CN111471727B - Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method - Google Patents
Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method Download PDFInfo
- Publication number
- CN111471727B CN111471727B CN202010298525.3A CN202010298525A CN111471727B CN 111471727 B CN111471727 B CN 111471727B CN 202010298525 A CN202010298525 A CN 202010298525A CN 111471727 B CN111471727 B CN 111471727B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- rhizopus oryzae
- water
- atractylodes
- spore
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a method for extracting atractylodes macrocephala polysaccharide by using a microbial fermentation method, which comprises the following steps: inoculating Rhizopus oryzae (Rhizopus oryzae) GDMCC No:60990 spore in Atractylodis rhizoma powder containing sterile water, fermenting at 28-30 deg.C for 2-3d, ultrasonic extracting the fermented product with water, and concentrating to obtain concentrate; precipitating the concentrate with ethanol and vacuum drying to obtain crude polysaccharide of Atractylodis rhizoma. The fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide. Rhizopus oryzae CH5 grows moderately in Atractylodis rhizoma powder to produce polysaccharide hydrolase, which can hydrolyze insoluble polysaccharide structure tissue of Atractylodes macrocephala, promote dissociation of soluble polysaccharide in binding state, and is more easily dissolved out during hot water ultrasonic extraction. Compared with the conventional method without fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by more than 50%.
Description
(I) technical field
The invention belongs to the technical field of bioengineering, and particularly relates to application of a microbial fermentation technology in extraction of bighead atractylodes rhizome polysaccharide.
(II) background of the invention
The Chinese medicinal material Atractylodis Rhizoma (Atractylodes macrocephala Koizoma), also known as Rhizoma Atractylodis, rhizoma Atractylodis Macrocephalae, thunberg Atractylodis, herba seu radix Cirsii Japonici, and Rhizoma Dioscoreae essence, is dried rhizome of Atractylodes macrocephala Koidz of Compositae. The bighead atractylodes rhizome is a traditional Chinese medicinal material used as both medicine and food, is collected in 2015 pharmacopoeia of China, and is bitter, sweet and warm in nature and taste; spleen and stomach meridians entered; has the effects of strengthening spleen, replenishing qi, eliminating dampness, inducing diuresis, suppressing sweating and preventing miscarriage. It can be used for treating deficiency-type food deficiency of spleen, abdominal distention, diarrhea, phlegm retention, dizziness, palpitation, edema, spontaneous perspiration, and threatened abortion. The application of the white atractylodes rhizome is wide, and the white atractylodes rhizome is an important raw material of more than 40 Chinese patent medicine preparations besides the traditional Chinese medicine prescription; in addition, the tea drink, the wine soaking, the cooking delicacies and the like are usually used as substitute tea in folk, and are good functional health food materials.
The main chemical components of the atractylodes include atractylodes macrocephala polysaccharide, atractylenolide, atractylol, atractylone and the like, wherein the activity of the atractylodes macrocephala polysaccharide is concerned by people. Modern pharmacological studies show that the atractylodes macrocephala polysaccharide has the functions of resisting tumor, resisting oxidation, resisting hypertension, reducing blood sugar, regulating immunity and the like. One of the active ingredients of the Chinese patent medicine Yupingfeng oral liquid is the atractylodes macrocephala polysaccharide extract, which has the efficacy of strengthening the spleen and replenishing qi. As a plant functional polysaccharide, the atractylodes polysaccharide has wide application prospect in medical treatment and health care.
At present, the common extraction methods of the atractylodes macrocephala polysaccharide mainly comprise a hot water extraction method, an ultrasonic extraction method, an enzyme extraction method and the like. Different extraction processes have respective advantages and disadvantages, and the yield of the polysaccharide is greatly different. The hot water extraction method has long time consumption and low polysaccharide extraction rate; although the enzyme extraction method can obtain higher polysaccharide yield, the cost of the enzyme is high, which limits the wide application of the enzyme; the ultrasonic extraction method is characterized in that ultrasonic extraction steps are added on the basis of hot water extraction, and the extraction yield is relatively high, so that the ultrasonic extraction method has relative advantages. In order to further improve the extraction yield of the atractylodes macrocephala polysaccharide, the invention improves the process of the ultrasonic extraction method of the atractylodes macrocephala polysaccharide and adds a microbial fermentation pretreatment step, thereby improving the extraction yield of the atractylodes macrocephala polysaccharide.
