技术领域technical field
本发明涉及微生物技术领域，具体涉及一种马比木内菌生木霉属菌株及其提取喜树碱的方法。The invention relates to the technical field of microorganisms, in particular to a Trichoderma genus strain of Trichoderma spp. and a method for extracting camptothecin thereof.
背景技术Background technique
目前全球各国已批准上市的抗癌药物大约有百余种。在众多抗癌药中，植物抗癌药是国际抗癌药物市场上一大类主导产品。其中喜树碱(Camptothecin，CPT)是著名的抗癌药的先导化合物之一，被认为是有发展和应用前景的植物天然产物抗癌药之一。At present, there are more than 100 kinds of anticancer drugs that have been approved for marketing in various countries around the world. Among many anticancer drugs, plant anticancer drugs are a major category of leading products in the international anticancer drug market. Among them, camptothecin (CPT) is one of the lead compounds of the famous anticancer drug, and is considered to be one of the plant natural product anticancer drugs with development and application prospects.
喜树碱属中性生物碱，喜树碱对许多肿瘤的生长有抑制作用，将其应用于消化道肿瘤、白血病、绒毛膜上皮癌、膀胱癌的治疗上有显著效果。目前，喜树碱是从喜树的果实中提取而来，其来源受限，并且含量低。随着研究的不断扩展，发现马比木的喜树碱含量较高，部分地区种植的马比木中喜树碱含量高达干重0.3％。Camptothecin is a neutral alkaloid. Camptothecin can inhibit the growth of many tumors. It has a significant effect on the treatment of digestive tract tumors, leukemia, choriocarcinoma, and bladder cancer. At present, camptothecin is extracted from the fruit of camptothecin, and its source is limited and its content is low. With the continuous expansion of the research, it was found that the content of camptothecin in mabi wood was relatively high, and the content of camptothecin in mabi wood planted in some areas was as high as 0.3% of dry weight.
传统的喜树碱提取方法，主要是采用离子交换树脂法直接从马比木中提取，其中，采用离子交换树脂柱层析，阳离子交换树脂对喜树碱吸附过强，需用强碱性的水液进行洗脱，强碱溶液下，部分氨基酸和蛋白质溶解性增大，吸附在柱内的这些大分子杂质也被洗脱下来，导致产品纯度低，而且洗脱液由于蛋白含量高，浓缩过程中产品易起泡，增加了浓缩难度。The traditional camptothecin extraction method mainly adopts the ion exchange resin method to directly extract from Mabi wood. Among them, the ion exchange resin column chromatography is used, and the cation exchange resin is too strong for the adsorption of camptothecin, so a strong alkaline solution is required. Eluting with water, under strong alkaline solution, the solubility of some amino acids and proteins increases, and these macromolecular impurities adsorbed in the column are also eluted, resulting in low product purity, and the eluent is concentrated due to high protein content. The product is prone to foaming during the process, which increases the difficulty of concentration.
在现有技术中，有出现了利用微生物发酵提取技术。如专利号为201110368489.4公开了一种利用微生物固态发酵技术提取马比木中喜树碱的方法，主要采用外源微生物进行发酵提取。In the prior art, there has been a technique for extracting by microbial fermentation. For example, Patent No. 201110368489.4 discloses a method for extracting camptothecin in mabi wood by using microbial solid-state fermentation technology, mainly using exogenous microorganisms for fermentation and extraction.
而关于马比木内生真菌产喜树碱的研究较少，进一步关于马比木内生真菌的 分离以及马比木内生真菌代谢产物的研究报道更为鲜有。However, there are few studies on the production of camptothecin by endophytic fungi in Mabiwood, and there are even fewer reports on the isolation of endophytic fungi in Mabiwood and the metabolites of endophytic fungi in Mabiwood.
发明内容Contents of the invention
本发明为解决上述技术问题，提供一种马比木内生菌木霉属菌株及其提取喜树碱的方法。In order to solve the above-mentioned technical problems, the present invention provides a Trichoderma strain of the endophyte Trichoderma and a method for extracting camptothecin.
