CN117025410A - Aspergillus aculeatus, microbial inoculum containing aspergillus aculeatus, application of aspergillus aculeatus and method for converting astragaloside - Google Patents
Aspergillus aculeatus, microbial inoculum containing aspergillus aculeatus, application of aspergillus aculeatus and method for converting astragaloside Download PDFInfo
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- CN117025410A CN117025410A CN202310951459.9A CN202310951459A CN117025410A CN 117025410 A CN117025410 A CN 117025410A CN 202310951459 A CN202310951459 A CN 202310951459A CN 117025410 A CN117025410 A CN 117025410A
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- 241000228215 Aspergillus aculeatus Species 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 33
- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 title claims abstract description 30
- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 title claims abstract description 29
- QMNWISYXSJWHRY-YLNUDOOFSA-N astragaloside IV Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)[C@H]4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)C[C@H]3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-YLNUDOOFSA-N 0.000 claims abstract description 62
- QMNWISYXSJWHRY-BCBPIKMJSA-N astragaloside IV Natural products CC(C)(O)[C@@H]1CC[C@@](C)(O1)[C@H]2[C@@H](O)C[C@@]3(C)[C@@H]4C[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@@H](O)[C@H]5O)[C@H]6C(C)(C)[C@H](CC[C@@]67C[C@@]47CC[C@]23C)O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O QMNWISYXSJWHRY-BCBPIKMJSA-N 0.000 claims abstract description 61
- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 claims abstract description 61
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
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- 238000011218 seed culture Methods 0.000 claims description 17
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
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- 241000233866 Fungi Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
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- 125000003147 glycosyl group Chemical group 0.000 description 2
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- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 2
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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
- C12N1/145—Fungal isolates
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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
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- 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
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
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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
- C12R2001/66—Aspergillus
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- Genetics & Genomics (AREA)
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Abstract
The invention relates to the technical field of microorganisms, and discloses aspergillus aculeatus, a microbial inoculum containing the aspergillus aculeatus, application of the aspergillus aculeatus and the microbial inoculum, and a method for converting astragaloside. The strain of the aspergillus aculeatus is named as aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1), and the preservation number is CCTCC NO: M20221645. The microbial inoculum provided by the invention contains the aspergillus aculeatus. The invention also provides application of the aspergillus aculeatus and the strain in preparation of beta-glucosidase and/or conversion of astragaloside IV. The method for converting astragaloside IV comprises the following steps: contacting the aspergillus aculeatus and/or the microbial inoculum with a raw material containing astragaloside IV. The aspergillus aculeatus provided by the invention can realize the efficient conversion from astragaloside to cycloastragaloside-6-O-beta-D-xyloside, and has the advantages of simple operation, mild reaction conditions and effective reduction of environmental pollution.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to aspergillus aculeatus, a microbial inoculum containing the same, application of the aspergillus aculeatus and the microbial inoculum and a method for converting astragaloside.
Background
Astragaloside IV is a flavonoid compound, and is extracted from dried root of Astragalus mongholicus bge of Leguminosae, and has antiaging and immunity enhancing effects, and its derivative formed by deglycosylation has better bioactivity and availability. The research shows that the astragaloside IV derivative also has important health care effects of promoting wound healing, activating telomerase, inhibiting natural aging of organisms, promoting nerve cell proliferation and the like.
At present, the astragaloside IV is used for producing the derivative thereof by a hydrolysis chemical method, which generally comprises an acid hydrolysis method and a Smith hydrolysis method, wherein a strong oxidant is required to be used for ring opening, hydrogenation is carried out, and then acid-specific hydrolysis is used for removing the ring-opened glycosyl, so that the hydrolysis chemical method has more reaction processes, is easy to produce byproducts and has low yield. In recent years, researchers at home and abroad begin to attempt to convert astragaloside IV by using microorganisms, and the method has the advantages of mild reaction conditions, safe and simple operation, high conversion rate and good specificity. According to the report of the related literature, some strains have been screened to have the potential of converting astragaloside, but the research progress of converting astragaloside by microorganisms still has a certain difficulty because astragaloside is difficult to dissolve in water and belongs to macromolecular natural products.
