CN105861614B - Application of aspergillus niger in preparation of astragaloside - Google Patents
Application of aspergillus niger in preparation of astragaloside Download PDFInfo
- Publication number
- CN105861614B CN105861614B CN201610313391.1A CN201610313391A CN105861614B CN 105861614 B CN105861614 B CN 105861614B CN 201610313391 A CN201610313391 A CN 201610313391A CN 105861614 B CN105861614 B CN 105861614B
- Authority
- CN
- China
- Prior art keywords
- aspergillus niger
- astragalus
- astragaloside
- residue
- dregs
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
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
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
Abstract
The invention discloses an application of aspergillus niger in preparation of astragaloside. The application can be specifically a method for preparing astragaloside IV. The method for preparing astragaloside IV comprises fermenting radix astragali residue with Aspergillus niger to obtain radix astragali residue Aspergillus niger fermented product, and extracting astragaloside IV from the radix astragali residue Aspergillus niger fermented product. Experiments prove that the yield of the astragaloside is improved after the astragalus residue is fermented by aspergillus niger: fermenting the astragalus residue with Aspergillus niger ACCC30583, and extracting to obtain astragaloside IV with yield 5.4 times of that of unfermented astragalus residue; the yield of the astragaloside extracted by the same extraction method after the astragalus dregs are fermented by aspergillus niger ACCC30784 is 2.7 times that of the astragalus dregs which are not fermented; the yield of astragaloside extracted by the same extraction method after the astragalus residue is fermented by the aspergillus niger ACCC 30177 is 4.8 times of that of the astragalus residue which is not fermented.
Description
Technical Field
The invention relates to an application of aspergillus niger in the field of microorganisms, in particular to an application of aspergillus niger in preparation of astragaloside IV.
Background
Traditional Chinese medicines are traditional medicines in China, with the continuous development of the traditional Chinese medicine industry in China in recent years, the market share of Chinese patent medicines in China and even internationally is larger and larger, so that the number of Chinese patent medicine production and Chinese medicine processing enterprises in China is increased continuously, Chinese medicine residues which are Chinese medicine wastes are increased in large quantity, and if the Chinese medicine residues cannot be treated in time, the events such as 'residue village' of Guizhou lark, 'pollution door' of Ha medicine and the like can occur, and serious pollution is caused to the ecological environment. Reports show that 1636 Chinese medicine enterprises consume 70 ten thousand tons of plant medicinal materials every year by 2010, and a large amount of traditional Chinese medicine dregs are generated. Due to the limitation of the production process of traditional Chinese medicines, the extraction rate of effective components of the traditional Chinese medicines is low for each traditional Chinese medicine enterprise, and the traditional Chinese medicine dregs also contain a large amount of effective components, so that the traditional Chinese medicine dregs have high utilization value, and how to correctly treat the traditional Chinese medicine dregs becomes the key point of the current research.
The traditional Chinese medicine dregs are residues after the effective components of the traditional Chinese medicines are extracted, and are generally treated as wastes in the traditional Chinese medicine extraction industry. At present, the treatment method of the traditional Chinese medicine dregs in industry mainly comprises stacking, landfill, incineration and the like, if the dregs are treated by adopting an incineration mode, a large amount of toxic and harmful gas can be generated in the incineration process of the dregs, the atmosphere is polluted, and wet materials can bring difficulty to incineration; if the medicine residues are treated by stacking, burying and other modes, the medicine residues can be decayed, a large amount of pungent smell is generated, and the surrounding environment is seriously polluted. The content of effective components in the traditional Chinese medicinal materials is low, and the traditional Chinese medicine production is laggard, so that a large amount of traditional Chinese medicine dregs are generated in the production of the traditional Chinese medicine, and the traditional Chinese medicine dregs have the characteristics of large volume, high yield and the like. Because the traditional Chinese medicine dregs have the characteristics of large volume and high yield, the traditional Chinese medicine dregs are mainly stacked and are stacked, so that the residual active ingredients in the dregs are wasted, the resource waste is caused, meanwhile, the surrounding water and soil resources are polluted, and the serious harm is brought to the production and life of people.
According to the regulation of Astragalus root in the first part of the pharmacopoeia of the people's republic of china, 2010, Astragalus root is the dried root of Astragalus mongholicus (Astragalus membranaceus, fish.) Bge. Saponins are one of the main active ingredients of astragalus mongholicus, and more than 40 saponins such as astragaloside, isoastragaloside and the like are separated from astragalus mongholicus at present. In recent years, with the continuous and intensive research on astragalosides, the astragalosides are found to have the effects of resisting virus, resisting tumor, reducing blood sugar, improving cardiovascular diseases and the like besides the function of regulating immunity. Wang et al have shown that astragaloside IV can promote the growth of mouse antibody and the proliferation of T, B lymphocyte, and improve the humoral immunity and cellular immunity of mouse.
