WO2013155862A1 - 一种利用蛹虫草液体发酵生产纤溶酶的方法 - Google Patents

一种利用蛹虫草液体发酵生产纤溶酶的方法 Download PDF

Info

Publication number
WO2013155862A1
WO2013155862A1 PCT/CN2013/000333 CN2013000333W WO2013155862A1 WO 2013155862 A1 WO2013155862 A1 WO 2013155862A1 CN 2013000333 W CN2013000333 W CN 2013000333W WO 2013155862 A1 WO2013155862 A1 WO 2013155862A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmin
cordyceps militaris
fermentation
protein
supernatant
Prior art date
Application number
PCT/CN2013/000333
Other languages
English (en)
French (fr)
Inventor
姚强
宫志远
刘兆辉
刘春华
潘登
肖丽
赵军胜
韩建东
刘晓
万鲁长
任鹏飞
孙涛
杨鹏
Original Assignee
济南亿安生物研究所
山东省农业科学院农业资源与环境研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 济南亿安生物研究所, 山东省农业科学院农业资源与环境研究所 filed Critical 济南亿安生物研究所
Priority to AU2013248822A priority Critical patent/AU2013248822B2/en
Priority to KR1020147008524A priority patent/KR101609603B1/ko
Publication of WO2013155862A1 publication Critical patent/WO2013155862A1/zh