Disclosure of the invention
The invention aims to apply the microbial fermentation technology to the ultrasonic extraction of the atractylodes macrocephalaon polysaccharide, thereby obviously improving the extraction yield of the atractylodes macrocephalaon polysaccharide.
The technical scheme adopted by the invention is as follows:
the invention provides a method for extracting atractylodes macrocephala polysaccharide by using a microbial fermentation method, which comprises the following steps: inoculating Rhizopus oryzae (Rhizopus oryzae) GDMCC No:60990 spore in Atractylodis rhizoma powder containing sterile water, fermenting at 28-30 deg.C for 2-3d, ultrasonic extracting the fermented product with water, and concentrating to obtain concentrate; precipitating the concentrate with ethanol and vacuum drying to obtain crude polysaccharide of Atractylodis rhizoma.
Further, the volume dosage of the sterile water is 2-3mL/g calculated by the weight of the rhizoma atractylodis macrocephalae powder; the inoculation amount of the Rhizopus oryzae CH5 spores is 2-5 multiplied by 10 in terms of the weight of the bighead atractylodes rhizome powder 7 Per gram; the white atractylodes rhizome powder is obtained by crushing white atractylodes rhizome and then sieving the crushed white atractylodes rhizome with a 60-mesh sieve.
Further, the rhizopus oryzae CH5 spores are added in the form of spore liquid, and the preparation method of the spore liquid comprises the following steps: inoculating Rhizopus oryzae CH5 preserved at low temperature into PDA plate culture medium, culturing at 28-30 deg.C for 2-3d, adding sterile water into culture dish, and suspending spore with inoculating ring under stirring to obtain spore solution; preferably, the spore liquid is transferred into a sterile test tube, and the spore concentration is adjusted to 2-5X 10 with sterile water 8 One per mL. The final concentration composition of the PDA plate culture medium (potato sucrose agar culture medium) is as follows: 200g/L of potatoes (filtering after boiling for 30min to leave juice), 20g/L of cane sugar, 20g/L of agar and a solvent of tap water, wherein the pH is natural (the actual measurement is 6.5).
Further, the preparation method of the concentrate comprises the following steps: adding deionized water into the fermented product, stirring, leaching in 80-90 deg.C constant temperature water bath for 3-5 hr, transferring into 80 deg.C ultrasonic cleaning machine, and ultrasonic extracting at 100-200W for 20-30min; filtering while hot after ultrasonic water extraction is finished, and concentrating the filtrate at 65 ℃ under reduced pressure to 1/4-1/5 of the original volume to obtain a concentrate; the volume addition of the deionized water is 20-30mL/g based on the weight of the bighead atractylodes rhizome powder before fermentation.
Further, the preparation method of the atractylodes macrocephala polysaccharide comprises the following steps: adding 3-4 times volume of 95% ethanol into the concentrate, standing at room temperature for precipitation for 10-16h, vacuum filtering with Buchner funnel, collecting polysaccharide precipitate, and vacuum drying at 60 deg.C to constant weight to obtain crude polysaccharide of Atractylodis rhizoma.
The Rhizopus oryzae (Rhizopus oryzae) CH5 strain is preserved in Guangdong province microorganism strain preservation center with the preservation number: GDMCC No. 60990, preservation date of 2020, 3 months and 27 days, address: building No. 59, building No. 5 of Jie No. 100 of the first Lianzhou city, guangdong province; zip code 510070.
Compared with the prior art, the invention has the following beneficial effects: the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide. Rhizopus oryzae CH5 grows moderately in Atractylodis rhizoma powder to generate polysaccharide hydrolase, which can hydrolyze insoluble polysaccharide structure tissue of Atractylodes macrocephala, promote dissociation of soluble polysaccharide in binding state, and dissolve out easily during hot water ultrasonic extraction. Compared with the conventional method without fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by more than 50%.