In addition, cycloastragaloside (CA) is taken as a derivative of astragaloside, has important medicinal value and health care effect, and cycloastragaloside-6-O-beta-D-xyloside is taken as a precursor substance of cycloastragaloside, is an important intermediate product for converting astragaloside into cycloastragaloside, but currently reported astragaloside conversion products are mainly cycloastragaloside-6-O-beta-D-glucoside, and no related report exists that the astragaloside is subjected to microbial conversion to break a glucoside bond so as to obtain the cycloastragaloside-6-O-beta-D-xyloside. If the traditional hydrolysis chemical method is adopted to prepare the cycloastragal-6-O-beta-D-glucoside, a large amount of strong acid, strong alkali and oxidant are needed, so that a large amount of pollution is caused to the environment, and the development is greatly limited by the higher cost and lower yield. Therefore, in recent years, the production of cycloastragal-6-O-beta-D-xyloside by using a microbial conversion method has received a great deal of attention, and screening of a microorganism capable of converting astragaloside IV to obtain cycloastragal-6-O-beta-D-xyloside has also become one of the important points of research.
Disclosure of Invention
The invention aims to solve the problems of difficult production operation, large pollution, high energy consumption and the like of the cycloastragal-6-O-beta-D-glucoside in the prior art, and provides aspergillus aculeatus, a microbial inoculum containing the microbial inoculum, application of the microbial inoculum and a method for converting astragaloside.
In order to achieve the above object, a first aspect of the present invention provides an aspergillus aculeatus (Aspergillus aculeatus) which is named as aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1) and has a preservation number of CCTCC NO: M20221645.
In a second aspect, the invention provides a microbial inoculum comprising aspergillus aculeatus as described above.
Preferably, the microbial inoculum contains living microbial cells of the aspergillus aculeatus; or the microbial inoculum is a liquid microbial inoculum or a solid microbial inoculum.
The third aspect of the invention provides application of aspergillus aculeatus and the microbial inoculum in preparation of beta-glucosidase and/or conversion of astragaloside IV.
Preferably, the beta-glucosidase has the activity of converting astragaloside IV into cycloastragaloside-6-O-beta-D-xyloside; the conversion of astragaloside IV is to convert astragaloside IV into cycloastragaloside-6-O-beta-D-xyloside.
In a fourth aspect, the present invention provides a method for converting astragaloside IV, the method comprising the steps of: contacting the aspergillus aculeatus and/or the microbial inoculum with a raw material containing astragaloside IV, wherein the contact conditions comprise: the temperature is 26-30deg.C, and pH is 3-7.
Preferably, the contacting comprises: inoculating the aspergillus aculeatus into a fermentation culture medium containing the raw material containing astragaloside IV, and performing fermentation culture under the conditions of the temperature of 26-30 ℃ and the pH value of 3-7.
Preferably, the aspergillus aculeatus is inoculated into the fermentation medium in the form of spore seed liquid, and the spore seed liquid is obtained by inoculating the aspergillus aculeatus into a seed medium for seed culture.
Preferably, the seed culture medium contains esculin 2-5g/L, peptone 2-10g/L, magnesium sulfate 0.04-1g/L and potassium dihydrogen phosphate 0.5-3g/L.
Preferably, the seed culture conditions include: the temperature is 26-30 ℃, the rotating speed is 180-250rpm, and the time is 32-48h.
Preferably, the inoculation amount of the spore seed liquid is 5-20mL relative to 1000mL of the fermentation medium, and the content of the raw material containing astragaloside IV is 1-5g.
Preferably, the raw material containing astragaloside IV is astragalus powder.
Preferably, the fermentation medium contains 2-5g/L of esculin, 2-10g/L of peptone, 0.04-1g/L of magnesium sulfate, 0.5-3g/L of monopotassium phosphate, 1-5g/L of astragalus powder and 1-50mL/L of dimethyl sulfoxide.
Preferably, the conditions of the fermentation culture further include: the rotation speed is 150-200rpm, and the time is 6-8 days.
Through the technical scheme, the beta-glucosidase produced by aspergillus aculeatus in the growth process has the activity of converting astragaloside into cycloastragaloside-6-O-beta-D-xyloside, can efficiently convert astragaloside into cycloastragaloside-6-O-beta-D-xyloside, has a mild conversion reaction process, does not need a strong oxidant or strong acid and strong alkali, and reduces environmental pollution compared with the traditional Smith degradation method and acidolysis method; wherein, the substrate astragaloside IV and the product cycloastragaloside-6-O-beta-D-xyloside are all pure natural products, and aspergillus aculeatus is adopted as a natural catalyst in the bioconversion process, so that the process is recyclable and environment-friendly.
