WO2022188162A1 - 一株银杏内生真菌及其在抗菌抗肿瘤中的应用 - Google Patents
一株银杏内生真菌及其在抗菌抗肿瘤中的应用 Download PDFInfo
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- WO2022188162A1 WO2022188162A1 PCT/CN2021/080512 CN2021080512W WO2022188162A1 WO 2022188162 A1 WO2022188162 A1 WO 2022188162A1 CN 2021080512 W CN2021080512 W CN 2021080512W WO 2022188162 A1 WO2022188162 A1 WO 2022188162A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the technical field of microorganisms and biomedicine, and particularly relates to an endophytic fungus of Ginkgo biloba and its application in antibacterial and antitumor.
- Cervical cancer is one of the most common gynecological malignancies. There are about 529,800 new cervical cancer cases and 275,000 deaths worldwide each year, which seriously threatens the health and life of women of childbearing age. Escherichia coli is a typical gram-negative bacillus, and the colibacillosis caused by it is a common disease. The harm caused by Escherichia coli in veterinary clinics is very serious, and it can cause disease all year round. Common and frequently-occurring diseases have caused serious economic losses to the poultry industry. In the course of treatment, drug resistance is easy to occur, and the drug resistance spectrum is wide and the drug resistance mechanism is complex, which brings great difficulties to the poultry industry to prevent and treat the disease.
- Staphylococcus aureus is one of the most common pathogens in hospital and out-of-hospital infections.
- the clinical bacterial strains obtained by the China Bacterial Resistance Monitoring Network in dozens of hospitals across the country over the years show that Staphylococcus aureus ranks among the top 5 clinical pathogens. bit.
- the bacteria can cause severe pneumonia, meningitis, and bone and joint infections, endocarditis, and bacteremia.
- patients with burn wound infection, acute liver failure and hematogenous nephritis are susceptible to infection with the bacteria.
- the emergence of Staphylococcus aureus with reduced susceptibility to some drugs will make humans face a situation where some infections are incurable. Therefore, it is of great significance to find an effective ingredient against Staphylococcus aureus.
- Ginkgo biloba is a deciduous tree of the Ginkgo family Ginkgoaceae, also known as ginkgo tree, Gongsun tree, etc. It is an important ornamental tree species, dried fruit tree species and medicinal tree species. The outer seed coat, leaves and fruits of Ginkgo biloba can be used as Chinese medicinal materials.
- Ginkgo biloba can be used to treat cold cough and phlegm, asthma, phlegm, cough, loss of voice, frequent urination, turbid urine, red vaginal discharge, intestinal 18 kinds of diseases such as blood under the wind, intestinal wind and dirty poison, chapped hands and feet, ringworm on the scalp and face, lower chancre sores, itching from pubic lice, sores caused by dog bites, ulceration of mammary abscesses and water boils and dark boils.
- Modern medical research shows that Ginkgo biloba extract can also be used to treat senile cardiovascular diseases, etc., and is known as "the whole body is a living fossil of treasure”.
- Ginkgo biloba grows slowly, and it takes more than 20 years from planting to fruiting under natural conditions, which restricts the development of its medicinal effects. Therefore, Ginkgo endophyte has become a research hotspot, and it is expected that it will become a new source of ginkgo drugs or its bioactive substances. new way.
- the present invention provides an endophytic fungus of Ginkgo biloba and its application in antibacterial and anti-tumor. Tests have proved that it has good anti-cervical cancer activity and also has inhibitory effect on various harmful bacteria, and has no toxic effect on human amniotic cell WISH, so it has good practical application value.
- the present invention relates to the following technical solutions:
- the first aspect of the present invention provides a strain of Fusarium oxysporum DZHQ2, which has been deposited in the China Center for Type Culture Collection on March 8, 2021 (address: China. Wuhan. Wuhan University), Its deposit number is CCTCC NO: M 2021211.
- the metabolites of F. oxysporum DZHQ2 also belong to the protection scope of the present invention.