Description of the drawings
FIG. 1 colony morphology of Rhizopus oryzae CH 5.
FIG. 2 is a standard curve for measuring polysaccharide by phenol-sulfuric acid method.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala Koidz) in the examples of the present invention is dried rhizome of Atractylodes macrocephala Koidz (Atractylodes macrocephala Koidz) belonging to Compositae family.
The room temperature of the invention is 25-30 ℃.
Example 1 screening and identification of fermentation strains
1. Screening of Rhizopus oryzae CH5 Strain
(1) Adding 20g of Ganoderma sinensis powder crushed and sieved by a 40-mesh sieve into a 250-mL triangular flask, adding 30mL of sterile water for wetting, and culturing at constant temperature of 28 ℃ for 4 days. Diluting the enriched culture of the mold with sterile water to 1 × 10 6 Coating on PDA plate culture medium, culturing at 28 deg.C for 3d, selecting mold colony with different colors and morphologies, transferring to fresh PDA culture medium, culturing at 28 deg.C for 3d to obtain 7 pure culture strains (numbered CH1-CH 7), inoculating to fresh PDA culture medium, standing at 28 deg.CAnd (5) culturing at constant temperature for 3d. To fresh plate cultures of 7 strains, 5mL of sterile water was added, and spores were suspended by stirring with an inoculating loop. Transferring the spore liquid into a sterile test tube, and adjusting the spore concentration to 2 × 10 with sterile water 8 one/mL.
(2) And (3) carrying out dry heat sterilization on a 250-mL triangular flask at 160 ℃ for 2h, adding 10g of pulverized 60-mesh-screened rhizoma atractylodis macrocephalae powder, adding 20mL of sterile water, inoculating 1mL of the spore liquid of rhizopus oryzae CH5 prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, and culturing at 28 deg.C for 2d to obtain fermented Atractylodis rhizoma powder.
(3) And (3) adding 200mL of deionized water into all the bighead atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a constant-temperature water bath kettle at 80 ℃, leaching for 3h, and shaking by hand once at intervals of 15 min. Then, the triangular flask is transferred into an ultrasonic cleaning machine with the water temperature of 80 ℃, and 100W ultrasonic extraction is carried out for 20min. And after the ultrasonic water extraction is finished, carrying out suction filtration by using a hot Buchner funnel, and concentrating the filtrate at 65 ℃ under reduced pressure to 50mL to obtain a concentrated solution.
(4) And (4) adding 150mL of 95% ethanol into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 10h, then carrying out suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, carrying out vacuum drying at 60 ℃ for 10h to obtain crude polysaccharide of the bighead atractylodes rhizome, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
Under the same conditions, when the non-inoculated spore liquid is taken as a control without fermentation, the extraction yield of the polysaccharide after the atractylodes macrocephala powder is fermented by different strains is shown in table 1.
TABLE 1 extraction yield of polysaccharide after fermentation of different bacterial strains
As can be seen from the data in Table 1, the extraction yield of polysaccharide is improved to the highest extent and reaches 42.2% compared with the non-fermented control after the white atractylodes rhizome is fermented by the strain CH5, and the strain is selected as the fermentation strain for improving the extraction yield of white atractylodes rhizome polysaccharide.
The Ganoderma is fruiting body of Ganoderma sinensis (Ganoderma sinense).
The plate culture medium is a potato sucrose agar culture medium (PDA) and is prepared according to the following components and methods: cleaning potato, peeling, cutting into small pieces, weighing 200g, adding 1000mL of tap water, boiling for 30min, filtering with 4 layers of gauze, removing residues, adding the filtrate to 1000mL, adding 20g of sucrose and 20g of agar, naturally adjusting pH (actually measuring for 6.5), heating until the agar is dissolved, placing in a triangular flask, sterilizing at 121 ℃ by high-pressure steam for 15min, pouring into a sterile culture dish with the diameter of 9cm, and cooling for later use.