Links

Classifications

    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the invention relates to a method for producing plasmin by liquid fermentation of Cordyceps militaris, and belongs to the field of biological fermentation engineering technology.
  • thromboembolic disease is one of the diseases that currently endanger human health and cause the highest mortality, including acute myocardial infarction, ischemic heart disease, cardiovascular disease, valvular heart disease, peripheral vascular disease, arrhythmia, hypertension, and cerebral embolism. , pulmonary embolism, etc. It is becoming a threat to human health. According to a study published in the American Heart Association's "Circulation: Cardiovascular Quality and Results", the annual incidence of cardiovascular disease worldwide will increase by 50% due to aging and population growth from 2010 to 2030. %. At present, the most effective and reliable means for treating thromboembolic diseases is thrombolytic therapy.
  • thrombolytic antithrombotic drugs has become one of the research hotspots at home and abroad.
  • the drugs used for the treatment of thrombotic diseases have the disadvantages of poor fibrin specificity, short half-life in vivo, high production cost, and need to be administered in large doses continuously. Therefore, experts and researchers at home and abroad have gradually turned their attention to finding some natural thrombolytic drugs with good safety, side effects and good curative effect.
  • Cordyceps sinensis is a traditional Chinese medicine of the Chinese genus, belonging to the genus Ascomycete s C0 ⁇ CO to), Ascomycetes, and the genus Pseudomonas ( Sordariomycetidae ypocreales, C vicipitaceae, Cordyceps sinensis) Is a Cordyceps Frey Link), an entomogenous fungus. In the folk, it is often called Cordyceps. Pharmacological studies have shown good efficacy in enhancing immunity, anti-tumor, anti-bacterial and anti-fungal. Cordyceps militaris has attracted much attention in recent years due to its similar biochemical structure and medicinal properties to the active constituents of Cordyceps sinensis.
  • Cordyceps militaris Due to the rapid increase in the demand for Cordyceps militaris, the wild Cordyceps resources are increasingly scarce due to their mad picking, so their widespread use is severely constrained by the source of the drug. It has been found that the active ingredients such as Cordyceps plasmin, which is directly extracted from the fruiting bodies and mycelium of Cordyceps militaris, are basically the same in biochemical structure and physiological action, and have a short culture period and easy production process. The advantages of control and easy extraction of active substances have been reported. The extraction content and efficiency of active substances such as plasmin in Cordyceps militaris have been much higher than those produced by artificial cultivation of Cordyceps. However, the current research and application of liquid culture of Cordyceps militaris is still in its infancy. Many fermentation technologies and processes are still immature, and the overall yield and fermentation level are low, which limits the development of industrial production.
  • Dilated proteins are a new class of proteins found in plant cell walls. Studies have shown that dilating proteins are involved in the entire development of plants: in addition to the function of increasing cells, dilated proteins in cell growth, seed germination, root hair formation, root growth, leaf primordium formation, leaf growth and development, fruit ripening, organ detachment , and the role of pollen tube growth plays an important role. Expanded proteins have been found in various plants such as Arabidopsis thaliana, tomato, strawberry, cotton, rice, and corn, and are thought to be ubiquitous in the cell walls of various dicotyledonous and monocotyledonous plants.
  • dilatation proteins are a class of cell wall enzyme proteins that induce acid-dependent cell wall elongation and pressure relaxation by breaking hydrogen bonds between cell wall polymers, which may be physiologically regulated and cell wall elongation during plant growth.
  • An important regulation Factor there is still no clear understanding about the mechanism of action of dilating proteins.
  • the application of dilating proteins to the liquid fermentation of the edible and medicinal fungus Cordyceps militaris for the production of secondary metabolites such as plasmin has not been reported at home and abroad.
  • the present invention is directed to the deficiencies of the prior art, and provides a method for improving the production of plasmin by liquid fermentation of Cordyceps militaris using expanded protein.
  • the technical solution of the present invention is specifically as follows:
  • a method for producing plasmin by liquid fermentation of Cordyceps militaris comprising the following steps:
  • the seed liquid obtained in the step (1) is inoculated into the liquid fermentation medium at a volume ratio of 5 to 15%, and the liquid is fermented for 20 to 30 hours at a temperature of 20 to 25 ° C, and then expanded.
  • the protein solution is adjusted to a concentration of 1.0 to 3.5 mg/mL, and then cultured for 120 to 168 hours to separate and remove the fibrinolytic enzyme fermentation supernatant of the Cordyceps militaris mycelium;
  • the PD liquid fermentation medium in the step (1) has the following components per liter: 200 g of potato, 20 g of glucose, and a volume of distilled water to 1000 mL.
  • the activation culture conditions in the step (1) are: a shaking speed of 100 to 160 r/min, a temperature of 20 to 25 V, and a dark culture activation of 24 to 72 h.
  • the seed fermentation medium in the step (1) is as follows per liter of the composition -
  • the number of bacteria balls can be increased by more than 60%, the diameter of the bacteria balls is reduced by more than 40%, and the hyphae are loose and uniform.
  • the liquid fermentation medium in the step (2) has the following composition per liter -
  • the concentration of the dilating protein is 1.5 to 3.5 mg/mL ; further preferably, the concentration of the dilating protein is 1.5 to 3.0 mg/niL ; most preferably, the step ( 2), the concentration of the dilated protein is 2.0 mg/mLo
  • the expanded protein solution in the step (2) can be prepared by referring to the prior art, such as using McQueen-Mason et al. in McQueen-Mason SJ, Durachko DM, Cosgrove D J. Two endogenous proteins that induce cell wall extension in plants. Plant Cell , 1992, 4: 1425-1433 prepared by the method described; can also be prepared as follows:
  • the broad bean or cucumber seeds are sterilized by 0.05 ⁇ 0.15 wt% HgCl 2 for 4 ⁇ 6 min, rinsed with running water for 5 ⁇ 7 h, planted into wet vermiculite, dark cultured at 25 ⁇ 28 °C for 4 ⁇ 6 days; cut seedling epicotyls Or roots 4 ⁇ 5 cm, pre-cooled at -20 °C for 1 ⁇ 2 h, After homogenization with homogenization buffer precooled to 0 ⁇ 4 °C, it is filtered through a nylon mesh with a pore size of 60 ⁇ 80 ⁇ , the filter residue is washed with homogenization buffer, and then the filter residue is added to the homogenization buffer and allowed to stand at room temperature.
  • the precipitate was reconstituted with acidic buffer, dialyzed in a dialysis bag with a molecular weight of 3000 Da at 4 ° C, and the dialysate was centrifuged at 20,000 g for 5 to 10 rniri, and the supernatant was taken for expansion. Protein solution.
  • the homogenization buffer component is: 25 mmol/L HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 3 mmol/L Na 2 S 2 0 5 , 1 mmol/ L EDTA (ethylenediaminetetraacetic acid), 0.1 wt% Triton X-100, pH 7.0;
  • the extract component is: 25 mmol/L 4-hydroxyethylpiperazineethanesulfonic acid, 1.0 mmol/L EDTA, 3 mmol/L Na 2 S 2 0 5 , 0.5 mol/L NaCl, pH 6.8;
  • the acidic buffer was prepared by dissolving 2.05 g of sodium acetate in water, adjusting the pH to 4.0 with glacial acetic acid, and diluting to 1 L with water.
  • step (2) is isolated in the 10 ⁇ 15 ⁇ centrifugation at 4 ° C 12000 r / mi n conditions.
  • the method for extracting extracellular plasmin of Cordyceps militaris in the step (3) is as follows - in the plasmin fermentation supernatant prepared in the step (2) at 0 ° C
  • the ground (NH 4 ) 2 S0 4 was added in an amount of 0.14 g/mL, and stirring was continued during the addition of (N) 2 S0 4 to prevent partial over-saturation of (NH 4 ) 2 S0 4 and static precipitation at 0 ° C.
  • the disodium hydrogen phosphate-citrate buffer solution is prepared by dissolving 0.69 g of sodium dihydrogen phosphate and 0.012 g of citric acid in distilled water to a volume of 1 L, pH 8.0o.
  • the invention has the following advantages:
  • the invention applies the plant dilating protein and the optimized fermentation method to the liquid fermentation production of the cordyceps fibrinolytic enzyme, and greatly increases the liquid fermentation yield of the cordyceps fibrinolytic enzyme, so that the yield reaches 93,070 U per liter of the fermentation liquid.
  • the traditional fermentation production method (Comparative Example 1) has a yield of 2.69 times or more, and has excellent industrial application prospects.
  • the invention adopts a liquid aerobic fermentation method, generally 6-8 days, and has high yield, short cycle, no need of static culture and induced synthesis products, and has high production efficiency compared with the prior art, and the production efficiency is high.
