNX-UHFFFAOYSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 4
- 229910052740 iodine Inorganic materials 0.000 claims description 4
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- 239000002075 main ingredient Substances 0.000 claims description 3
- 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 claims description 3
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- MCPLVIGCWWTHFH-UHFFFAOYSA-L methyl blue Chemical compound [Na+].[Na+].C1=CC(S(=O)(=O)[O-])=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[NH+]C=2C=CC(=CC=2)S([O-])(=O)=O)C=2C=CC(NC=3C=CC(=CC=3)S([O-])(=O)=O)=CC=2)C=C1 MCPLVIGCWWTHFH-UHFFFAOYSA-L 0.000 claims description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 2
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- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- RWJXTYSZCDAMCD-UHFFFAOYSA-N 8-sulfanylcyclohepta[c]oxathiole-3,4-dione Chemical compound SC1=CC=CC(=O)C2=C1SOC2=O RWJXTYSZCDAMCD-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- HEFAKSOPZZHPHW-JZJYNLBNSA-N CC/C(/c1c(C)cccc1)=C(\C=C(c1c[nH]c2ccc(CC)cc12)N1)/C1=[ClH] Chemical compound CC/C(/c1c(C)cccc1)=C(\C=C(c1c[nH]c2ccc(CC)cc12)N1)/C1=[ClH] HEFAKSOPZZHPHW-JZJYNLBNSA-N 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 241001300810 Cochlodinium Species 0.000 description 1
- 241000199914 Dinophyceae Species 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241001518925 Raphidophyceae Species 0.000 description 1
- 241000206732 Skeletonema costatum Species 0.000 description 1
- DQGOJKVIMNNGAH-UHFFFAOYSA-N Thiotropocin Natural products OC1=CC=CC(=S)C2=C1C(=O)OS2 DQGOJKVIMNNGAH-UHFFFAOYSA-N 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- UGAPHEBNTGUMBB-UHFFFAOYSA-N acetic acid;ethyl acetate Chemical compound CC(O)=O.CCOC(C)=O UGAPHEBNTGUMBB-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 241000395133 bacterium A3 Species 0.000 description 1