- a second aspect of the present invention provides a fermentation production method of the above-mentioned Fusarium oxysporum DZHQ2, the fermentation production method comprising inoculating the Fusarium oxysporum DZHQ2 into a liquid fermentation medium for fermentation culture to obtain a fermentation broth.
- a microbial inoculum which contains the F. oxysporum DZHQ2 and/or the metabolite of F. oxysporum-containing DZHQ2.
- the harmful bacteria inhibitor may be a drug for use by living organisms, or may also be a harmful bacteria inhibitory agent for inhibiting harmful bacteria in the environment.
- the harmful bacteria include, but are not limited to, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and drug-resistant Mycobacterium tuberculosis.
- the tumor includes cervical cancer.
- the above technical solution provides a ginkgo endophyte Fusarium oxysporum DZHQ2, and the detection results of MTT and flow cytometry all show that the crude extract of the fermentation broth of the above strain has a good inhibitory effect on cervical cancer cell Hela in vitro, and is resistant to cervical cancer in vivo.
- the results of the cancer test were also consistent with the above results.
- the crude extract of the fermentation broth of the strain has good bacteriostatic effect on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and drug-resistant Mycobacterium tuberculosis, and the crude extract of the fermentation broth of the strain has no toxic effect on normal cell WISH .
- the detection results of liquid chromatography-mass spectrometry showed that resveratrol, scopolamine, taxifolin, etc. in the crude extract of fermentation broth have anti-tumor or antibacterial activities, which have wide application value, and the method of optimizing culture conditions can be adopted , to increase the yield of the above substances.
- the strain DZHQ2 has good antibacterial and anti-cervical cancer activities and has value for further development.
- Fig. 1 is the anti-cervical cancer activity detection of fermented liquid crude extract in the embodiment of the present invention (processing 24h); Wherein, A: bacterial strain A-3 fermentation liquid crude extract anti-cervical cancer activity detection; B: cyclophosphamide anti-cervical cancer activity detection.
- Figure 2 is the toxicity test of the crude extract of the fermentation broth of strain A-3 to normal cells in the embodiment of the present invention (treatment for 24 hours).
- FIG. 3 is a graph showing the anti-cervical cancer activity of the crude extract of the fermentation broth of strain A-3 in the embodiment of the present invention.
- FIG. 4 is a comparison diagram of solid tumors in the embodiment of the present invention, wherein the first row is the blank control group, and the second row is the cyclophosphamide group and the A-3 group from left to right.
- a strain of Fusarium oxysporum DZHQ2 is provided, which has been deposited in the China Center for Type Culture Collection on March 8, 2021 (address: China. Wuhan. Wuhan University) , whose deposit number is CCTCC NO: M 2021211.
- the metabolites of F. oxysporum DZHQ2 also belong to the protection scope of the present invention.
- the metabolite of Fusarium oxysporum DZHQ2 can be prepared according to the following method: inoculate the Fusarium oxysporum DZHQ2 in a liquid fermentation medium for fermentation culture, and remove the Fusarium oxysporum DZHQ2 in the liquid culture (fermentation broth) to obtain Metabolites of F. oxysporum DZHQ2.
- the metabolite of F. oxysporum DZHQ2 is prepared by the following method: after inoculating F. oxysporum DZHQ2 mycelial block in liquid medium and culturing for 5-8 d (preferably 7 d) , add ethyl acetate to the liquid medium with a volume ratio of 0.5 to 2:1 (preferably 1:1) for 3 to 5 days, filter and remove the hyphae, and recover the organic phase containing the secondary metabolites of endophytic fungi of Ginkgo biloba.
- the ethyl acetate part the material obtained by concentrating and drying the ethyl acetate part and dissolving in DMSO is the metabolite of Fusarium oxysporum DZHQ2.
- Tests have shown that the above metabolites of Fusarium oxysporum DZHQ2 have a good inhibitory effect on cervical cancer cells, and at the same time have a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and drug-resistant Mycobacterium tuberculosis. .