The aspergillus aculeatus screened by the invention ensures the safety of the conversion process of astragaloside IV and cycloastragaloside-6-O-beta-D-xyloside, has strong capability of converting into cycloastragaloside-6-O-beta-D-xyloside, and has important significance for efficient, large-scale and low-cost production of cycloastragaloside-6-O-beta-D-xyloside and cycloastragaloside.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Preservation of organisms
The strain provided by the invention is aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1) and is preserved in China center for type culture collection (address: eight-channel 299-No. Wuhan university in Wuchang district of Wuhan, hubei province, 10 months and 24 days, china center for type culture collection 211 room, postal code: 430072, abbreviated as CCTCC of the preservation unit), and the preservation number is CCTCC NO: M20221645.
Drawings
FIG. 1 is a graph of the single colony of the black hydrolysis circles evident on the esculin plate of example 1;
FIG. 2 is a colony morphology of strain Q1 of example 2;
FIG. 3 is a microscopic view (20X) of strain Q1 in example 2;
FIG. 4 is a high performance liquid chromatogram of the fermentation broth of example 3 at 12h of fermentation.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
A first aspect of the invention provides an Aspergillus aculeatus (Aspergillus aculeatus) which has the strain named Aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1) and the preservation number of the Aspergillus aculeatus is CCTCC NO: M20221645.
The aspergillus aculeatus provided by the invention is separated from the wine yeast Daqu (Luzhou in collection), and the aspergillus aculeatus can be separated by a method for separating new strains, which is conventional in the field, for example, a liquid phase enrichment method, which is a "fractional screening" strategy.
The liquid phase enrichment method specifically can include:
preparing a suspension of the brewing yeast, namely preparing the brewing yeast into tiny particles, suspending the tiny particles in water to ensure that microorganisms in the particles can be fully distributed in the suspension for coating on a screening flat plate, specifically, crushing and screening the brewing yeast, mixing a proper amount of yeast powder with sterile water or physiological saline, and standing for 0.5-1.5h at the temperature of 28-32 ℃ and the rotating speed of 180-250rpm to prepare the yeast suspension;
enrichment of astragaloside IV transformed strain, specifically adding 0.5-2 vol% of Daqu suspension into the sub-packed enrichment medium in an ultra clean bench, and culturing for 1-3h at 26-30deg.C and rotation speed of 180-250 rpm;
the primary screening of astragaloside IV converting strain is to suck 1mL of the enriched liquid in an ultra clean bench, dilute the liquid 10 times in a test tube filled with 9mL of sterile water, and then dilute the liquid stepwise to 10 -1 -10 -5 The gradient of the multiple is that the enrichment culture solution with different dilution gradients is coated on a esculin plate separation culture medium, 2-4 plates are coated on each dilution, and the culture medium is placed in a biochemical incubator at 26-30 ℃ for 2-3d for observation;
separating and purifying the astragaloside IV transformed strain, specifically taking out a cultured esculin plate, observing colony characteristics, selecting a single colony with obvious black hydrolysis circle for purification, streaking on a PDA culture medium plate by using an inoculating needle, culturing for 24-48h in an incubator at 26-30 ℃, and repeating until the single colony is obtained by purification;
rescreening of astragaloside IV transformed strains: culturing the separated and purified strain on PDA culture medium for 24-48h until spores are mature, scraping part of thalli, inoculating to PDA liquid culture medium, and shake culturing at 26-30deg.C and rotation speed of 180-250 rpm; after 2-3 days of culture, adding sterilized astragaloside IV aqueous suspension into a shake flask to enable the final concentration of astragaloside IV to be 2g/L, continuously culturing for 3-5 days, taking 2-5mL of fermentation liquor, adding 2-4mL of n-butanol to extract to obtain n-butanol extract, drying the n-butanol extract, adding a small amount of methanol to redissolve, and filtering with a 0.22 mu m organic phase filter membrane for detection;
the detection conditions of the high performance liquid chromatography are as follows: using a Dalian Nepeter C18 chromatographic column, a PDA detector, detecting the wavelength of 204nm, the mobile phase of 60-80% methanol, the flow rate of 0.6-1mL/min, the detection time of 20-30min, and the column temperature of 30-40 ℃; detecting that the peak time of astragaloside IV is 12.6min, that of cycloastragaloside-6-O-beta-D-xyloside is 14.0min, and that of cycloastragaloside is 17.7min; and detecting the yields of the cycloastragaloside-6-O-beta-D-xyloside and the cycloastragaloside in the sample to be detected obtained by re-screening, and screening out the strain with the highest activity on astragaloside conversion.
