CN111471727B - Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method - Google Patents

Abstract

The invention discloses a method for extracting Atractylodes atractylodes polysaccharides by using a microbial fermentation method. The method comprises: inoculating spores of Rhizopus oryzae (Rhizopus oryzae) GDMCC No: 60990 into Atractylodes atractylodes powder containing sterile water, and inoculating them at 28-30°C After 2-3 days of fermentation, the fermented product was concentrated by ultrasonic water extraction to obtain a concentrate; the concentrate was subjected to alcohol precipitation and vacuum drying to obtain Atractylodes macrocephala crude polysaccharide. In advance of Atractylodes polysaccharide ultrasonic water, the fermentation pretreatment of Rhizopus oryzae CH5 was added. Rhizopus oryzae CH5 grows moderately in Atractylodes macrocephala powder, and produces some polysaccharide hydrolase, which can hydrolyze the insoluble polysaccharide structure of Atractylodes macrocephala, promote the dissociation of bound soluble polysaccharides, and dissolve more easily in hot water ultrasonic extraction. Compared with the conventional method without fermentation pretreatment, the extraction yield of Atractylodes macrocephala polysaccharide can be increased by more than 50%.

Description

A method for extracting Atractylodes macrocephala polysaccharides by microbial fermentation

(1) Technical field

The invention belongs to the technical field of bioengineering, and in particular relates to the application of microbial fermentation technology in the extraction of Atractylodes macrocephala polysaccharide.

(2) Background technology

Atractylodes macrocephalae Rhizoma (Atractylodes macrocephalae Rhizoma), also known as Atractylodes macrocephalae Rhizoma, is the dried rhizome of Atractylodes macrocephala Koidz in the family Compositae. Atractylodes are traditional Chinese medicinal materials for medicines and foods. They are collected in the 2015 Chinese Pharmacopoeia. Its taste, sweetness, warmth; returning spleen and stomach meridians; The effect of the fetus. Indications for spleen deficiency, lack of food, abdominal distension and diarrhea, phlegm retention, dizziness, palpitations, edema, spontaneous sweating, restless fetal movement. Atractylodes macrocephala has a wide range of uses. In addition to traditional Chinese medicine prescriptions, it is also an important raw material for more than 40 kinds of Chinese patent medicine preparations. In addition, folks usually use it to replace tea, brew wine, and cook delicacies. It is a good functional health food material.

The main chemical components of Atractylodes Rhizome include Atractylodes Rhizoma Atractylodes Polysaccharide, Atractylodes Lactone, Atractylodes Alcohol, Atractylodes Ketone, etc. Among them, the activity of Atractylodes Rhizome polysaccharide has attracted much attention. Modern pharmacological studies have shown that Atractylodes polysaccharides have anti-tumor, anti-oxidation, anti-hypertensive, hypoglycemic and immune-regulating activities. One of the effective components of the Chinese patent medicine Yupingfeng Oral Liquid is Atractylodes macrocephala polysaccharide extract, which has the effect of invigorating the spleen and replenishing qi. As a plant functional polysaccharide, Atractylodes macrocephala polysaccharide has broad application prospects in medical and health care.

At present, the common extraction methods of Atractylodes macrocephala polysaccharides mainly include hot water extraction, ultrasonic extraction and enzyme extraction. Different extraction processes have their own advantages and disadvantages, and the yield of polysaccharides also varies greatly. The hot water extraction method takes a long time and the polysaccharide extraction rate is low; although the enzyme extraction method can obtain a higher polysaccharide yield, the high cost of the enzyme limits its wide application; the ultrasonic extraction method is extracted in hot water. On the basis of this method, the ultrasonic extraction step is added, and the extraction yield is relatively high, so it has relative advantages. In order to further improve the extraction yield of the Atractylodes Rhizome polysaccharide, the invention improves the ultrasonic extraction process of the Atractylodes Rhizoma Atractylodes Polysaccharide, and adds a microbial fermentation pretreatment step, thereby improving the extraction yield of the Atractylodes Rhizoma Atractylodes Polysaccharide.

(3) Contents of the invention

The object of the present invention is to apply the microbial fermentation technology to the ultrasonic extraction of Atractylodes Rhizoma Polysaccharides, thereby significantly improving the extraction yield of Atractylodes Rhizoma Polysaccharides.