Disclosure of Invention
The invention aims to solve the technical problem of how to prepare astragaloside by taking astragalus dregs as raw materials.
In order to solve the technical problems, the invention provides the application of aspergillus niger in the preparation of astragaloside.
In the application, the aspergillus niger can be aspergillus niger ACCC30583, aspergillus niger ACCC 30177 or aspergillus niger ACCC 30784.
In order to solve the technical problems, the invention provides a method for preparing astragaloside by using aspergillus niger.
The method for preparing the astragaloside comprises the steps of fermenting astragalus dregs with aspergillus niger to obtain an aspergillus niger fermented substance of the astragalus dregs, and extracting the astragaloside from the aspergillus niger fermented substance of the astragalus dregs.
In the method, the Aspergillus niger is used for fermenting the astragalus dregs to obtain the Aspergillus niger fermented product of the astragalus dregs.
In the above method, the Aspergillus niger can be Aspergillus niger ACCC30583, Aspergillus niger ACCC 30177 or Aspergillus niger ACCC 30784.
In the above method, the fermentation medium containing astragalus residue can be prepared as follows: sterilizing the material with the water content of 50-60% to obtain the fermentation culture medium containing the astragalus mongholicus dregs; the material consists of the following raw materials in parts by mass: 10 parts by mass of astragalus root dregs, 0.03-0.12 part by mass (such as 0.03-0.06 part by mass or 0.06 part by mass) of nitrogen source and 1.2 parts by mass of KH2PO4And 0.6 part by mass of MgSO4·7H2O。
In the above methodThe nitrogen source can be urea or KNO3、(NH4)2SO4Yeast powder, beef extract or/and peptone.
In the above method, the culturing is carried out at 28 ℃ for 5 to 8 days or 5 days.
In order to solve the above technical problems, the present invention provides a kit for preparing astragaloside IV.
The complete set of products for preparing the astragaloside provided by the invention comprise the Aspergillus niger and the fermentation culture medium containing the astragalus residue; and the aspergillus niger and the fermentation medium containing the astragalus residue are independently packaged.
The fermentation medium containing the astragalus residue also belongs to the protection scope of the invention.
In the fermentation culture medium containing radix astragali residue, the particle diameter (diameter) of radix astragali residue can be 0.8-1.2cm or 2 mm-4 mm.
The astragalus residue of the invention refers to residue left after extracting the active ingredients of astragalus by water. The radix astragali residue can be prepared by boiling radix astragali in water for 2-3 times, filtering, and collecting residue, which is radix astragali residue.
Experiments prove that the yield of the astragaloside is improved after the astragalus residue is fermented by aspergillus niger: fermenting the astragalus residue with Aspergillus niger ACCC30583, and extracting to obtain astragaloside IV with yield 5.4 times of that of unfermented astragalus residue; the yield of the astragaloside extracted by the same extraction method after the astragalus dregs are fermented by aspergillus niger ACCC30784 is 2.7 times that of the astragalus dregs which are not fermented; the yield of astragaloside extracted by the same extraction method after the astragalus residue is fermented by the aspergillus niger ACCC 30177 is 4.8 times of that of the astragalus residue which is not fermented.
Drawings
FIG. 1 is an HP L C chromatogram of an astragaloside standard substance, and an arrow shows an astragaloside chromatographic peak.
FIG. 2 is a HP L C spectrum of an astragaloside IV sample extracted from Aspergillus niger ACCC30583 radix astragali residue solid fermentation product, and an arrow shows the astragaloside IV chromatographic peak.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Aspergillus niger ACCC30784 in the following examples was collected at 11.2006 in 11.5.7 by the agricultural microorganism center of China Committee for culture Collection of microorganisms (ACCC for short, address: southern Avenue 12 of Guancun, Haizhou, Beijing, institute of agricultural resources and agricultural divisions, Japan, Zip code 100081), and was publicly available from the agricultural microorganism center of the China Committee for culture Collection on the day of collection. Aspergillus niger (Aspergillus niger) ACCC30784 is hereinafter referred to as Aspergillus niger ACCC 30784.
Aspergillus niger ACCC 30177 in the following examples was collected at the agricultural microorganism center of China Committee for culture Collection of microorganisms (ACCC for short, address: southern Avenue 12 of Guancun, in Haizu, Beijing, academy of agricultural resources and agriculture, Pod. code 100081) on 6.8.1989, and was publicly available from the agricultural microorganism center of the China Committee for culture Collection on the day of collection. Aspergillus niger (Aspergillus niger) ACCC 30177 is hereinafter referred to as Aspergillus niger ACCC 30177.