According to the invention, preferably, the enrichment medium contains: 2-10g/L cellobiose, 5-15g/L peptone, 2-5g/L beef powder and 2-10g/L sodium chloride; the esculin plate isolation medium contained: 2-5g/L of esculin, 0.2-1g/L of ferric citrate, 2-10g/L of peptone, 0.2-1g/L of magnesium sulfate heptahydrate, 0.5-2g/L of potassium dihydrogen phosphate and 15-25g/L of agar; the preparation process of the PDA culture medium comprises the following steps: selecting fresh potatoes with good state, cleaning and peeling, weighing 200g, chopping, boiling in boiling water for 30min, filtering with warp cloth, adding 20g of glucose and 15-20g of agar, and fixing the volume to 1000mL.
Filling the culture medium into bottles, wrapping, sterilizing at 121deg.C for 20min, and packaging into sterilized conical bottles or pouring solid flat plates for use.
The inventor of the invention obtains a strain Q1 with highest astragaloside IV conversion activity through the screening method, and performs ITSDNA extraction and sequencing, and the identification result shows that the ITSDNA sequence of the strain is highly similar to that of aspergillus aculeatus (Aspergillus aculeatus), is relatively close in evolution distance, can be determined to be aspergillus aculeatus, is named aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1), and is preserved in China Center for Type Culture Collection (CCTCC) No. M20221645 in 10 and 24 days of 2022.
The aspergillus aculeatus provided by the invention can produce a large amount of living thalli and/or fermentation products of the aspergillus aculeatus after being cultured. The culture method of the present invention is not particularly limited as long as the koji mold can be proliferated in a large amount by the culture method, and for example, a spore suspension of the koji mold can be inoculated in a culture medium in an amount of not less than 0.5% by volume and cultured at a temperature of 26 to 30℃for not less than 48 hours to obtain a culture solution.
The present invention may further isolate the cells of aspergillus aculeatus in the above-mentioned culture solution, and the method of the isolation is not particularly limited as long as the cells can be enriched from the culture solution, for example, by centrifugation and/or filtration, and the conditions of the centrifugation and the filtration may be conventional conditions in the art, which are well known to those skilled in the art, and are not described herein.
In a second aspect, the invention provides a microbial inoculum comprising aspergillus aculeatus as described above.
According to the present invention, preferably, the microbial inoculum contains living cells of the aspergillus aculeatus. The preparation form of the microbial inoculum is not particularly limited, and can be prepared into different preparation forms according to different preset purposes, and components such as corresponding excipients and the like are added, for example, the microbial inoculum can be liquid microbial inoculum (for example, can be bacterial liquid of aspergillus aculeatus) or solid microbial inoculum (for example, can be powder prepared by drying aspergillus aculeatus or can be prepared into a high-purity preparation through steps of separation, purification and the like). The addition of excipients to the bacterial agents in which the dosage forms are described herein is well known to those skilled in the art and will not be described in detail herein.
The beta-glucosidase produced in the growth process of aspergillus aculeatus provided by the invention has the activity of converting astragaloside into cycloastragaloside-6-O-beta-D-xyloside, and can efficiently convert astragaloside into cycloastragaloside-6-O-beta-D-xyloside. Based on the above, the third aspect of the invention provides the application of the aspergillus aculeatus and the microbial inoculum in preparing beta-glucosidase and/or converting astragaloside.
According to the present invention, preferably, the beta-glucosidase has an activity of converting astragaloside IV into cycloastragaloside-6-O-beta-D-xyloside; the conversion of astragaloside IV is to convert astragaloside IV into cycloastragaloside-6-O-beta-D-xyloside.
In the invention, the thallus obtained after fermentation culture of aspergillus aculeatus can be crushed, extracted and the like to obtain beta-glucosidase, and can be further used for preparing an enzyme preparation of the beta-glucosidase and applied to converting astragaloside IV.
In a fourth aspect, the present invention provides a method for converting astragaloside IV, the method comprising the steps of: contacting the aspergillus aculeatus and/or the microbial inoculum with a raw material containing astragaloside IV, wherein the contact conditions comprise: the temperature is 26-30deg.C, and pH is 3-7.