具体通过以下方案得以实现：Specifically, it is achieved through the following schemes:
一种马比木内生菌木霉属菌株，是从马比木中分离得到的，命名为木霉属(Trichoderma sp.)GZU-BCEC-GX8菌株，保藏于中国典型培养物保藏中心，保藏日期为2015年9月14日，保藏编号为CCTCC NO:M2015529；保藏单位地址：湖北省武汉市武昌区八一路299号武汉大学校内，武汉大学保藏中心。A strain of Trichoderma sp., an endophytic bacterium of Mabi, is isolated from Mabi, named Trichoderma (Trichoderma sp.) GZU-BCEC-GX8 strain, preserved in China Center for Type Culture Collection, date of preservation On September 14, 2015, the deposit number is CCTCC NO: M2015529; the address of the depository unit: Wuhan University Preservation Center, Wuhan University Campus, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
所述的木霉属(Trichoderma sp.)GZU-BCECX-GX8菌株，其中，该菌株的分离培养方法包括以下步骤：Described Trichoderma sp. (Trichoderma sp.) GZU-BCECX-GX8 bacterial strain, wherein, the isolation culture method of this bacterial strain comprises the following steps:
(1)组织的准备：取马比木新鲜的幼茎、叶、叶柄、芽、根皮、根芯之一种或几种，用自来水冲洗，除去样品表面的表生微生物；(1) Tissue preparation: take one or more of the fresh young stems, leaves, petioles, buds, root bark, and root cores of Mabi wood, rinse them with tap water, and remove the surface microorganisms on the surface of the sample;
(2)表面消毒及无菌检测：将组织置于超净工作台上，用1‰的吐温-80无菌水将材料洗2～3次，再用无菌水洗至无泡沫，用无菌滤纸吸去组织表面水分后，浸泡于75％乙醇溶液3min，再用无菌水冲洗2-3次，然后采用0.1％HgCl2溶液浸泡1～3min，然后用无菌水冲至冲洗水无菌，用无菌滤纸吸干组织段表面残留水滴后，切成长为0.5cm，宽为0.5cm的组织切段；(2) Surface disinfection and sterility testing: Place the tissue on an ultra-clean workbench, wash the material 2 to 3 times with 1‰ Tween-80 sterile water, then wash with sterile water until there is no foam, and use sterile After the bacterial filter paper absorbs the water on the surface of the tissue, soak it in 75% ethanol solution for 3 minutes, then rinse it with sterile water for 2-3 times, then soak it in 0.1% HgCl2 solution for 1-3 minutes, and then rinse it with sterile water until the rinse water is dry. Bacteria, dry the remaining water droplets on the surface of the tissue section with sterile filter paper, then cut into 0.5cm in length and 0.5cm in width tissue section;
(3)内生真菌的分离纯化：将步骤(2)消毒后的组织切段接种于双抗PDA++培养基平板上，置于25-30℃条件下恒温培养4～7天，至菌丝体从组织块中长出，采用边缘菌丝挑取法，将菌丝转移至PDA培养基斜面上，于25-30℃条件下进行恒温培养，待菌落长出，再转接2～3次，以保证所得菌落为纯培养，即得马比木内生真菌。(3) Isolation and purification of endophytic fungi: inoculate the tissue sections after disinfection in step (2) on a double-antibody PDA++ medium plate, and place them at 25-30°C for constant temperature cultivation for 4-7 days until the bacteria The filament grows from the tissue block, and the mycelium is transferred to the slope of the PDA medium by picking the edge mycelium, and cultured at a constant temperature at 25-30°C. After the colony grows, transfer it 2 to 3 times , to ensure that the obtained colony is a pure culture, that is, the endophytic fungus Mabimu.
所述PDA培养基为去皮马铃薯200g，葡萄糖20g，琼脂20g。将马铃薯切成小块，加水煮沸30min，四层纱布过滤，将滤液加蒸馏水补足至1L，121℃，灭菌30min。The PDA medium is 200 g of peeled potatoes, 20 g of glucose, and 20 g of agar. Cut the potatoes into small pieces, add water to boil for 30 minutes, filter through four layers of gauze, add distilled water to the filtrate to make up to 1L, sterilize at 121°C for 30 minutes.
所述双抗PDA++培养基含有浓度为40U/mL的链霉素和浓度为30U/mL的青 霉素。The double-antibody PDA++ medium contains streptomycin at a concentration of 40 U/mL and penicillin at a concentration of 30 U/mL.