- the liquid fermentation medium can be a PDA medium
- the fermentation culture conditions include: a rotational speed of 160-200 r/min, preferably 180 r/min; an inoculation temperature of 20-28°C, preferably 25°C.
- a fermentation production method of the above-mentioned Fusarium oxysporum DZHQ2 comprising inoculating the Fusarium oxysporum DZHQ2 into a liquid fermentation medium for fermentation culture, that is, to obtain fermentation culture liquid.
- the liquid fermentation medium can be a PDA medium
- Fermentation culture conditions include: rotating speed of 100-150r/min, preferably 120r/min; inoculation temperature of 20-28°C, preferably 25°C; culture time of 5-8d, preferably 7d.
- a microbial inoculum which contains the F. oxysporum DZHQ2 and/or the metabolite of F. oxysporum-containing DZHQ2.
- the microbial inoculum in addition to the active ingredient, also contains a carrier.
- the carrier may be a carrier commonly used in the field of microbial formulations and which is biologically inert.
- the carrier can be a solid carrier or a liquid carrier
- the solid carrier can be mineral material, plant material and/or polymer compound;
- the mineral material can be clay, talc, medical stone, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth. at least one;
- the plant material can be at least one of corn meal, soybean meal, rice husk meal and starch;
- the polymer compound can be polyvinyl alcohol or/and polyglycol;
- the liquid carrier can be an organic solvent, vegetable oil, mineral oil or water; the organic solvent can be decane or/and dodecane.
- the dosage form of the bacterial agent can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule; preferably powder.
- surfactants such as Tween 20, Tween 80, etc.
- adhesives such as Tween 20, Tween 80, etc.
- stabilizers such as antioxidants
- pH adjusters etc.
- the harmful bacteria inhibitor may be a drug for use by living organisms, or may also be a harmful bacteria inhibitory agent for inhibiting harmful bacteria in the environment.
- the harmful bacteria include, but are not limited to, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and drug-resistant Mycobacterium tuberculosis.
- the tumor includes cervical cancer.
- A-2 and A-3 Two endophytic fungi (A-2 and A-3, named after the inventor) were isolated and purified from the bark of Ginkgo biloba. Based on sequence similarity and phylogenetic analysis, combined with their physiological and biochemical indicators, they were identified as A-2 is Fusarium proliferatum, A-3 is Fusarium oxysporum.
- Preparation of crude extract of Ginkgo endophyte fermentation broth inoculate Ginkgo endophyte A-2 and A-3 mycelial blocks in a conical flask containing 250ml PDA liquid medium, shake at 25°C and 120r/min. Culture for 7d. Then, ethyl acetate was added to each culture flask at a volume ratio of 1:1, and the culture was continued for 4 days in a shaker. The fermentation broth was filtered through 4 layers of gauze to remove mycelium, and layered with a separatory funnel to obtain an organic phase containing secondary metabolites of endophytic fungi of Ginkgo biloba.
- the results of the determination of the anti-cervical cancer activity of the crude extract of the fermentation broth are shown in Figure 1.
- the inhibition rate of strain A-3 on cervical cancer cells was 65%, while the inhibition rate of A-2 was only 23%. Cervical cancer cells were inhibited by 75%.
- strain A-3 had better antibacterial effects on Escherichia coli, Staphylococcus aureus and Bacillus subtilis (the diameter of the inhibition zone was 3.1cm, 3.2cm and 1.8cm, respectively); Staphylococcus aureus has a certain inhibitory effect (the diameter of the inhibition zone is 2.9cm), but has no inhibitory effect on Escherichia coli and Bacillus subtilis.
- the results of MTT assay showed that the MIC values of the crude extracts from the fermentation broth of strains A-2 and A-3 against drug-resistant Mycobacterium tuberculosis were 64 ⁇ g/mL and 32 ⁇ g/mL, respectively.