  • the plasmin active ingredient of Cordyceps can be directly used for the preparation of drugs for thromboembolic diseases such as cardiovascular diseases, hypertension, cerebral embolism and pulmonary embolism;
  • the expanded protein of the present invention can be extracted from most dicotyledonous and monocotyledonous plants, has a wide range of sources and low cost, and the preparation method of the present invention is relatively simple after the optimization, the yield is high, and the scale can be extracted and produced. Fermentation production of Cordyceps militaris active substances has a good promotion and enhancement effect;
  • the liquid fermentation process of Cordyceps militaris used in the invention is simple, environmentally friendly and non-toxic, the raw material cost is low, and the whole fermentation process is controllable, and is not restricted by external environmental conditions, and is very suitable for industrial scale fermentation tank culture production. This method is also suitable Used in other common mushroom cultivars
  • the present invention optimizes the formulation of the liquid fermentation medium and the seed fermentation medium, and can greatly increase the yield of the plasmin active ingredient of the Cordyceps militaris.
  • Figure 1 is a graph showing the effect of different concentrations of dilated protein solution on the production of plasmin in Cordyceps militaris;
  • the Corifyce/w miYton's fermenting strains described in the examples are CGMCC No. 5.700 and CGMCC No. 3.4655, and are purchased from the General Microorganisms Collection and Management Center of China.
  • the thrombin, fibrinogen agar, and urokinase standards in the examples were purchased from Jinan Shengwei Biotechnology Co., Ltd.;
  • Seeds of broad bean (Viciafaba L.; purchased from Jinan Maofeng Seedling Co., Ltd.) or cucumber (Cucumis sativus L. CV. Jinnian No. 6; purchased from Jinan Weili Seed Co., Ltd.) were sterilized by 0.15 wt% HgCl 2 Min, rinsed with running water for 6 h, planted in wet vermiculite, and cultured at 27 °C for 4 d. Cut the seedling hypocotyl 4 ⁇ 5 cm, that is, the growth area is about 100 g, pre-cool for 1 h at -20 °C, add the homogenization buffer pre-cooled to 4 °C, and homogenize with 70 ⁇ nylon after high-speed homogenization.
  • the filter residue is washed by the homogenization buffer, and then the filter residue is added to the homogenization buffer, and allowed to stand at room temperature for 2 h to obtain a static solution; the extract is added to the static solution, and extracted at 4 ° C for 24 h, filtered.
  • the filtrate was slowly added (NH 4 ) 2 S0 4 to the filtrate according to the addition amount of 0.4 g/mL, and the mixture was continuously stirred during the addition of (N3 ⁇ 4) 2 S0 4 to prevent partial over-saturation of (NH 4 ) 2 S0 4 and then allowed to stand.
  • the above homogenization buffer components were: 25 mmol/L HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 3 mmol/L Na 2 S 2 0 5 , 1 mmol/L EDTA, 0.1 wt % Triton X- 100, pH 7.0;
  • the above extract components are: 25 mmol / L HEPES, 1.0 mmol / L EDTA, 3 mmol / L Na 2 S 2 0 5 , 0.5 mol / L NaCl, pH 6.8;
  • the acidic buffer is prepared per liter as follows:
  • the method for determining the concentration of the dilated protein solution is detected by the Coomassie Brilliant Blue method, and can be specifically referred to by the Coomassie Brilliant Blue method described in the Guide to Fine Protein Science Experiments, ISBN: 703018086, publication date 1900-1-1.
  • Bovine serum albumin was used as a standard curve, and the concentration of the expanded protein in the above expanded protein solution was determined to be 0.31 g/mL.
  • the PD liquid fermentation medium described in the examples has the following composition per liter:
  • composition per liter is as follows:
  • liquid fermentation medium described in the examples the composition per liter is as follows:
  • the disodium hydrogen phosphate-citrate buffer solution described in the examples is prepared by dissolving 0.69 g of sodium dihydrogen phosphate and 0.012 g of citric acid in distilled water to a volume of 1 L, pHS.
  • a liquid fermentation method for using a dilating protein to increase the yield of a plasmin component of Cordyceps militaris comprising the following steps -
  • the seed liquid obtained in the step (1) is inoculated into the liquid fermentation medium at a volume ratio of 15%, and the liquid is fermented for 24 hours at a temperature of 25 V, and then the dilating protein solution is added to increase the concentration of the expanded protein. 1.5mg/mL, then continue to culture for 144 h, centrifuged for 10 min at 12000 r/min, and remove the hyphae to obtain the fermentation supernatant;
  • the precipitate was dissolved in disodium hydrogen phosphate-citrate buffer; dialyzed against a dialysis bag with a molecular weight cut off of 3000 Da. Then, ultrafiltration concentration is carried out using a dialysis membrane with a molecular weight cut off of 3000 Da to remove small molecules of the protein, thereby obtaining extracellular plasmin of Cordyceps militaris.
  • the plasmin prepared by the 1 L fermentation broth was made up to 10 mL with disodium hydrogen phosphate-citric acid buffer, and 100 ⁇ L of the solution was diluted to 5 mL to obtain a test sample of Cordyceps militaris.
  • the thrombin is mixed with a fibrinogen agar solution at 37 ° C to form fibrin, which is immediately inverted, and is a fibrin plate after solidification.
  • the gradient concentration of the urokinase standard was incubated at 37 ° C for 18 h, and the diameter of the dissolution circle was measured.
  • the log ( C m 2 ) was linearly correlated with log (U/mL). According to the size of the circle of the sample to be tested, the activity unit of the urokinase of the test sample of the cordyceps militaris was calculated to represent the fibrinolytic activity of the test sample of the cordyceps militaris.
  • urokinase Prepare a standard solution of urokinase at a concentration of 100 U/mL, 200 U/tnL, 300 U/mL > 400 U/mL, 500 U/mL.
  • concentrations of urokinase standard solution 100 were spotted on fibrin plates, cultured for 37 h at 37 °C, and the vernier calipers were used to measure the two perpendicular diameters of the hydrolyzed circle.
  • the logarithm of the product of the vertical diameter of the hydrolysis zone is taken as the abscissa, and the logarithm of the urokinase concentration is plotted on the ordinate, and the urokinase standard curve is used.
  • step (2) the expanded protein solution is added at a temperature of 25 ° C to make the concentration of the expanded protein 2.0 mg/mL, and then the culture is continued. h.
  • the activity of the plasmin component per milliliter of Cordyceps militaris fermentation broth was 93.27 U, that is, the plasmin activity of the fermentation broth of the Cordyceps militaris was 93270 U; the results are shown in Fig. 1.
  • step (2) the dilating protein solution is added at a temperature of 25 ° C to make the concentration of the expanded protein 2.5 mg / mL, and then continue to culture 144 h.
  • the plasmin activity of the fermentation broth of the Cordyceps militaris was 81.32 U, that is, the plasmin activity of the fermentation broth of the Cordyceps militaris was 81320 U; the results are shown in Fig. 1.
  • step (2) The liquid fermentation method as described in Example 1, except that in step (2), the dilating protein solution is added at a temperature of 25 Torr to make the concentration of the expanded protein 3.0 mg/mL, and then the culture is continued for 144 h. .
  • the activity of the plasmin component per ml of the fermentation liquid of Cordyceps militaris was 82.02 U, that is, the plasmin activity of the fermentation broth of the Cordyceps militaris was 82020 U; the results are shown in Fig. 1.
  • step (2) the dilating protein solution is added at a temperature of 25 ° C to make the concentration of the expanded protein 3.5 mg / mL, and then continue to culture 144 h.
  • the plasmin activity of the fermentation broth of milligrams of Cordyceps militaris is 77.75 U, which is the fermentation broth of wormwood per liter.
  • the plasmin activity was 77,750 U ; the results are shown in Figure 1.
  • the liquid fermentation method as described in Example 1 differs in that the C ceps militarist strain number used for fermentation is CGMCC No. 3.4655, which is purchased from the China General Microorganisms Collection and Management Center.
  • the dilating protein solution was added at a temperature of 25 ° C to make the concentration of the dilating protein 2.0 mg/mL, and then the culture was continued for 144 h.
  • the plasmin activity of the fermentation broth of the Cordyceps militaris was 89.84 U, that is, the plasmin activity of the fermentation broth of the Cordyceps militaris was 89840 11 .
  • the activity of the plasmin component per milliliter of Cordyceps militaris fermentation broth was 34.66 U, that is, the plasmin activity of the fermentation broth of the Cordyceps militaris was 34660 U; the results are shown in Fig. 1.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种利用蛹虫草的液体发酵生产纤溶酶的方法，包括（1）将蛹虫草菌种接种到PD液体发酵培养基中，进行活化培养，然后转接到种子发酵培养基中，进行种子培养，得到种子液；（2）将种子液按5-15%的体积比接种于液体发酵培养基中，液体发酵培养20-30h，然后加入扩张蛋白溶液，继续培养120-168h，分离去除蛹虫草菌丝体得纤溶酶发酵上清液；（3）从纤溶酶发酵上清液中提取蛹虫草胞外纤溶酶，得到蛹虫草纤溶酶。本发明将植物扩张蛋白和优化后的发酵方法应用于蛹虫草纤溶酶的液体发酵生产，大幅提高了蛹虫草纤溶酶的液体发酵产量，使产量达到每升发酵液93280U，是传统发酵生产方法产量的2.69倍以上，具有很好的工业化应用前景。