The content of the atractylodes macrocephala polysaccharide is determined by adopting a phenol-sulfuric acid method, and the specific method comprises the following steps: the crude polysaccharide of the largehead atractylodes rhizome is prepared into an aqueous solution with the concentration of 0.1 mg/mL. Sucking 2mL of sample solution into a 25mL colorimetric tube, adding 1mL of 5% phenol aqueous solution, shaking uniformly, quickly dropwise adding 2.5mL of concentrated sulfuric acid, shaking uniformly, standing at room temperature for 10min, heating in a boiling water bath for 15min, and cooling to room temperature by running water. Absorbance A was measured with reference to the same treatment with 1mL of distilled water as a blank 490 . Determination of A for samples of different glucose concentrations in the same way 490 Plotting the glucose concentration-A 490 And (3) obtaining a regression equation by using a standard curve (figure 2), and calculating and measuring the polysaccharide content in the crude polysaccharide sample of the bighead atractylodes rhizome by using the regression equation.
The extraction yield of the atractylodes macrocephala polysaccharide is calculated according to the following formula:
2. identification of Rhizopus oryzae CH5 Strain
Culturing Rhizopus oryzae CH5 strain on potato agar plate culture medium at 28 deg.C to obtain white fine villus at the early stage of colony, and gradually turning into grey brown after 1 day; the rhizoid is developed and brown; the cyst stalks grow from creeping hypha at the positions of the pseudorhizomes, are upright or slightly bent, and are clustered by 3-5 branches; the sporangium is spherical or elliptical, dark brown, and most conidia are elliptical, with diameter of 7.0-10.0 μm, and brown. A photograph of a colony of the Rhizopus oryzae CH5 strain cultured on a PDA plate medium for 3 days at 28 ℃ is shown in figure 1.
The strain CH5 is handed to a biological engineering (Shanghai) limited company for identification, the nucleotide sequence of rDNA ribose in vivo transcribed spacer (rDNA-ITS) is determined to be shown in SEQ ID NO.1, and the biological classification position of the strain CH5 is determined according to the bacterial colony characteristics of the strain CH5 and the nucleotide sequence comparison of rDNA-ITS (refer to Mycobank, http:// www.mycobank.org): kingdom fungoides (Fungi), phylum mucor (mucomyceta), class Mucorales (mucomycetes), order Mucorales (Mucorales), family Rhizopus (Rhizopus), genus Rhizopus (Rhizopus), rhizopus oryzae (Rhizopus oryzae). Namely, the strain is rhizopus oryzae (rhizopus oryzae) CH5, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number: GDMCC No. 60990, preservation date 2020, 3 months and 27 days.
Example 2 application of Rhizopus oryzae CH5 to extraction of polysaccharide from Atractylodes macrocephala
(1) Preparation of rhizopus oryzae CH5 spore liquid: inoculating Rhizopus oryzae CH5 plate colony stored at 4 deg.C in fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5mL sterile water into the plate culture, suspending spore with inoculating loop under stirring, transferring spore solution into sterile test tube, adjusting spore concentration to 3 × 10 with sterile water 8 One per mL. The PDA plate medium components and preparation method are the same as example 2.
(2) And (3) carrying out dry heat sterilization on a 500-mL triangular flask at 160 ℃ for 2h, adding 20g of pulverized 60-mesh-screened rhizoma atractylodis macrocephalae powder, adding 50mL of sterile water, inoculating 2mL of the CH5 spore solution prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, and culturing at 30 deg.C for 2d to obtain fermented Atractylodis rhizoma powder.
(3) Adding 400mL of deionized water into all the bighead atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a constant-temperature water bath kettle at 85 ℃, leaching for 4h, and shaking by hand once at intervals of 15 min. Then, the triangular flask is transferred into an ultrasonic cleaning machine with the water temperature of 80 ℃, and 100W ultrasonic extraction is carried out for 30min. And after the ultrasonic water extraction is finished, carrying out suction filtration by using a hot Buchner funnel, and concentrating the filtrate at 65 ℃ under reduced pressure to 100mL to obtain a concentrated solution.
(4) And (3) adding 300mL of 95% ethanol into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 12h, then carrying out suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, carrying out vacuum drying at 60 ℃ for 10h to obtain crude rhizoma atractylodis macrocephalae polysaccharide, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 4.23g of crude polysaccharide of the largehead atractylodes rhizome is obtained, the content of the polysaccharide is 80.5 percent, and 3.41g of polysaccharide of the largehead atractylodes rhizome is obtained, the extraction yield is 17.1 percent, and the product is off-white powder.