所述的木霉属(Trichoderma sp.)GZU-BCECX-GX8代谢产物中喜树碱的提取方法，包括以下步骤：The extraction method of camptothecin in the described Trichoderma (Trichoderma sp.) GZU-BCECX-GX8 metabolite comprises the following steps:
A：液体发酵培养：将分离保存的木霉属GZU-BCECX-GX8菌株接种于PDA培养基上，在28℃条件下培养5d，然后将其接种于装有PD培养基或发酵培养基的三角瓶中，然后将三角瓶置于摇床上，在120±3rpm、28±2℃条件下培养，得菌株发酵产物；A: Liquid fermentation culture: the isolated and preserved Trichoderma GZU-BCECX-GX8 strain was inoculated on PDA medium, cultured at 28°C for 5 days, and then inoculated in triangles filled with PD medium or fermentation medium bottle, and then place the Erlenmeyer flask on a shaker, and cultivate it under the conditions of 120±3rpm and 28±2°C to obtain the fermentation product of the strain;
B：代谢产物中提取喜树碱的方法：B: Method for extracting camptothecin from metabolites:
①将步骤(1)所得的菌株发酵产物采用布氏漏斗真空抽滤，分离得菌丝体和发酵液；用蒸馏水冲洗菌丝体，将菌丝体置于温度为45±3℃的条件下烘干，经研磨、过60目筛，得菌丝体粉；将发酵液在温度为45±3℃的条件下真空浓缩至原体积的10％-20％，得浓缩发酵液；①Use the Buchner funnel to vacuum-filter the fermentation product of the strain obtained in step (1) to separate the mycelium and fermentation liquid; rinse the mycelium with distilled water, and place the mycelium at a temperature of 45±3°C Drying, grinding, and passing through a 60-mesh sieve to obtain mycelium powder; vacuum-concentrating the fermentation broth to 10%-20% of the original volume at a temperature of 45±3°C to obtain a concentrated fermentation broth;
②在浓缩发酵液中加3倍体积的95％乙醇，醇沉，低温下静置过夜，布氏漏斗抽滤去除大分子沉淀，取上清液于45±3℃真空浓缩至原浓缩体积，得浓缩除杂发酵液；② Add 3 times the volume of 95% ethanol to the concentrated fermentation broth, ethanol precipitation, stand overnight at low temperature, remove the macromolecular precipitate by Buchner funnel suction filtration, take the supernatant and concentrate it in vacuum at 45±3°C to the original concentrated volume, Obtain concentrated impurity-removing fermented liquid;
③取1mL浓缩除杂发酵液，加入萃取剂2-4mL，超声萃取30min后，避光振荡12h，再超声萃取30min，静置，萃取液与发酵液分离后，获得萃取液A；再向发酵液中加入萃取剂2-4mL，超声萃取30min，静置，萃取液与发酵液分离后，获得萃取液B；③Take 1mL of concentrated impurity-removing fermentation broth, add 2-4mL of extractant, ultrasonically extract for 30 minutes, shake in the dark for 12 hours, then ultrasonically extract for 30 minutes, let stand, and separate the extract from the fermentation broth to obtain extract A; Add 2-4mL of extractant to the liquid, ultrasonically extract for 30min, let it stand still, and separate the extract from the fermentation liquid to obtain the extract B;
④取1g菌丝体粉，加入萃取剂2-5mL，置于超声萃取30min后，避光静置12h，再超声萃取30min；过滤，获得萃取液C和滤渣；取滤渣，加入萃取剂2-5mL，超声萃取30min，过滤，获得萃取液D；④Take 1g of mycelium powder, add 2-5mL extractant, put it in ultrasonic extraction for 30min, keep it in the dark for 12h, and then extract ultrasonically for 30min; filter to obtain extract C and filter residue; take filter residue, add extractant 2- 5mL, ultrasonic extraction for 30min, filtered to obtain extract D;
⑤将萃取液A、B、C和D混合后即得木霉属(Trichoderma sp.)GZU-BCECX-GX8菌株代谢产物中喜树碱的提取物。⑤ After mixing the extracts A, B, C and D, the extract of camptothecin in the metabolites of the Trichoderma sp. GZU-BCECX-GX8 strain was obtained.
