WO2021093299A1 - 黄芪-蝉拟青霉发酵菌质、制备方法及用途 - Google Patents

黄芪-蝉拟青霉发酵菌质、制备方法及用途 Download PDF

Info

Publication number
WO2021093299A1
WO2021093299A1 PCT/CN2020/091617 CN2020091617W WO2021093299A1 WO 2021093299 A1 WO2021093299 A1 WO 2021093299A1 CN 2020091617 W CN2020091617 W CN 2020091617W WO 2021093299 A1 WO2021093299 A1 WO 2021093299A1
Authority
WO
WIPO (PCT)
Prior art keywords
astragalus
paecilomyces
preparation
paecilomyces cicadae
fungal substance
Prior art date
Application number
PCT/CN2020/091617
Other languages
English (en)
French (fr)
Inventor
张加余
代龙
王少平
耿子凯
王喻淇
刘子菡
Original Assignee
滨州医学院
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 滨州医学院 filed Critical 滨州医学院
Publication of WO2021093299A1 publication Critical patent/WO2021093299A1/zh

Links

Images

Classifications

    • 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
    • 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
    • A61K36/062—Ascomycota
    • A61K36/066—Clavicipitaceae
    • A61K36/068—Cordyceps
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • C12N1/145—Fungal isolates
    • 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

