CN115927057B - 一种链霉菌菌株yp1及其应用 - Google Patents
一种链霉菌菌株yp1及其应用 Download PDFInfo
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- CN115927057B CN115927057B CN202211015138.XA CN202211015138A CN115927057B CN 115927057 B CN115927057 B CN 115927057B CN 202211015138 A CN202211015138 A CN 202211015138A CN 115927057 B CN115927057 B CN 115927057B
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请涉及一种链霉菌菌株YP1及其应用，属于微生物技术领域。本申请从土壤中分离出一种能产榴菌素的菌株，命名为YP1，并提供使用YP1产榴菌素的应用。本申请的链霉菌菌株YP1已在中国微生物菌种保藏管理委员会普通微生物中心进行保藏，保藏登记编号为CGMCC No.23383，保藏日期为2021年9月10日。
Description
技术领域
本申请涉及一种链霉菌菌株YP1及其应用，属于微生物技术领域。
背景技术
榴菌素(granaticin，CAS号：19879-06-2)是苯并异色烷醌家族(benzoisochromanequinones，BIQs)抗生素中的一员，属于芳香聚酮类抗生素。其分子式为C22H20O10，分子量为444.38800，在一个标准大气压下沸点为748.3℃，闪点是266.8℃，熔点在223-225℃之间，正常情况下密度为1.77g/cm3，折射率是1.745。榴菌素是深红色晶体，溶解性在不同的溶液中有所不同，其可溶于水、甲醇和乙醇等极性溶液，但在非极性溶液中不具有溶解性，并且在水中的溶解度受pH的影响，与pH值呈正相关，且在酸性水溶液中为红色，碱性水溶液中为蓝色。
榴菌素第一次被发现是在橄榄色链霉菌(S.olivaceus)中。之后，人们相继从粤蓝链霉菌GIMV4.0001(S.vietnamensisg IMV4.0001)、紫红链霉菌(S.violaceoruber)、砖红链霉菌(S.lateritius)中分离出了榴菌素。同时也从部分菌株中分离得到了榴菌素类似物，如榴菌素B，二氢榴菌素等。
榴菌素可作为一种极具潜力的抑菌和抗癌抗生素，但是目前发现的能产榴菌素的菌株种类有限，远远不能满足实际需要。
发明内容
为了解决能产榴菌素的菌株种类较少的技术问题、丰富能产榴菌素的菌株的种类；本申请从土壤中分离出一种能产榴菌素的菌株，命名为YP1,并提供使用YP1产榴菌素的应用。
本申请提供的菌株YP1是从中国河南省郑州市(34°48'00.00"N，113°39'00.00"E)收集的土壤中分离出来的。具体的是将从中国河南省郑州市(34°48'00.00"N，113°39'00.00"E)收集的土壤，配置为土壤悬液，然后稀释成10-3，涂布于高氏培养基平板进行培养，于28℃培养。取具有产色特征的菌种并在显微镜下检查，证明为单一菌体后，通过反复分离和大量菌株重复性培养筛选，获得YP1菌株。本申请的链霉菌菌株YP1已在中国微生物菌种保藏管理委员会普通微生物中心进行保藏，保藏登记编号为CGMCCNo.23383，保藏日期为2021年9月10日。
本申请提供的菌株YP1的形态学特征：
本菌株菌落呈乳白或淡黄色，类圆形，以不透明为主，菌落表面平滑或略向上凸起。孢子丝直、柔曲、螺旋；孢子表面光滑，呈粉末状
另外，本申请的菌株YP1是一种革兰氏阳性菌好氧菌；培养条件为：pH5-8、20-40℃；其中，最佳pH为7.0，最佳温度为28℃；NaCl耐受性0-5％(w/v)。
本申请提供的菌株YP1的生理生化学特征：