- 239000001055 blue pigment Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
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- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
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- 230000001590 oxidative effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
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- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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Abstract
抑藻活性物质紫色杆菌素及其制备方法,涉及一株抑藻细菌及抑藻活性物质。抑藻活性化合物的菌株为Rugamonas sp.A3。抑藻活性物质紫色杆菌素的分子式为C18H12O2N6,分子量为343.1。通过分离筛选得到的Rugamonas sp.A3制备发酵液,离心后获得菌体,萃取,离心,取上清蒸干,萃取,分离去除杂质,旋干后得粗提物;取甲醇溶解粗提物,上样,洗脱并收集不同颜色组分,蒸干后进行抑藻活性验证,选取具有抑藻效果的组分;将有效组分过柱洗脱,收集不同组分进行层析,显色,合并相似组分,蒸干后验证抑藻活性,选取具有抑藻活性的组分;检测纯度后,用质谱、1H-NMR检测得到的抑藻活性组分。
Description
技术领域
本发明涉及一株抑藻细菌及抑藻活性物质,特别涉及一种具有强效抑藻活性物质紫色杆菌素及其制备方法。
背景技术
海洋中的浮游微藻、原生动物或细菌等在适宜的环境条件下大量繁殖或聚集,引起水质变坏、海水变色的海洋生态异常现象被称之为赤潮,又称有害藻华[1]。针胞藻纲(Raphidophytes)赤潮异弯藻(Heterosigma akashiwo)是一种引起鱼类死亡的典型有害赤潮藻种,在世界南北半球的许多国家都发生过赤潮异弯藻赤潮,造成过很大的经济损失[2-3]。
当前的赤潮治理方法主要有:物理方法、化学方法和生物方法[4]。物理方法主要有隔离法、超声波法、微滤机除藻、活性炭吸附及气浮法等。由于物理方法对于低密度或底层藻类的灭杀效果不好、费用又高,因此难于大面积使用通常只是一种应急措施。化学方法主要是向水体中投入特定的化学药物:如硫酸铜、高锰酸钾、漂白粉等来杀灭或抑制赤潮藻。化学方法具有操作简单、见效快等优点,但是成本高,化学物质难以去除,特别是所施用的化学药剂会给环境造成二次污染。因此,人们开始关注用生物方法来治理赤潮。
由于藻类水华爆发过程常常导致菌群结构的变化,使得菌藻关系的研究成为指导学界开始利用微生物对赤潮藻进行调控的重要思路[5-7]。利用微生物治理赤潮,主要基于特殊微生物的溶藻能力。微生物的溶藻功能主要包括两种方式:一种为直接溶藻,即溶藻菌与藻细胞表面直接接触,甚至侵入藻细胞内,从而抑制藻细胞的生长或引起藻细胞裂解死亡;另一种为间接溶藻,即间接进攻宿主,主要包括细菌同藻类竞争有限营养和通过分泌胞外代谢物质溶藻[8]。目前报道的多数功能溶藻细菌的溶藻作用方式属于后者,该溶藻方式是至今发现的溶藻功能细菌的主要作用方式。Wang[9]等发现从厦门海域分离出的溶藻菌DHQ25,可通过分泌胞外蛋白来抑制和杀死赤潮藻。Jeong[10]等从具有强烈溶藻活性的海洋芽抱杆菌SY一菌株中分离出1种新的多肤类物质Bacillamide,它对有害甲藻(多环旋沟藻)有特异性的杀灭作用。Kawan[11]等从菌株PK654中分离出1种抗生素(thiotropocin),对中肋骨条藻和赤潮异湾藻均有明显的抑制作用。
参考文献:
[1]汪艳涛,高强,姜少慧.赤潮灾害监测预报与减灾对策研究进展[J].中国渔业经济,2013,04:161-167.
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发明内容
本发明的第一目的是提供一种抑藻活性化合物的菌株及其筛选方法。
本发明的第二目的是提供一种抑藻活性物质紫色杆菌素及其制备方法。
本发明的第三目的是提供所述抑藻活性物质紫色杆菌素在制备抑藻剂中的应用。
所述抑藻活性化合物的菌株为Rugamonas sp.A3,该菌株已于2015年01月06日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮编:100101,保藏中心登记入册编号:CGMCC No.10281。
菌株Rugamonas sp.A3的筛选方法包括以下步骤:
1)取福建九龙江流域江水样品,溶解于高压灭菌的90mL高氏I号培养基,置于150~200rpm摇床震荡20~30min,使样品均匀分散;所述高氏I号培养基的组成为:可溶性淀粉2g,KNO30.1g,K2HPO40.05g,MgSO4·7H2O 0.05g,NaCl 0.05g,FeSO4·7H2O 0.001g,蒸馏水100mL,pH7.2~7.4;
2)用10倍稀释法,涂布均匀分散的样品于高氏I号固体平板,置于27℃温度下培养3~5d;
3)挑取不同类型单菌落划线于高氏I号固体平板,置于27℃温度下培养3~5d,验证是否纯培养,重复该步骤直到得到纯培养;
4)接种分离出的纯培养物单菌落于4mL高氏I号液体培养基,置于27℃摇床,180rpm震荡培养3~5d;
5)将1mL发酵液加入到20mL指数期赤潮异弯藻培养液中,于20±1℃,12h光照,12h黑暗,50μmol photons m-2s-1光照强度条件下培养3天,高氏I号培养基为对照加入藻液中,分别设置3个平行;观察赤潮异弯藻藻细胞是否死亡,从而筛选出抑藻活性菌株。将抑藻菌株于-80℃保藏,待用。
所述抑藻活性物质紫色杆菌素的分子式为C18H12O2N6,分子量为343.1,结构式如下:
所述抑藻活性物质紫色杆菌素的制备方法,包括以下步骤:
1)将菌株Rugamonas sp.A3接种于高氏I号平板上划线分离培养,待平板上的单菌落开始有白色变为蓝色时,挑取仍为白色的单菌落接种于蒸馏水配制的高氏I号液体培养基中培养,待菌液完全变蓝即为所需的发酵液;
2)将步骤1)中得到的发酵液离心,去除上清液,用无水乙醇萃取获得的蓝色菌体,超声振荡后离心,将所得的上清蒸干,加入乙酸乙酯和蒸馏水萃取过夜,再去除水相杂质,重新旋干后得到粗提取物A;
3)将步骤2)所得的粗提物A溶于甲醇中,上样于葡聚糖凝胶柱,以甲醇为流动相洗脱,根据颜色不同收集洗脱液,获得不同组分,置于旋转蒸发仪下旋干后称重,再溶解于DMSO中进行抑藻活性验证,确定抑藻活性组分B;
4)将步骤3)所得的抑藻活性组分B利用高效液相色谱(High Performance LiquidChromatography,HPLC)分析;
5)将步骤3)所得的抑藻活性组分B溶于乙酸乙酯中,然后上样于硅胶柱,采用流动相梯度浓度洗脱,蓝色和***混合,将***条带与蓝色组分分离并洗脱下蓝色组分,收集洗脱液进行层析,采用碘蒸气显色和硫酸铵乙醇显色,然后合并相似组分,得到两个主要组分,置于旋转蒸发仪下旋干后称重,再溶解于DMSO验证抑藻活性,选取经验证后具有抑藻活性组分的活性物质C;
6)将步骤5)中所得的活性物质C通过Q Exactive LC-MS/MS***和1H-NMR检测对化合物进行鉴定,该活性物质C即为本发明所述抑藻活性物质紫色杆菌素,分子式如下:
在步骤1)中,所述划线分离培养的条件可于27℃下培养2~3d;所述接种于蒸馏水配制的高氏I号液体培养基中培养的条件可于27℃下180rpm培养2~3d。
在步骤2)中,所述发酵液离心的条件可于12000~14000g离心10~20min;所述超声振荡后离心的条件可超声振荡1h后,于12000~14000g离心10~20min;所述蒸干的条件可置于旋转蒸发仪下30℃真空蒸干;所述乙酸乙酯和蒸馏水的体积比可为2∶1;所述去除水相杂质可采用分液漏斗去除水相杂质。
在步骤3)中,所述葡聚糖凝胶柱可采用Sephadex LH-20。