If the fermentation process of step (2) in this example is not performed, and other steps are performed according to the method in this example, 20g of rhizoma atractylodis macrocephalae powder can be extracted to obtain 2.81g of rhizoma atractylodis macrocephalae crude polysaccharide, the content of the polysaccharide is 82.2%, and then 2.31g of rhizoma atractylodis macrocephalae polysaccharide is obtained, and the extraction yield is 11.6%. Therefore, in the embodiment, the fermentation pretreatment of rhizopus oryzae CH5 is added before the ultrasonic water treatment of the atractylodes macrocephala polysaccharide is performed, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by 47.4%.
Example 3 application of rhizopus oryzae CH5 to extraction of atractylodes macrocephala polysaccharides:
(1) Preparing rhizopus oryzae CH5 spore liquid: inoculating Rhizopus oryzae CH5 plate colony preserved at 4 deg.C in fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5mL sterile water into the plate culture, stirring with inoculating loop to suspend spore, transferring spore solution into sterile test tube, adjusting spore concentration to 4 × 10 with sterile water 8 One per mL. The components and preparation method of the PDA plate culture medium are the same as those in example 1.
(2) And (2) performing dry heat sterilization on the mixture for 2 hours in a 2-L triangular flask at 160 ℃, adding 50g of pulverized 60-mesh-screened rhizoma atractylodis macrocephalae powder, adding 150mL of sterile water, inoculating 5mL of the rhizopus oryzae CH5 spore solution prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, and culturing at 30 deg.C for 3d to obtain fermented Atractylodis rhizoma powder.
(3) Adding 1250mL of deionized water into all the bighead atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a 90-DEG C constant-temperature water bath kettle, leaching for 5 hours, and shaking by hand once at intervals of 15 min. Then, the triangular flask is transferred into an ultrasonic cleaning machine with the water temperature of 80 ℃, and the ultrasonic extraction is carried out for 30min at 200W. And after the ultrasonic water extraction is finished, carrying out suction filtration by using a hot Buchner funnel, and concentrating the filtrate at 65 ℃ under reduced pressure to 250mL to obtain a concentrated solution.
(4) And (4) adding 1000mL of 95% ethanol into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 14h, then performing suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, performing vacuum drying at 60 ℃ for 10h to obtain crude rhizoma atractylodis macrocephalae polysaccharide, and determining the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 11.5g of crude polysaccharide of the largehead atractylodes rhizome is obtained, the content of the polysaccharide is 80.8 percent, 9.29g of polysaccharide of the largehead atractylodes rhizome is obtained, the extraction yield is 18.6 percent, and the product is off-white powder.
If the fermentation process of step (2) in this example is not performed, 50g of Atractylodis rhizoma powder can be extracted to obtain 7.48g of crude polysaccharide of Atractylodis rhizoma with polysaccharide content of 81.1% according to the method in this example, to obtain 6.07g of polysaccharide of Atractylodis rhizoma with extraction yield of 12.1%. Therefore, in the embodiment, the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by 53.0%.
Example 4 application of Rhizopus oryzae CH5 to extraction of polysaccharide from Atractylodes macrocephala
(1) Preparation of rhizopus oryzae CH5 spore liquid: inoculating Rhizopus oryzae CH5 plate colony at 4 deg.C in fresh PDA plate culture medium, culturing at 28 deg.C for 3 days, adding 5mL sterile water into the plate culture, suspending spore with inoculating loop under stirring, transferring spore solution into sterile test tube, adjusting spore concentration to 5 × 10 with sterile water 8 One per mL. The components and preparation method of the PDA plate culture medium are the same as those of example 2.
(2) And (3) carrying out dry heat sterilization on a 5-L triangular flask at 160 ℃ for 2h, adding 100g of pulverized 60-mesh-screened rhizoma atractylodis macrocephalae powder, adding 300mL of sterile water, inoculating 10mL of the CH5 spore solution prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, and culturing at 30 deg.C for 3d to obtain fermented Atractylodis rhizoma powder.