本菌株具有接触酶、氧化酶、β半乳糖苷酶、精氨酸双水解酶活性，不具有赖氨酸、鸟氨酸脱羧酶和脲酶活性。能够水解明胶、还原硝酸盐，不能水解吐温40，不能产生H2S和吲哚，VP反应和甲基红实验为阴性，不能利用柠檬酸。能够利用糊精、麦芽糖、海藻糖、蔗糖、水苏糖、棉子糖、乳糖等碳源，不能利用岩藻糖糖、山梨醇、甘露醇、阿糖醇、肌醇作为碳源。不能利用水样苷、丝氨酸、丙氨酸、精氨酸、天冬氨酸，能够利用果胶，半乳糖醛酸、L-半乳糖内酯、葡糖酸、奎宁酸、γ-氨基丁酸、α-羟丁酸、β-羟基-D,L-丁酸，不能利用D-葡糖-6-磷酸、葡糖醛酰胺、D-果糖-6-磷酸、粘酸、p-羟基苯乙酸、丙酮酸甲酯、D-乳酸甲酯、L-乳酸、柠檬酸、α-酮戊二酸、D-苹果酸、L-苹果酸。对醋竹桃霉素、利福霉素SV、二甲胺四环素、万古霉素敏感。
本申请提供的菌株YP1的基因特征：
与近型菌株沙阿链霉菌(Streptomyceszaomyceticus)、近型菌株砖红链霉菌(Streptomyceslateritius)的dDH值分别为28.5％和33.9％；远低于细菌物种的70％的阈值；
与近型菌株沙阿链霉菌(Streptomyceszaomyceticus)、近型菌株砖红链霉菌(Streptomyceslateritius)的ANI值分别为83.95％和87.56％，远低于建议划界物种的95-96％的阈值；是一种新的链霉菌菌种。
通过形态学特征、生理生化特征和基因特征分析，YP1菌株符合链霉菌的特征，属于链霉菌，是一种新的链霉菌(拉丁文Streptomycesvilmorinianum)菌种。
本申请还提供的链霉菌菌株YP1在产榴菌素中的应用。
本申请的链霉菌菌株YP1产榴菌素，包括以下步骤：
(1)平板培养：将菌株YP1划线于平板固体培养基上，于36-38℃恒温中培养1-3天；
(2)种子液培养：从平板固体培养基上挑取生长良好的YP1菌体，接入高温灭菌好的增殖培养基中，放入恒温摇床中培养1-3天，摇床转速为170-190r/min，培养温度为36-38℃；获得菌株YP1种子液；
(3)发酵培养：将菌株YP1种子液以4-6％v/v的接种量接入经高温灭菌的发酵培养基中，将发酵培养基置入恒温摇床中培养3-5天，摇床转速为170-190r/min，培养温度为36-38℃；获得蓝色的菌株YP1发酵液；
(4)对菌株YP1发酵液进行离心、旋蒸浓缩、纯化即得榴菌素；
平板固体培养基：琼脂20g、乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min；
增殖培养基：乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min；
发酵培养基：乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min。
在一些实施方案中，所述步骤(1)的培养条件为：37℃恒温中培养2天；在一些实施方案中，所述步骤(2)的培养条件为：恒温摇床中培养2天，摇床转速为180r/min，培养温度为37℃；
在一些实施方案中，所述步骤(3)的培养条件为：恒温摇床中培养4天，摇床转速为180r/min，培养温度为37℃。
在一些实施方案中，所述纯化包括以下操作：
(1)用大孔吸附树脂纯化旋蒸浓缩获得的样品，选择乙醇—水体系进行冲洗，收集90％和100％浓度乙醇冲出的样品作为粗纯物A；
(2)将粗纯物A溶解到40％甲醇中，过滤后上半制备高效液相色谱仪，选择甲醇—水体系洗脱样品，收集60％甲醇冲出的样品即可；
所述％为质量百分数。
有益效果
本申请提供的链霉菌菌株YP1，是链霉菌菌属下的新菌株，丰富了链霉菌菌属的种类；
本申请提供的链霉菌菌株YP1，能够产榴菌素，为天然榴菌素的获取提供了新途径；
本申请提供的链霉菌菌株YP1产榴菌素的产量为180U/mL。
保藏信息