Aspergillus niger ACCC30583 in the following examples was collected from China center for microorganisms and cultures Collection, agricultural microorganisms (ACCC, address: southern street 12, Ministry of agricultural resources and agriculture, division, Inc. of China academy of agricultural sciences, postal code 100081) at 7.21.2006, and was publicly available from the agricultural microorganisms center of China Committee for culture Collection of microorganisms from the date of collection. Aspergillus niger (Aspergillus niger) ACCC30583 is hereinafter referred to as Aspergillus niger ACCC 30583.
Example 1 preparation of Astragaloside IV Using Aspergillus niger
The media used in this example were as follows:
PDA, peeling potato, cutting 200g into small pieces, adding water, boiling for 30min, filtering with 4 layers of gauze, adding 20g glucose and 17g agar, adding distilled water to 1000m L, boiling, mixing, and sterilizing at 121 deg.C for 20 min to obtain PDA culture medium.
PD, peeling potato, cutting 200g into small pieces, adding water, boiling for 30min, filtering with 4 layers of gauze, adding 20g glucose, adding distilled water to a constant volume of 1000m L, boiling, mixing, and sterilizing at 121 deg.C for 20 min to obtain PD culture medium.
The astragalus residue solid fermentation culture medium: 10.0g (dry weight basis) of radix astragali residue with diameter of 2mm to 4mm, 0.03g of urea, KH2PO41.2g，MgSO4·7H2O0.6 g, adjusting the water content of the materials to 60 percent and the pH value to 7.0, and sterilizing for 20 minutes at 121 ℃ to obtain the astragalus residue solid fermentation culture medium.
The astragalus dregs used in this example are all prepared according to the following method:
placing 100g radix astragali decoction pieces (Hefeile Jia old shop Chinese medicinal decoction pieces Co., Ltd., Sichuan of production area, lot number 13060501) in a beaker, adding cold pure water, soaking for 1h (the amount of water is based on the fact that the medicinal materials are just soaked completely), adding cold pure water (about 1000m L) 10 times of the total amount of the medicinal materials during decoction, boiling with strong fire, boiling, keeping boiling with slow fire for 60min, filtering to obtain medicinal liquid, adding warm water at about 50 ℃ into the residue, decocting for the second time, filtering to obtain medicinal liquid, and keeping the residue to obtain radix astragali residue.
1. Preparation of aspergillus niger astragalus residue solid fermentation product by aspergillus niger fermentation of astragalus residue
In this example, aspergillus niger ACCC30583, aspergillus niger ACCC30784 and aspergillus niger ACCC 30177 are used to ferment astragalus membranaceus dregs respectively under the same conditions, so as to obtain three aspergillus niger ACCC30583 astragalus membranaceus dreg solid fermentations, aspergillus niger ACCC30784 astragalus membranaceus dreg solid fermentations and aspergillus niger ACCC 30177 astragalus membranaceus dreg solid fermentations, wherein the preparation methods of the three aspergillus niger astragalus membranaceus dreg solid fermentations are different only in the used aspergillus niger strains. The preparation method of the three aspergillus niger astragalus residue solid fermentation products comprises the following steps:
1.1 preparation of Aspergillus niger ACCC30583 radix astragali residue solid fermentation product
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC30583 on PDA plate, inoculating loop to pick Aspergillus niger ACCC30583 spore, diluting with sterile water to 1 × 108Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ for 24 hours at 160rpm to obtain a seed solution, inoculating the seed solution into a 500ml conical flask filled with 50g of astragalus residue solid fermentation medium at the inoculation amount of 1% (V/V) to culture at 28 ℃ for 5 days, collecting all substances in the culture vessel to obtain aspergillus niger ACCC30583 astragalus residue solid fermentation product, and repeating the steps for 10 conical flasks each time.
1.2 preparation of Aspergillus niger ACCC30784 radix astragali residue solid fermentation product
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC30784 to PDA plate, inoculating to inoculating loop, picking Aspergillus niger ACCC30784 spore, diluting with sterile water to 1 × 108Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ for 24 hours at 160rpm to obtain a seed solution, inoculating the seed solution into a 500ml conical flask filled with 50g of astragalus residue solid fermentation medium at the inoculation amount of 1% (V/V) to culture at 28 ℃ for 5 days, collecting all substances in the culture vessel to obtain aspergillus niger ACCC30784 astragalus residue solid fermentation product, and repeating the steps for 10 conical flasks each time.
1.3 preparation of Aspergillus niger ACCC 30177 radix astragali residue solid fermentation product
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC 30177 on PDA plate, inoculating loop, picking Aspergillus niger ACCC 30177 spore, diluting with sterile water to 1 × 108Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ for 24 hours at 160rpm to obtain a seed solution, inoculating the seed solution into a 500ml conical flask filled with 50g of astragalus residue solid fermentation medium at the inoculation amount of 1% (V/V) to culture at 28 ℃ for 5 days, collecting all substances in the culture vessel to obtain aspergillus niger ACCC 30177 astragalus residue solid fermentation product, and repeating the steps for 10 conical flasks each time.