(3) Adding 3000mL of deionized water into all the bighead atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a 90 ℃ constant-temperature water bath kettle, leaching for 5h, and shaking by hand once at intervals of 15 min. Then, the triangular flask is transferred into an ultrasonic cleaning machine with the water temperature of 80 ℃, and the ultrasonic extraction is carried out for 30min at 200W. And after the ultrasonic water extraction is finished, carrying out suction filtration by using a hot Buchner funnel, and concentrating the filtrate at 65 ℃ under reduced pressure to 600mL to obtain a concentrated solution.
(4) And (3) adding 2400mL of 95% ethanol into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 16h, performing suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, performing vacuum drying at 60 ℃ for 10h to obtain crude rhizoma atractylodis macrocephalae polysaccharide, and determining the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 22.2g of crude polysaccharide of the largehead atractylodes rhizome is obtained, the content of the polysaccharide is 80.6 percent, 17.9g of polysaccharide of the largehead atractylodes rhizome is obtained, the extraction yield is 17.9 percent, and the product is off-white powder.
If the fermentation process of step (2) in this example is not performed, and other steps are performed according to the method in this example, 100g of rhizoma atractylodis macrocephalae powder can be extracted to obtain 14.4g of rhizoma atractylodis macrocephalae crude polysaccharide with the polysaccharide content of 81.7%, namely 11.8g of rhizoma atractylodis macrocephalae polysaccharide, and the extraction yield is 11.8%. Therefore, in the embodiment, the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by 51.7%.
Sequence listing
<110> Zhejiang Tree college (Zhejiang Tree university)
<120> a method for extracting atractylodes macrocephalaon polysaccharide by using microbial fermentation method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 598
<212> DNA
<213> Rhizopus oryzae (Rhizopus oryzae)
<400> 1
cggaaggatc attaattatg ttaaagcgcc ttaccttagg gtttcctctg gggtaagtga 60
ttgcttctac actgtgaaaa tttggctgag agactcagac tggtcatggg tagacctatc 120
tggggtttga tcgatgccac tcctggtttc aggagcaccc ttcataataa acctagaaat 180
tcagtattat aaagtttaat aaaaaacaac ttttaacaat ggatctcttg gttctcgcat 240
cgatgaagaa cgtagcaaag tgcgataact agtgtgaatt gcatattcag tgaatcatcg 300
agtctttgaa cgcagcttgc actctatggt ttttctatag agtacgcctg cttcagtatc 360
atcacaaacc cacacataac atttgtttat gtggtaatgg gtcgcatcgc tgttttatta 420
cagtgagcac ctaaaatgtg tgtgattttc tgtctggctt gctaggcagg aatattacgc 480
tggtctcagg atctttttct ttggttcgcc caggaagtaa agtacaagag tataatccag 540
caactttcaa actatgatct gaagtcaggt gggattaccc gctgaactta agcatatc 598
Claims (6)
1. A method for extracting atractylodes macrocephalaon polysaccharide by using microbial fermentation method is characterized by comprising the following steps: rhizopus oryzae (A) and (B)Rhizopus oryzae) Inoculating GDMCC No. 60990 spore in Atractylodis rhizoma powder containing sterile water, fermenting at 28-30 deg.C for 2-3d, ultrasonic extracting the fermented product with water, and concentrating to obtain concentrate; precipitating the concentrate with ethanol and vacuum drying to obtain crude polysaccharide of Atractylodis rhizoma.
2. The method according to claim 1, wherein the sterile water is used in a volume of 2-3mL/g based on the weight of the powder of atractylodes macrocephala koidz.
3. The method of claim 1, wherein the amount of inoculated rhizopus oryzae GDMCC No 60990 spore is 2-5 x 10 by weight of the powder of Atractylodes macrocephala koidz 7 Per gram.
4. The method of claim 1, wherein the spores of rhizopus oryzae GDMCC No 60990 are added as a spore liquid prepared by: inoculating Rhizopus oryzae GDMCC No. 60990 preserved at low temperature to PDA plate culture medium, culturing at 28-30 deg.C for 2-3d, adding sterile water, and stirring with inoculating loop to suspend spore to obtain spore solution; the final concentration composition of the PDA plate culture medium is as follows: 200g/L of potato, 20g/L of cane sugar, 20g/L of agar and a natural pH value, wherein the solvent is tap water.