保藏单位：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保藏日期：2021年9月10日
保藏编号：CGMCC No.23383
分类命名：Streptomyces vilmorinianum。
附图说明
图1是本申请提供的链霉菌菌株YP1在ISP2培养基上的菌落形态照片；
图2是本申请提供的链霉菌菌株YP1在ISP3培养基上的菌落形态照片；
图3是本申请提供的链霉菌菌株YP1在ISP4培养基上的菌落形态照片；
图4是本申请提供的链霉菌菌株YP1在ISP5培养基上的菌落形态照片；
图5是本申请提供的链霉菌菌株YP1在ISP6培养基上的菌落形态照片；
图6是本申请提供的链霉菌菌株YP1在ISP7培养基上的菌落形态照片；
图7是本申请的菌株YP1的基因序列通过基因库(Genebank)序列比对，利用MEGA6.0建立***发育树。
具体实施方式
在下面所述的具体实施例中，如无特殊说明，均为本领域常用方法。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本申请中，如果没有特殊说明，所述％为质量百分数。
本申请所用缩略语说明：
d：天；
dDDH值：基因组数据杂交值(digital DNA–DNA hybridization)
ANI值：平均核苷酸一致性(average nucleotide identity)
MEGA6：软件--分子进化遗传学分析6.0版本(Moleccular EvolutionarygeneticsAnalysis)
MK-n(Hm)：甲基萘醌，n表示异戊二烯单元的数量，m表示取代异戊二烯的氢原子数量
anteiso-c18:0：14-甲基-十六烷酸
anteiso-C16:0：12-甲基-十四烷酸
iso-C17:0：14-甲基-十五烷酸
本申请所用各种培养基及其成分、pH如下：
ISP2培养基：酵母浸粉4g、麦芽提取物10g、葡萄糖4g、去离子水1L和固体加琼脂粉20g，pH＝7.3；
ISP3培养基：燕麦粉20g、琼脂18g、七水硫酸亚铁0.001g、氯化锰0.001g、硫酸锌0.001g和去离子水1L，pH＝7.3±0.2；
ISP4培养基：可溶性淀粉10g、磷酸二氢钾1g、硫酸镁1g、氯化钠1g、硫酸铵2g、硫酸钙2g、七水硫酸亚铁0.001g、氯化锰0.001g、硫酸锌0.001g、琼脂15g和去离子水1L，pH＝7.2±0.2；
ISP5培养基：天门冬氨酸1g、磷酸二氢钾1g、七水硫酸亚铁0.001g、氯化锰0.001g、硫酸锌0.001g、琼脂15g和去离子水1L，pH＝7.4±0.2；
ISP6培养基：蛋白胨15g、胨5g、酵母浸粉1g、柠檬酸铁铵0.5g、磷酸氢二钾1g、硫代硫酸钠0.08g、琼脂15g和去离子水1L，pH＝6.7±0.2；
ISP7培养基：甘油15g、酪氨酸0.5g、天冬氨酸1g、磷酸氢二钾0.5g、硫酸镁0.5g、氯化钠0.5g、硫酸亚铁0.5g、七水硫酸亚铁0.001g、氯化锰0.001g、硫酸锌0.001g、琼脂20g和去离子水1L，pH＝7.3±0.1；
平板(斜面)培养基：琼脂20g、乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min；
增殖/发酵培养基：乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min；
LB培养基：胰蛋白10g、酵母浸粉5g、NaCl10g和蒸馏水1000ml，自然pH，115℃灭菌30min；
YPD培养基：葡萄糖20、胰蛋白胨20、酵母浸粉10和蒸馏水1000ml，自然pH，115℃灭菌30min；
实施例1菌株YP1的鉴定
1.1形态学特征
将获得的菌株YP1纯培养体分别接种到ISP2、ISP3、ISP4、ISP5、ISP6和ISP7培养基中进行培养，观察形态特征：结果如下：