The technical scheme adopted in the present invention is:

The invention provides a method for extracting Atractylodes macrocephala polysaccharides by microbial fermentation. The method is as follows: inoculate Rhizopusoryzae (Rhizopusoryzae) GDMCC No: 60990 spores in Atractylodes macrocephala powder containing sterile water, and ferment at 28-30°C for 2 -3d, the fermented product was concentrated by ultrasonic water extraction to obtain a concentrate; the concentrate was subjected to alcohol precipitation and vacuum drying to obtain Atractylodes macrocephala crude polysaccharide.

Further, the volume dosage of the sterile water is 2-3mL/g based on the weight of the Atractylodes macrocephala powder; the inoculum amount of the Rhizopus oryzae CH5 spores is 2-5× ¹⁰⁷ /g based on the weight of the Atractylodes macrocephala powder; It is obtained by crushing Atractylodes macrocephala through 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 is: inoculate the low-temperature-preserved Rhizopus oryzae CH5 on PDA plate medium, and culture it at a constant temperature of 28-30°C for 2-3 days. , add sterile water to the petri dish, stir with an inoculation loop to suspend the spores, and obtain the spore liquid; preferably transfer the spore liquid to a sterile test tube, and use sterile water to adjust the spore concentration to 2–5× ¹⁰⁸ /mL . The final concentration of described PDA plate culture medium (potato sucrose agar medium) consists of: potato 200g/L (filtering juice after boiling for 30min), sucrose 20g/L, agar 20g/L, solvent is tap water, and pH is natural (actual measurement 6.5).

Further, the preparation method of the concentrate is: adding deionized water to the fermented product, stirring evenly, placing it in a constant temperature water bath at 80-90°C for 3-5 hours, and then transferring it to an ultrasonic cleaning machine with a water temperature of 80°C. -200W ultrasonic extraction for 20-30min; after the ultrasonic water extraction is completed, filter while it is hot, and concentrate the filtrate under reduced pressure at 65°C to 1/4-1/5 of the original volume to obtain a concentrate; the volume of deionized water added is The weight of Atractylodes macrocephala powder before fermentation is 20-30mL/g.

Further, the preparation method of the Atractylodes macrocephala polysaccharide is as follows: add 3-4 times the volume of 95% ethanol to the concentrate, let it stand for precipitation at room temperature for 10-16 hours, filter it with a Buchner funnel, collect the polysaccharide precipitate, and dry it in vacuum at 60°C To constant weight, Atractylodes macrocephala crude polysaccharide was obtained.

The Rhizopus oryzae CH5 strain described in the present invention is preserved in Guangdong Microbial Culture Collection Center, preservation number: GDMCC No: 60990, preservation date March 27, 2020, address: Xianlie Middle Road, Guangzhou City, Guangdong Province 5th Floor, Building 59, No. 100 Compound; Zip code 510070.

Compared with the prior art, the beneficial effect of the present invention is mainly reflected in that the fermentation pretreatment of Rhizopus oryzae CH5 is added in advance of the Atractylodes macrocephala polysaccharide ultrasonic water. Rhizopus oryzae CH5 grows moderately in Atractylodes rhizome powder, and produces some polysaccharide hydrolase, which can hydrolyze the insoluble polysaccharide structure of Atractylodes macrocephala, promote the dissociation of bound soluble polysaccharides, and dissolve more easily in hot water ultrasonic extraction. Compared with the conventional method without fermentation pretreatment, the extraction yield of Atractylodes macrocephala polysaccharide can be increased by more than 50%.

(4) Description of drawings

Fig. 1 Colony morphology of Rhizopus oryzae CH5.

Figure 2 The standard curve for the determination of polysaccharides by the phenol-sulfuric acid method.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Atractylodes macrocephalae Rhizoma described in the embodiment of the present invention is the dry rhizome of Atractylodes macrocephala Koidz.

The room temperature mentioned in the present invention refers to 25-30°C.