Description

说 明 书
一种利用蛹虫草液体发酵生产纤溶酶的方法 技术领域
本发明涉及一种利用蛹虫草的液体发酵生产纤溶酶的方法， 属于生物发酵工程技术领 域。
背景技术
血栓栓塞性疾病是当前危害人类健康、 导致死亡率最高的疾病之一， 包括急性心肌梗 死、 缺血性心脏病、 心血管疾病、 心脏瓣膜病、 周边血管疾病、 心律失常、 高血压、 脑栓 塞、 肺栓塞等。 日渐成胁着人类的健康。 根据发表在美国心脏协会的 《循环： 心血管质量 与结果》 杂志上的一项研究预计， 仅仅由于 2010年到 2030年的衰老和人口增长， 世界范 围内的年心血管疾病发病率将增长 50 %。 目前治疗血栓栓塞疾病的最为有效并且可靠的手 段是溶栓疗法， 因此，溶栓抗栓药物的研制开发已成为国内外研究热点之一。 目前用于血栓 性疾病治疗的药物都不同程度具有纤维蛋白特异性差、 体内半衰期短、 生产成本高、 需大 剂量连续用药等缺点。于是国内外专家学者逐渐将目光投向寻找一些安全性好、副作用小、 疗效好的天然来源的溶栓药物。 虫草菌是我国传统名贵中药， 属子囊菌门 sC0^COto)、 子囊菌纲 (Ascomycetes ) , 粪壳菌亚纲 ( Sordariomycetidae 肉座菌目 ypocreales、、 麦 角菌科 ( C vicipitaceae)、 虫草菌属 Cordyceps Frey Link ) , 是一种虫生真菌。 在民间， 常常被称为冬虫夏草。 药理学研究表明在增强免疫力、 抗肿瘤、 抗细菌和抗真菌等方面具 有良好功效。 蛹虫草 C ceps militaris) 因与冬虫夏草的活性成分上有着相似的生化结 构和药用功能， 近年来备受各界关注。
由于对蛹虫草的需求增长迅速， 野生蛹虫草资源被疯狂采摘而日益稀缺， 因此其广泛 应用受到药源资源的严重制约。 研究发现， 通过液体发酵生产与从蛹虫草子实体、 菌丝体 中直接提取得到的虫草纤溶酶等活性成分在生化结构、 生理作用上基本是相同的， 而且具 有培养周期短、 生产流程易控制、 活性物质易于提取等优点， 己有报道蛹虫草的纤溶酶等 活性物质的提取含量及效率都要远高于利用人工栽培虫草进行生产和提取。 但是目前的蛹 虫草液体培养研究和应用仍是起步阶段， 许多发酵技术和工艺仍不成熟， 整体产量和发酵 水平较低， 限制了其工业化生产的发展。
扩张蛋白是在植物细胞壁中发现的一类新蛋白种类。 研究表明， 扩张蛋白几乎参与了 植物的整个发育过程： 除具有增大细胞的功能外， 扩张蛋白在细胞生长、 种子萌发、 根毛 形成、 根系生长、 叶原基形成、 叶子生长发育、 果实成熟、 器官脱离， 以及花粉管生长方 面起着重要的作用。 在拟南芥、 番茄、 草莓、 棉花、 水稻和玉米等多种植物中均已发现扩 张蛋白， 被认为其普遍存在于各种双子叶和单子叶植物的细胞壁中。 试验表明扩张蛋白是 一类细胞壁酶蛋白， 能够通过打断细胞壁多聚物之间的氢键诱导酸依赖的细胞壁延展和压 力松弛， 进而可能还是在植物生长过程中起生理调控和细胞壁延伸过程中的一个重要调控 因子。 然而， 关于扩张蛋白的作用机制目前仍未有明确定论， 将扩张蛋白应用于食药用菌 蛹虫草的液体发酵进行纤溶酶等次生代谢产物的生产， 国内外目前均未见报道。
发明内容
本发明针对现有技术的不足， 提供一种利用扩张蛋白进行提高蛹虫草液体发酵生产纤 溶酶产量的方法。 本发明技术方案具体如下：
一种利用蛹虫草的液体发酵生产纤溶酶的方法， 包括如下步骤：
( 1 )将蛹虫草菌种接种到 PD液体发酵培养基中， 进行活化培养， 然后按体积百分数 10〜15 %的接种量转接到种子发酵培养基中， 进行种子培养， 种子培养条件为： 温度 20〜 25 V , 摇床培养 24〜72 h， 得种子液；
(2) 将步骤 （1 ) 制得种子液按 5〜15 %的体积比接种于液体发酵培养基中， 在温度 20〜25 °C的条件下， 液体发酵培养 20〜30 h， 然后加入扩张蛋白溶液， 使扩张蛋白的浓度 为 1.0〜3.5 mg/mL， 然后继续培养 120〜168 h， 分离去除蛹虫草菌丝体得纤溶酶发酵上清 液；
(3 ) 从步骤 （2) 制得的纤溶酶发酵上清液中提取蛹虫草胞外纤溶酶， 即得蛹虫草纤 溶酶。
根据本发明优选的， 所述步骤 （1 ) 中的 PD液体发酵培养基， 每升组分如下： 马铃薯 200 g、 葡萄糖 20 g， 蒸馏水定容至 1000 mL。
根据本发明优选的， 所述步骤 （1 ) 中的活化培养条件为： 摇床转速 100〜160 r/min， 温度 20〜25 V， 暗培养活化 24〜72 h。
根据本发明优选的， 所述步骤 （1 ) 中的种子发酵培养基， 每升组分如下-
3 g葡萄糖、 2.5 g酵母粉上清液、 0.1 g MgS04、 0.03 g CaCl2、 0.1 g K¾P04、 0.1 g
K2HP04, 2〜5颗直径 3〜6 mm的玻璃珠。经种子发酵培养基培养后，菌球数量可提高 60 % 以上、 菌球直径缩小 40 %以上， 菌丝松散均匀。
根据本发明优选的， 所述步骤 （2) 中的液体发酵培养基， 每升组分如下-
3 g乳糖、 2 g糖蜜、 3 g豆粕粉、3 g 蛋白胨，0.2 g MgS04、 0.03 g CaCl2、 0.3 g KH2P04、
0.3 g K2HPO4。
根据本发明优选的， 所述步骤（2) 中， 扩张蛋白的浓度为 1.5〜3.5 mg/mL; 进一步优 选的， 扩张蛋白的浓度为 1.5〜3.0 mg/niL; 最优选的， 所述步骤 （2) 中， 扩张蛋白的浓度 为 2.0 mg/mLo
所述步骤 （2) 中的扩张蛋白溶液可参照现有技术制备， 如采用 McQueen-Mason等在 McQueen-Mason S J, Durachko D M, Cosgrove D J. Two endogenous proteins that induce cell wall extension in plants. Plant Cell, 1992, 4: 1425-1433中的记载的方法制备； 也可以按照如 下方法制备扩张蛋白溶液：
将蚕豆或黄瓜种子经 0.05〜0.15 wt% HgCl2消毒 4〜6 min, 流水冲洗 5〜7 h， 栽入湿 蛭石， 25〜28 °C暗培养 4〜6天； 剪取幼苗上胚轴或根系 4〜5 cm, 置 -20 °C预冷 1〜2 h， 加预冷至 0〜4 °C的匀浆缓冲液匀浆后， 用孔径 60〜80 μιη的尼龙网过滤， 滤渣经匀浆缓冲 液洗涤， 然后将滤渣加入匀桨缓冲液中， 室温静置 〜 3 h， 得静置液； 向静置液中加入提 取液， 0〜4 °C下提取 24〜30 h，过滤，按 0.3〜0.5 g/ mL的添加量向滤液中缓慢添加（N¾) 2S04， 添加 （N ) 2S04过程中不断搅拌， 防止 （N ) 2S04局部过饱和， 然后静置 24〜 30 h， 4 °。条件下25000 8离心5〜10 111 ， 沉淀用酸性缓冲液复溶， 4 °C下分子量 3000 Da 的透析袋透析， 透析液经 20000 g离心 5〜10 rniri, 取上清液即为制备的扩张蛋白溶液。