在步骤4)中,所述高效液相色谱分析的条件可为:分析柱采用SunFireTm C18 5μm(4.6mm×250mm Column),流动相为甲醇和水,甲醇和水的体积比可为7∶3,流速为1mL/min,柱温为30℃,检测波长为575nm,每次进样为20μL。
在步骤5)中,所述硅胶柱可采用170mm×30mm,200~300目的硅胶柱;所述采用流动相梯度浓度洗脱的洗脱剂可为三氯甲烷和甲醇,三氯甲烷和甲醇的初始体积比可为50∶1,逐步过渡到最终体积比可为15∶1;洗脱程序如下:三氯甲烷和甲醇的体积比50∶1,蓝色和***混合,三氯甲烷和甲醇的体积比30∶1,***条带与蓝色组分分离并洗脱,三氯甲烷和甲醇的体积比15∶1,洗脱下蓝色组分,洗脱流速:1mL/min;在洗脱过程中,最好间隔收集洗脱液。
所述抑藻活性物质紫色杆菌素可在制备抑藻剂中应用。
为控制赤潮异弯藻的生长,本发明采集不同生境下的环境样品分离筛选菌株,从福建漳州九龙江口的江水中分离到一株产蓝色素菌株,该菌株被鉴定为Rugamonas菌属,其所产蓝色素主成分紫色杆菌素对赤潮异弯藻的生长具有明显的抑制效果,且特异性较高,目前尚未见关于紫色杆菌素具有抑制赤潮异弯藻的报道,菌株A3所产紫色杆菌素对赤潮异弯藻的溶藻机理具有重要的研究意义,也将为赤潮异弯藻抑藻制剂的研发提供的理论支持,具有重要的实用价值和研究意义。
本发明经筛选获得了一株Rugamonas sp.A3,通过发酵培养,获得含有强抑藻活性化合物的发酵液,将所述发酵液离心,乙醇萃取蓝色菌体,然后离心后将上清液进行分离纯化,获得具有强抑藻活性的化合物。所述活性物质能够高效、专一地杀灭藻细胞,具有开发成抑藻剂的潜能,在生物降解藻类,治理赤潮方面具有广泛的应用。
附图说明
图1为菌株Rugamonas sp.A3的电镜照片图(A3在高氏I号平板上27℃培养24h后观察)。
图2为菌株Rugamonas sp.A3的进化树(将与细菌A3相似性最近的20株细菌的16S rRNA序列进行临接法(Neighbour-joining)构建进化树,只保留bootstrap values>70%)。在图2中,标尺为0.01核苷酸替换率(Knuc)。
图3为活性组分高效液相图谱。
图4为本发明所述抑藻活性物质紫色杆菌素的质谱图谱。在图4中,横坐标代表质荷比(m/z),纵坐标代表各个峰的相对丰度。
具体实施方式
以下实施例是对本发明的进一步说明,但本发明不限于下述实施例。
实施例1:抑藻细菌菌株的分离筛选
1)取福建九龙江流域江水样品,溶解于高压灭菌的90mL高氏I号培养基(可溶性淀粉2g,KNO30.1g,K2HPO40.05g,MgSO4·7H2O 0.05g,NaCl 0.05g,FeSO4·7H2O 0.001g,蒸馏水100mL,pH7.2~7.4),置于150~200rpm摇床震荡20~30min,使样品均匀分散;
2)10倍稀释法,涂布均匀分散的样品于高氏I号固体平板,于27℃温度下培养3~5d;
3)挑取不同类型单菌落划线于高氏I号固体平板,置于27℃温度下培养3~5d,验证是否纯培养,重复该步骤直到得到纯培养;
4)接种分离出的纯培养物单菌落于4mL高氏I号液体培养基,置于27℃摇床,180rpm震荡培养3~5d;
5)将1mL发酵液加入到20mL指数期赤潮异弯藻培养液中,于20±1℃,12h光照,12h黑暗,50μmol photons m-2s-1光照强度条件下培养3天,高氏I号培养基为对照加入藻液中,分别设置3个平行;观察赤潮异弯藻藻细胞是否死亡,从而筛选出抑藻活性菌株。将抑藻菌株于-80℃保藏,待用。
实施例2:抑藻效果测定方法
1)赤潮异弯藻在20±1℃,12h光照,12h黑暗,50μmol photons m-2s-1光照强度条件下,于三角瓶中培养至指数期,然后分装到24孔细胞培养板中,每孔分装2mL藻细胞悬液,适应生长1天;
2)定量待测样品加入24孔板;
3)每隔一定时间取赤潮异弯藻培养液,200μL样品于24孔板,用酶标仪检测440nm激发光激发下680nm处的荧光强度,根据测定的对照组和处理组的藻细胞荧光强度,同时藻细胞形态变化进行观察。
实施例3:抑藻活性物质的分离和鉴定