所述步骤A中的PD培养基为PDA培养基中去除琼脂成分；发酵培养基为碳源10-25g，氮源0.5-2.5g，K2HPO4 1g，MgSO4 1g，蒸馏水补足1L；所述PD 培养基或发酵培养基在三角瓶中的装液量为三角瓶体积的30％。The PD medium in the step A is the removal of agar components from the PDA medium; the fermentation medium is 10-25g of carbon source, 0.5-2.5g of nitrogen source, K2 HPO4 1g, MgSO4 1g, and distilled water is added to 1L; The filling volume of the PD medium or fermentation medium in the Erlenmeyer flask is 30% of the volume of the Erlenmeyer flask.
所述步骤A中发酵培养基的碳源为可溶性淀粉、葡萄糖和甘露醇中的任意一种，氮源为(NH4)2SO4、蛋白胨和NH4Cl中的任意一种。The carbon source of the fermentation medium in step A is any one of soluble starch, glucose and mannitol, and the nitrogen source is any one of (NH4 )2 SO4 , peptone and NH4 Cl.
所述步骤A中培养时间为7～14天。The culture time in the step A is 7-14 days.
所述萃取剂由氯仿、甲醇按体积比为(1-4)︰1的比例制成。The extractant is made of chloroform and methanol in a volume ratio of (1-4):1.
所述超声萃取，其功率为90W、频率为59KHz。The ultrasonic extraction has a power of 90W and a frequency of 59KHz.
本发明的有益效果Beneficial effects of the present invention
一是本发明从马比木中分离筛选出代谢物有生产喜树碱作用的木霉属(Trichoderma sp.)GZU-BCECX-GX8菌株，这可做为喜树碱及其衍生物的另外一条来源途径，这在医药上抗肿瘤新药源的开拓提供了一种微生物菌株。One is that the present invention isolates and screens out the Trichoderma sp. (Trichoderma sp.) GZU-BCECX-GX8 bacterial strain whose metabolites have the effect of producing camptothecin from Mabi wood, which can be used as another one of camptothecin and its derivatives Source approach, which provides a microbial strain in the development of new anti-tumor drug sources in medicine.
二是本发明提供了利用木霉属(Trichoderma sp.)GZU-BCECX-GX8代谢产物提取喜树碱的方法，而喜树碱可应用于抗肿瘤药物的制备，为医用药物新来源的开发增添了新的途径。The second is that the present invention provides a method for extracting camptothecin using Trichoderma sp. (Trichoderma sp.) GZU-BCECX-GX8 metabolites, and camptothecin can be applied to the preparation of antineoplastic drugs, adding new resources to the development of new sources of medical drugs. a new way.
附图说明Description of drawings
图1：菌株GZU-BCEC-GX8的菌落形态；Figure 1: Colony morphology of strain GZU-BCEC-GX8;
图2：菌株GZU-BCEC-GX8的产孢结构。Figure 2: Sporulation structure of strain GZU-BCEC-GX8.
具体实施方式detailed description
下面结合具体的实施方式来对本发明的技术方案做进一步的限定，但要求保护的范围不仅局限于所作的描述。The technical solutions of the present invention will be further limited below in conjunction with specific embodiments, but the scope of protection is not limited to the descriptions made.
实施例1(木霉属(Trichoderma sp.)GZU-BCECX-GX8的分离)Example 1 (Isolation of Trichoderma sp. GZU-BCECX-GX8)
本发明于2014年3月在贵州省贵阳市郊区采集药用植物——马比木根芯，经表面消毒、内生真菌分离、培养、发酵、形态观察以及筛选等步骤，从中获得木霉属(Trichodermasp.)GZU-BCECX-GX8，保存。In March 2014, the present invention collected the medicinal plant—Mabi wood root core in the suburbs of Guiyang City, Guizhou Province, and obtained Trichoderma ( Trichoderma sp.) GZU-BCECX-GX8, preserved.