- the strain A-3 with good anti-tumor and antibacterial properties was selected for preservation, and the preservation name is Fusarium oxysporum DZHQ2.
- This strain has been deposited in the China Center for Type Culture Collection on March 8, 2021 (address: China . Wuhan. Wuhan University), its deposit number is CCTCC NO: M 2021211.
- the cell cycle and apoptosis detection kit was used to detect the effect of the crude extract of the fermentation broth on the apoptosis cycle of cervical cancer cells.
- the results are as follows: It can be seen from the detection results that strain A-3 has no effect on the cycle of cervical cancer cells, while strain A has no effect on the cycle of cervical cancer cells.
- the crude extract of fermentation broth of -2 can reduce the proportion of Hela cells in S phase.
- mice were subcutaneously inoculated with human cervical cancer cells Hela, subcutaneous tumors with a diameter of about 5-10 mm that were visible to the naked eye began to appear. After peeling off the tumors in each group, it was found that the surface of the tumor was completely wrapped with a capsule, and the boundary with the surrounding tissue was clear and easy to peel. , the solid tumors of each group of mice are shown in Figure 4.
- the first row is blank control group, and the second row is cyclophosphamide group and A-3 group from left to right.
- the tumor volume in the cyclophosphamide group and the strain A3 fermentation broth crude extract treatment group was significantly reduced (see Table 2) (P ⁇ 0.05). It is suggested that the crude extract of the fermentation broth of strain A3 may also have the effect of anti-cervical cancer in vivo.
- strain A-3 has good antibacterial and anti-cervical cancer activities, and has value for further development.