Definitions

  • the invention relates to a traditional Chinese medicine fermentation fungal substance, a preparation method and application, and in particular to a traditional Chinese medicine Astragalus-Paecilomyces cicada fermentation fungal substance, a preparation method and application.
  • Fermentation of traditional Chinese medicine is based on the action of enzymes and microorganisms. Under certain environmental conditions (such as temperature, humidity, air, moisture, etc.), the fermentation process allows the medicine to change its original performance, enhance or produce new functions, and expand the variety of medicines. , In order to meet the needs of clinical medication.
  • Traditional Chinese medicine fermentation is a microbial fermentation technology, which refers to a method in which the medicine is cleaned or processed, under certain temperature and humidity conditions, with the help of microorganisms and enzymes to catalyze the decomposition to foam the medicine and produce yellow and white mold coats. Because the types and quantities of bacteria involved in fermentation of traditional Chinese medicine will be affected by geographical environment and seasonal changes, and the fermentation process is mostly judged and controlled by human subjective experience, it is difficult to ensure the safety, effectiveness and stability of fermented Chinese medicine products. Controllability.
  • Modern Chinese medicine fermentation technology is based on inheriting traditional Chinese medicine fermentation and processing methods, combined with modern microecology, bioengineering, fermentation engineering technology and other disciplines, forming a new modern Chinese medicine pharmaceutical technology.
  • the two-way fermentation technology uses Chinese medicinal materials with active ingredients as the substrate to be fermented by beneficial medicinal fungi.
  • Astragalus is a commonly used traditional Chinese medicine, which has the functions of replenishing qi and raising yang, strengthening the surface and antiperspirant, and relieving swelling.
  • Astragalus Polysaccharide is the main effective component of Astragalus in regulating immune function. It also has many functions such as anti-fatigue, regulating blood sugar and anti-tumor. It has been successfully developed into products, such as Astragalus Polysaccharide Injection.
  • Astragalus Polysaccharide Injection Regarding the modern fermentation research of Astragalus, Lu Tan et al.
  • An object of the present invention is to provide Astragalus-Paecilomyces cicadae fermented fungal substance, the content of Astragalus polysaccharide in the fermented fungal substance is significantly increased.
  • Another object of the present invention is to provide a method for preparing the fermented fungal substance.
  • Another object of the present invention is to provide the use of the fermented fungal substance.
  • the present invention provides an Astragalus-Paecilomyces cicada fermented fungal substance, which uses an astragalus enzymatic hydrolysate as a fermentation substrate, and Paecilomyces cicadae as a fermentation strain, prepared by liquid fermentation; wherein the astragalus enzymatic hydrolysate is Astragalus medicinal material is obtained by enzymatic hydrolysis with cellulase and pectinase.
  • the present invention also provides a method for preparing the fermented fungal substance of Astragalus-Paecilomyces cicadae, which includes the following steps:
  • Fermentation step inoculate Paecilomyces cicadae in the enzymatic hydrolysate of astragalus, and culture it in liquid to obtain Paecilomyces cicadaicum-astragalus fermented fungal substance.
  • the astragalus medicinal material is used in the form of astragalus medicinal powder, the particle size of the astragalus medicinal powder is 60-120 mesh sieve; and the weight of the astragalus medicinal powder and water The ratio is 1:1.5 ⁇ 10.
  • the astragalus medicinal material is pretreated by an enzymatic hydrolysis method, and the enzymatic hydrolysis step has an important effect on increasing the yield of astragalus polysaccharide and the final fermentative fungal substance to treat hyperuricemia and triglycerides.
  • the amount of cellulase is 0.5-5 wt% of the weight of the astragalus medicinal material; the amount of pectinase is 0.5-5 wt% of the weight of the astragalus medicinal material
  • the enzymolysis temperature of the enzymolysis process is 40-60°C, and the enzymolysis time is 70-110 min.
  • the Paecilomyces cicadae is obtained by culturing the species of Paecilomyces cicada through activation.
  • the method for activating and cultivating the Paecilomyces cicadae strain is: inoculating the Paecilomyces cicadae strain on a potato dextrose agar (PDA) slant medium, and placing it at 27°C and relative humidity 80% constant temperature and humidity box was activated and cultured for 5 days to obtain activated Paecilomyces cicadae; then the activated Paecilomyces cicadae was picked up with an inoculation loop and placed in a potato dextrose liquid medium at 25°C, 140r ⁇ min Cultivate for 7 days under a shaker condition of -1 to obtain Paecilomyces cicadae and seed liquid.
  • PDA potato dextrose agar
  • step (2) the seed solution of Paecilomyces cicadae is also inoculated into the astragalus enzymatic hydrolysate.
  • the temperature of the liquid culture is 26-30°C, and the time of the liquid culture is 3-20 days.
  • the present invention also provides the use of the fermented fungal substance of Astragalus-Paecilomyces cicadae for preparing astragalus polysaccharides.
  • the present invention also provides the use of the fermented fungal substance of Astragalus-Paecilomyces cicadae for preparing a medicine for treating hyperuricemia and/or hypertriglyceridemia.
  • the fermented fungal substance of Astragalus-Paecilomyces cicadae of the present invention has a significant increase in total polysaccharide content, and can be used to prepare Astragalus polysaccharides, thereby increasing the yield of Astragalus polysaccharides.
  • the fermented fungal substance of Astragalus-Paecilomyces cicadae of the present invention is significantly more effective in treating hyperuricemia and hypertriglyceridemia.
  • the method for preparing Astragalus-Paecilomyces cicadae fermented fungal substance of the present invention firstly performs enzymatic hydrolysis treatment on Astragalus medicinal materials, which has a significant effect on the increase in the yield of Astragalus polysaccharides and the medicinal effect.
  • Figure 1 is a comparison chart of serum UA levels in hyperuricemia rats.
  • Figure 2 is a comparison chart of serum BUN levels in hyperuricemia rats.
  • Figure 3 is a comparison chart of serum CRE levels in hyperuricemia rats.
  • Figure 4 is a comparison chart of TG levels in hyperuricemia rats.
  • the present invention provides an Astragalus-Paecilomyces cicadae fermented fungal substance and a preparation method and application thereof.
  • the embodiments of the present invention will be described in detail below.
  • a preparation method of Astragalus-Paecilomyces cicadae fermented fungal substance including the following steps:
  • Fermentation step inoculate Paecilomyces cicadae in the astragalus enzymatic hydrolysate and culture it in liquid to obtain Paecilomyces cicadae-astragalus fermented bacterium substance.
  • the astragalus medicinal material is preferably used in the form of astragalus medicinal material powder.
  • the particle size of the astragalus medicinal powder is passed through a 60-120 mesh sieve, preferably through an 80-120 mesh sieve, and more preferably through a 100 mesh sieve. Adopting the particle size of the astragalus medicinal powder is beneficial to the enzymatic hydrolysis of the astragalus medicinal material and the dissolution of chemical components, and saves the amount of enzyme and the enzymolysis time.
  • the weight ratio of astragalus medicinal powder to water is 1:1.5-10, preferably 1:2-8, more preferably 1:3-6. Using the above ratio is beneficial to obtain a suitable concentration of astragalus enzymatic hydrolysate, so that Paecilomyces cicadae and Astragalus can be fully contacted, and a better fermentation effect can be obtained.
  • the weight ratio of astragalus medicinal powder to water is in the range of 1:3-6, and the obtained astragalus enzymatic hydrolysate is more suitable for the growth of Paecilomyces cicada, the growth rate of Paecilomyces cicadae and the growth rate of Paecilomyces cicadae-Astragalus fermentative bacteria The yield rate of quality is higher.
  • the dosage of the cellulase is 0.5-5 wt% of the weight of the astragalus medicinal material, preferably 0.8-4 wt%, more preferably 1-3 wt%.
  • the amount of the pectinase is 0.5-5 wt% of the weight of the astragalus medicinal material, preferably 0.8-4 wt%, more preferably 1.2-3.5 wt%.
  • the present invention adopts the dosage of the enzyme, which can effectively enzymolyze the astragalus medicinal material while avoiding the waste of the enzyme.
  • the enzymolysis temperature of the enzymolysis process is 40-60°C, preferably 45-55°C, more preferably 48-52°C; enzymolysis time is 70-110min, preferably 80-100min , More preferably 85 to 95 min.
  • the enzymatic hydrolysis temperature of the enzymatic hydrolysis process is 48-52°C, and the enzymatic hydrolysis time is 85-95 min. Adopting the above enzymatic hydrolysis conditions can realize full enzymatic hydrolysis and save time.
  • step (1) in the cooling step, cooling to room temperature is sufficient.
  • the Paecilomyces cicadae is obtained by culturing the species of Paecilomyces cicada through activation.
  • the species of Paecilomyces cicadae is a known species, which can be provided by the China Forestry Microorganism Collection, and the number is cfcc81169.
  • the activation culture method can be a conventional activation method in the field without any limitation. According to one embodiment of the present invention, the activation culture method is: inoculate Paecilomyces cicadae on a potato dextrose agar (PDA) slant medium, and place it in a constant temperature and humidity box at 27° C.
  • PDA potato dextrose agar
  • the inoculation amount of Paecilomyces cicadae is 5-20 wt% of the weight of the astragalus medicinal material, preferably 8-15 wt%, more preferably 8-12 wt%.