2. Extracting astragaloside from Aspergillus niger radix astragali residue solid fermentation product
2.1 extraction of Astragaloside IV from Aspergillus niger ACCC30583 radix astragali residue solid fermentation product
The experiment is repeated for 3 times, each time the method comprises taking 10g of aspergillus niger ACCC30583 radix astragali residue solid fermentation product, adding 100m L of distilled water, soaking for 30min, boiling and keeping slightly boiling for 30min, filtering and collecting filtrate (the filtrate is called as filtrate 1), adding 100m L of distilled water into filter residue, boiling and keeping slightly boiling for 30min, filtering and collecting filtrate (the filtrate is called as filtrate 2), combining filtrate 1 and filtrate 2, centrifuging for 10min at 4000r/min, taking supernatant, concentrating to 10m L by using a rotary evaporator, shaking and extracting for 4 times by using water saturated n-butanol, 40m L each time, combining n-butanol phases, fully washing for 2 times by using ammonia liquid, 40m L each time, discarding ammonia liquid, evaporating the n-butanol liquid to dryness, adding 5m L of water to the residue to dissolve, passing through a D101 type macroporous adsorption resin column (inner diameter is 1.5cm, column height is 12cm), eluting by using 50m L of water, discarding water, eluting with 40% of ethanol solution, discarding L m of water solution, eluting, discarding 3580% of the aspergillus niger eluting solid fermentation residue, eluting, collecting an ethanol eluent, eluting a sample, eluting with 3580, and detecting and eluting with 3680% of ethanol, and collecting an aspergillus niger eluent (3680).
2.2 extracting astragaloside IV from Aspergillus niger ACCC30784 radix astragali residue solid fermentation product
The only difference from step 2.1 is that the aspergillus niger ACCC30583 radix astragali residue solid fermentation product of step 2.1 is replaced by the aspergillus niger ACCC30784 radix astragali residue solid fermentation product.
2.3 extracting astragaloside IV from Aspergillus niger ACCC 30177 radix astragali residue solid fermentation product
The only difference from step 2.1 is that the aspergillus niger ACCC30583 radix astragali residue solid fermentation product of step 2.1 is replaced by the aspergillus niger ACCC 30177 radix astragali residue solid fermentation product.
3. Extracting astragaloside from radix astragali residue
And simultaneously extracting astragaloside from astragalus dregs according to the method in the step 2.1 as a control. The difference from the step 2.1 is only that the aspergillus niger ACCC30583 radix astragali residue solid fermentation product in the step 2.1 is replaced by radix astragali residue.
4. Detection of astragaloside by HP L C
Astragaloside IV (standard substance, batch No. 110781-.
HP L C chromatographic conditions are that an Agilent 1100 liquid chromatographic workstation is adopted, a chromatographic column is ZORBAX SB-C18250 × 4.6.6 mm 5 mu m C18880975-902, a mobile phase comprises acetonitrile and water (volume ratio) 30:70, a flow rate is 1m L/min, a detection wavelength is 210nm, a column temperature is 30 ℃, and a sample feeding amount is 10 mu L.
The result shows that the astragalus residue is not fermented, and 0.131 +/-0.004 mg of astragaloside can be obtained per gram of astragalus residue on the basis of dry weight; fermenting radix astragali residue with Aspergillus niger ACCC30583 to obtain 0.713 + -0.003 mg astragaloside IV per gram radix astragali residue by fermenting with Aspergillus niger ACCC 30583; aspergillus niger ACCC30784 fermenting radix astragali residue to obtain 0.354 + -0.004 mg of astragaloside IV per gram of radix astragali residue by fermenting with Aspergillus niger ACCC 30784; aspergillus niger ACCC 30177 fermenting radix astragali residue to obtain 0.632 + -0.002 mg astragaloside IV per gram radix astragali residue by Aspergillus niger ACCC 30177 fermentation. Fermenting the astragalus residue with Aspergillus niger ACCC30583, and extracting to obtain astragaloside IV with yield 5.4 times of that of unfermented astragalus residue; the yield of the astragaloside extracted by the same extraction method after the astragalus dregs are fermented by aspergillus niger ACCC30784 is 2.7 times that of the astragalus dregs which are not fermented; the yield of astragaloside extracted by the same extraction method after the astragalus residue is fermented by the aspergillus niger ACCC 30177 is 4.8 times of that of the astragalus residue which is not fermented. The method is characterized in that the yield of the astragaloside is improved after the astragalus dregs are fermented by aspergillus niger, the same astragalus dregs are fermented by different aspergillus niger strains under the same condition to generate aspergillus niger astragaloside solid fermentation products, the mass of the astragaloside extracted by the same extraction method is different, and the mass of the astragaloside extracted after the astragalus dregs per gram are fermented by aspergillus niger ACCC30583 is 1.13 times of that of aspergillus niger ACCC 30177 and 2.01 times of that of aspergillus niger ACCC 30784.