5. The method of claim 1, wherein the concentrate is prepared by: adding deionized water into the fermented product, stirring, leaching in 80-90 deg.C constant temperature water bath for 3-5 hr, transferring into ultrasonic cleaning machine with water temperature of 80 deg.C, and performing 100-200W ultrasonic extraction for 20-30min; after ultrasonic water extraction is finished, filtering while hot, and concentrating the filtrate at 65 ℃ under reduced pressure to 1/4-1/5 of the original volume to obtain a concentrate; the volume addition amount of the deionized water is 20-30mL/g based on the weight of the bighead atractylodes rhizome powder before fermentation.
6. The method according to claim 1, wherein the preparation method of the atractylodes macrocephalaon polysaccharide comprises the following steps: adding 3-4 times volume of 95% ethanol into the concentrate, standing at room temperature for precipitation for 10-16h, vacuum filtering with Buchner funnel, collecting polysaccharide precipitate, and vacuum drying at 60 deg.C to constant weight to obtain Atractylodis rhizoma crude polysaccharide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010298525.3A CN111471727B (en) | 2020-04-16 | 2020-04-16 | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010298525.3A CN111471727B (en) | 2020-04-16 | 2020-04-16 | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111471727A CN111471727A (en) | 2020-07-31 |
| CN111471727B true CN111471727B (en) | 2023-01-31 |
Family
ID=71754136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010298525.3A Active CN111471727B (en) | 2020-04-16 | 2020-04-16 | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111471727B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394258B (en) * | 2020-04-02 | 2022-03-18 | 浙江工业大学 | Rhizopus stolonifer FL-3 and application thereof in extracting pachyman |
| CN113185618B (en) * | 2021-04-28 | 2022-06-07 | 南京中医药大学 | Anti-alcoholic liver injury rhizoma atractylodis macrocephalae polysaccharide and preparation method and application thereof |
| CN113564212B (en) * | 2021-07-26 | 2024-01-16 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by utilizing microbial fermentation method |
| CN114452357A (en) * | 2022-01-17 | 2022-05-10 | 广西健铧健康产业有限公司 | Liupao tea-containing preparation with blood fat and blood sugar reducing effects and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102366024A (en) * | 2011-09-16 | 2012-03-07 | 汪松林 | Production method of pig feed additive containing traditional Chinese medicine and active probiotics |
| CN113559022A (en) * | 2021-07-30 | 2021-10-29 | 徐晓英 | Fermentation method for fermenting traditional Chinese medicine 'Xinqibai' by using probiotics |
| CN111334440B (en) * | 2020-04-16 | 2022-07-08 | 浙江树人学院（浙江树人大学） | Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide |
-
2020
- 2020-04-16 CN CN202010298525.3A patent/CN111471727B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102366024A (en) * | 2011-09-16 | 2012-03-07 | 汪松林 | Production method of pig feed additive containing traditional Chinese medicine and active probiotics |
| CN111334440B (en) * | 2020-04-16 | 2022-07-08 | 浙江树人学院（浙江树人大学） | Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide |
| CN113559022A (en) * | 2021-07-30 | 2021-10-29 | 徐晓英 | Fermentation method for fermenting traditional Chinese medicine 'Xinqibai' by using probiotics |
Non-Patent Citations (3)
| Title |
|---|
| Polysaccharide of Atractylodes macrocephala Koidz (PAMK) Relieves Immunosuppression in Cyclophosphamide-Treated Geese by Maintaining a Humoral and Cellular Immune Balance;Wanyan Li et al.;《Molecules》;20180417;文献号:932 * |
| 平菇粗多糖的提取及活性研究;朱振元 等;《食品安全质量检测学报》;20160225;第656-661页 * |
| 白术多糖提取方法的筛选及其对樱桃谷鸭饲用价值的研究;王洁;《中国优秀硕士学位论文全文数据库 农业科技辑》;20080215;第D050-150页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111471727A (en) | 2020-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111471727B (en) | Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method | |