  • the growth rate of Paecilomyces cicadae and the yield of Paecilomyces cicadae-astragalus fermentation fungus are higher, and the amount of Paecilomyces cicadae can be saved.
  • the seed solution of Paecilomyces cicadae in addition to inoculating Paecilomyces cicadae in the astragalus enzymatic hydrolysate, can also be inoculated into the enzymatic hydrolysate of Astragalus.
  • the inoculation amount of the seed liquid may be 1-10 vol% of the astragalus enzymatic hydrolysate, preferably 2-8 vol%, more preferably 3-6 vol%.
  • inoculation of seed liquid is conducive to the growth of fungal substance.
  • the growth rate of Paecilomyces cicadae and the yield of P. cicadae-astragalus fermentation fungus are higher.
  • the temperature of the liquid culture is 26-30°C, preferably 27-29°C, more preferably 28°C; the liquid culture time is 3-20 days, preferably 5-15 days, more preferably For 6 to 10 days. According to a preferred embodiment of the present invention, the temperature of the liquid culture is 26-30°C, and the time of the liquid culture is 6-10 days.
  • the liquid culture can be carried out under stirring or under the rotation of a shaker. According to one embodiment of the present invention, the liquid culture is cultured on a shaker, and the rotation speed of the shaker is 60 to 180 r ⁇ min -1 , preferably 80 to 160 r ⁇ min -1 , more preferably 100 to 140 r ⁇ min -1 .
  • the Astragalus-Paecilomyces cicada fermented fungal substance for the treatment of hyperuricemia and hypertriglyceridemia of the present invention uses an astragalus enzymatic hydrolysate as a fermentation substrate, and Paecilomyces cicada as a fermentation strain, prepared by liquid fermentation Obtained; wherein the astragalus enzymatic hydrolysis solution is obtained by enzymatically hydrolyzing astragalus medicinal materials through cellulase and pectinase.
  • the fermented fungal substance is prepared by the above-mentioned preparation method.
  • the content of astragalus polysaccharide in the fermented fungal substance of the present invention is significantly increased, and can be used to prepare astragalus polysaccharides, thereby increasing its output.
  • the fermented fungal substance of the present invention has a significantly enhanced effect on treating hyperuricemia and hypertriglyceridemia.
  • the fermented fungal substance of Astragalus-Paecilomyces cicadae of the present invention has a significant increase in total polysaccharide content, and can be used to prepare Astragalus polysaccharides, thereby increasing the yield of Astragalus polysaccharides. Therefore, the present invention provides the use of the fermented fungal substance of Astragalus-Paecilomyces cicadae for preparing Astragalus polysaccharides.
  • the fermented fungal substance of Astragalus-Paecilomyces cicadae of the present invention is significantly more effective in treating hyperuricemia and hypertriglyceridemia. Therefore, the present invention also provides the use of the fermented fungal substance of Astragalus-Paecilomyces cicadae for preparing a medicine for treating hyperuricemia and/or hypertriglyceridemia. Preferably, the present invention also provides the use of the fermented fungal substance of Astragalus-Paecilomyces cicadae for preparing a medicine for treating hyperuricemia complicated with hypertriglyceridemia.
  • the species of Paecilomyces cicadae was provided by the China Forestry Microorganism Collection, numbered cfcc81169.
  • the Paecilomyces cicadae strain was inoculated on potato dextrose agar (PDA) slant medium and placed in a constant temperature and humidity box at 27° C. and a relative humidity of 80% for activation and culture for 5 days to obtain activated Paecilomyces cicadae.
  • the activated Paecilomyces cicadae was picked up by an inoculation loop, placed in a potato dextrose liquid medium, and cultured for 7 days under a shaker condition of 140 r ⁇ min -1 at 25° C. to obtain Paecilomyces cicadae and seed liquid.
  • Potassium oxonate was purchased from Sigma, USA; Narcarin was purchased from Excella GmbH, Germany.
  • the calibration solution, quality control solution and detection kit were purchased from Beijing Leadman Biochemical Technology Co., Ltd.
  • Triglyceride determination kit batch number: 112293K. Kit for determination of uric acid, batch number: 00275. Kit for determination of urea nitrogen, batch number: 01049; kit for determination of creatinine, batch number: 04210.
  • CX-4Pro automatic biochemical analyzer is a product of American Beckman Company
  • R200D electronic analytical balance one in 100,000
  • Millipore Synergy UV ultrapure water machine is a product of American Millipore Company
  • the -30R high-pressure steam sterilizer is the product of Xi’an Yichuang Laboratory Equipment Co., Ltd.
  • the UV-2000 ultraviolet-visible spectrophotometer is the product of Beijing Ruili Analytical Instrument Co., Ltd.
  • the FW-100 high-speed grinder is the product of Tianjin Test Instrument Co., Ltd. Company product
  • LHS-80HC-II constant temperature and humidity incubator is a product of Shanghai Yiheng Technology Co., Ltd.
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • Mix Astragalus medicinal powder with 5 times the amount (weight) of distilled water of the medicinal powder, and add 1.5wt% cellulase by weight of Astragalus medicinal powder and pectinase 1.5wt% by weight of Astragalus medicinal powder in turn, and enzyme at 50°C.
  • mycoplasma A Paecilomyces cicadae-Astragalus fermented fungal substance A
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • Mix Astragalus medicinal powder with 5 times the amount (weight) of distilled water of the medicinal powder, and add 1.5wt% cellulase by weight of Astragalus medicinal powder and pectinase 1.5wt% by weight of Astragalus medicinal powder in turn, and enzyme at 50°C.
  • Dissolve for 90 minutes sterilize in a steam sterilizer at 121°C for 30 minutes, and cool to room temperature to obtain the astragalus enzymatic hydrolysate; inoculate Paecilomyces cicadae and the seed liquid into the enzymatic hydrolysate of Astragalus.
  • the weight of the medicinal material is 10wt%, the seed liquid inoculation amount is 4vol%, and the culture is cultured for 7 days at 28°C and 120r ⁇ min -1 in a shaker to obtain Paecilomyces cicadae-Astragalus fermented fungal substance B (hereinafter referred to as "Fungal substance B” ).
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • Mix Astragalus medicinal powder with 5 times the amount (weight) of distilled water of the medicinal powder, and add 1.5wt% cellulase by weight of Astragalus medicinal powder and pectinase 1.5wt% by weight of Astragalus medicinal powder in turn, and enzyme at 50°C.
  • Mycelium D Paecilomyces cicadaicum-Astragalus fermented mycelium D
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • Mix Astragalus medicinal powder with 5 times the amount (weight) of distilled water of the medicinal powder, and add 1.5wt% cellulase by weight of Astragalus medicinal powder and pectinase 1.5wt% by weight of Astragalus medicinal powder in turn, and enzyme at 50°C.
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • Mycoplasma F Paecilomyces cicadae-Astragalus fermented fungal substance F
  • the astragalus medicinal material is crushed and passed through a 100-mesh sieve to obtain the astragalus medicinal material powder.
  • the fungal substance A of Example 1, the fungal substance F of Comparative Example 4, and the total polysaccharides in Astragalus medicinal powder of Comparative Example 5 were respectively extracted, and the sulfuric acid-phenol colorimetric method was used (the method is the same as in the following literature: Yang Li et al., Astragalus in Comparison of methods for determination of Astragalus polysaccharide content, Chinese Journal of Pharmaceutical Industry [J], 2005, 36(9): 562-563 pages)
  • the total flavonoids in the fungal substance A of Example 1, the fungal substance F of Comparative Example 4, and the Astragalus medicinal powder of Comparative Example 5 were extracted with ethyl acetate, and the NaNO 2 -A1(NO 3 ) 3 colorimetric method was used (the method is the same).
  • the total polysaccharide content in fungal substance A was significantly increased, which was 1.37 times that of the astragalus medicinal powder group; the flavonoid content was significantly decreased, only 1/3 of the astragalus group.
  • the content of total polysaccharides in the bacterioplasm F increased, and the content of total flavonoids decreased, which were very significantly different from the bacterioplasm A group (P ⁇ 0.001).
  • each group was fed with normal feed.
  • the normal group was given distilled water by gavage; the other groups were given 300mg ⁇ kg -1 oxoxazine potassium salt by gavage every day for modeling.
  • the positive control group was given 20mg ⁇ kg -1 benzbroma by gavage.
  • the bacterioplasm A group was given 300mg ⁇ kg -1 bacterioplasm A
  • the bacterioplasm F group was given 300mg ⁇ kg -1 bacterioplasm F, continuously administered for 14 days, twice a day, the gastric volume was 15mL ⁇ kg -1 .
  • an automatic biochemical analyzer is used to detect uric acid (UA), urea nitrogen (BUN), creatinine (CRE) and triglyceride (TG) in serum Content.
  • the serum BUN level of rats in the hyperuricemia model group induced by oxoxazine potassium salt was significantly increased (P ⁇ 0.01); compared with the model group, the bacterioplasm A group
  • the bacterioplasm F group reduced the serum UA level of model rats; among them, the bacterioplasm A group significantly reduced the serum UA level of model rats (P ⁇ 0.001), which was significantly better than the bacterioplasm F group (P ⁇ 0.05) ,
  • the positive drug benzbromarone had no significant effect on serum BUN levels.
  • Hyperuricemia is usually complicated by hypertriglyceridemia.
  • the serum TG level of rats in the hyperuricemia model group induced by oxoxazine potassium salt was extremely significantly increased (P ⁇ 0.001); compared with the model group, the positive drug benzene Bromalone group, bacterioplasm A group, bacterioplasm F group can reduce the serum TG level of model rats (P ⁇ 0.01 or P ⁇ 0.05), and the bacterioplasm A group has the strongest TG-lowering effect, which is significantly better than that of bacterioplasm Group F.