ISP2培养基，28℃，培养2d；菌落乳白色、类圆形，不透明，菌落表面略向上凸起，不分泌色素；显微镜下观察，孢子丝及孢子的形态如图1所示；
ISP3培养基，28℃，培养2d，菌落乳白色，类圆形，不透明，菌落表面略向下凹陷，初始分泌褐色色素，最后分泌蓝紫色色素；显微镜下观察，孢子丝及孢子的形态如图2所示；
ISP4培养基，28℃，培养2d；菌落淡黄色，圆形，不透明，菌落表面平滑，分泌粉色色素；显微镜下观察，孢子丝及孢子的形态如图3所示；
ISP5培养基，28℃，培养2d；菌落白色，圆形，不透明，菌落表面平滑，不分泌色素；显微镜下观察，孢子丝及孢子的形态如图4所示；
ISP6培养基，28℃，培养2d；菌落乳白色，圆形，微透明，菌落表面向上突起，分泌黑色色素；显微镜下观察，孢子丝及孢子的形态如图5所示；
ISP7培养基，28℃，培养2d；菌落淡乳白色，类圆形，透明，菌落表面略向上凸起、分泌灰蓝紫色色素；显微镜下观察，孢子丝及孢子的形态如图6所示；
菌株YP1的培养特征，如表1所示；
表1
菌株YP1的形态特征表明，菌株YP1具有链霉菌属的典型特征，是一种链霉菌。
另外，菌株YP1经革兰氏染色呈革兰氏阳性是革兰氏阳性菌；在培养基不变的情况下，将摇床以低转速(小于100rpm/min)培养(溶氧量低)菌株YP1，菌体生长较差，说明菌株YP1是好氧菌。
对菌株YP1的培养条件进行优化实验。实验结果表明：菌株YP1的培养条件为：pH5-8、20-40℃；其中，最佳pH为7.0，最佳温度为28℃。NaCl耐受性0-5％(w/v)。
1.2生理生化特征
检测方法：菌株的生理生化特征检测方法参照《伯杰细菌鉴定手册》和《常见细菌***鉴定手册》进行。
检测结果如表2所示。
检测结果说明：“-”是指阴性(未检测到)，“+”是指阳性(检测到)“W”是指无法检出。
表2
1.3基因特征
对本申请的菌株YP1进行DNA测序，采用如SEQ ID NO.2和SEQ ID NO.3所示的引物对其16SrRNA基因序列进行扩增，然后进行测序，其16SrRNA基因序列如SEQ ID NO.1所示。该基因序列已经加入GenBank/EMBL加入编号为MK801227.1。本申请的链霉菌菌株YP1基因组序列数据显示，其DNA中G+C含量为71.5％(物质的量含量)。本申请的链霉菌菌株YP1与近型菌株沙阿链霉菌(Streptomyceszaomyceticus，NBRC13348)、近型菌株砖红链霉菌(Streptomyceslateritius，LMG19282)的dDH值分别为28.5％和33.9％；远低于迈尔-科尔托夫等人推荐的细菌物种的70％的阈值。本申请的链霉菌菌株YP1与近型菌株沙阿链霉菌(Streptomyceszaomyceticus，NBRC13348)、近型菌株砖红链霉菌(Streptomyceslateritius，LMG19282)的ANI值分别为83.95％和87.56％，远低于建议划界物种的95-96％的阈值。将本申请的菌株YP1的基因序列通过基因库(Genebank)序列比对，利用MEGA6.0建立***发育树，结果见图7。通过16SrDNA序列***发育分析，菌株YP1鉴定为链霉菌(Streptomyces vilmorinianumgroup)。
SEQ ID NO.2:27F(5′-AGAGTTTGATCCTGGCTCAG-3′)；
SEQ ID NO.3:1492R(5′-TACGACTTAACCCCAATCGC-3′)。
1.4化学特征
细胞壁水解：将15mg菌株YP1的冻干菌体置入带有螺纹盖的小瓶中，加入1ml1mol/L的H2SO4混匀，于100℃水解2h，加入饱和氢氧化钡调pH至5.2-5.5，旋蒸至干燥，加入0.3ml水复溶；获得YP1菌株的细胞壁水解产物。