The aspergillus aculeatus provided by the invention generates beta-glucosidase in the growth process under specific temperature and pH conditions, so as to convert astragaloside IV into cycloastragaloside-6-O-beta-D-xyloside. Preferably, the contacting comprises: inoculating the aspergillus aculeatus into a fermentation culture medium containing the raw material containing astragaloside IV, and performing fermentation culture under the conditions of the temperature of 26-30 ℃ and the pH value of 3-7.
According to the present invention, preferably, the aspergillus aculeatus is inoculated into the fermentation medium in the form of a spore seed liquid obtained by inoculating the aspergillus aculeatus into a seed medium for seed culture.
According to the present invention, the seed medium may be a medium commonly known in the art, preferably the seed medium contains esculin, peptone, magnesium sulfate and potassium dihydrogen phosphate. Illustratively, the seed culture medium specifically comprises: esculin 2-5g/L, peptone 2-10g/L, magnesium sulfate 0.04-1g/L and potassium dihydrogen phosphate 0.5-3g/L. The inventors have found that in this preferred embodiment, the growth of Aspergillus aculeatus can be promoted and the efficiency of converting astragaloside to cycloastragaloside-6-O-beta-D-xyloside can be improved.
According to the present invention, preferably, the seed culture conditions include: the temperature is 26-30 ℃, the rotating speed is 180-250rpm, and the time is 32-48h. The inventors found that in this preferred embodiment, the growth rate of aspergillus aculeatus can be increased, thereby increasing the conversion efficiency of astragaloside.
According to the present invention, preferably, the spore seed liquid is inoculated in an amount of 5 to 20mL and the astragaloside IV-containing material is contained in an amount of 1 to 5g with respect to 1000mL of the fermentation medium.
According to the invention, any material containing astragaloside can be used as the material containing astragaloside. Preferably, the raw material containing astragaloside is astragalus powder, which can be obtained commercially, and the astragalus powder can be an astragalus crude extract with the astragaloside content of about 30 wt% and no cycloastragaloside-6-O-beta-D-xyloside.
According to the invention, the fermentation medium contains esculin, peptone, magnesium sulfate, potassium dihydrogen phosphate, astragalus powder and dimethyl sulfoxide, and a raw material containing astragaloside is added on the basis. Illustratively, the fermentation medium contains esculin 2-5g/L, peptone 2-10g/L, magnesium sulfate 0.04-1g/L, potassium dihydrogen phosphate 0.5-3g/L, astragaloside-containing raw material 1-5g/L, dimethyl sulfoxide 1-50mL/L. The inventors have found that in this preferred embodiment, the growth rate of Aspergillus aculeatus can be increased, thereby increasing the rate of production and conversion of cycloastragenol-6-O-beta-D-xyloside.
Specifically, the preparation process of the fermentation medium comprises the following steps: mixing esculin, peptone, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, and raw material containing astragaloside IV with water, sterilizing with 121 deg.C high pressure steam for 20min, and adding dimethyl sulfoxide 0.1-5 vol%.
According to the present invention, preferably, the conditions of the fermentation culture include: the rotation speed is 150-200rpm, and the time is 6-8 days. The inventors found that the production rate and conversion rate of cycloastragenol-6-O-beta-D-xyloside can be effectively improved under the preferred specific embodiment.
According to a particularly preferred embodiment of the present invention, the method for converting astragaloside IV comprises the steps of:
inoculating aspergillus aculeatus to a PDA solid slant culture medium for culturing for 2-5 days until spores are mature, scraping off spores on the surface of a strain to prepare a spore suspension with a certain concentration, inoculating the spore suspension into a seed culture medium with an inoculum size of 1-4 vol%, and culturing for 32-48 hours at a culture temperature of 26-30 ℃ and a rotating speed of 180-250rpm to obtain spore seed liquid; inoculating spore seed solution with inoculum size of 0.5-2 vol% into fermentation medium, and culturing at 26-30deg.C, rotation speed of 150-200rpm and pH of 3-7 for 6-8 days;
the seed culture medium specifically contains: 2-5g/L of esculin, 2-10g/L of peptone, 0.04-1g/L of magnesium sulfate and 0.5-3g/L of monopotassium phosphate; the fermentation medium contains: 2-5g/L of esculin, 2-10g/L of peptone, 0.04-1g/L of magnesium sulfate, 0.5-3g/L of monopotassium phosphate, 1-5g/L of raw material containing astragaloside IV and 1-50mL/L of dimethyl sulfoxide.
The present invention will be described in detail by examples.