Abstract

提供了一种黄芪-蝉拟青霉发酵菌质及其制备方法和用途。本发明的发酵菌质以黄芪酶解液作为发酵底物，蝉拟青霉作为发酵菌种，通过液体发酵制备得到；其中，黄芪酶解液是将黄芪药材经过纤维素酶和果胶酶酶解得到的。本发明的黄芪-蝉拟青霉发酵菌质中黄芪多糖含量显著提高，且具有治疗高尿酸血症和/或高甘油三酯血症的作用。

Description

黄芪-蝉拟青霉发酵菌质、制备方法及用途 技术领域
本发明涉及一种中药发酵菌质、制备方法及用途，具体来说，涉及中药黄芪-蝉拟青霉发酵菌质、制备方法及用途。
背景技术
中药发酵是借助于酶和微生物的作用，在一定的环境条件下(如温度、湿度、空气、水分等)，使药物通过发酵过程改变其原有性能、增强或产生新的功效，扩大用药品种，以适应临床用药的需要。
传统中药发酵为微生物发酵技术，是指药物经过净制或处理后，在一定的温度和湿度条件下，借助微生物和酶的催化分解作用，使药物发泡，产生黄白色霉衣的方法。由于传统中药发酵中参与发酵的菌种种类及数量会受到地理环境和季节变化的影响，而且发酵工艺过程多凭人为主观经验进行判断和控制，因此难以保证发酵中药产品的安全有效性和稳定可控性。
现代中药发酵技术，是在继承传统中药发酵炮制方法的基础上，结合现代微生态学、生物工程学、发酵工程技术等学科，形成的中药现代化制药新技术。双向发酵技术是采用具有活性成分的中药材作为基质被有益的药用真菌发酵，它们之间由于真菌的生长过程，会发生一系列复杂的分解合成代谢，产生新成分和新功能，使发酵的作用从原来单向型的仅由农副产品构成的营养基质提供真菌生长所需要的碳、氮等养分，发展到应用真菌的生理活动使药性基质中的有效成分发生转化，产生新的成分，从而产生新的性味和功能，这就具有双向型，即双向型发酵。
黄芪是常用中药，具有补气升阳、固表止汗、利水消肿等功效。 现代研究表明，黄芪多糖是黄芪中调节免疫功能的主要有效成份，另外具有抗疲劳、调节血糖、抗肿瘤等多种功效，目前已成功开发成产品，如黄芪多糖注射液。关于黄芪的现代发酵研究，陆潭等报道了以灵芝作为发酵菌种、以黄芪药渣作为基质进行固体发酵后，得到的发酵产物具有抗高尿酸血症活性(一种黄芪药渣发酵产物的抗高尿酸血症活性，江苏农业科学[J]，2013年第41卷第9期，288-291页)。赵崇妍等建立了蝉拟青霉-黄芪药材的双向发酵体系，并发现，经发酵后，黄芪药材菌质中多糖、总皂苷含量减少，黄酮含量增加(蝉拟青霉-黄芪双向发酵体系建立及成分研究，世界中医药，2018年12月第13卷第12期，3195-3198页)。
因此，需要一种能够有效提高黄芪多糖产量的黄芪发酵方法。
发明内容
本发明的一个目的在于提供黄芪-蝉拟青霉发酵菌质，该发酵菌质黄芪多糖含量明显提高。
本发明的另一目的在于提供所述发酵菌质的制备方法。
本发明的再一目的在于提供所述发酵菌质的用途。
本发明的目的是通过如下技术方案实现的。
本发明提供一种黄芪-蝉拟青霉发酵菌质，其以黄芪酶解液作为发酵底物，蝉拟青霉作为发酵菌种，通过液体发酵制备得到；其中，所述黄芪酶解液是将黄芪药材经过纤维素酶和果胶酶酶解得到的。
本发明还提供所述黄芪-蝉拟青霉发酵菌质的制备方法，包括如下步骤：
(1)黄芪酶解液的制备步骤：将黄芪药材与水混合，加入纤维素酶和果胶酶进行酶解，灭菌，冷却，得到黄芪酶解液；
(2)发酵步骤：将蝉拟青霉接种于所述黄芪酶解液中，液体培养，得到蝉拟青霉-黄芪发酵菌质。
本发明中，优选地，步骤(1)中，所述黄芪药材以黄芪药材粉末的形式使用，所述黄芪药材粉末的粒度为过60～120目筛；且所述黄芪药材粉末与水的重量比为1:1.5～10。
本发明中，采用酶解方法对黄芪药材进行预处理，酶解步骤对于提高黄芪多糖产量和最终的发酵菌质发挥治疗高尿酸血症和甘油三酯症具有重要影响。
本发明中，优选地，步骤(1)中，所述纤维素酶的用量为所述黄芪药材重量的0.5～5wt％；所述果胶酶的用量为所述黄芪药材重量的0.5～5wt％；所述酶解过程的酶解温度为40～60℃，酶解时间为70～110min。
本发明中，优选地，步骤(2)中，所述蝉拟青霉为将蝉拟青霉菌种经过活化培养后得到的。
本发明中，优选地，步骤(2)中，蝉拟青霉菌种的活化培养方法为：将蝉拟青霉菌种接种于马铃薯葡萄糖琼脂(PDA)斜面培养基上，置于27℃、相对湿度80％的恒温恒湿箱活化培养5天，得到活化后的蝉拟青霉；然后用接种环挑取活化后的蝉拟青霉置于马铃薯葡萄糖液体培养基中，于25℃、140r·min -1的摇床条件下培养7天，得到蝉拟青霉和种子液。
本发明中，优选地，步骤(2)中，还将所述蝉拟青霉种子液接种于所述黄芪酶解液中。
本发明中，优选地，步骤(2)中，所述液体培养的温度为26～30℃，液体培养的时间为3～20天。
本发明还提供所述黄芪-蝉拟青霉发酵菌质用于制备黄芪多糖的用途。
本发明还提供所述黄芪-蝉拟青霉发酵菌质用于制备治疗高尿酸血症和/或高甘油三酯血症的药物的用途。
本发明的黄芪-蝉拟青霉发酵菌质与黄芪药材相比，总多糖含量显著上升，可用于制备黄芪多糖，从而提高黄芪多糖的产量。另外，与黄芪药材相比，本发明的黄芪-蝉拟青霉发酵菌质治疗高尿酸血症和高甘油三酯血症效果显著增强。本发明的黄芪-蝉拟青霉发酵菌质的制备方法，对黄芪药材首先进行酶解处理，对黄芪多糖产量的增加和药效影响显著。
附图说明
图1为高尿酸血症大鼠血清UA水平对比图。
图2为高尿酸血症大鼠血清BUN水平对比图。
图3为高尿酸血症大鼠血清CRE水平对比图。
图4为高尿酸血症大鼠TG水平对比图。
注：与正常组相比，***表示具有极显著差异(P<0.001)，**表示具有显著差异(P<0.01)，*表示具有统计学差异(P<0.05)；与模型组相比， +++表示具有极显著差异(P<0.001)， ++表示具有显著差异(P<0.01)， +表示具有统计学差异(P<0.05)。
具体实施方式
本发明提供了一种黄芪-蝉拟青霉发酵菌质及其制备方法和用途，下面对本发明的实施方式作具体说明。
＜制备方法＞
一种黄芪-蝉拟青霉发酵菌质的制备方法，包括如下步骤：
(1)黄芪酶解液的制备步骤：将黄芪药材与水混合，加入纤维素酶和果胶酶进行酶解，灭菌，冷却，得到黄芪酶解液；
(2)发酵步骤：将蝉拟青霉接种于所述黄芪酶解液中，液体培 养，得到蝉拟青霉-黄芪发酵菌质。
步骤(1)中，所述黄芪药材优选以黄芪药材粉末的形式使用。根据本发明优选的实施方式，所述黄芪药材粉末的粒度为过60～120目筛，优选为过80～120目筛，更优选为过100目筛。采用所述的黄芪药材粉末的粒度，有利于黄芪药材的酶解和化学成分的溶出，节约酶的用量和酶解时间。
步骤(1)中，黄芪药材粉末与水的重量比为1:1.5～10，优选为1:2～8，更优选为1:3～6。采用上述比例，有利于获得合适浓度的黄芪酶解液，以使蝉拟青霉与黄芪充分接触，取得更佳发酵效果。特别是黄芪药材粉末与水的重量比为1:3～6范围内，获得的黄芪酶解液更适合蝉拟青霉的生长，蝉拟青霉的生长率和蝉拟青霉-黄芪发酵菌质的得率更高。