培养基的准备：Culture medium preparation:
PDA培养基：马铃薯200g，葡萄糖20g，琼脂20g；将去皮后的马铃薯切成小块，加水煮沸30min，四层纱布过滤，将滤液加蒸馏水补足至1L；所述灭菌条件：121℃，30min；PDA medium: 200g of potatoes, 20g of glucose, 20g of agar; cut the peeled potatoes into small pieces, add water to boil for 30min, filter through four layers of gauze, and add distilled water to the filtrate to make up to 1L; the sterilization conditions: 121°C, 30min;
双抗PDA++培养基：PDA培养基灭菌之后分装前冷却到45℃，在未凝固前按链霉素40U/mL，青霉素30U/mL用量加入PDA培养基，混匀倒平板；所述灭菌条件：121℃，30min；Double-antibody PDA++ medium: After the PDA medium is sterilized, cool to 45°C before subpackaging, add streptomycin 40U/mL and penicillin 30U/mL before solidification, add PDA medium, mix well and pour the plate; Bacteria conditions: 121°C, 30min;
查氏固体培养基：NaNO3 2g，K2HPO4 1g，KCl 0.5g，MgSO4 0.5g，FeSO4 0.01g，蔗糖30g，琼脂20g，蒸馏水1L，pH值自然；所述灭菌条件：121℃，30min；Chase solid medium: NaNO3 2g, K2 HPO4 1g, KCl 0.5g, MgSO4 0.5g, FeSO4 0.01g, sucrose 30g, agar 20g, distilled water 1L, pH natural; the sterilization conditions: 121 ℃, 30min;
具体的分离培养方法为：The specific isolation and cultivation methods are:
(1)组织的准备：取马比木新鲜的幼茎、叶、叶柄、芽、根皮、根芯之一种或几种，用自来水冲洗，除去样品表面的表生微生物；(1) Tissue preparation: take one or more of the fresh young stems, leaves, petioles, buds, root bark, and root cores of Mabi wood, rinse them with tap water, and remove the surface microorganisms on the surface of the sample;
(2)表面消毒及无菌检测：将组织置于超净工作台上，用1‰的吐温-80无菌水将材料洗2次，再用无菌水洗至无泡沫，用无菌滤纸吸去组织表面水分后，浸泡于75％乙醇溶液3min，再用无菌水冲洗3次，然后采用0.1％HgCl2溶液浸泡2min，然后用无菌水冲洗至冲洗水无菌，用无菌滤纸吸干组织段表面残留水滴后，切成长为0.5cm，宽为0.5cm的组织切段；(2) Surface disinfection and sterility testing: Place the tissue on an ultra-clean workbench, wash the material twice with 1‰ Tween-80 sterile water, and then wash with sterile water until there is no foam, and then clean it with sterile filter paper. After absorbing the water on the tissue surface, soak in 75% ethanol solution for 3 minutes, then rinse with sterile water for 3 times, then soak in 0.1% HgCl2 solution for 2 minutes, then rinse with sterile water until the rinse water is sterile, and use sterile filter paper After absorbing the remaining water droplets on the surface of the tissue section, cut into 0.5cm in length and 0.5cm in width;
(3)内生真菌的分离纯化：将步骤(2)消毒后的组织切段接种于双抗PDA++培养基平板上，所述双抗PDA++培养基是将PDA培养基灭菌之后分装前冷却到45℃，在未凝固前按链霉素40U/mL，青霉素30U/mL用量加入PDA培养基，混匀倒平板；置于28℃恒温培养7天，至菌丝体从组织块中长出，采用边缘菌丝挑取法，将菌丝转移至PDA培养基斜面上，于28℃条件下进行恒温培养，待菌落长出，再转接2～3次，以保证所得菌落为纯培养，即得马比木内生真菌；(3) Isolation and purification of endophytic fungi: inoculate the tissue sections after disinfection in step (2) on the double-antibody PDA++ medium plate, and the double-antibody PDA++ medium is after the PDA medium is sterilized Cool to 45°C before dispensing, add streptomycin 40U/mL and penicillin 30U/mL before solidification, add PDA medium, mix well and pour plate; place at 28°C for 7 days, until the mycelium grows from the tissue The mycelium was grown in the block, and the mycelium was transferred to the slope of the PDA medium by picking the edge mycelium, and the constant temperature culture was carried out at 28°C. After the colony grew, it was transferred 2 to 3 times to ensure that the obtained colony was Pure culture, that is, the endophytic fungus Mabi wood;
菌株GZU-BCEC-GX8的形态观察：Morphological observation of strain GZU-BCEC-GX8:
将菌株接种于查氏固体培养基上，以点植法接种，即用接种针挑取少量菌丝 点植在平板的中心位置，然后于28℃培养7d或14d进行观察；Inoculate the strains on the Chase solid medium, and inoculate by spotting method, that is, pick a small amount of hyphae with an inoculation needle and plant them on the center of the plate, and then culture them at 28°C for 7 days or 14 days for observation;
观察结果：菌株GZU-BCEC-GX8在查氏培养基上最初为白色基质菌丝，生长迅速，而后出现绿斑状密实产孢丛束区，菌落反面无色，菌丝透明，有隔，分枝繁复，分生孢子梗无色，分生孢子梗为菌丝的短侧枝，分生孢子为球形，在显微镜下单个孢子淡绿色，因此菌株GZU-BCEC-GX8的形态特征和木霉属(Trichoderma)真菌形态特征较吻合，菌株GZU-BCEC-GX8的菌落形态和产孢结构分别如图1和图2所示。Observation results: The strain GZU-BCEC-GX8 was initially a white matrix hyphae on Chase's medium, which grew rapidly, and then appeared a green spot-like dense spore-forming bundle area. Complex, the conidiophores are colorless, the conidiophores are short lateral branches of mycelia, the conidia are spherical, and single spores are light green under the microscope, so the morphological characteristics of strain GZU-BCEC-GX8 are similar to those of Trichoderma genus ) fungal morphological characteristics are relatively consistent, the colony morphology and sporulation structure of the strain GZU-BCEC-GX8 are shown in Figure 1 and Figure 2, respectively.