Screening and identification of embodiment 1 fermentation strain

1. Screening of Rhizopus oryzae CH5 strain

(1) Add 20g of Zizhi powder crushed through a 40-mesh sieve into a 250-mL Erlenmeyer flask, then add 30mL of sterile water to moisten it, and incubate at 28°C for 4 days. Dilute the mold-covered enriched culture 1× ¹⁰⁶ times with sterile water, spread it on the PDA plate medium, and culture it at 28°C for 3 days, pick mold colonies with different colors and shapes and transfer them to fresh PDA culture Cultured at a constant temperature of 28°C for 3 days to obtain 7 purely cultured strains (CH1-CH7), which were inoculated with fresh PDA medium and cultured at a constant temperature of 28°C for 3 days. Add 5 mL of sterile water to each of the fresh plate cultures of the 7 strains, and stir with an inoculating loop to suspend the spores. Transfer the spore liquid to a sterile test tube, and adjust the spore concentration to 2× ¹⁰⁸ /mL with sterile water.

(2) 250-mL triangular flask is sterilized by dry heat at 160°C for 2 hours, add 10 g of Atractylodes macrocephala powder that has been crushed through a 60-mesh sieve, add 20 mL of sterile water, and inoculate 1 mL of spores of Rhizopus oryzae CH5 prepared in step (1) liquid, stir well. The mouth of the triangular flask was tied with eight layers of gauze, and cultured at 28°C for 2 days to obtain fermented Atractylodes macrocephala powder.

(3) Add 200 mL of deionized water to all the Atractylodes macrocephala powder obtained in step (2), stir evenly, place in a constant temperature water bath at 80°C, extract for 3 hours, and shake by hand once every 15 minutes. Afterwards, the flask was transferred to an ultrasonic cleaning machine with a water temperature of 80°C, and 100W ultrasonic extraction was performed for 20 minutes. After the ultrasonic water extraction was completed, the Buchner funnel was suction-filtered while it was hot, and the filtrate was concentrated to 50 mL under reduced pressure at 65° C. to obtain a concentrated solution.

(4) Add 150mL of 95% ethanol to the entire concentrated solution obtained in step (3), and after standing for precipitation at room temperature for 10 hours, suction filter through a Buchner funnel, collect the polysaccharide precipitate, and vacuum-dry at 60°C for 10 hours to obtain the crude polysaccharide of Atractylodes macrocephala. The content of polysaccharides in crude polysaccharides was determined by the phenol-sulfuric acid method.

Under the same conditions, with no inoculation of spore liquid as the unfermented control, the extraction yields of polysaccharides of Atractylodes macrocephala powder fermented by different strains are shown in Table 1.

Table 1 The polysaccharide extraction yield of different bacterial strains after fermentation

As can be seen from the data in Table 1, after Atractylodes Rhizome is fermented by bacterial strain CH5, compared with the unfermented control, the extraction yield of polysaccharides is the highest, reaching 42.2%. rate of fermentation strains.

The Zizhi is the fruiting body of the fungus Ganoderma sinense.

The plate medium is potato sucrose agar medium (PDA), prepared according to the following composition and method: wash and peel the potatoes and cut them into small pieces, weigh 200g, add 1000mL of tap water, boil for 30min, filter through 4 layers of gauze to remove slag , the filtrate was made up to 1000mL, then 20g of sucrose and 20g of agar were added, the pH was natural (measured at 6.5), heated until the agar melted, placed in a triangular flask, sterilized by high-pressure steam at 121°C for 15min, and then poured into a sterile petri dish with a diameter of 9cm. Let cool and set aside.

The content of the Atractylodes Rhizome polysaccharide is determined by the phenol-sulfuric acid method, and the specific method is: the Atractylodes Rhizome crude polysaccharide is prepared into an aqueous solution with a concentration of 0.1 mg/mL. Draw 2mL of the sample solution into a 25mL colorimetric tube, add 1mL of 5% phenol aqueous solution, shake well, then quickly add 2.5mL concentrated sulfuric acid dropwise, shake well, place at room temperature for 10min, heat in a boiling water bath for 15min, and cool to room temperature under running water. The absorbance A ₄₉₀ was measured with 1 mL of distilled water as the blank control with the same treatment as a reference. Measure A ₄₉₀ of samples with different glucose concentrations in the same way, draw the glucose concentration-A ₄₉₀ standard curve (Figure 2), and obtain the regression equation, which is used to calculate and determine the polysaccharide content in Atractylodes macrocephala crude polysaccharide samples.