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Abstract
提供一株银杏内生真菌尖孢镰刀菌DZHQ2及其在抗菌抗肿瘤中的应用。该尖孢镰刀菌的保藏编号为CCTCC No. M 2021211。该菌株的发酵液粗提物在体外对宫颈癌细胞Hela具有良好的抑制效果,对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结核分枝杆菌均有良好的抑菌效果,且对正常细胞WISH无毒性作用。
Description
本发明属于微生物和生物医药技术领域,具体涉及一株银杏内生真菌及其在抗菌抗肿瘤中的应用。
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
宫颈癌是最常见的妇科恶性肿瘤之一,全球每年新发宫颈癌病例约52.98万,死亡病例约27.5万,严重威胁着广大育龄妇女的健康和生命。大肠杆菌是典型的革兰氏阴性杆菌,其引起的大肠杆菌病是一种常见的疾病,兽医临床上大肠杆菌造成的危害十分严重,一年四季均可致病,一直是困扰养殖业发展的常见病、多发病,给养禽业造成了严重的经济损失。在治疗过程中容易产生耐药性,且耐药谱广,耐药机制复杂,给养禽业预防和治疗该病带来了很大困难。因此寻找一种对抗大肠杆菌有效的成分成为近年来的研究热点。金黄色葡萄球菌是医院内、外感染最常见的病原菌之一,中国细菌耐药监测网多年来在全国数十所医院获得的临床分离细菌菌株表明,金黄色葡萄球菌占临床致病菌前5位。该菌可以引起严重肺炎、脑膜炎以及骨关节感染、心内膜炎和菌血症。尤其是烧伤创面感染、急性肝功能衰竭和血源性肾炎等疾病患者易感染该菌。近年来,对一些药物敏感性降低的金黄色葡萄球菌的出现,将使人类面临某些感染无药可治的局面。因此,寻找一种对抗金黄色葡萄球菌有效的成分具有重要意义。
银杏为裸子植物银杏科银杏属落叶乔木,别名有白果树、公孙树等,是重要的观赏树种、干果树种和药用树种。银杏的外种皮、叶子和果实都可以作为中药材,《本草纲目》中提及银杏可用于治疗寒嗽痰喘、哮喘痰嗽、咳嗽失声、小便频数、小便白浊、赤白带下、肠风下血、肠风脏毒、手足皲裂、头面癣疮、下部疳疮、阴虱作痒、狗咬成疮、乳痈溃烂和水疔暗疔等18种疾病。现代医学研究表明,银杏叶提取物还可用于治疗老年性心血管疾病等,被称为“全身都是宝的活化石”。然而银杏生长缓慢,自然条件下从栽种到结果要20年以上,制约其药用功效的开发,所以银杏内生菌成为研究的热点,期待其成为银杏类药物的新来源或其生物活性物质的新途径。发明人发现,银杏内生菌发酵产物的体内抗肿瘤研究国内外均未见报道。
发明内容
基于上述现有技术的不足,本发明提供一株银杏内生真菌及其在抗菌抗肿瘤中的应用,该真菌是从银杏材料中通过筛选、纯化及鉴定等技术获得的尖孢镰刀菌,经试验证明,其具有良好的抗宫颈癌活性同时兼具对多种有害菌的抑制作用,且对人羊膜细胞WISH无毒性作用,因此具有良好的实际应用之价值。
为实现上述技术目的,本发明涉及以下技术方案:
本发明的第一个方面,提供一株尖孢镰刀菌(Fusarium oxysporum)DZHQ2,该菌株已于2021年3月8日保藏于中国典型培养物保藏中心(地址:中国.武汉.武汉大学),其保藏编号为CCTCC NO:M 2021211。
所述尖孢镰刀菌DZHQ2的代谢物也属于本发明的保护范围。
本发明的第二个方面,提供上述尖孢镰刀菌DZHQ2的发酵生产方法,所述发酵生产方法包括将所述尖孢镰刀菌DZHQ2接种至液体发酵培养基进行发酵培养,即得发酵培养液。
本发明的第三个方面,提供一种微生物菌剂,其含有所述尖孢镰刀菌DZHQ2和/或含尖孢镰刀菌DZHQ2的代谢物。
本发明的第四个方面,上述尖孢镰刀菌DZHQ2、尖孢镰刀菌DZHQ2的代谢物和/或微生物菌剂在如下1)-2)中全部或部分中的应用也是本发明保护的范围:
1)在抑制有害菌和/或在制备有害菌抑制剂中的应用;
2)在抗肿瘤和/或制备抗肿瘤产品中的应用。
其中,所述有害菌抑制剂可以是药物,供生物体使用,或者,也可以是有害菌抑制试剂,用于抑制环境中的有害菌。