对YP1菌株的细胞壁水解产物进行分析，水解产物含有二氨基庚二酸(DAP)、葡萄糖和核糖。对细胞醌类进行检测，检测到的醌类物质为MK-9(H6)(56％)、MK-9(H8)(38％)、MK-8(H6)(3％)、MK-9(H4)(1％)和MK-9(H2)(1％)。对极性脂质和脂肪酸进行测定，检测到的极性脂质为：磷脂酰乙醇胺(PE)、磷脂酰肌醇(PI)、磷脂酰肌醇甘露糖苷(PIM)、一种不明磷脂；主要脂肪酸(>10％)为iso-c16：0和C18：0。
细胞壁水解产物：细胞的呼吸醌、细胞糖类型、极性脂质和脂肪酸能够标志某一类微生物的存在。细胞醌类是指好氧菌常见的呼吸醌型(甲基萘醌、泛醌)，链霉菌应只能检测出甲基萘醌类，而不能检测出泛醌(辅酶Q)。细胞糖可以将放线菌分为四类，如表3所示。极性脂质可将放线菌分为五类，如表4所示。根据细胞的脂肪酸组成，细菌可分为两个大组。一组包含支链和/或反支链脂肪酸作为主要成分。另一组由直链脂肪酸酸(包括单不饱和酸)组成。革兰氏阳性菌脂肪酸的主要成分只含有顺式支链或反式支链脂肪酸。
细胞壁水解产物中含有DAP说明为革兰氏阳性菌，其细胞糖为葡萄糖和核糖，可知本菌株属于C型放线菌。需氧性细菌的呼吸醌中含有甲基萘醌和泛醌两类化合物，其中革兰氏阳性菌只含有甲基萘醌化合物，革兰氏阴性菌及兼性厌氧菌则会含有泛醌类化合物，故本菌株属于革兰氏阳性好氧菌。本菌株测定的标志性极性脂质为PE，属于PⅡ型放线菌。
表3放线菌全细胞壁的主要糖型
| 半乳糖 | ***糖 | 木糖 | 马杜罗塘 | |
| A型 | + | + | - | - |
| B型 | - | - | - | + |
| C型 | - | - | - | - |
| D型 | - | + | + | - |
+表示检出；-表示未检出。
表4放线菌的极性脂类型
+表示检出；-表示未检出；PE:磷脂酰乙醇胺、PC:磷脂酰胆碱、PME:磷脂酰甲基乙醇胺、PG:磷脂酰甘油、GluNus：含葡萄糖胺未知结构的磷脂；
实施例2菌株YP1发酵液的抑菌性能
平板培养：从冰箱中拿出保存好的菌株YP1的甘油管，常温自然解冻，用接种环将菌株YP1甘油管划线于凝固好的平板固体培养基上，于37℃恒温培养箱中培养2d。经平板活化的菌株在平板上产生色泽优美的蓝色物质。
种子液培养：从产生蓝色色素的平板固体培养基上挑取生长良好的YP1菌体，于超净台中接入高温灭菌好的增殖培养基中，将装有50ml增殖培养基的锥形瓶(容量为250ml)放入恒温摇床中培养2d，摇床转速为180r/min，培养温度为37℃。获得菌株YP1种子液。
发酵培养：在超净台中将处于对数生长中后期的菌株YP1种子液以5％(v/v)的接种量接入经高温灭菌的发酵培养基中，将装有的50ml发酵培养基的锥形瓶置入恒温摇床中培养4d，摇床转速为180r/min，培养温度为37℃。获得蓝色菌株YP1发酵液。对发酵液进行离心、旋蒸浓缩，蒸去多余液体，得***浓缩样品。
采用牛津杯法测定菌株YP1的发酵液对4种受试菌(菌浓为107CFU/mL)的体外抑制作用，结果表明，菌株YP1发酵液对革兰氏阳性菌即金黄色葡萄球菌和枯草芽孢杆菌有抑制作用，对革兰氏阴性菌即大肠杆菌和真菌白色念珠菌无抑制作用，抑菌结果与榴菌素抑菌特性基本吻合，初步判断菌株YP1发酵液中存在榴菌素及其类似物。具体结果如表5所示：
表5牛津杯测定链霉菌菌株YP1发酵液的抑菌效果
| 菌株 | 抑菌圈直径/mm |
| 金黄色葡萄球菌 | 18.98±0.18 |
| 枯草芽孢杆菌 | 18.55±0.24 |
| 大肠杆菌 | - |
| 白色念珠菌 | - |
注：“-”表示无抑菌直径；抑菌圈直径＞20极敏感，15-20高敏感，10-14中敏感，＜10低敏感，0不敏感。
实施例3菌株YP1发酵液产物的鉴定
将实施例2获得***浓缩样品进行下述分离纯化。同时对分离纯化的纯物质进行分子量的确定和结构的鉴定，具体操作如下：
(1)用大孔吸附树脂纯化浓缩样品，选择乙醇—水体系进行实验，以去离子水，10％、30％、47％、75％、90％、100％乙醇梯度冲样品，选择90％和100％高浓度乙醇冲出的样品作为粗纯物A，选择水冲出的样品作为粗纯物B；