Example 2 optimization of conditions for Aspergillus niger ACCC30583 fermentation of Astragalus membranaceus residue
1. Influence of nitrogen source species on astragaloside yield
The nitrogen source variety used for optimizing the astragalus residue solid fermentation medium is as follows: 10.0g (dry weight basis) of radix astragali residue with diameter of 0.8-1.2cm, 1.2g of nitrogen source, KH2PO41.2g，MgSO4·7H2Adjusting the water content of the materials to 50 percent and the pH value to 7.0, and sterilizing the materials for 20 minutes at the temperature of 121 ℃ to obtain the nitrogen source type optimized astragalus residue solid fermentation culture medium. Wherein the nitrogen source is (NH)4)2SO4、KNO3Urea, beef extract, yeast powder or peptone.
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC30583 on PDA plate, inoculating loop to pick Aspergillus niger ACCC30583 spore, diluting with sterile water to 1 × 108Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ for 24 hours at 160rpm to obtain a seed solution, then inoculating the seed solution into a 500ml conical flask filled with 50g of nitrogen source type optimized astragalus residue solid fermentation medium at the inoculation amount of 1% (V/V), culturing at 28 ℃ for 5 days, collecting all substances in a culture container to obtain aspergillus niger ACCC30583 astragalus residue solid fermentation product, and repeatedly arranging 10 conical flasks at each time.
The method for extracting the astragaloside from the aspergillus niger ACCC30583 radix astragali residue solid fermentation product is carried out according to the method of step 2 in the example 1, and the astragaloside is detected according to the method HP L C in the step 4 in the example 1. the result shows that the aspergillus niger ACCC30583 can produce the highest astragaloside yield of the fermented radix astragali residue in the radix astragali residue solid fermentation culture medium optimized by the nitrogen source type taking urea as the nitrogen source, and each gram of radix astragali residue can produce 0.673mg of the astragaloside (table 1) on the basis of dry weight.
TABLE 1 influence of nitrogen source species on Astragaloside IV production
| Serial number | Nitrogen source of astragalus residue solid fermentation medium | Yield of Astragaloside IV (mg/g) |
| 1 | KNO3 | 0.541±0.001 |
| 2 | Urea | 0.673±0.002 |
| 3 | Ammonium sulfate | 0.492±0.003 |
| 4 | Beef extract | 0.352±0.006 |
| 5 | Peptone | 0.304±0.002 |
| 6 | Yeast powder | 0.503±0.001 |
2. Influence of Urea content on Astragaloside IV yield
The used urea content-optimized astragalus residue solid fermentation medium is as follows: 10.0g (dry weight basis) of radix astragali dregs with diameter of 0.8-1.2cm, urea KH2PO41.2g，MgSO4·7H2Adjusting the water content of the materials to 50 percent and the pH value to 7.0, and sterilizing the materials for 20 minutes at 121 ℃ to obtain the astragalus residue solid fermentation culture medium with optimized urea content. WhereinThe mass of the urea is 0.01g (being 0.1 percent of the mass of the astragalus mongholicus dregs), 0.02g (being 0.2 percent of the mass of the astragalus mongholicus dregs), 0.03g (being 0.3 percent of the mass of the astragalus mongholicus dregs), 0.04g (being 0.4 percent of the mass of the astragalus mongholicus dregs), 0.05g (being 0.5 percent of the mass of the astragalus mongholicus dregs) or 0.06g (being 0.6 percent of the mass of the astragalus mongholicus dregs). each time, 10 erlenmeyer flasks are repeatedly arranged, the experiment is repeated for 3 times, and the method for repeating each time comprises the steps of inoculating aspergillus niger ACCC30583 to a PDA flat plate, inoculating and picking out aspergillus niger ACCC 83 spores, diluting the aspergillus niger8Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ and 160rpm for 24 hours to obtain a seed solution, inoculating the seed solution into a 500ml conical flask containing 50g of urea content-optimized astragalus residue solid fermentation medium at the inoculation amount of 1% (V/V) for culturing at 28 ℃ for 5 days, and collecting all substances in a culture container to obtain the aspergillus niger ACCC30583 astragalus residue solid fermentation product.
The method for extracting the astragaloside from the aspergillus niger ACCC30583 radix astragali residue solid fermentation product is carried out according to the method in the step 2 of the example 1, and the astragaloside is detected according to the method HP L C in the step 4 of the example 1. the result shows that the yield of the astragaloside in the fermented radix astragali residue in the radix astragali residue solid fermentation culture medium is higher when the amount of urea is 0.3-0.6% of the mass of the radix astragali residue, and the astragaloside can be generated in an amount of 0.668-0.684mg per gram of the radix astragali residue (Table 2).