所述有害菌包括但不限于大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结核分枝杆菌。
所述肿瘤包括宫颈癌。
上述一个或多个技术方案的有益技术效果:
上述技术方案提供一株银杏内生菌尖孢镰刀菌DZHQ2,MTT和流式细胞术检测结果均表明上述菌株的发酵液粗提物在体外对宫颈癌细胞Hela具有良好的抑制效果,体内抗宫颈癌试验的结果也与上述结果一致。
另外,该菌株发酵液粗提物对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结 核分枝杆菌均有良好的抑菌效果,且菌株发酵液粗提物对正常细胞WISH无毒性作用。最后,液质联用检测结果显示发酵液粗提物中的白藜芦醇、东莨菪素、花旗松素等均有抗肿瘤或抗菌活性,具有广泛应用的价值,可采用优化培养条件的方法,提高上述物质的产量。总之,菌株DZHQ2具有良好的抗菌和抗宫颈癌活性,具有进一步开发的价值。
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例中发酵液粗提物抗宫颈癌活性检测(处理24h);其中,A:菌株A-3发酵液粗提物抗宫颈癌活性检测;B:环磷酰胺抗宫颈癌活性检测。
图2为本发明实施例中菌株A-3发酵液粗提物对正常细胞的毒性检测(处理24h)。
图3为本发明实施例中菌株A-3发酵液粗提物抗宫颈癌活性图。
图4为本发明实施例中实体瘤对比图,其中第一行均为空白对照组,第二行从左至右分别为环磷酰胺组、A-3组。
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本发明的一个具体实施方式中,提供一株尖孢镰刀菌(Fusarium oxysporum)DZHQ2,该菌株已于2021年3月8日保藏于中国典型培养物保藏中心(地址:中国.武汉.武汉大学),其保藏编号为CCTCC NO:M 2021211。
所述尖孢镰刀菌DZHQ2的代谢物也属于本发明的保护范围。
尖孢镰刀菌DZHQ2的代谢物可按照如下方法制备:将所述尖孢镰刀菌DZHQ2接种于液体发酵培养基中进行发酵培养,除去液体培养物(发酵液)中的尖孢镰刀菌DZHQ2即得到尖孢镰刀菌DZHQ2的代谢物。
本发明的又一具体实施方式中,所述尖孢镰刀菌DZHQ2的代谢物采用如下方法制备得到:将尖孢镰刀菌DZHQ2菌丝块接种于液体培养基后培养5-8d(优选7d)后,以体 积比0.5~2:1(优选为1:1)向液体培养基中加入乙酸乙酯培养3~5d,过滤除去菌丝后,回收含有银杏内生真菌次级代谢产物有机相中的乙酸乙酯部位,将乙酸乙酯部位浓缩并干燥后溶于DMSO中得到的物质即为尖孢镰刀菌DZHQ2的代谢物。经试验证明,上述尖孢镰刀菌DZHQ2的代谢物对于宫颈癌细胞有较好的抑制作用,同时对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结核分枝杆菌均有良好的抑制效果。
本发明的又一具体实施方式中,所述液体发酵培养基可以为PDA培养基;
本发明的又一具体实施方式中,发酵培养条件包括:转速160-200r/min,优选为180r/min;接种温度20-28℃,优选为25℃。
本发明的又一具体实施方式中,提供上述尖孢镰刀菌DZHQ2的发酵生产方法,所述发酵生产方法包括将所述尖孢镰刀菌DZHQ2接种至液体发酵培养基进行发酵培养,即得发酵培养液。
本发明的又一具体实施方式中,所述液体发酵培养基可以为PDA培养基;
发酵培养条件包括:转速100-150r/min,优选为120r/min;接种温度20-28℃,优选为25℃;培养时间为5-8d,优选为7d。
本发明的又一具体实施方式中,提供一种微生物菌剂,其含有所述尖孢镰刀菌DZHQ2和/或含尖孢镰刀菌DZHQ2的代谢物。
本发明的又一具体实施方式中,所述微生物菌剂中,除含活性成分外,还含有载体。所述载体可为微生物制剂领域常用的且在生物学上是惰性的载体。
所述载体可为固体载体或液体载体;
所述固体载体可为矿物材料、植物材料和/或高分子化合物;所述矿物材料可为粘土、滑石、麦饭石、高岭土、蒙脱石、白碳、沸石、硅石和硅藻土中的至少一种;所述植物材料可为玉米粉、豆粉、稻壳粉和淀粉中的至少一种;所述高分子化合物可为聚乙烯醇或/和聚二醇;