上述扩张蛋白溶液制备方法中， 所述匀浆缓冲液组分为： 25 mmol/L HEPES (4-羟乙基 哌嗪乙磺酸）， 3 mmol/L Na2S205, 1 mmol/ L EDTA (乙二胺四乙酸）， 0.1 wt% Triton X- 100， pH 7.0； 所述提取液组分为： 25 mmol/ L 4-羟乙基哌嗪乙磺酸， 1.0 mmol/ L EDTA, 3 mmol/ LNa2S205, 0.5 mol/LNaCl, pH 6.8； 所述酸性缓冲液配制是： 将 2.05 g醋酸钠溶于水中， 用冰醋酸调节 pH至 4.0， 水定容至 1 L。
根据本发明优选的，步骤（2)中所述的分离是在 4 °C 12000 r/min条件下离心分离 10〜 15 ηώι。
根据本发明优选的， 所述步骤 （3 ) 中提取蛹虫草胞外纤溶酶的方法， 步骤如下- 在 0 °C条件下， 向步骤（2)制得的纤溶酶发酵上清液中按 0.14 g/ mL的添加量添加研 磨后的 （NH4) 2S04, 添加 （N ) 2S04过程中不断搅拌， 防止 （NH4) 2S04局部过饱和， 0 °C静置沉淀 4〜8 h去除杂蛋白， 4 °C 12000 rpm离心 10 min， 弃沉淀， 按 0.51 g/ mL的添 加量将研磨后的硫酸铵加入上清液， 静置 4〜8 h; 4 °C条件下， 12000 rpm离心 lO min, 弃 上清液，沉淀溶解于磷酸氢二钠 -柠檬酸缓冲液中；用截留分子量为 3000 Da的透析袋透析， 然后用截留分子量为 3000 Da的透析膜进行超滤浓缩， 除去小分子的蛋白质， 即得蛹虫草 胞外纤溶酶。
所述磷酸氢二钠-柠檬酸缓冲液配制方法是： 将 0.69 g磷酸二氢钠、 0.012 g柠檬酸溶于 蒸熘水中， 定容至 1 L， pH 8.0o
本发明同现有技术相比有如下优点：
1、 本发明将植物扩张蛋白和优化后的发酵方法应用于蛹虫草纤溶酶的液体发酵生产， 大幅度提高了虫草纤溶酶的液体发酵产量， 使产量达到每升发酵液 93270 U， 是传统发酵 生产方法 （对比例 1 ) 产量的 2.69倍以上， 具有极好的工业化应用前景。
2、 本发明采用液体好氧发酵方法， 一般为 6〜8天， 相对于现有技术， 产量高， 周期 短， 无需静置培养及诱导合成产物等过程， 生产效率较高， 所生产的蛹虫草的纤溶酶活性 成分可直接用于治疗心血管疾病、 高血压、 脑栓塞、 肺栓塞等血栓栓塞性疾病药物的制备；
3、本发明所述扩张蛋白可从大多数双子叶和单子叶植物中提取，来源广泛，成本较低， 本发明的制备方法经优化后步骤也相对简单， 产量高， 可规模提取生产， 对蛹虫草纤溶酶 类活性物质的发酵生产具有很好的促进和提升效果；
4、 本发明所采用的蛹虫草液体发酵工艺简单， 环保无毒， 原材料成本低廉， 而且全发 酵过程可控， 不受外部环境条件限制， 非常适合工业规模化发酵罐培养生产。 本方法也适 用于其它普通香菇栽培品种
5、本发明对液体发酵培养基和种子发酵培养基的配方进行了优化， 能够大幅度提高蛹 虫草的纤溶酶活性成分的产量。
附图说明
图 1是不同浓度的扩张蛋白溶液对蛹虫草的纤溶酶产量的影响曲线；
具体实施方式
下面结合实施例对本发明作详细说明， 但本发明所保护范围不限于此。
原料及培养基
实施例中所述的蛹虫草（Corifyce/w miYton's)发酵菌种，菌种保藏号为 CGMCC No.5.700 和 CGMCC No. 3.4655， 购自中国普通微生物菌种保藏管理中心。
实施例中的凝血酶、 纤维蛋白原琼脂、 尿激酶标准品均购自济南盛伟生物科技有限公 司；
实施例中所述的扩张蛋白溶液的制备步骤如下：
将蚕豆 ( Viciafaba L. ; 购自济南茂丰种苗有限公司） 或黄瓜 （Cucumis sativus L. CV. Jinnian No. 6; 购自济南伟丽种业有限公司）种子经 0.15 wt% HgCl2消毒 6 min， 流水冲洗 6 h， 栽入湿蛭石中， 27 °C暗培养 4 d。剪取幼苗上胚轴 4〜5 cm, 即生长区约 100 g， 置 -20 °C 冰箱预冷 l h， 加预冷至 4 °C的匀浆缓冲液， 高速匀浆后， 用 70 μιη尼龙网过滤， 滤渣经匀 浆缓冲液洗涤， 然后将滤渣加入匀浆缓冲液中， 室温静置 2 h， 得静置液； 向静置液中加入 提取液， 4 °C下提取 24 h, 过滤， 滤液按 0.4 g/mL的添加量向滤液中缓慢添加（NH4) 2S04， 添加 （N¾) 2S04过程中不断搅拌， 防止 （NH4) 2S04局部过饱和， 然后静置 24 h， 4 °C条 件下 25000 g离心 5 min, 沉淀用酸性缓冲液复溶， 4 Ό条件下用截留分子量为 3000 Da的 聚偏氟乙烯 （PVDF) 透析袋 （购自北京博润莱特科技有限公司） 透析， 透析液经 20000 g 离心 5 min, 取上清液即为制备的扩张蛋白溶液， 置 4 °C下保存。 其他未描述步骤可参照 McQueen-Mason等在 McQueen-Mason S J, Durachko D M, Cosgrove D J. Two endogenous proteins that induce cell wall ext ension in plants. Plant Cell, 1992, 4: 1425-1433 以及 https://homes.bio.psu.edu/expansins/Protocols/Extraction.htm中的描述进行。
上述匀浆缓冲液组分为：25 mmol/L HEPES (4-羟乙基哌嗪乙磺酸），3 mmol/ L Na2S205， 1 mmol/ L EDTA, 0.1 wt % Triton X- 100， pH 7.0；
上述提取液组分为： 25 mmol/ L HEPES, 1.0 mmol/ L EDTA, 3 mmol/ L Na2S205, 0.5 mol/L NaCl, pH 6.8；
所述酸性缓冲液每升按如下方法配制：
将 2.05 g醋酸钠溶于水中， 用冰醋酸调节 pH至 4.0， 水定容至 1 L。
扩张蛋白溶液浓度的测定方法采用考马斯亮蓝法检测， 具体可参照（《精编蛋白质科学 实验指南》， ISBN: 703018086, 出版日期 1900-1-1 ) 中记载的考马斯亮蓝法进行操作， 以 牛血清白蛋白作标准曲线， 经检测上述扩张蛋白溶液中扩张蛋白浓度为 0.31 g/mL。 实施例中所述的 PD液体发酵培养基， 每升组分如下：
马铃薯 200 g、 葡萄糖 20g， 蒸熘水定容至 1000 mL。
实施例中所述的种子发酵培养基， 每升组分如下：