TABLE 2 influence of Urea content on Astragaloside IV production
| Serial number | The mass of urea is the mass percent of astragalus root dregs | Yield of Astragaloside IV (mg/g) |
| 1 | 0.1 | 0.341±0.01 |
| 2 | 0.2 | 0.523±0.01 |
| 3 | 0.3 | 0.674±0.02 |
| 4 | 0.4 | 0.668±0.03 |
| 5 | 0.5 | 0.674±0.02 |
| 6 | 0.6 | 0.684±0.05 |
3. Influence of fermentation time on Astragaloside IV yield
The used astragalus residue solid fermentation culture medium comprises the following components: radix astragali residue with diameter of 0.8-1.2cm 10.0g (dry basis), (NH)4)2SO41.2g，KH2PO41.2g，MgSO4·7H2O0.6 g, adjusting the water content of the materials to 50 percent and the pH value to 7.0, and sterilizing at 121 ℃ for 20 minutes to obtain the astragalus residue solid fermentation culture medium.
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC30583 on PDA plate, inoculating loop to pick Aspergillus niger ACCC30583 spore, diluting with sterile water to 1 × 108Inoculating PD into cfu/m L suspension at 1% (V/V) inoculum size, culturing at 28 deg.C and 160rpm for 24 hr to obtain seed solution, inoculating the seed solution at 1% (V/V) inoculum size into optimized yellow containing 50g ureaCulturing the astragalus residue solid fermentation culture medium in a 500ml conical flask at 28 ℃ for 3 days, 4 days, 5 days, 6 days, 7 days or 8 days respectively, and collecting all substances in a culture container to obtain the aspergillus niger ACCC30583 astragalus residue solid fermentation product. Each time, 10 erlenmeyer flasks were set repeatedly.
Astragaloside IV is extracted from the aspergillus niger ACCC30583 astragalus mongholicus residue solid fermentation product according to the method of the step 2 in the example 1, and the astragaside IV is detected according to the method HP L C in the step 4 in the example 1. the result shows that the yield of the astragaside IV of the aspergillus niger ACCC30583 fermented astragalus mongholicus residue in the astragalus mongholicus residue solid fermentation culture medium for 5-8 days is higher, and each gram of the astragalus mongholicus residue can generate 0.671-0.682mg of the astragaside IV (table 3) on the basis of dry weight.
TABLE 3 influence of fermentation time on Astragaloside IV production
| Serial number | Fermentation time (Tian) | Yield of Astragaloside IV (mg/g) |
| 1 | 3 | 0.152±0.001 |
| 2 | 4 | 0.443±0.002 |
| 3 | 5 | 0.682±0.001 |
| 4 | 6 | 0.671±0.004 |
| 5 | 7 | 0.682±0.004 |
| 6 | 8 | 0.674±0.002 |
4. Influence of radix astragali residue granularity on astragaloside yield
The solid fermentation medium for optimizing the granularity of the astragalus residue is as follows: 10.0g (dry weight basis) of astragalus residue, (NH)4)2SO41.2g，KH2PO41.2g，MgSO4·7H2Adjusting the water content of the materials to 50 percent and the pH value to 7.0, and sterilizing the materials for 20 minutes at 121 ℃ to obtain the astragalus mongholicus decoction dreg granularity optimized solid fermentation culture medium. Wherein the granularity of the astragalus residue is 0.8-1.2cm, 2mm to 4mm or less than 2 mm.
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC30583 on PDA plate, inoculating loop to pick Aspergillus niger ACCC30583 spore, diluting with sterile water to 1 × 108Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ for 24 hours at 160rpm to obtain a seed solution, then inoculating the seed solution into 500ml conical flasks filled with 50g of astragalus residue granularity optimization solid fermentation medium at the inoculation amount of 1% (V/V) to respectively culture for 5 days at 28 ℃, collecting all substances in the culture containers to obtain aspergillus niger ACCC30583 astragalus residue solid fermentation product, and repeatedly arranging 10 conical flasks at each time.
The method for extracting the astragaloside from the aspergillus niger ACCC30583 radix astragali residue solid fermentation product according to the step 2 in the example 1 is adopted, and the astragaloside is detected according to the method HP L C in the step 4 in the example 1, the result shows that the yield of the astragaloside of the aspergillus niger ACCC30583 in the radix astragali residue granularity optimized solid fermentation culture medium with the diameter of 0.8-1.2cm and the diameter of 2 mm-4 mm of the radix astragali residue is higher, and 0.624-0.673mg of the astragaloside can be generated per gram of the radix astragali residue on the basis of dry weight (Table 4).