所述液体载体可为有机溶剂、植物油、矿物油或水;所述有机溶剂可为癸烷或/和十二烷。
所述菌剂的剂型可为多种剂型,如液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂;优选为粉剂。
根据需要,所述菌剂中还可添加表面活性剂(如吐温20、吐温80等)、粘合剂、稳定剂(如抗氧化剂)、pH调节剂等。
本发明的又一具体实施方式中,上述尖孢镰刀菌DZHQ2、尖孢镰刀菌DZHQ2的代谢物和/或微生物菌剂在如下1)-2)中全部或部分中的应用也是本发明保护的范围:
1)在抑制有害菌和/或在制备有害菌抑制剂中的应用;
2)在抗肿瘤和/或制备抗肿瘤产品中的应用。
其中,所述有害菌抑制剂可以是药物,供生物体使用,或者,也可以是有害菌抑制试剂,用于抑制环境中的有害菌。
所述有害菌包括但不限于大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结核分枝杆菌。
所述肿瘤包括宫颈癌。
以下通过实施例对本发明做进一步解释说明,但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例
1.银杏内生真菌的筛选及鉴定
从银杏树皮中分离纯化获得两株内生真菌(分别为A-2,A-3,为发明人的自命名),基于序列相似性和系统发育分析,并结合其生理生化指标,分别认定A-2为层生镰刀菌(Fusarium proliferatum),A-3为尖孢镰刀菌(Fusarium oxysporum)。
2.MTT法检测发酵液粗提物的抗肿瘤活性
银杏内生菌发酵液粗提物的制备:将银杏内生菌A-2、A-3菌丝块分别接种于装有250ml PDA液体培养基的三角瓶中,25℃、120r/min摇床培养7d。然后向每个培养瓶中按1:1的体积比加入乙酸乙酯,继续在摇床中培养4d。发酵液经4层纱布过滤后除菌丝体,用分液漏斗进行分层,获得含有银杏内生真菌次级代谢产物的有机相。利用旋转蒸发仪将有机相中大多数乙酸乙酯回收,得到含有银杏内生真菌次级代谢产物的浓缩液。最后,利用真空浓缩干燥器将浓缩液完全干燥,溶于DMSO中即为银杏内生菌发酵液粗提物。
利用MTT法检测了两株银杏内生菌发酵液粗提物的抗宫颈癌活性,按下式计算抑制率:抑制率=(阴性对照OD值-实验组OD值)/(阴性对照OD值-空白对照OD值)×100%。发酵液粗提物抗宫颈癌活性测定结果如图1所示,菌株A-3对宫颈癌细胞的抑制率为65%, 而A-2的抑制率仅为23%,阳性药物环磷酰胺对宫颈癌细胞的抑制率为75%。
3.发酵液粗提物的抗菌活性检测
抑菌圈检测结果显示,菌株A-3对大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌抑菌效果都较好(抑菌圈直径分别3.1cm、3.2cm和1.8cm);菌株A-2对金黄色葡萄球菌有一定抑制效果(抑菌圈直径为2.9cm),对大肠杆菌及枯草芽孢杆菌均无抑制效果。另外,MTT检测结果显示,菌株A-2和A-3发酵液粗提物对耐药结核分枝杆菌的MIC值分别为64μg/mL和32μg/mL。从上述结果可以看出,菌株A-3发酵液粗提物对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结核分枝杆菌均有良好的抑制效果。
选取具有良好抗肿瘤抗菌性能的菌株A-3进行保藏,保藏名称为尖孢镰刀菌(Fusarium oxysporum)DZHQ2,该菌株已于2021年3月8日保藏于中国典型培养物保藏中心(地址:中国.武汉.武汉大学),其保藏编号为CCTCC NO:M 2021211。
4.菌株A-3发酵液粗提物对正常细胞的毒性检测
MTT检测结果显示,菌株A-3发酵液粗提物在抗菌和抗肿瘤作用浓度范围内对WISH细胞无毒性作用。
5.流式细胞术检测发酵液粗提物的抗宫颈癌活性
从流式细胞术的检测结果可以看出,菌株A-3的发酵液粗提物对于宫颈癌细胞有较好的抑制作用,菌株A-2对宫颈癌细胞的抑制效果不明显,这与之前MTT的检测结果一致。