步骤(1)中，所述纤维素酶的用量为所述黄芪药材重量的0.5～5wt％，优选为0.8～4wt％，更优选为1～3wt％。所述果胶酶的用量为所述黄芪药材重量的0.5～5wt％，优选为0.8～4wt％，更优选为1.2～3.5wt％。本发明采用所述酶用量，能够将黄芪药材有效酶解，同时避免酶的浪费。
步骤(1)中，所述酶解过程的酶解温度为40～60℃，优选为45～55℃，更优选多为48～52℃；酶解时间为70～110min，优选为80～100min，更优选为85～95min。根据本发明一个优选的实施方式，所述酶解过程的酶解温度为48～52℃，酶解时间为85～95min。采用上述酶解条件，能够实现充分酶解，且节约时间。
步骤(1)中，所述冷却步骤，冷却至室温即可。
步骤(2)中，所述蝉拟青霉为将蝉拟青霉菌种经过活化培养后得到的。所述蝉拟青霉菌种为已知菌种，可以为中国林业微生物保藏中心提供的菌种，编号为cfcc81169。活化培养方法可以为本领域常规 活化方法，不做任何限制。根据本发明的一个实施方式，活化培养方法为：将蝉拟青霉菌种接种于马铃薯葡萄糖琼脂(PDA)斜面培养基上，置于27℃、相对湿度80％的恒温恒湿箱活化培养5天，得到活化后的蝉拟青霉；然后用接种环挑取活化后的蝉拟青霉置于马铃薯葡萄糖液体培养基中，于25℃、140r·min -1的摇床条件下培养7天，得到蝉拟青霉和种子液。
步骤(2)中，所述蝉拟青霉的接种量为所述黄芪药材重量的5～20wt％，优选为8～15wt％，更优选为8～12wt％。采用上述接种量，蝉拟青霉的生长率和蝉拟青霉-黄芪发酵菌质的得率更高，且节约蝉拟青霉的用量。
步骤(2)中，除了将蝉拟青霉接种于所述黄芪酶解液中，还可以将所述蝉拟青霉种子液接种于所述黄芪酶解液中。所述种子液的接种量可以为所述黄芪酶解液的1～10vol％，优选为2～8vol％，更优选为3～6vol％。同时接种种子液，有利于菌质的生长，蝉拟青霉的生长率和蝉拟青霉-黄芪发酵菌质的得率更高。
步骤(2)中，所述液体培养的温度为26～30℃，优选为27～29℃，更优选为28℃；液体培养的时间为3～20天，优选为5～15天，更优选为6～10天。根据本发明一个优选的实施方式，所述液体培养的温度为26～30℃，液体培养的时间为6～10天。所述液体培养可以在搅拌下进行或者摇床旋转下进行。根据本发明的一个实施方式，所述液体培养在摇床进行培养，摇床转速为60～180r·min -1，优选为80～160r·min -1，更优选为100～140r·min -1。
＜发酵菌质＞
本发明的治疗高尿酸血症和高甘油三酯血症的黄芪-蝉拟青霉发酵菌质，其以黄芪酶解液作为发酵底物，蝉拟青霉作为发酵菌种，通 过液体发酵制备得到；其中，所述黄芪酶解液是将黄芪药材经过纤维素酶和果胶酶酶解得到的。具体地，所述发酵菌质由上述制备方法制备得到。
与黄芪药材相比，本发明的发酵菌质中黄芪多糖含量显著上升，可用于制备黄芪多糖，从而提高其产量。另外，与黄芪药材相比，本发明的发酵菌质治疗高尿酸血症和高甘油三酯血症效果显著增强。
＜用途＞
本发明的黄芪-蝉拟青霉发酵菌质与黄芪药材相比，总多糖含量显著上升，可用于制备黄芪多糖，从而提高黄芪多糖的产量。因此，本发明提供所述黄芪-蝉拟青霉发酵菌质用于制备黄芪多糖的用途。
另外，与黄芪药材相比，本发明的黄芪-蝉拟青霉发酵菌质治疗高尿酸血症和高甘油三酯血症效果显著增强。因此，本发明还提供所述黄芪-蝉拟青霉发酵菌质用于制备治疗高尿酸血症和/或高甘油三酯血症的药物的用途。优选地，本发明还提供所述黄芪-蝉拟青霉发酵菌质用于制备治疗高尿酸血症并发高甘油三酯血症的药物的用途。
以下通过具体实施例对本发明的实施方式做进一步说明。
以下实施例中，蝉拟青霉菌种由中国林业微生物保藏中心提供，编号cfcc81169。将该蝉拟青霉菌种接种于马铃薯葡萄糖琼脂(PDA)斜面培养基上，置于27℃、相对湿度80％的恒温恒湿箱活化培养5天，得到活化后的蝉拟青霉。用接种环挑取活化后的蝉拟青霉置于马铃薯葡萄糖液体培养基中，于25℃、140r·min -1的摇床条件下培养7天，得到蝉拟青霉和种子液。
试剂：氧嗪酸钾盐(Potassium oxonate)购自美国Sigma公司；苯溴马隆片(Narcarin)购自德国Excella GmbH公司。定标液、质控液和 检测试剂盒均购于北京利德曼生化技术有限公司。甘油三酯测定试剂盒，批号：112293K。尿酸测定试剂盒，批号：00275。尿素氮测定试剂盒，批号：01049；肌酐测定试剂盒，批号：04210。
动物：Sprague Dawley(SD)大鼠，雄性，体质量220～250g，购于北京维通利华实验动物技术有限公司，许可证号为SCXK(京)2016-0006。
仪器：CX-4Pro型全自动生化分析仪为美国Beckman公司产品；R200D型电子分析天平(十万分之一)为德国Sartorius公司产品；Millipore Synergy UV型超纯水机为美国Millipore公司产品；BXM-30R高压蒸汽灭菌锅为西安仪创实验室仪器设备有限公司产品；UV-2000紫外可见分光光度计为北京瑞利分析仪器公司产品；FW-100高速粉碎机为天津市泰斯特仪器有限公司产品；LHS-80HC-Ⅱ型恒温恒湿培养箱为上海一恒科技有限公司产品。
实施例1
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。将黄芪药材粉末与该药材粉末5倍量(重量)的蒸馏水混合均匀，依次加入黄芪药材粉末重量1.5wt％的纤维素酶和黄芪药材粉末重量1.5wt％的果胶酶，于50℃下酶解90min，置于121℃蒸汽灭菌锅中灭菌30min，冷却至室温，得到黄芪酶解液；将蝉拟青霉接种至黄芪酶解液中，接种量为黄芪药材重量的10wt％，在28℃、120r·min -1摇床条件下培养7天，得到蝉拟青霉-黄芪发酵菌质A(以下简称“菌质A”)。
实施例2
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。将黄芪药材粉末与该药材粉末5倍量(重量)的蒸馏水混合均匀，依次加入黄芪 药材粉末重量1.5wt％的纤维素酶和黄芪药材粉末重量1.5wt％的果胶酶，于50℃下酶解90min，置于121℃蒸汽灭菌锅中灭菌30min，冷却至室温，得到黄芪酶解液；将蝉拟青霉和种子液接种至黄芪酶解液中，蝉拟青霉接种量为黄芪药材重量的10wt％，种子液接种量为4vol％，在28℃、120r·min -1摇床条件下培养7天，得到蝉拟青霉-黄芪发酵菌质B(以下简称“菌质B”)。
对比例1