3 g葡萄糖、 2.5 g酵母粉上清液、 0.1 g MgS04、 0.03 g CaCl2、 0.1 g K¾P04、 0.1 g K2HP04， 2〜5颗直径 3〜6 mm的玻璃珠。
实施例中所述的液体发酵培养基， 每升组分如下：
3g乳糖、 2g糖蜜、 3 g豆粕粉、3 g 蛋白胨，0.2 gMgS04、 0.03 gCaCl2、 0.3 gKH2P04、 0.3 g/LK2HP0
实施例中所述的磷酸氢二钠-柠檬酸缓冲液配制方法是： 将 0.69 g磷酸二氢钠、 0.012 g 柠檬酸溶于蒸馏水中， 定容至 1 L， pHS
实施例
一种利用扩张蛋白提高蛹虫草的纤溶酶成分产量的液体发酵方法， 包括如下步骤-
(1)将蛹虫草 Cordyceps militaris)菌种， 菌种编号为 CGMCC No.5.700， 接种到 PD 液体发酵培养基中，进行活化培养，在温度 25 、摇床转速 150 r/min的条件下暗培养 48 h， 然后按体积百分数 15%的接种量转接到种子发酵培养基中， 进行种子培养， 种子培养条件 为： 温度 25 °C， 摇床培养 48 h， 得种子液；
(2) 将步骤 （1) 制得种子液按 15 %体积比接种于液体发酵培养基中， 在温度 25 V 的条件下， 液体发酵培养 24 h， 然后加入扩张蛋白溶液， 使扩张蛋白的浓度为 1.5mg/mL， 然后继续培养 144 h， 12000 r/min条件下离心分离 10 min， 去菌丝沉淀得到发酵上清液；
(3) 从步骤 （2) 制得的蛹虫草发酵上清液中提取蛹虫草胞外纤溶酶的方法， 步骤如 下：
在 0 °C条件下， 向步骤（2)制得的纤溶酶发酵上清液中按 0.14 g/mL的添加量添加研 磨后的 （N ) 2S04, 添加 （NH4) 2S04过程中不断搅拌， 防止 （N ) 2S04局部过饱和， 0 °C静置沉淀 4h去除杂蛋白， 4 °C 12000rpm离心 10min， 弃沉淀， 按 0.51 g/mL的添加 量将研磨后的硫酸铵加入上清液， 静置 6 h; 4 °C条件下， 12000 rpm离心 lOmi 弃上清 液， 沉淀溶解于磷酸氢二钠 -柠檬酸缓冲液中； 用截留分子量为 3000 Da的透析袋透析， 然 后用截留分子量为 3000 Da的透析膜进行超滤浓缩， 除去小分子的蛋白质， 即得蛹虫草胞 外纤溶酶。
蛹虫草纤溶酶待测样品的制备
将通过 1 L发酵液制得的纤溶酶用磷酸氢二钠-柠檬酸缓冲液定容至 10 mL， 取 100 μL 稀释至 5 mL得蛹虫草纤溶酶待测样品。
虫草纤溶酶活性成分的测定
采用本领域常规的纤维蛋白平板法， 测定方法参见文献（AstrupS, Mullertz. The fibrin Plate method for estimating of fibrinolytic activity [J]. Archives of Biochemistry and Biophysics, 1952, 40:346-351)： ( 1 ) 纤维蛋白平板的制作：
将凝血酶与 37°C的纤维蛋白原琼脂溶液混合， 即生成纤维蛋白， 立即倒平板， 待凝固 后即为纤维蛋白平板。 点样梯度浓度的尿激酶标准品， 37°C恒温孵育 18 h， 测其溶解圈直 径， log(Cm2)与 log (U/mL)呈线性相关。 根据待测样品的溶圈面积大小计算出蛹虫草纤溶 酶待测样品相当尿激酶的活力单位， 来表示蛹虫草纤溶酶待测样品的纤溶活性。
(2) 尿激酶标准曲线的测定：
配制浓度为 100 U/mL、 200 U/tnL、 300 U/mL > 400 U/mL、 500 U/mL的尿激酶标准溶 液。 取以上各浓度的尿激酶标准溶液 100 点样于纤维蛋白平板， 37Ό培养 6 h， 游标卡 尺测量水解圈的两垂直直径。 以水解圈垂直直径的乘积的对数作为横坐标， 尿激酶浓度的 对数为纵坐标， 做尿激酶标准曲线。
(3 ) 样品纤溶活性的测定：
取 100 蛹虫草纤溶酶待测样品点样于纤维蛋白平板的牛津杯小孔内， 37°C培养 6 h， 游标卡尺测量水解圈的两垂直直径。 参照尿激酶标准曲线的方程计算待测样品的纤溶活性 为 114.98 U/mL
每升蛹虫草发酵液对应的纤溶酶活力为： 114.98 U/mLx (5 mL/100 L) xl0 mL=57490 U; 结果如图 1所示。
实施例 2:
如实施例 1所述的液体发酵方法， 不同之处在于， 步骤 （2) 中在温度 25 °C的条件下， 加入扩张蛋白溶液， 使扩张蛋白的浓度为 2.0 mg/mL， 然后继续培养 144 h。
经检测计算， 每毫升蛹虫草发酵液的纤溶酶成分活力为 93.27 U， 即每升蛹虫草发酵液 的纤溶酶成分活力为 93270 U; 结果如图 1所示。
实施例 3:
如实施例 1所述的液体发酵方法， 不同之处在于， 步骤（2) 中在温度 25 °C的条件下， 加入扩张蛋白溶液， 使扩张蛋白的浓度为 2.5 mg/mL， 然后继续培养 144 h。
经检测计算， 每毫升蛹虫草发酵液的纤溶酶成分活力为 81.32 U， 即每升蛹虫草发酵液 的纤溶酶成分活力为 81320 U; 结果如图 1所示。
实施例 4
如实施例 1所述的液体发酵方法， 不同之处在于， 步骤（2) 中在温度 25 Ό的条件下， 加入扩张蛋白溶液， 使扩张蛋白的浓度为 3.0 mg/mL， 然后继续培养 144 h。
经检测计算， 每毫升蛹虫草发酵液的纤溶酶成分活力为 82.02 U， 即每升蛹虫草发酵液 的纤溶酶成分活力为 82020 U; 结果如图 1所示。
实施例 5:
如实施例 1所述的液体发酵方法， 不同之处在于， 步骤（2) 中在温度 25 °C的条件下， 加入扩张蛋白溶液， 使扩张蛋白的浓度为 3.5 mg/mL， 然后继续培养 144 h。
经检测计算， 每毫升蛹虫草发酵液的纤溶酶成分活力为 77.75 U， 即每升蛹虫草发酵液 的纤溶酶成分活力为 77750 U; 结果如图 1所示。
实施例 6：
如实施例 1 所述的液体发酵方法， 不同之处在于， 发酵所用的蛹虫草 C ceps militarist 菌种保藏号为 CGMCC No. 3.4655， 购自中国普通微生物菌种保藏管理中心。 步 骤（2 )中在温度 25 °C的条件下， 加入扩张蛋白溶液， 使扩张蛋白的浓度为 2.0 mg/mL, 然 后继续培养 144 h。
经检测计算， 每毫升蛹虫草发酵液的纤溶酶成分活力为 89.84 U， 即每升蛹虫草发酵液 的纤溶酶成分活力为 89840 11。
对比例 1
如实施例 1所述的液体发酵方法， 不同之处在于， 所述步骤（2 ) 中用不含扩张蛋白的 酸性缓冲液替代实施例加入的扩张蛋白溶液， 温度 25 °C的条件下， 液体发酵培养 168 h。
经检测计算， 每毫升蛹虫草发酵液的纤溶酶成分活力为 34.66 U， 即每升蛹虫草发酵液 的纤溶酶成分活力为 34660 U; 结果如图 1所示。