In the following examples, the brewing yeast is taken from Luzhou Laojiao Co., ltd, the astragalus powder is purchased from Nanjing spring and autumn bioengineering Co., ltd, the astragalus powder belongs to a crude astragalus extract, the content of astragaloside IV is about 30% by weight, the Astragaloside IV does not contain cyclo-Astragaloside-6-O-beta-D-xyloside, and the esculin is purchased from Shanghai source leaf Biotechnology Co., ltd, and the product model is S30576; without any particular description, the remaining materials or reagents are all conventional analytically pure or chemically pure commercial products.
In the following examples, the formulation of the enrichment medium was: cellobiose 5g/L, peptone 10g/L, beef powder 3g/L and sodium chloride 5g/L;
the formula of the esculin plate separation medium is as follows: esculin 3g/L, ferric citrate 0.5g/L, peptone 5g/L, magnesium sulfate heptahydrate 0.5g/L, potassium dihydrogen phosphate 1g/L, and agar 20g/L;
the PDA culture medium is prepared through the following steps: selecting fresh potatoes with good states, cleaning and peeling, weighing 200g, chopping, boiling in boiling water for 30min, filtering with warp cloth, adding 20g of glucose and 15g of agar, and fixing the volume to 1000mL.
The conditions for detecting astragaloside IV, cycloastragaloside-6-O-beta-D-xyloside and cycloastragaloside by high performance liquid chromatography are as follows: using a Dalian Nepeter C18 chromatographic column, a PDA detector, a detection wavelength of 204nm, a mobile phase of 75% methanol, a flow rate of 1mL/min, a detection time of 20-30min, and a column temperature of 30-40 ℃; wherein, the peak time of astragaloside IV is 12.6min, the peak time of cycloastragaloside-6-O-beta-D-xyloside is 14.0min, and the peak time of cycloastragaloside is 17.7min.
Example 1
Preparing a suspension of brewing Daqu: pulverizing the collected brewing yeast, sieving with a 40-mesh sieve to obtain yeast powder, weighing 5g of yeast powder, mixing with 45mL of sterile water (feed-liquid ratio is 1:9), and standing for 1h at 30deg.C and 220rpm to obtain yeast suspension;
enrichment of Astragaloside IV transformed strains: adding 1 volume percent of Daqu suspension into 50mL of packed enrichment culture medium in an ultra-clean bench, and culturing for 2h at 28 ℃ and 220rpm to obtain enrichment culture liquid;
primary screening of astragaloside IV transformed strains: sucking 1mL of enrichment culture liquid in an ultra-clean bench, adding into a test tube filled with 9mL of sterile water for dilution by 10 times, and then gradually diluting to 10 -1 -10 -5 Doubling, namely 0.2mL of enrichment culture solution with different dilution gradients is coated on a esculin plate separation culture medium, 3 plates are coated on each dilution, and the culture medium is placed in a biochemical incubator at 30 ℃ for 2-3d for observation;
separating and purifying the astragaloside IV transformation strain: taking out the esculin plate after primary screening culture, observing colony characteristics, selecting single colony with obvious black hydrolysis ring (as shown in figure 1, the colony can see obvious black fungus ring, which shows that the fungus has obvious glycosyl hydrolase activity), purifying, streaking on a PDA culture medium plate by using an inoculating needle, culturing for 24-48h in a 30 ℃ incubator, and repeating until the single colony is obtained after purification;
rescreening of astragaloside IV transformed strains: culturing the single colony obtained by separation and purification on a PDA culture medium for 24-48 hours until spores are mature, scraping part of thalli, inoculating the thalli into the PDA liquid culture medium, and performing shake culture under the conditions that the temperature is 28 ℃ and the rotating speed is 220 rpm; after 2-3 days of culture, adding sterilized astragaloside IV aqueous suspension into a shake flask to enable the final concentration of astragaloside IV to be 2g/L, continuously culturing for 3-5 days to obtain fermentation liquor, taking 2-5mL of fermentation liquor, adding 2-4mL of n-butanol to extract to obtain n-butanol extract, drying the n-butanol extract, adding a small amount of methanol to re-dissolve, filtering with a 0.22 mu m organic phase filter membrane, and performing high performance liquid chromatography detection to obtain a strain with highest yield of cycloastragaloside-6-O-beta-D-xyloside and cycloastragaloside IV, wherein the strain has highest conversion activity to astragaloside IV, and the strain is named as Q1.