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。将黄芪药材粉末与该药材粉末15倍量(重量)的蒸馏水混合均匀，依次加入黄芪药材粉末重量1.5wt％的纤维素酶和黄芪药材粉末重量1.5wt％的果胶酶，于50℃下酶解90min，置于121℃蒸汽灭菌锅中灭菌30min，冷却至室温，得到黄芪酶解液；将蝉拟青霉接种至黄芪酶解液中，接种量为黄芪药材重量的10wt％，在28℃、120r·min -1摇床条件下培养7天，得到蝉拟青霉-黄芪发酵菌质C(以下简称“菌质C”)。
对比例2
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。将黄芪药材粉末与该药材粉末5倍量(重量)的蒸馏水混合均匀，依次加入黄芪药材粉末重量1.5wt％的纤维素酶和黄芪药材粉末重量1.5wt％的果胶酶，于50℃下酶解90min，置于121℃蒸汽灭菌锅中灭菌30min，冷却至室温，得到黄芪酶解液；将蝉拟青霉接种至黄芪酶解液中，接种量为黄芪药材重量的4wt％，在28℃、120r·min -1摇床条件下培养7天，得到蝉拟青霉-黄芪发酵菌质D(以下简称“菌质D”)。
对比例3
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。将黄芪药材粉末与该药材粉末5倍量(重量)的蒸馏水混合均匀，依次加入黄芪药材粉末重量1.5wt％的纤维素酶和黄芪药材粉末重量1.5wt％的果胶酶，于50℃下酶解90min，置于121℃蒸汽灭菌锅中灭菌30min，冷却至室温，得到黄芪酶解液；将蝉拟青霉接种至黄芪酶解液中，接种量为黄芪药材重量的20wt％，在28℃、120r·min -1摇床条件下培养7天，得到蝉拟青霉-黄芪发酵菌质E(以下简称“菌质E”)。
对比例4
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。将黄芪药材粉末与该药材粉末5倍量(重量)的蒸馏水混合均匀，置于121℃蒸汽灭菌锅中灭菌30min，冷却至室温，得到黄芪底物；将蝉拟青霉接种至黄芪底物中，接种量为黄芪药材重量的10wt％，在28℃、120r·min -1摇床条件下培养7天，得到蝉拟青霉-黄芪发酵菌质F(以下简称“菌质F”)。
对比例5
将黄芪药材粉碎，过100目筛，得到黄芪药材粉末。
实验例1 菌质生长率测定
于发酵第7天取样，测定菌丝湿重，将发酵液经8层纱布过滤后获得菌丝体，菌丝体用蒸馏水冲洗3次，然后置60℃烘箱中烘干至恒重，称重。结果如表1所示。
表1
Figure PCTCN2020091617-appb-000001
实验例2 有效成分含量测定
分别提取实施例1的菌质A、对比例4的菌质F和对比例5的黄芪药材粉末中的总多糖，并采用硫酸-苯酚比色法(方法同如下文献：杨莉等，黄芪中黄芪多糖含量测定方法的比较，中国医药工业杂志[J]，2005，36(9)：562-563页)分别测定总多糖的含量，得回归方程:y＝0.0119x+0.0025，R 2＝0.9972。
采用乙酸乙酯萃取实施例1的菌质A、对比例4的菌质F和对比例5的黄芪药材粉末中的总黄酮，并采用NaNO 2-A1(NO 3) 3比色法(方法同如下文献：房树标等，紫外分光光度法测定蒙古黄芪中总黄酮的含量，中国药物与临床[J]，2007，7(12)：899-901)分别测定总黄酮的含量，得回归方程:y＝13.839x+0.0297，R 2＝0.9997。
以上测定均平行取三份样品，测定含量，取平均值。
测定结果见表2。
表2
Figure PCTCN2020091617-appb-000002
注：*表示与黄芪药材粉末组相比有显著差异(P<0.05)；***表示与黄芪药材粉末组相比有极显著差异(P<0.001)； +++表示与发酵菌质A组相比有极显著差异(P<0.001)
与黄芪药材粉末组相比，菌质A中的总多糖含量明显增加，为黄芪药材粉末组的1.37倍；黄酮的含量则明显下降，仅为黄芪组的1/3。与黄芪药材粉末组相比，菌质F中总多糖含量升高，总黄酮含量下降，均极显著区别于菌质A组(P<0.001)。
实验例3 药效试验
1.造模及给药
取SD大鼠，按体质量随机分为正常组、HUA模型组、阳性药对照组、菌质A组及菌质F组，每组8只，动物房适应性饲养1周后用于实验。
各组均给予正常饲料喂养。此外，正常组灌胃给予蒸馏水；其余各组每天首先灌胃给予300mg·kg -1氧嗪酸钾盐进行造模，1小时后，阳性药对照组灌胃给予20mg·kg -1苯溴马隆，菌质A组灌胃给予300mg·kg -1菌质A，菌质F组给予300mg·kg -1菌质F，连续给药14天，每天两次，灌胃量均为15mL·kg -1。
2.血清样品的收集
最后一天给药处理后4小时，对大鼠进行眼眶静脉丛取血，血样4℃条件下静置过夜后，离心取血清，用于生化指标的测定。
3.生化指标的测定
采用全自动生化分析仪在常规定标、质量控制后，检测血清中尿酸(uric acid，UA)、尿素氮(urea nitrogen，BUN)、肌酐(creatinine，CRE)和甘油三酯(triglyceride，TG)的含量。
4.数据分析
所有试验结果以“均值±标准差”的形式表示。采用Excel和SPSS17.0软件进行计算和统计，所有数据经正态分布和方差齐性检验后，进行单因素方差分析，两组均数间的差异采用t检验，P<0.05时认为差异具有统计学意义。
5.结果
5.1对高尿酸血症大鼠血清UA水平的影响
如图1所示，与正常组相比，氧嗪酸钾盐诱导的高尿酸血症模型组大鼠的血清UA水平极显著升高(P<0.001)，提示大鼠高尿酸血症模型塑造成功；与模型组相比，阳性药苯溴马隆组、菌质A组、菌质F组均极显著地降低了模型大鼠血清UA水平，且菌质A组的作用效果最强。
5.2对高尿酸血症大鼠血清BUN水平的影响
如图2所示，与正常组相比，氧嗪酸钾盐诱导的高尿酸血症模型组大鼠的血清BUN水平显著升高(P<0.01)；与模型组相比，菌质A组、菌质F组均降低了模型大鼠血清UA水平；其中，菌质A组极显著地降低了模型大鼠血清UA水平(P<0.001)，明显优于菌质F组(P<0.05)，而阳性药苯溴马隆对血清BUN水平无明显影响。
5.3对高尿酸血症大鼠血清CRE水平的影响
如图3所示，不同分组大鼠的血清肌酐水平无明显差异。但是，与模型组相比，阳性药苯溴马隆组、菌质A组、菌质F组有降低血清CRE水平的趋势，且菌质A组作用效果最强。
5.4对高尿酸血症大鼠TG水平的影响
高尿酸血症通常并发高甘油三脂血症。如图4所示，与正常组相比，氧嗪酸钾盐诱导的高尿酸血症模型组大鼠的血清TG水平极显著升高(P<0.001)；与模型组相比，阳性药苯溴马隆组、菌质A组、菌质F组均能降低模型大鼠血清TG水平(P<0.01或P<0.05)，且菌质A组的降TG作用最强，明显优于菌质F组。
本发明并不限于上述实施方式，在不背离本发明的实质内容的情况下，本领域技术人员可以想到的任何变形、改进、替换均落入本发明的范围。