1)将菌株Rugamonas sp.A3接种于高氏I号平板上划线分离,于27℃下培养2~3d,待平板上的单菌落开始有白色变为蓝色时,挑取仍为白色的单菌落接种于蒸馏水配制的高氏I号液体培养基中,于27℃下180rpm培养2~3d,待菌液完全变蓝即为所需的发酵液;
2)将步骤1)中的发酵液于12000~14000g离心10~20min;
3)去掉步骤2)中的上清液,用无水乙醇萃取步骤2)中获得的蓝色菌体,超声振荡1h后,于12000~14000g离心10~20min;
4)将在步骤3)所得的上清置于旋转蒸发仪下30℃真空蒸干,加入一定量的乙酸乙酯和蒸馏水(比例为2∶1)萃取过夜,分液漏斗去除水相杂质,重新旋干后得到粗提取物A;
5)将步骤4)所得的粗提物A溶于少量甲醇中,上样于葡聚糖凝胶柱(Sephadex LH-20),以甲醇为流动相洗脱,根据颜色不同收集洗脱液,获得不同组分;
6)将步骤5)中获得的不同组分置于旋转蒸发仪下旋干后称重,按一定比例溶解于DMSO中进行抑藻活性验证,确定抑藻活性组分B;
7)将步骤6)所得的抑藻活性组分B利用高效液相色谱(High Performance LiquidChromatography,HPLC)分析,分析柱SunFireTm C18 5μm(4.6mm×250mm Column);流动相:甲醇∶水(体积比7∶3);流速:1mL/min;柱温:30℃;检测波长575nm;每次进样20μL。
8)将步骤6)所得的组分B溶于乙酸乙酯中,然后上样于硅胶柱(170mm×30mm,200~300目),采用流动相梯度浓度洗脱,洗脱剂为三氯甲烷和甲醇(初始比例为50∶1,逐步过渡到最终比例为15∶1),洗脱程序:体积比50∶1,蓝色和***混合,30∶1,***条带与蓝色组分分离并洗脱下来,15∶1,蓝色组分洗脱下来,洗脱流速:1mL/min;在洗脱过程中,间隔收集洗脱液,将洗脱液进行层析,采用碘蒸气显色和硫酸铵乙醇显色,然后合并相似组分,得到两个主要组分,然后将两个组分置于旋转蒸发仪下旋干后称重,再溶解于DMSO验证抑藻活性,选取经验证后具有抑藻活性组分的活性物质C;
9)将步骤8)中所得的活性物质C通过Q Exactive LC-MS/MS***和1H-NMR检测对化合物进行鉴定,该活性物质C即为本发明所述的紫色杆菌素,分子式如下:
表1
表1给出实验检测有效组分的化学位移与已报道的紫色杆菌素化学位移对比。
本发明所抑藻活性物质紫色杆菌素为蓝黑色色素,由两个色氨酸分子氧化缩合而成,其分子量为343.1,其分子式是C18H12O2N6。通过分离筛选得到的Rugamonas菌属菌株A3制备发酵液,离心后获得菌体,用无水乙醇进行萃取,离心后取上清蒸干,加入乙酸乙酯萃取过夜,分液漏斗分离去除杂质,将乙酸乙酯萃取液旋干后获得粗提物;取少量甲醇溶解粗提物,上样于葡聚糖凝胶柱,洗脱并收集不同颜色组分,蒸干后溶解于DMSO中进行抑藻活性验证,选取具有抑藻效果的组分,实现大量杂质的去除;将有效组分过硅胶柱进行洗脱,收集不同组分进行层析分析,采用碘蒸气显色和硫酸铵乙醇显色,合并相似组分,蒸干后溶解于DMSO验证抑藻活性,选取具有抑藻活性的组分;利用HPLC检测纯度后,用质谱、1H-NMR检测得到的抑藻活性组分。该抑藻活性物质能高效、快速抑制有害藻华藻赤潮异弯藻的生长,在赤潮灾害防治和海洋活性物质开发利用等方面具有重要意义和实用价值。
Claims (10)
1.菌株Rugamonas sp.A3,已于2015年01月06日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏中心登记入册编号:CGMCC No.10281。
2.如权利要求1所述菌株Rugamonas sp.A3的筛选方法,其特征在于包括以下步骤:
1)取福建九龙江流域江水样品,溶解于高压灭菌的90mL高氏I号培养基,置于150~200rpm摇床震荡20~30min,使样品均匀分散;所述高氏I号培养基的组成为:可溶性淀粉2g,KNO30.1g,K2HPO40.05g,MgSO4·7H2O 0.05g,NaCl 0.05g,FeSO4·7H2O 0.001g,蒸馏水100mL,pH7.2~7.4;
2)用10倍稀释法,涂布均匀分散的样品于高氏I号固体平板,置于27℃温度下培养3~5d;