实施例2(木霉属(Trichoderma sp.)GZU-BCECX-GX8代谢产物中提取喜树碱的方法)Example 2 (method for extracting camptothecin from Trichoderma sp. GZU-BCECX-GX8 metabolites)
(1)液体发酵培养：将分离保存的木霉属GZU-BCECX-GX8菌株接种于PDA培养基上，在28℃的条件下恒温培养5d，将其接种于装有75mL发酵培养基的250mL三角瓶中，然后将三角瓶置于摇床上，在120±3rpm、28±2℃条件下培养12天；(1) Liquid fermentation culture: Inoculate the isolated and preserved Trichoderma GZU-BCECX-GX8 strain on PDA medium, culture it at a constant temperature at 28°C for 5 days, and inoculate it in a 250mL triangle containing 75mL fermentation medium. flask, then place the flask on a shaker, and cultivate it for 12 days at 120±3rpm, 28±2°C;
(2)代谢产物中提取喜树碱的方法：(2) The method for extracting camptothecin from metabolites:
①将步骤(1)所得的菌株发酵产物采用布氏漏斗真空抽滤，分离得菌丝体和发酵液；用蒸馏水冲洗菌丝体，将菌丝体置于温度为45±3℃的条件下烘干，经研磨、过60目筛，得菌丝体粉，将发酵液在温度为45±3℃的条件下真空浓缩至原体积的20％，得浓缩发酵液；①Use the Buchner funnel to vacuum-filter the fermentation product of the strain obtained in step (1) to separate the mycelium and fermentation liquid; rinse the mycelium with distilled water, and place the mycelium at a temperature of 45±3°C Drying, grinding, and passing through a 60-mesh sieve to obtain mycelium powder, and vacuum-concentrating the fermentation broth to 20% of the original volume at a temperature of 45±3°C to obtain a concentrated fermentation broth;
②在浓缩发酵液中加3倍体积的95％乙醇，醇沉，低温下静置过夜，布氏漏斗抽滤去除大分子沉淀，取上清液于45±3℃真空浓缩至原浓缩体积，得浓缩除杂发酵液；② Add 3 times the volume of 95% ethanol to the concentrated fermentation broth, ethanol precipitation, stand overnight at low temperature, remove the macromolecular precipitate by Buchner funnel suction filtration, take the supernatant and concentrate it in vacuum at 45±3°C to the original concentrated volume, Obtain concentrated impurity-removing fermented liquid;
③取1mL浓缩除杂发酵液，加入萃取剂2mL，超声萃取30min后，避光振荡12h，再超声萃取30min，静置，萃取液与发酵液分离后，获得萃取液A；再向发酵液加入萃取剂2mL，超声萃取30min，静置，萃取液与发酵液分离后，收集获得萃取液B；③Take 1mL of concentrated impurity-removing fermentation broth, add 2mL of extractant, after ultrasonic extraction for 30min, shake in the dark for 12h, then ultrasonically extract for 30min, let it stand, after the extract and fermentation broth are separated, extract A is obtained; then add to the fermentation broth Extraction agent 2mL, ultrasonic extraction for 30min, stand still, extract liquid and fermentation liquid separated, collect and obtain extract B;
④取1g菌丝体粉，加入萃取剂2mL，置于超声萃取30min后，避光静置12h， 再超声萃取30min；过滤，获得萃取液C和滤渣；取滤渣，加入萃取剂2mL，超声萃取30min，过滤，获得萃取液D；④Take 1g of mycelium powder, add 2mL of extractant, put it in ultrasonic extraction for 30min, keep it in the dark for 12h, and then extract with ultrasonic for 30min; filter to obtain extract C and filter residue; take filter residue, add 2mL of extractant, and ultrasonically extract 30min, filtered to obtain extract D;