Claims (10)

  1. 一种黄芪-蝉拟青霉发酵菌质，其以黄芪酶解液作为发酵底物，蝉拟青霉作为发酵菌种，通过液体发酵制备得到；其中，所述黄芪酶解液是将黄芪药材经过纤维素酶和果胶酶酶解得到的。
  2. 根据权利要求1所述的黄芪-蝉拟青霉发酵菌质的制备方法，包括如下步骤：
    (1)黄芪酶解液的制备步骤：将黄芪药材与水混合，加入纤维素酶和果胶酶进行酶解，灭菌，冷却，得到黄芪酶解液；
    (2)发酵步骤：将蝉拟青霉接种于所述黄芪酶解液中，液体培养，得到蝉拟青霉-黄芪发酵菌质。
  3. 根据权利要求2所述的制备方法，其特征在于，步骤(1)中，所述黄芪药材以黄芪药材粉末的形式使用，所述黄芪药材粉末的粒度为过60～120目筛；且所述黄芪药材粉末与水的重量比为1:1.5～10。
  4. 根据权利要求2所述的制备方法，其特征在于，步骤(1)中，所述纤维素酶的用量为所述黄芪药材重量的0.5～5wt％；所述果胶酶的用量为所述黄芪药材重量的0.5～5wt％；所述酶解过程的酶解温度为40～60℃，酶解时间为70～110min。
  5. 根据权利要求2所述的制备方法，其特征在于，步骤(2)中，所述蝉拟青霉为将蝉拟青霉菌种经过活化培养后得到的。
  6. 根据权利要求5所述的制备方法，其特征在于，步骤(2)中，蝉拟青霉菌种的活化培养方法为：将蝉拟青霉菌种接种于马铃薯葡萄糖琼脂(PDA)斜面培养基上，置于27℃、相对湿度80％的恒温恒湿箱活化培养5天，得到活化后的蝉拟青霉；然后用接种环挑取活化后的蝉拟青霉置于马铃薯葡萄糖液体培养基中，于25℃、140r·min -1的摇床条件下培养7天，得到蝉拟青霉和种子液。
  7. 根据权利要求6所述的制备方法，其特征在于，步骤(2)中， 还将所述蝉拟青霉种子液接种于所述黄芪酶解液中。
  8. 根据权利要求2～7任一项所述的制备方法，其特征在于，步骤(2)中，所述液体培养的温度为26～30℃，液体培养的时间为3～20天。
  9. 根据权利要求1所述的黄芪-蝉拟青霉发酵菌质或根据权利要求2～7任一项所述的制备方法制得的黄芪-蝉拟青霉发酵菌质用于制备黄芪多糖的用途。
  10. 根据权利要求1所述的黄芪-蝉拟青霉发酵菌质或根据权利要求2～7任一项所述的制备方法制得的黄芪-蝉拟青霉发酵菌质用于制备治疗高尿酸血症和高甘油三酯血症的药物的用途。
PCT/CN2020/091617 2019-11-15 2020-05-21 黄芪-蝉拟青霉发酵菌质、制备方法及用途 WO2021093299A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911120901.3 2019-11-15
CN201911120901.3A CN110656130B (zh) 2019-11-15 2019-11-15 黄芪-蝉拟青霉发酵菌质、制备方法及用途