3)挑取不同类型单菌落划线于高氏I号固体平板,置于27℃温度下培养3~5d,验证是否纯培养,重复该步骤直到得到纯培养;
4)接种分离出的纯培养物单菌落于4mL高氏I号液体培养基,置于27℃摇床,180rpm震荡培养3~5d;
5)将1mL发酵液加入到20mL指数期赤潮异弯藻培养液中,于20±1℃,12h光照,12h黑暗,50μmol photons m-2s-1光照强度条件下培养3天,高氏I号培养基为对照加入藻液中,分别设置3个平行;观察赤潮异弯藻藻细胞是否死亡,从而筛选出抑藻活性菌株;将抑藻菌株于-80℃保藏,待用。
3.抑藻活性物质紫色杆菌素,其特征在于其分子式为C18H12O2N6,分子量为343.1,结构式如下:
4.如权利要求3所述抑藻活性物质紫色杆菌素的制备方法,其特征在于包括以下步骤:
1)将菌株Rugamonas sp.A3接种于高氏I号平板上划线分离培养,待平板上的单菌落开始有白色变为蓝色时,挑取仍为白色的单菌落接种于蒸馏水配制的高氏I号液体培养基中培养,待菌液完全变蓝即为所需的发酵液;
2)将步骤1)中得到的发酵液离心,去除上清液,用无水乙醇萃取获得的蓝色菌体,超声振荡后离心,将所得的上清蒸干,加入乙酸乙酯和蒸馏水萃取过夜,再去除水相杂质,重新旋干后得到粗提取物A;
3)将步骤2)所得的粗提物A溶于甲醇中,上样于葡聚糖凝胶柱,以甲醇为流动相洗脱,根据颜色不同收集洗脱液,获得不同组分,置于旋转蒸发仪下旋干后称重,再溶解于DMSO中进行抑藻活性验证,确定抑藻活性组分B;
4)将步骤3)所得的抑藻活性组分B利用高效液相色谱(High Performance LiquidChromatography,HPLC)分析;
5)将步骤3)所得的抑藻活性组分B溶于乙酸乙酯中,然后上样于硅胶柱,采用流动相梯度浓度洗脱,蓝色和***混合,将***条带与蓝色组分分离并洗脱下蓝色组分,收集洗脱液进行层析,采用碘蒸气显色和硫酸铵乙醇显色,然后合并相似组分,得到两个主要组分,置于旋转蒸发仪下旋干后称重,再溶解于DMSO验证抑藻活性,选取经验证后具有抑藻活性组分的活性物质C;
6)将步骤5)中所得的活性物质C通过Q Exactive LC-MS/MS***和1H-NMR检测对化合物进行鉴定,该活性物质C即为本发明所述抑藻活性物质紫色杆菌素,分子式如下:
5.如权利要求4所述抑藻活性物质紫色杆菌素的制备方法,其特征在于在步骤1)中,所述划线分离培养的条件是于27℃下培养2~3d;所述接种于蒸馏水配制的高氏I号液体培养基中培养的条件可于27℃下180rpm培养2~3d。
6.如权利要求4所述抑藻活性物质紫色杆菌素的制备方法,其特征在于在步骤2)中,所述发酵液离心的条件是于12000~14000g离心10~20min;所述超声振荡后离心的条件可超声振荡1h后,于12000~14000g离心10~20min;所述蒸干的条件可置于旋转蒸发仪下30℃真空蒸干;所述乙酸乙酯和蒸馏水的体积比可为2∶1;所述去除水相杂质可采用分液漏斗去除水相杂质。
7.如权利要求4所述抑藻活性物质紫色杆菌素的制备方法,其特征在于在步骤3)中,所述葡聚糖凝胶柱采用Sephadex LH-20。
8.如权利要求4所述抑藻活性物质紫色杆菌素的制备方法,其特征在于在步骤4)中,所述高效液相色谱分析的条件为:分析柱采用SunFireTm C185μm,4.6mm×250mmColumn,流动相为甲醇和水,甲醇和水的体积比可为7∶3,流速为1mL/min,柱温为30℃,检测波长为575nm,每次进样为20μL。
9.如权利要求4所述抑藻活性物质紫色杆菌素的制备方法,其特征在于在步骤5)中,所述硅胶柱采用170mm×30mm,200~300目的硅胶柱;所述采用流动相梯度浓度洗脱的洗脱剂可为三氯甲烷和甲醇,三氯甲烷和甲醇的初始体积比可为50∶1,逐步过渡到最终体积比可为15∶1;洗脱程序如下:三氯甲烷和甲醇的体积比50∶1,蓝色和***混合,三氯甲烷和甲醇的体积比30∶1,***条带与蓝色组分分离并洗脱,三氯甲烷和甲醇的体积比15∶1,洗脱下蓝色组分,洗脱流速:1mL/min;在洗脱过程中,最好间隔收集洗脱液。
10.如权利要求3所述抑藻活性物质紫色杆菌素在制备抑藻剂中应用。
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