⑤将萃取液A、B、C和D混合后即得木霉属(Trichoderma sp.)GZU-BCECX-GX8菌株代谢产物中喜树碱的提取物；⑤ After mixing the extracts A, B, C and D to obtain the extract of camptothecin in the metabolites of Trichoderma sp. GZU-BCECX-GX8 strain;
所述的发酵培养基为可溶性淀粉20g，硫酸铵1.5g，K2HPO4 1g，MgSO4 1g，蒸馏水补足1L；The fermentation medium is 20 g of soluble starch, 1.5 g of ammonium sulfate, 1 g of K2 HPO4 , 1 g of MgSO4 , and 1 L of distilled water;
所述萃取剂由氯仿、甲醇按体积比为4:1的比例制成；Described extractant is made by the ratio of 4:1 by volume ratio by chloroform, methanol;
所述超声萃取，其功率为90W、频率为59KHz；The ultrasonic extraction has a power of 90W and a frequency of 59KHz;
将萃取液A、B、C和D混合所得的GZU-BCEC-GX8菌株萃取液，送于贵州省中国科学院天然产物重点实验室和贵州大学精细化工中心，通过对喜树碱标准品、GZU-BCEC-GX8菌株萃取液、及GZU-BCEC-GX8菌株萃取液与喜树碱标准品混合样进行HPLC-MS分析，质谱条件如下：The GZU-BCEC-GX8 strain extract obtained by mixing the extracts A, B, C and D was sent to the Key Laboratory of Natural Products of the Chinese Academy of Sciences in Guizhou Province and the Fine Chemical Center of Guizhou University. BCEC-GX8 strain extract, and the mixed sample of GZU-BCEC-GX8 strain extract and camptothecin standard were analyzed by HPLC-MS, and the mass spectrometry conditions were as follows:
正离子模式；毛细管压3.5kV；脱溶剂气温度350℃；喷雾器压力50PSI；流量10L/h；Positive ion mode; capillary pressure 3.5kV; desolvation temperature 350°C; nebulizer pressure 50PSI; flow rate 10L/h;
结果：喜树碱标准品的出峰时间为T标＝4.345min，GZU-BCEC-GX8菌株萃取液的保留时间为TGX8＝4.512min，GZU-BCEC-GX8菌株萃取液相出峰时间与喜树碱标准品出峰时间相近，并且通过混合进样检测，GZU-BCEC-GX8菌株萃取液的样品峰与喜树碱的标准品峰重合，保留时间为TGX8+标＝4.394min，再通过GZU-BCEC-GX8菌株萃取液的API-ES图谱与喜树碱标准品的API-ES图谱中对比，GZU-BCEC-GX8菌株萃取液的API-ES图谱中也发现了349.4[M+H]+的分子量的物质存在，说明：GZU-BCEC-GX8菌株明显具有代谢产生喜树碱类化合物的能力。Result: the elution time of camptothecin standard substance is Tstandard =4.345min, and the retention time of GZU-BCEC-GX8 bacterial strain extract is TGX8 =4.512min, and GZU-BCEC-GX8 bacterial strain extract liquid phase elution time and hi The peak time of the standard product of camptothecin is similar, and through the mixed sampling detection, the sample peak of the GZU-BCEC-GX8 strain extract coincides with the standard product peak of camptothecin, and the retention time is TGX8 + standard = 4.394min, and then passed The API-ES spectrum of the GZU-BCEC-GX8 strain extract was compared with the API-ES spectrum of the standard camptothecin, and 349.4[M+H] was also found in the API-ES spectrum of the GZU-BCEC-GX8 strain extract The presence of substances with a molecular weight of+ indicates that the GZU-BCEC-GX8 strain obviously has the ability to metabolize camptothecin compounds.
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