Example 2
2.1 morphological identification of Astragaloside IV transformant strain Q1
Inoculating the strain Q1 obtained in the example 1 on a PDA plate, culturing for 24-48 hours until the colony condition is good, observing and recording, and the result is shown in figure 2; hyphae were picked up on a glass slide, fixed, placed under a microscope to observe the morphology of the hyphae and sporophores, observed and recorded, and the results are shown in fig. 3.
As shown in FIG. 2, after 2d incubation of strain Q1 on PDA medium at 28℃the colony was round mycelium with a central brown front, black after maturation, white surrounding, loose texture and central bulge. As shown in FIG. 3, strain Q1 was observed under a microscope, and the spore peduncles were typical of Aspergillus appearance and the spores were spherical.
2.2 molecular characterization of Astragaloside IV transformant strain Q1
The strain Q1 is sent to a biological company for sequencing, the ITSDNA sequence of the strain is shown as SEQ ID NO.1, the sequencing result is subjected to BLAST analysis in a Genebank database to analyze the similarity and consistency of the sequence, the strain Q1 is determined to be aspergillus aculeatus (Aspergillus aculeatus), the strain is named aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1), and the strain is preserved in China center for type culture collection (CCTCC NO: M20221645) at 10 months of 2022.
Example 3
(1) The formula of the seed culture medium is as follows: esculin 3g/L, peptone 5g/L, magnesium sulfate heptahydrate 0.5g/L, potassium dihydrogen phosphate 1g/L; the preparation process of the fermentation medium comprises the following steps: mixing esculin 3g, peptone 5g, magnesium sulfate heptahydrate 0.5g, potassium dihydrogen phosphate 1g, radix astragali powder 2g with 1L water, sterilizing with 121 deg.C high pressure steam for 20min, and adding dimethyl sulfoxide 20mL;
(2) Inoculating Aspergillus aculeatus XXLS-Q1 obtained in example 1 onto PDA solid slant culture medium, culturing for 4 days until spore is mature, scraping off spores on strain surface to obtain 5×10 6 Adding 1mL of spore suspension into 50mL of seed culture medium, and culturing for 40h at the culture temperature of 28 ℃ and the rotating speed of 220rpm to obtain spore seed liquid;
(3) The spore seed solution was transferred to a fermentation medium at an inoculum size of 1 vol%, and cultured at 28℃for 7 days at 180rpm and pH 5 to give a fermentation broth.
Example 4
(1) The formula of the seed culture medium is as follows: 2g/L of esculin, 10g/L of peptone, 0.1g/L of magnesium sulfate heptahydrate and 0.5g/L of potassium dihydrogen phosphate; the preparation process of the fermentation medium comprises the following steps: mixing 2g of esculin, 10g of peptone, 0.1g of magnesium sulfate heptahydrate, 0.5g of potassium dihydrogen phosphate and 5g of astragalus powder with 1L of water, sterilizing by high-pressure steam at 121 ℃ for 20min, and adding 1mL of dimethyl sulfoxide;
(2) Inoculating Aspergillus aculeatus XXLS-Q1 obtained in example 1 onto PDA solid slant culture medium, culturing for 2 days until spore is mature, scraping off spores on strain surface to obtain 1×10 6 Adding 0.5mL of spore suspension into 50mL of seed culture medium, and culturing for 48 hours at the culture temperature of 26 ℃ and the rotating speed of 250rpm to obtain spore seed liquid;
(3) The spore seed solution was transferred to a fermentation medium at an inoculum size of 0.5 vol%, and cultured at a culture temperature of 26℃at 200rpm for 8 days at pH 3 to give a fermentation broth.
Example 5
(1) The formula of the seed culture medium is as follows: 5g/L of esculin, 2g/L of peptone, 1g/L of magnesium sulfate heptahydrate and 3g/L of potassium dihydrogen phosphate; the preparation process of the fermentation medium comprises the following steps: mixing esculin 5g, peptone 2g, magnesium sulfate heptahydrate 1g, potassium dihydrogen phosphate 3g, radix astragali powder 1g with 1L water, sterilizing with 121 deg.C high pressure steam for 20min, and adding dimethyl sulfoxide 50mL;
(2) Aspergillus aculeatus XXLS-Q1 obtained in example 1Inoculating to PDA solid slant culture medium, culturing for 5 days until spore is mature, scraping off spore on strain surface to obtain 1×10 strain 7 Adding 2mL of spore suspension into 50mL of seed culture medium, and culturing for 32h at the culture temperature of 30 ℃ and the rotating speed of 180rpm to obtain spore seed liquid;
(3) The spore seed solution was transferred to a fermentation medium at an inoculum size of 2 vol%, and cultured at a culture temperature of 30℃at 150rpm for 6 days at pH 7 to obtain a fermentation broth.