Described Atractylodes macrocephala polysaccharide extraction yield is calculated according to the following formula:

2. Identification of Rhizopus oryzae CH5 strain

Rhizopus oryzae CH5 strain was cultivated on potato agar plate medium at 28°C. The colony was white and finely fluffy at the beginning, and gradually turned grayish brown after 1 day; the rhizoids were developed and brown; The creeping hyphae grow, erect or slightly curved, clustered with 3–5 branches; the sporangia are spherical or elliptical, dark brown, and most conidia are elliptical, with a diameter of 7.0–10.0 μm and brown. See accompanying drawing 1 for the colony photo of Rhizopus oryzae CH5 strain cultured at 28 DEG C for 3 days on the PDA plate medium.

The bacterial strain CH5 was identified by Sangon Bioengineering (Shanghai) Co., Ltd., and the nucleotide sequence of its rDNA ribosome transcribed spacer (rDNA-ITS) was determined as shown in SEQ ID NO.1. According to the characteristics of the bacterial strain CH5 colonies and The rDNA-ITS nucleotide sequence comparison determined the biological taxonomic position of the strain CH5 (refer to Mycobank, http://www.mycobank.org): Fungi (Fungi), Mucoromycota (Mucoromycota) Mucorales ( Mucoromycetes), Mucorales, Rhizopodaceae, Rhizopus, Rhizopusoryzae. That is, the strain is Rhizopus oryzae (Rhizopusoryzae) CH5, which was preserved in the Guangdong Microbial Culture Collection Center, with a preservation number: GDMCC No: 60990, and a preservation date of March 27, 2020.

Embodiment 2 Rhizopus oryzae CH5 is applied to the extraction of Atractylodes macrocephala polysaccharide

(1) Preparation of Rhizopus oryzae CH5 spore liquid: Plate colonies of Rhizopus oryzae CH5 preserved at 4°C were inoculated on fresh PDA plate medium, cultured at a constant temperature of 28°C for 3 days, added 5 mL of sterile water to the plate culture, and used Agitate the inoculation loop to suspend the spores, transfer the spore liquid to a sterile test tube, and adjust the spore concentration to 3×10 ⁸ /mL with sterile water. Described PDA plate medium composition and preparation method are the same as embodiment 2.

(2) 500-mL triangular flask was sterilized by dry heat at 160°C for 2 hours, added 20 g of Atractylodes macrocephala powder crushed through a 60-mesh sieve, added 50 mL of sterile water, and then inoculated with 2 mL of CH5 spore liquid prepared in step (1), and stirred uniform. The mouth of the triangular flask was tied with eight layers of gauze, and cultured at 30°C for 2 days to obtain fermented Atractylodes macrocephala powder.

(3) Add 400 mL of deionized water to all the Atractylodes macrocephala powder obtained in step (2), stir evenly, place in a constant temperature water bath at 85°C, extract for 4 hours, and shake once every 15 minutes by hand. Afterwards, the flask was transferred to an ultrasonic cleaning machine with a water temperature of 80°C, and 100W ultrasonic extraction was performed for 30 minutes. After the ultrasonic water extraction was completed, the Buchner funnel was suction-filtered while it was hot, and the filtrate was concentrated to 100 mL under reduced pressure at 65° C. to obtain a concentrated solution.

(4) Add 300mL of 95% ethanol to the entire concentrated solution obtained in step (3), and after standing for precipitation at room temperature for 12 hours, suction filter through a Buchner funnel, collect the polysaccharide precipitate, and vacuum-dry at 60°C for 10 hours to obtain the crude polysaccharide of Atractylodes macrocephala. Determination of polysaccharide content in crude polysaccharide by phenol-sulfuric acid method.

According to the above steps, 4.23 g of Atractylodes Rhizoma Atractylodes Polysaccharides was obtained, the polysaccharide content was 80.5%, that is, 3.41 g of Atractylodes Rhizoma Polysaccharides was obtained, the extraction yield was 17.1%, and the product was off-white powder.

If the fermentation process of step (2) of this embodiment is not carried out, and other steps are carried out according to the method of this embodiment, 20 g of Atractylodes Rhizome powder can be extracted to obtain 2.81 g of Atractylodes Rhizoma Atractylodes Polysaccharide, and the polysaccharide content is 82.2%. The rate was 11.6%. It can be seen that in this embodiment, the fermentation pretreatment of Rhizopus oryzae CH5 is added in advance of the ultrasonic water of Atractylodes macrocephala polysaccharide, and the extraction yield of Atractylodes macrocephala polysaccharide can be increased by 47.4% compared with the conventional method without fermentation pretreatment.