TABLE 4 influence of the granularity of the astragalus residue on the yield of astragaloside
| Serial number | Granularity of astragalus root dregs | Yield of Astragaloside IV (mg/g) |
| 1 | The diameter is 0.8-1.2cm | 0.624±0.001 |
| 2 | The diameter is 2mm to 4mm | 0.673±0.002 |
| 3 | The diameter is less than 2mm | 0.378±0.003 |
5. Influence of water content of fermentation medium on astragaloside yield
The water content optimization astragalus residue solid fermentation culture medium comprises the following components: radix astragali residue with diameter of 0.8-1.2cm 10.0g (dry basis), (NH)4)2SO41.2g，KH2PO41.2g，MgSO4·7H2Adjusting the water content of the materials to be 30%, 40%, 50%, 60%, 70% or 80%, adjusting the pH value of the materials to be 7.0, and sterilizing at 121 ℃ for 20 minutes to obtain the astragalus residue solid fermentation culture medium with the optimized water content. Each time10 erlenmeyer flasks were repeated.
The experiment was repeated 3 times, each time by inoculating Aspergillus niger ACCC30583 on PDA plate, inoculating loop to pick Aspergillus niger ACCC30583 spore, diluting with sterile water to 1 × 108Inoculating PD into a suspension of cfu/m L at an inoculation amount of 1% (V/V), culturing at 28 ℃ and 160rpm for 24 hours to obtain a seed solution, inoculating the seed solution into a 500ml conical flask containing 50g of astragalus residue solid fermentation medium with optimized water content at the inoculation amount of 1% (V/V) for culturing at 28 ℃ for 5 days, and collecting all substances in a culture container to obtain the aspergillus niger ACCC30583 astragalus residue solid fermentation product.
The method for extracting the astragaloside from the aspergillus niger ACCC30583 radix astragali residue solid fermentation product is carried out according to the method of step 2 in the example 1, and the astragaloside is detected according to the method HP L C in the step 4 in the example 1. the result shows that the aspergillus niger ACCC30583 can produce the highest astragaloside yield of the fermented radix astragali residue in the radix astragali residue solid fermentation culture medium with the water content of 60 percent and can produce 0.672mg of the astragaloside per gram of the radix astragali residue on the basis of dry weight (Table 5).
TABLE 5 influence of the Water content of the fermentation Medium on the yield of Astragaloside IV
| Serial number | Fermentation Medium Water content (%) | Yield of Astragaloside IV (mg/g) |
| 1 | 30 | 0.273±0.001 |
| 2 | 40 | 0.334±0.002 |
| 3 | 50 | 0.463±0.003 |
| 4 | 60 | 0.672±0.002 |
| 5 | 70 | 0.445±0.001 |
| 6 | 80 | 0.117±0.002 |
Claims (7)
1. The method for preparing the astragaloside comprises the steps of fermenting astragalus dregs with aspergillus niger to obtain an aspergillus niger fermented product of the astragalus dregs, and extracting the astragaloside from the aspergillus niger fermented product of the astragalus dregs, wherein the aspergillus niger is aspergillus niger ACCC 30583.
2. The method of claim 1, wherein: the method for fermenting the astragalus mongholicus dregs and the aspergillus niger fermented matter by using the aspergillus niger comprises the step of inoculating the aspergillus niger into a fermentation culture medium containing the astragalus mongholicus dregs, and culturing to obtain the astragalus mongholicus dregs and the aspergillus niger fermented matter.
3. The method according to claim 1 or 2, characterized in that: the fermentation medium containing the astragalus residue is prepared according to the following method: sterilizing the material with the water content of 50-60% to obtain the fermentation culture medium containing the astragalus mongholicus dregs; the material consists of the following raw materials in parts by mass: 10 parts by mass of astragalus mongholicus dregs, 0.03-0.12 part by mass of nitrogen source and 1.2 parts by mass of KH2PO4And 0.6 parts by mass of MgSO4·7H2O。
4. The method of claim 3, wherein: the nitrogen source is urea and KNO3、(NH4)2SO4Yeast powder, beef extract or/and peptone.