6.流式细胞术检测宫颈癌细胞的凋亡周期
利用细胞周期与凋亡检测试剂盒检测发酵液粗提物对宫颈癌细胞凋亡周期的影响,结果如下:从检测结果可以看出,菌株A-3对宫颈癌细胞周期无影响,而菌株A-2的发酵液粗提物可使S期Hela细胞的比例降低。
7.发酵液粗提物中活性化合物单体的初步分析
液质联用检测结果显示菌株A-2和A-3的发酵液粗提物中均含有180多种化合物。通过文献检索,发现目前已知的具有明确抗菌和抗肿瘤活性的主要物质,如槲皮素,白藜芦醇,柚皮素,迷迭香酸,茉莉酸甲酯,花旗松素等。
表1发酵液粗提物中目前已知的主要活性物质
+表示通过文献检索该物质具有此活性。
8.菌株A-3发酵液粗提物的体内抗宫颈癌作用
小鼠皮下接种人宫颈癌细胞Hela后,开始出现肉眼可见的直径约5~10mm左右的皮下肿瘤,剥离各组瘤体发现,瘤体表面有包膜完整包裹,与周围组织界限清晰,易剥离,各组小鼠实体瘤见图4。
注:第一行均为空白对照组,第二行从左至右分别为环磷酰胺组、A-3组。
表2各组实体瘤体积及抑瘤率对比
注:与空白对照组相比较,a P<0.05
与空白对照组相比,环磷酰胺组和菌株A3发酵液粗提物处理组瘤体体积显著减少(见表2)(P<0.05)。提示菌株A3发酵液粗提物在体内可能也具有抗宫颈癌的作用。
总之,以上结果显示,菌株A-3具有良好的抗菌和抗宫颈癌活性,具有进一步开发的价值。
最后应该说明的是,以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。上述虽然对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (10)
- 一株尖孢镰刀菌(Fusarium oxysporum)DZHQ2,该菌株已于2021年3月8日保藏于中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 2021211。
- 权利要求1所述尖孢镰刀菌DZHQ2的代谢物,其特征在于,其制备方法包括:将所述尖孢镰刀菌DZHQ2接种于液体发酵培养基中进行发酵培养,除去液体培养物中的尖孢镰刀菌DZHQ2即得到尖孢镰刀菌DZHQ2的代谢物。
- 如权利要求2所述的尖孢镰刀菌DZHQ2的代谢物,其特征在于,所述尖孢镰刀菌DZHQ2的代谢物采用如下方法制备得到:将尖孢镰刀菌DZHQ2菌丝块接种于液体培养基后培养5-8d(优选7d)后,以体积比0.5~2:1(优选为1:1)向液体培养基中加入乙酸乙酯培养3~5d,过滤除去菌丝后,回收含有银杏内生真菌次级代谢产物有机相中的乙酸乙酯部位,将乙酸乙酯部位浓缩并干燥后溶于DMSO中得到的物质即为尖孢镰刀菌DZHQ2的代谢物。
- 权利要求1所述的尖孢镰刀菌DZHQ2的发酵生产方法,其特征在于,所述发酵生产方法包括将所述尖孢镰刀菌DZHQ2接种至液体发酵培养基进行发酵培养。
- 如权利要求4所述的发酵生产方法,其特征在于,所述液体发酵培养基为PDA培养基。
- 如权利要求4所述的发酵生产方法,其特征在于,发酵培养条件包括:转速100-150r/min,优选为120r/min;接种温度20-28℃,优选为25℃;培养时间为5-8d,优选为7d。
- 一种微生物菌剂,其特征在于,其含有权利要求1所述尖孢镰刀菌DZHQ2和/或含权利要求2或3所述尖孢镰刀菌DZHQ2的代谢物。
- 如权利要求7所述的微生物菌剂,其特征在于,所述微生物菌剂还含有载体;优选的,所述载体为微生物制剂领域常用的且在生物学上是惰性的载体;所述载体为固体载体或液体载体。
- 权利要求1所述尖孢镰刀菌DZHQ2、权利要求2或3所述尖孢镰刀菌DZHQ2的代谢物和/或微生物菌剂在如下1)-2)中全部或部分中的应用:1)在抑制有害菌和/或在制备有害菌抑制剂中的应用;2)在抗肿瘤和/或制备抗肿瘤产品中的应用。
- 如权利要求9所述应用,其特征在于,所述有害菌包括大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和耐药结核分枝杆菌;所述肿瘤包括宫颈癌。
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