(2)将粗纯物A溶解到40％甲醇中，过滤后上半制备高效液相色谱仪；选择甲醇—水体系洗脱样品，以40％、60％、70％、80％、96％甲醇梯度冲样，将60％甲醇冲出的样品重新用60％甲醇溶解，继续上半制备高效液相色谱仪分离纯化，选择甲醇—水体系洗脱样品。分别用60％甲醇和高浓度甲醇(94％)冲样，选择60％甲醇冲出来的、组分单一的样品合并，蒸干称重记作X1；选择94％甲醇冲出来的、组分单一的样品合并，蒸干称重记作X2。通过液质联用仪确定X1D的纯度与分子量。纯物质的核磁测样溶剂选择CD3OD，选用Varian 500MHz光谱仪进行实验。鉴定结果为：X1为榴菌素，X2为榴菌素类似物：4,8,13-三羟基-6,11-二酮-三氢榴菌素A(4,8,13-trihydroxy-6,11-dione-trihydrogranaticinsA)，即TDTA。
(3)粗纯物B进行红外光谱扫描，红外光谱图在3271.55cm-1、1635.99cm-1、1589.87cm-1处有特征吸收峰，具有典型的黑色素特征吸收峰；是黑色素。
Claims (9)
1.一种链霉菌菌株Streptomyces vilmorinianum YP1，简称YP1，其保藏号为CGMCC.23383。
2.根据权利要求1所述的链霉菌菌株YP1，其16S rRNA基因序列如SEQ ID NO.1所示。
3.根据权利要求1或2所述的链霉菌菌株YP1，耐受0-5％w/v的NaCl。
4.一种权利要求1、2或3任意一项所述链霉菌菌株YP1在产榴菌素中的应用。
5.根据权利要求4所述的应用，包括以下步骤：
(1)平板培养：将菌株YP1划线于平板固体培养基上，于36-38℃恒温中培养1-3天；
(2)种子液培养：从平板固体培养基上挑取生长良好的YP1菌体，接入高温灭菌好的增殖培养基中，放入恒温摇床中培养1-3天，摇床转速为170-190r/min，培养温度为36-38℃；获得菌株YP1种子液；
(3)发酵培养：将菌株YP1种子液以4-6％v/v的接种量接入经高温灭菌的发酵培养基中，将发酵培养基置入恒温摇床中培养3-5天，摇床转速为170-190r/min，培养温度为36-38℃；获得蓝色的菌株YP1发酵液；
(4)对菌株YP1发酵液进行离心、旋蒸浓缩、纯化即得榴菌素；
平板固体培养基：琼脂20g、乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl 4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min；
增殖培养基：乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl 4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min；
发酵培养基：乳糖10g、胰蛋白胨5g、酵母浸粉10g、NaCl 4g和蒸馏水1000ml，pH＝7.0，121℃灭菌20min。
6.根据权利要求5所述的应用，所述步骤(1)的培养条件为：37℃恒温中培养2天。
7.根据权利要求5所述的应用，所述步骤(2)的培养条件为：恒温摇床中培养2天，摇床转速为180r/min，培养温度为37℃。
8.根据权利要求5所述的应用，所述步骤(3)的培养条件为：恒温摇床中培养4天，摇床转速为180r/min，培养温度为37℃。
9.根据权利要求5、6、7或8所述的应用，所述纯化包括以下操作：
(1)用大孔吸附树脂纯化旋蒸浓缩获得的样品，选择乙醇—水体系进行冲洗，收集90％和100％浓度乙醇冲出的样品作为粗纯物A；
(2)将粗纯物A溶解到40％甲醇中，过滤后上半制备高效液相色谱仪，选择甲醇—水体系洗脱样品，收集60％甲醇冲出的样品即可；
所述％为质量百分数。
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