Claims

杈 利 要 求 书
1、 一种利用蛹虫草的液体发酵生产纤溶酶的方法， 其特征在于， 包括如下步骤：
(1) 将蛹虫草菌种接种到 PD液体发酵培养基中， 进行活化培养， 然后按体积百分数 10〜15%的接种量转接到种子发酵培养基中， 进行种子培养， 种子培养条件为： 温度 20〜 25 V, 摇床培养 24〜72h， 得种子液；
(2) 将步骤 （1) 制得种子液按 5〜15 %的体积比接种于液体发酵培养基中， 在温度 20〜25°C的条件下， 液体发酵培养 20〜30h， 然后加入扩张蛋白溶液， 使扩张蛋白的浓度 为 1.0〜3.5 mg/mL， 然后继续培养 120〜168 h， 分离去除蛹虫草菌丝体得纤溶酶发酵上清 液；
(3) 从步骤 （2) 制得的纤溶酶发酵上清液中提取蛹虫草胞外纤溶酶， 即得蛹虫草纤 溶酶；
所述步骤 （2) 中的扩张蛋白溶液按照如下方法制备：
将蚕豆或黄瓜种子经 0.05〜0.15 wt% HgCl2消毒 4〜6 min, 流水冲洗 5〜7 h, 栽入湿 蛭石， 25〜28 °C暗培养 4〜6天； 剪取幼苗上胚轴或根系 4〜5 cm， 置 -20 °C预冷 1〜2 h， 加预冷至 0〜4°C的匀浆缓冲液匀浆后，用孔径 60〜80μιη的尼龙网过滤， 滤渣经匀浆缓冲 液洗涤， 然后将滤渣加入匀浆缓冲液中， 室温静置 l〜3 h， 得静置液； 向静置液中加入提 取液， 0〜4 Ό下提取 24〜30 h，过滤，按 0.3〜0.5 g/mL的添加量向滤液中缓慢添加（NH4) 2S04， 添加 （N ) 2S04过程中不断搅拌， 防止 （N ) 2S04局部过饱和， 然后静置 24〜 30 h， 4 °C条件下 25000g离心 5〜10min， 沉淀用酸性缓冲液复溶， 4 °C下分子量 3000 Da 的透析袋透析， 透析液经 20000g离心 5〜10min， 取上清液即为制备的扩张蛋白溶液； 上述匀浆缓冲液组分为： 25 mmol/L 4-羟乙基哌嗪乙磺酸， 3 mmol/L Na2S205， 1 mmol/ L EDTA, 0.1 wt% Triton X-100, pH 7.0； 所述提取液组分为： 25 mmol/ L 4-羟乙基哌嗪乙 磺酸， 1.0 mmol/L 乙二胺四乙酸， 3 mmol/ L Na2S205 , 0.5 mol/LNaCl, pH 6.8； 所述酸性 缓冲液配制是： 将 2.05 g醋酸钠溶于水中， 用冰醋酸调节 pH至 4.0， 水定容至 1L。
2、 如权利要求 1所述的方法， 其特征在于， 所述步骤 （1) 中的活化培养条件为： 摇 床转速 100〜160r/min， 温度 20〜25°C， 暗培养活化 24〜72h。
3、 如权利要求 1所述的方法， 其特征在于， 所述步骤 （1) 中的种子发酵培养基， 每 升组分如下-
3 g葡萄糖、 2.5 g酵母粉上清液、 0.1 g MgS04、 0.03 g CaCl2、 0.1 g KH2P04、 0.1 g K2HP04, 2〜5颗直径 3〜6 mm的玻璃珠。
4、 如权利要求 1所述的方法， 其特征在于， 所述步骤 （2) 中的液体发酵培养基， 每 升组分如下-
3g乳糖、 2g糖蜜、 3g 豆粕粉、3g 蛋白胨，0.2 gMgS04、 0.03 gCaCl2、 0.3 gK¾P04、 0.3gK2HPO 5、如权利要求 1所述的方法，其特征在于，所述步骤（2)中，扩张蛋白的浓度为 1.
5〜
Figure imgf000011_0001
6、 如权利要求 5所述的方法， 其特征在于， 扩张蛋白的浓度为 1.5〜3.0mg/mL。
7、 如权利要求 6所述的方法， 其特征在于， 扩张蛋白的浓度为 2.0mg/mL。
8、如权利要求 1所述的方法，其特征在于，步骤（2)中所述的分离是在 4 °C 12000 r/min 条件下离心分离 10〜15 min。
9、 如权利要求 1所述的方法， 其特征在于， 所述步骤 （3) 中提取蛹虫草胞外纤溶酶 的方法， 步骤如下：
在 0 条件下， 向步骤（2)制得的纤溶酶发酵上清液中按 0.14 g/mL的添加量添加研 磨后的 （N ) 2S04, 添加 （NH4) 2S04过程中不断搅拌， 防止 （N ) 2S04局部过饱和， 0°C静置沉淀 4〜8h去除杂蛋白， 4 °C 12000rpm离心 10min， 弃沉淀， 按 0.51 g/mL的添 加量将研磨后的硫酸铵加入上清液， 静置 4〜8h; 4°C条件下， 12000rpm离心 10min， 弃 上清液，沉淀溶解于磷酸氢二钠 -柠檬酸缓冲液中；用截留分子量为 3000 Da的透析袋透析， 然后用截留分子量为 3000 Da的透析膜进行超滤浓缩， 除去小分子的蛋白质， 即得蛹虫草 胞外纤溶酶。
10、 如权利要求 9所述的方法， 其特征在于， 所述磷酸氢二钠-柠檬酸缓冲液配制方法 是： 将 0.69g磷酸二氢钠、 0.012 g柠檬酸溶于蒸馏水中， 定容至 1L， pH8.0。
PCT/CN2013/000333 2012-04-16 2013-03-22 一种利用蛹虫草液体发酵生产纤溶酶的方法 WO2013155862A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2013248822A AU2013248822B2 (en) 2012-04-16 2013-03-22 Method for producing plasmin by liquid fermentation of Cordyceps militaris
KR1020147008524A KR101609603B1 (ko) 2012-04-16 2013-03-22 번데기동충하초 액체발효를 이용하여 혈전용해효소를 생산하는 방법

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210111230.6 2012-04-16
CN 201210111230 CN102604919B (zh) 2012-04-16 2012-04-16 一种利用蛹虫草液体发酵生产纤溶酶的方法

Publications (1)

Publication Number Publication Date
WO2013155862A1 true WO2013155862A1 (zh) 2013-10-24

Family

ID=46522692

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/000333 WO2013155862A1 (zh) 2012-04-16 2013-03-22 一种利用蛹虫草液体发酵生产纤溶酶的方法

Country Status (4)

Country Link
KR (1) KR101609603B1 (zh)
CN (1) CN102604919B (zh)
AU (1) AU2013248822B2 (zh)
WO (1) WO2013155862A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058119A (zh) * 2016-12-29 2017-08-18 徐州鸿宇农业科技有限公司 一种提高蛹虫草液态发酵生产虫草素和耐热蛋白酶产量的方法