Comparative example 1
Astragaloside IV was converted by fermentation as in example 3, except that the pH adjustment in step (3) was replaced with 9.
Comparative example 2
Astragaloside IV was converted by fermentation in the same manner as in example 3 except that the temperature of the culture in step (3) was changed to 35 ℃.
Test example 1
Taking 3mL of fermentation liquor obtained in the embodiment 3-embodiment 5 and the comparative embodiment 1-comparative embodiment 2 respectively, adding 3mL of n-butanol for extraction to obtain n-butanol extract, drying the n-butanol extract, adding a small amount of methanol for re-dissolution, filtering with a 0.22 mu m organic phase filter membrane, performing high performance liquid chromatography detection to obtain the concentration of cycloastragaloside-6-O-beta-D-xyloside in the fermentation liquor, calculating the conversion rate of astragaloside IV, wherein the calculation formula is shown in (I), and the result is shown in Table 1; the high performance liquid chromatogram of the fermentation broth obtained in example 3 at 12h of fermentation is shown in FIG. 4.
TABLE 1
As can be seen from the results in Table 1, the aspergillus aculeatus XXLS-Q1 provided by the invention can realize the efficient conversion from astragaloside IV to cycloastragaloside-6-O-beta-D-xyloside, and has the advantages of simple operation, mild reaction conditions and effective reduction of environmental pollution.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. A strain of aspergillus aculeatus (Aspergillus aculeatus) is named as aspergillus aculeatus XXLS-Q1 (Aspergillus aculeatus XXLS-Q1), and the preservation number of the aspergillus aculeatus is CCTCC NO: M20221645.
2. A microbial inoculum, characterized in that the microbial inoculum contains the Aspergillus aculeatus according to claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent contains viable cells of aspergillus aculeatus; or,
the microbial inoculum is a liquid microbial inoculum or a solid microbial inoculum.
4. Use of aspergillus aculeatus as claimed in claim 1, the microbial inoculum as claimed in claim 2 or 3 for the preparation of beta-glucosidase and/or for the conversion of astragaloside.
5. The use according to claim 4, wherein the beta-glucosidase has the activity of converting astragaloside IV to cycloastragaloside-6-O-beta-D-xyloside;
the conversion of astragaloside IV is to convert astragaloside IV into cycloastragaloside-6-O-beta-D-xyloside.
6. A method for converting astragaloside iv, comprising the steps of: contacting aspergillus aculeatus of claim 1 and/or the microbial inoculum of claim 2 or 3 with a astragaloside-containing feedstock, the conditions of said contacting comprising: the temperature is 26-30deg.C, and pH is 3-7.
7. The method of claim 6, wherein the contacting comprises: inoculating the aspergillus aculeatus into a fermentation culture medium containing the raw material containing astragaloside IV, and performing fermentation culture under the conditions of the temperature of 26-30 ℃ and the pH value of 3-7.
8. The method according to claim 7, wherein the aspergillus aculeatus is inoculated into the fermentation medium in the form of a spore seed liquid obtained by inoculating the aspergillus aculeatus into a seed medium for seed culture;
preferably, the seed culture medium contains 2-5g/L of esculin, 2-10g/L of peptone, 0.04-1g/L of magnesium sulfate and 0.5-3g/L of potassium dihydrogen phosphate;
preferably, the seed culture conditions include: the temperature is 26-30 ℃, the rotating speed is 180-250rpm, and the time is 32-48h.
9. The method according to claim 8, wherein the spore seed liquid is inoculated in an amount of 5 to 20mL relative to 1000mL of the fermentation medium, and the astragaloside-containing raw material is contained in an amount of 1 to 5g;
preferably, the raw material containing astragaloside IV is astragalus powder.
10. The method according to any one of claims 7 to 9, wherein the fermentation medium contains esculin 2-5g/L, peptone 2-10g/L, magnesium sulfate 0.04-1g/L, potassium dihydrogen phosphate 0.5-3g/L, astragalus powder 1-5g/L, dimethyl sulfoxide 1-50mL/L;
preferably, the conditions of the fermentation culture further include: the rotation speed is 150-200rpm, and the time is 6-8 days.
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