| CN103907987B (en) | A kind of multi-cultur es mixing is continuously fermented and is prepared the method for Haw Apple Vinegar Drink | |
| CN112481134B (en) | Method for extracting mulberry leaf polysaccharide by using microbial fermentation method | |
| CN113564212B (en) | Method for extracting eucommia ulmoides leaf polysaccharide by utilizing microbial fermentation method | |
| CN111334440B (en) | Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide | |
| CN109536560A (en) | A method of improving rare saponin content in ginseng water extract | |
| CN101463326B (en) | Production method of Guangdong Cordyceps sinensismycelium | |
| CN110507681A (en) | A kind of technique of higher value application flower of Panax ginseng | |
| CN106119121B (en) | Selenium-rich cordyceps bacterium and its cultural method, selenium-enriched yellow wine and preparation method thereof | |
| CN105995330A (en) | High-cellulose functional red date fermented beverage and preparation method thereof | |
| CN118374357A (en) | Mucor racemosus SJ6-19 and application thereof in extraction of total flavonoids of sea buckthorn leaves | |
| CN116549507B (en) | Method for preparing radix puerariae extract by microbial fermentation method | |
| CN105535035B (en) | A kind of Inonotus obliquus fermented and cultured composition and preparation method thereof | |
| CN117137131A (en) | Polygonatum sibiricum and wolfberry enzyme powder and preparation method thereof | |
| CN114933973B (en) | Mucor lassitanum HZ-6-27 and application thereof in extraction of rhizoma polygonati polysaccharide | |
| CN111394258B (en) | Rhizopus stolonifer FL-3 and application thereof in extracting pachyman | |
| CN111607621A (en) | Yeast capable of producing rose fragrance and application of yeast in Lingwu jujube enzyme | |
| KR100643735B1 (en) | Fermented Wine made from Fruit of Araliaceous Shrubs, and Method for producing Thereof | |
| CN114606137A (en) | Aspergillus japonicus HY-8-25 and application thereof in rosemary essential oil extraction | |
| CN110607332B (en) | Culture medium for improving content of functional red yeast rice Monacolin K and fermentation method | |
| CN113564055B (en) | Felt Mao Qingmei DZ-9-67 and application thereof in extraction of total flavonoids of eucommia ulmoides leaves | |
| CN116115675B (en) | Method for extracting loquat leaf total flavonoids by microbial enzymolysis | |
| CN116041561B (en) | Method for extracting lotus leaf polysaccharide with assistance of microbial fermentation | |
| CN117467725B (en) | Fermentation production process of paecilomyces hepiali mycelium powder with high nucleoside content | |
| CN105567545A (en) | Golden cordyceps and lotus root thick vinegar |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 312028 No. 2016 Jiangxia Road, Yang Xun Qiao, Keqiao District, Shaoxing, Zhejiang. Patentee after: ZHEJIANG SHUREN University Address before: 312028 No. 2016 Jiangxia Road, Yang Xun Qiao, Keqiao District, Shaoxing, Zhejiang. Patentee before: ZHEJIANG SHUREN College (ZHEJIANG SHUREN University) |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230914 Address after: 509 Kangrui Times Square, Keyuan Business Building, 39 Huarong Road, Gaofeng Community, Dalang Street, Longhua District, Shenzhen, Guangdong Province, 518000 Patentee after: Shenzhen Litong Information Technology Co.,Ltd. Address before: 312028 No. 2016 Jiangxia Road, Yang Xun Qiao, Keqiao District, Shaoxing, Zhejiang. Patentee before: ZHEJIANG SHUREN University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230928 Address after: 5 / F, 277 Huqingping Road, Minhang District, Shanghai, 201105 Patentee after: Shanghai Zhiyu Biotechnology Co.,Ltd. Address before: 509 Kangrui Times Square, Keyuan Business Building, 39 Huarong Road, Gaofeng Community, Dalang Street, Longhua District, Shenzhen, Guangdong Province, 518000 Patentee before: Shenzhen Litong Information Technology Co.,Ltd. |
|
| TR01 | Transfer of patent right |