5. The method of claim 4, wherein: the culture is carried out at 28 ℃ for 5-8 days.
6. The method of claim 5, wherein: the 5-8 days are 5 days.
7. A kit for the preparation of astragaloside comprising aspergillus niger ACCC30583 and the fermentation medium containing astragalus mongholicus dregs in the method of any one of claims 2-4; the Aspergillus niger ACCC30583 and the fermentation culture medium containing radix astragali residue are packaged independently.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610313391.1A CN105861614B (en) | 2016-05-12 | 2016-05-12 | Application of aspergillus niger in preparation of astragaloside |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610313391.1A CN105861614B (en) | 2016-05-12 | 2016-05-12 | Application of aspergillus niger in preparation of astragaloside |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105861614A CN105861614A (en) | 2016-08-17 |
| CN105861614B true CN105861614B (en) | 2020-08-04 |
Family
ID=56632010
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610313391.1A Active CN105861614B (en) | 2016-05-12 | 2016-05-12 | Application of aspergillus niger in preparation of astragaloside |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105861614B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111920843A (en) * | 2019-05-13 | 2020-11-13 | 北京中医药大学 | Astragalus membranaceus-paecilomyces cicadae fermentation product, and preparation method and application thereof |
| CN111671790A (en) * | 2020-07-10 | 2020-09-18 | 武倩 | Chinese herbal medicine micro-molecule fermentation extraction process |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130802A (en) * | 2007-07-30 | 2008-02-27 | 金凤燮 | Method for preparing astragaloside iv by enzymic hydrolysis for astragalus saponin glycosyl |
| CN102559828A (en) * | 2010-12-30 | 2012-07-11 | 复旦大学 | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms |
-
2016
- 2016-05-12 CN CN201610313391.1A patent/CN105861614B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130802A (en) * | 2007-07-30 | 2008-02-27 | 金凤燮 | Method for preparing astragaloside iv by enzymic hydrolysis for astragalus saponin glycosyl |
| CN102559828A (en) * | 2010-12-30 | 2012-07-11 | 复旦大学 | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms |
Non-Patent Citations (1)
| Title |
|---|
| 固体发酵黄芪药渣工陈华艺条件优化;陈华;《安徽农业大学学位论文》;20160501;摘要、第18/40页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105861614A (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020101570A4 (en) | A Method for liquid fermentation of Cordyceps militaris strain | |
| CN106581083A (en) | Extraction method for ganoderma lucidum components, biological feed and preparation method thereof | |
| CN105647832A (en) | High-temperature-resistant garden waste decomposition bacterium FHM1 and application thereof | |
| CN105331668B (en) | A kind of method that bioconversion arasaponin prepares ginsenoside Rd | |
| CN105624200A (en) | Recycling method of hirsutella sinensis fermentation filtrate | |
| CN104996729B (en) | Comprehensive recycling technology of radix ophiopogonis decoction dregs | |
| CN115336503B (en) | Preparation method of substitute bacteria for gastrodia elata cultivation | |
| CN101492706A (en) | Method for improving cordyceps sinensis bacterium native volume of production with cordyceps militaris link liquid fermentation | |
| CN111617120B (en) | Preparation method of ginseng cordyceps sinensis fermentation extract | |
| CN106967775B (en) | Method for preparing diosgenin through biocatalysis and microbial inoculum used by same | |
| CN114208616B (en) | Organic planting method of gastrodia elata | |
| CN105861614B (en) | Application of aspergillus niger in preparation of astragaloside | |
| CN107509915B (en) | Method for reducing cyanide content of cassava residue | |
| CN101531968A (en) | Method for improving output of cordyceps militars fruiting body and cordycepin by adopting red yeast rice synergistic fermentation | |
| CN113480345A (en) | Bio-organic fertilizer based on municipal biogas residues and livestock and poultry manure and preparation method thereof | |
| Zhang et al. | Enhancement of diosgenin production in Dioscorea zingiberensis cell culture by oligosaccharide elicitor from its endophytic fungus Fusarium oxysporum Dzf17 | |
| CN102199548A (en) | Polygonum cuspidatum root endophytic strain G3 for efficiently transforming polydatin into resveratrol | |
| CN111996223A (en) | Method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing bidirectional fermentation technology | |
| CN102405764B (en) | The fermentation process of suolunbguan bacterin | |
| CN113502230B (en) | Hericium erinaceus strain and culture method thereof, hericium erinaceus-ginseng bidirectional solid fermentation method and method for efficiently converting rare ginsenoside | |
| CN112451554B (en) | Preparation method and application of pseudo-ginseng stem and leaf extract | |
| CN107513504A (en) | A kind of saccharomyces cerevisiae mutant bacterial and its mutagenesis and screening technique | |
| CN112552121A (en) | Cow dung bio-organic fertilizer and preparation method and application thereof | |
| CN106390937A (en) | Traditional Chinese medicine liquorice root residue water treatment agent and preparation method thereof | |
| CN111991479A (en) | Bidirectional solid fermentation method of radix isatidis and folium isatidis dregs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210713 Address after: 028011 west section of Qinghe street, Tongliao Economic and Technological Development Zone, Inner Mongolia Autonomous Region (400m west of the State Administration of Taxation) Patentee after: INNER MONGOLIA HANEN BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 100081 agricultural resources and agricultural regionalization Research Institute, Chinese Academy of Agricultural Sciences, 12 South Avenue, Beijing, Haidian District, Zhongguancun Patentee before: INSTITUTE OF AGRICULTURAL RESOURCES AND REGIONAL PLANNING, CHINESE ACADEMY OF AGRICULTURAL SCIENCES |