Publications (1)

Publication Number Publication Date
WO2021093299A1 true WO2021093299A1 (zh) 2021-05-20

Family

ID=69043690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/091617 WO2021093299A1 (zh) 2019-11-15 2020-05-21 黄芪-蝉拟青霉发酵菌质、制备方法及用途

Country Status (2)

Country Link
CN (1) CN110656130B (zh)
WO (1) WO2021093299A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110656130B (zh) * 2019-11-15 2021-05-11 滨州医学院 黄芪-蝉拟青霉发酵菌质、制备方法及用途
CN114259507B (zh) * 2021-12-27 2022-09-16 暨南大学 一种小克里斯滕森氏菌在制备治疗和/或预防高尿酸相关疾病的药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1891239A (zh) * 2005-07-07 2007-01-10 福州安素酶工程开发有限公司 利用酶制剂制备中草药的方法
CN110656130A (zh) * 2019-11-15 2020-01-07 滨州医学院 黄芪-蝉拟青霉发酵菌质、制备方法及用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1262669C (zh) * 2002-12-24 2006-07-05 中国农业大学 利用酶处理提取黄芪多糖的方法
CN104046570B (zh) * 2014-06-13 2016-01-27 上海百岁谷健康管理咨询有限公司 一种提高蝉拟青霉液体发酵产物产量及产物活性的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1891239A (zh) * 2005-07-07 2007-01-10 福州安素酶工程开发有限公司 利用酶制剂制备中草药的方法
CN110656130A (zh) * 2019-11-15 2020-01-07 滨州医学院 黄芪-蝉拟青霉发酵菌质、制备方法及用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LIU XIAORUI ,LI JIAN: "Antihypertensive and lipid-lowering effects of active ingredients of Astragalus", CHINESE JOURNAL OF GERONTOLOGY, vol. 35, no. 17, 10 September 2015 (2015-09-10), pages 4800 - 4801, XP055812018, ISSN: 1005-9202, DOI: 10.3969/j.issn.1005-9202.2015.17.029 *
LU TAN , ZHANG LIYANG , CHEN YUSHENG: "Anti-hyperuricemia activity of a fermented product of astragalus medicinal residue", JIANGSU AGRICULTURAL SCIENCES, vol. 41, no. 9, 25 September 2013 (2013-09-25), pages 288 - 291, XP055812013, ISSN: 1002-1302, DOI: 10.15889/j.issn.1002-1302.2013.09.040 *
WANG YU-QI ,LIU ZI-HAN ,WANG SHAO-PING ,DONG FAN, WANG ZHI-BIN ,DAI LONG ,ZHANG JIA-YU: "Effect of Bidirectional Fermentation System of Paecilomyces cicadae /Astragalus Membranaceus of in Hyperuricemia Models and Study on Its Components", MODERN CHINESE MEDICINE, 3 April 2020 (2020-04-03), pages 463 - 471, XP055812000, ISSN: 1673-4890, DOI: 10.26914/c.cnkihy.2019.070768 *
ZHAO CHONGYAN,YANG FANG,QU QINGSONG,LIU ZIYAO,GAO PENGFEI,SHI XINYUAN: "Establishment of Bidirectional Fermentation System of Paecilomyces cicadae/Astragalus Membranaceus and Study on Its Components", WORLD CHINESE MEDICINE, vol. 13, no. 12, 25 December 2018 (2018-12-25), pages 3195 - 3198, XP055812009, ISSN: 1673-7202, DOI: 10.3969/j.issn.1673-7202.2018.12.057 *

Also Published As

Publication number Publication date
CN110656130B (zh) 2021-05-11
CN110656130A (zh) 2020-01-07

Similar Documents

Publication Publication Date Title
US9394513B2 (en) Method for fermentation and cultivation, fermented plant extract, fermented plant extract powder, and composition containing the extract of fermented plant
CN104770575B (zh) 一种黄芪微生态制剂及其制备方法和应用
WO2021093299A1 (zh) 黄芪-蝉拟青霉发酵菌质、制备方法及用途
CN107156638B (zh) 一种降脂红曲粉的制备方法
CN103315220B (zh) 一种富硒红曲的制作方法
WO2021093425A1 (zh) 黄芪-蝉拟青霉发酵菌质及其用途
CN103948023B (zh) 一种增强免疫力及改善睡眠的保健食品及其二步发酵制备方法
CN108713447A (zh) 一种高海拔地区太岁肉灵芝养殖方法及其保健品制备方法
CN103315359B (zh) 一种灰树花固态发酵功能饮品及制备方法
CN105559068B (zh) 一种利用食药真菌发酵三叶青获得的组合物及其制备方法
CN112691125B (zh) 一种用于美白或者抗衰老的药物组合物及其制备方法、护肤品
CN111449239B (zh) 一种灵芝菌发酵沙棘籽粕的功能性食品添加物及其制备方法
CN113143812A (zh) 一种卡瓦胡椒发酵物的制备方法和产品及其在化妆品中的应用
CN116355816A (zh) 一种发酵翅果油种子的微生物及其降血脂组合物
CN105535035A (zh) 一种桦褐孔菌发酵培养组合物及其制备方法
AU2021102561A4 (en) Astragalus membranaceus-Paecilomyces cicadae fermentation germ plasm and application thereof
CN112079938A (zh) 一种青稞多糖的提取方法、青稞多糖提取物及其应用
CN109350648B (zh) 一种连翘发酵原液及其制备方法
CN109984249A (zh) 一种发酵免疫增强剂及其制备方法和应用
CN109793699A (zh) 一种天麻面膜乳的制备方法
CN110387334A (zh) 一种快速培养牛樟芝菌丝体的方法
CN117547572B (zh) 一种组合物的复合乳酸菌发酵品的制备方法、产品及应用
AU2020102037A4 (en) A method of efficiently increasing the alpha-glucosidase inhibitor content in fresh mulberry leaves by the solid-state fermentation
CN117695345A (zh) 一种赪桐提取物及其制备方法和应用
CN106860781A (zh) 一种米根霉固态发酵提取物、制备方法及其抗癌功效的应用

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: 20887636

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20887636

Country of ref document: EP

Kind code of ref document: A1