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102604919B (zh) * 2012-04-16 2013-10-16 山东省农业科学院农业资源与环境研究所 一种利用蛹虫草液体发酵生产纤溶酶的方法
CN102605005B (zh) * 2012-04-16 2013-09-18 山东省农业科学院农业资源与环境研究所 一种利用蛹虫草液体发酵生产虫草酸的方法
CN103340909B8 (zh) * 2013-06-07 2022-07-29 浙江五养堂药业有限公司 一种灵芝孢子粉的生物破壁方法
CN104152360B (zh) * 2014-08-15 2017-08-25 河南省科学院生物研究所有限责任公司 一种生产具有纤溶和抗氧化功能的蛹虫草菌丝粉的蛹虫草及其应用
CN104498367B (zh) * 2014-11-28 2017-06-20 沈阳源康生物技术发展有限公司 一种用于蛹虫草液体深层发酵的培养基及其使用方法
CN104911169B (zh) * 2015-04-07 2018-04-24 齐齐哈尔大学 一种培制蛹虫草菌丝体和纤溶酶的方法
CN105706742B (zh) * 2016-04-18 2018-10-02 程致远 一种虫草贝和贝虫草的培养方法
CN109504725B (zh) * 2018-12-26 2021-07-02 华熙生物科技股份有限公司 一种发酵猴头菌制备高纯度猴头菌多糖的方法和发酵培养基

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050021146A (ko) * 2003-08-26 2005-03-07 김승 동충하초 추출물 유래의 혈전용해 효소
CN101503682A (zh) * 2008-11-12 2009-08-12 齐齐哈尔大学 一种真菌纤溶酶及其培制方法
CN101962637A (zh) * 2009-12-04 2011-02-02 齐齐哈尔大学 一种蛹虫草纤溶酶及其培制方法
CN102604919A (zh) * 2012-04-16 2012-07-25 山东省农业科学院农业资源与环境研究所 一种利用蛹虫草液体发酵生产纤溶酶的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050021146A (ko) * 2003-08-26 2005-03-07 김승 동충하초 추출물 유래의 혈전용해 효소
CN101503682A (zh) * 2008-11-12 2009-08-12 齐齐哈尔大学 一种真菌纤溶酶及其培制方法
CN101962637A (zh) * 2009-12-04 2011-02-02 齐齐哈尔大学 一种蛹虫草纤溶酶及其培制方法
CN102604919A (zh) * 2012-04-16 2012-07-25 山东省农业科学院农业资源与环境研究所 一种利用蛹虫草液体发酵生产纤溶酶的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KIM, J.S. ET AL.: "A Fibrinolytic Enzyme from the Medicinal Mushroom Cordyceps militaris", THE JOURNAL OF MICROBIOLOGY, vol. 44, no. 6, 31 December 2006 (2006-12-31), pages 622 - 631 *
LI, C. ET AL.: "A novel fibrinolytic enzyme from Cordyceps militaris, a Chinese traditional medicinal mushroom", WORLD J MICROBIOL BIOTECHNOL, 2008, pages 483 - 489 *
MCQUEEN, S.M. ET AL.: "Two Endogenous Proteins That Induce Cell Wall extension in plants", THE PLANT CELL, vol. 4, 31 December 1992 (1992-12-31), pages 1425 - 1433 *
WANG, LEI ET AL.: "Optimization on Liquid Fermentation Conditions of Fibrinolytic Enzyme from Cordyceps Militaris", JOURNAL OF XINXIANG UNIVERSITY (NATURAL SCIENCE EDITION), vol. 27, no. 6, 31 December 2010 (2010-12-31) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058119A (zh) * 2016-12-29 2017-08-18 徐州鸿宇农业科技有限公司 一种提高蛹虫草液态发酵生产虫草素和耐热蛋白酶产量的方法
CN107058119B (zh) * 2016-12-29 2020-03-17 徐州鸿宇农业科技有限公司 一种提高蛹虫草液态发酵生产虫草素和耐热蛋白酶产量的方法

Also Published As

Publication number Publication date
AU2013248822B2 (en) 2016-12-22
KR101609603B1 (ko) 2016-04-06
CN102604919A (zh) 2012-07-25
KR20140081811A (ko) 2014-07-01
CN102604919B (zh) 2013-10-16
AU2013248822A1 (en) 2014-06-12

Similar Documents

Publication Publication Date Title
WO2013155862A1 (zh) 一种利用蛹虫草液体发酵生产纤溶酶的方法
WO2013155866A1 (zh) 一种利用扩张蛋白提高虫草多糖产量的液体发酵方法
US9284585B2 (en) Method for increasing yield of total flavonoids in Ganoderma lucidum mycelium
CN113564212B (zh) 一种利用微生物发酵法提取杜仲叶多糖的方法
WO2013155864A1 (zh) 一种利用蛹虫草液体发酵生产虫草酸的方法
CN108330072A (zh) 桦褐孔菌液态深层发酵培养组合物及其制备方法及应用
CN106754411B (zh) 一株高产β-D-呋喃果糖苷酶的黑曲霉菌株及其液态发酵产酶方法
CN105695340B (zh) 一种米曲霉及其应用
CN105779299B (zh) 一株产腺苷及甘露醇类物质的蝙蝠蛾拟青霉菌株及应用
CN108865895A (zh) 蝙蝠蛾拟青霉zjb18001及其应用
CN111334440B (zh) 米根霉ch5及其在提取紫芝多糖中的应用
WO2020134688A1 (zh) 一种发酵猴头菌制备高纯度猴头菌多糖的方法和发酵培养基
CN108823110B (zh) 一株产灰黄霉素的菌株及其应用
CN110551636B (zh) 一种紫红曲霉my-21菌株及其应用
CN109609482B (zh) 一种地衣芽孢杆菌凝乳酶的制备方法
CN111394258B (zh) 匍枝根霉fl-3及其在提取茯苓多糖中的应用
WO2013155865A1 (zh) 一种提高桑黄总三萜类化合物产量的方法
CN114525211B (zh) 一株杂色曲霉zlh-1、蛋白酶及其制备方法和应用
CN114958898B (zh) Peg介导的菜豆壳球孢遗传转化体系的建立方法及其应用
CN110982767B (zh) 一个细胞融合菌株及其应用
CN113637588B (zh) 一种简便快速分离荔枝霜疫霉菌的方法
CN110257266B (zh) 一种近玫色锁掷酵母菌株及其在生产乳糖酶中的应用
CN110195083B (zh) 一种提高桦褐孔菌液态发酵桦褐孔菌醇产量的方法
CN114806956A (zh) 贝莱斯芽孢杆菌db219及其应用
CN116042407A (zh) 少根根霉as7-34及其在提取当归多糖中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13777891

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 20147008524

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2013248822

Country of ref document: AU

Date of ref document: 20130322

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, FORM 1205A DATED 03-03-2015

122 Ep: pct application non-entry in european phase

Ref document number: 13777891

Country of ref document: EP

Kind code of ref document: A1