Embodiment 3 Rhizopus oryzae CH5 is applied to the extraction of Atractylodes macrocephala polysaccharide:

(1) Preparation of Rhizopus oryzae CH5 spore liquid: Plate colonies of Rhizopus oryzae CH5 preserved at 4°C were inoculated on fresh PDA plate medium, cultured at a constant temperature of 28°C for 3 days, added 5 mL of sterile water to the plate culture, and used Agitate the inoculation loop to suspend the spores, transfer the spore liquid to a sterile test tube, and adjust the spore concentration to 4×10 ⁸ /mL with sterile water. Described PDA plate medium composition and preparation method are the same as embodiment 1.

(2) 2-L triangular flask was sterilized by dry heat at 160°C for 2 hours, added 50 g of Atractylodes macrocephala powder crushed through a 60-mesh sieve, added 150 mL of sterile water, and then inoculated with 5 mL of Rhizopus oryzae CH5 spore liquid prepared in step (1) , stir well. The mouth of the triangular flask was tied with eight layers of gauze, and cultured at 30°C for 3 days to obtain fermented Atractylodes macrocephala powder.

(3) Add 1250 mL of deionized water to all the Atractylodes macrocephala powder obtained in step (2), stir evenly, place in a 90°C constant temperature water bath, extract for 5 hours, and shake it by hand once every 15 minutes. Afterwards, the flask was transferred to an ultrasonic cleaning machine with a water temperature of 80°C, and 200W ultrasonic extraction was performed for 30 minutes. After the ultrasonic water extraction was completed, the Buchner funnel was suction-filtered while it was hot, and the filtrate was concentrated to 250 mL under reduced pressure at 65° C. to obtain a concentrated solution.

(4) Add 1000 mL of 95% ethanol to the entire concentrated solution obtained in step (3), and after standing for precipitation at room temperature for 14 hours, suction filter through a Buchner funnel, collect the polysaccharide precipitate, and vacuum-dry at 60° C. for 10 hours to obtain the crude polysaccharide of Atractylodes macrocephala. Determination of polysaccharide content in crude polysaccharide by phenol-sulfuric acid method.

According to the above steps, 11.5 g of Atractylodes Rhizoma Atractylodes Polysaccharides was obtained, the polysaccharide content was 80.8%, that is, 9.29 g of Atractylodes Rhizoma Atractylodes Polysaccharides was obtained, the extraction yield was 18.6%, and the product was off-white powder.

If the fermentation process of step (2) of this embodiment is not carried out, and other steps are carried out according to the method of this embodiment, 50 g of Atractylodes Rhizome powder can be extracted to obtain 7.48 g of Atractylodes Rhizoma Atractylodes Polysaccharide, and the polysaccharide content is 81.1%. The rate was 12.1%. It can be seen that in this embodiment, the fermentation pretreatment of Rhizopus oryzae CH5 is added in advance of the ultrasonic water of Atractylodes macrocephala polysaccharide, and the extraction yield of Atractylodes macrocephala polysaccharide can be increased by 53.0% compared with the conventional method without fermentation pretreatment.

Embodiment 4 Rhizopus oryzae CH5 is applied to the extraction of Atractylodes macrocephala polysaccharide

(1) Preparation of Rhizopus oryzae CH5 spore liquid: Inoculate plate colonies of Rhizopus oryzae CH5 plate cultured at 4°C on fresh PDA plate medium, cultivate at 28°C for 3 days, add 5 mL of sterile water to the plate culture, Stir with an inoculation loop to suspend the spores, transfer the spore liquid to a sterile test tube, and adjust the spore concentration to 5×10 ⁸ /mL with sterile water. Described PDA plate medium composition and preparation method are the same as embodiment 2.

(2) 5-L triangular flask was sterilized by dry heat at 160°C for 2 hours, added 100g of Atractylodes macrocephala powder crushed through a 60-mesh sieve, added 300mL of sterile water, and then inoculated with 10mL of CH5 spore liquid prepared in step (1), and stirred uniform. The mouth of the triangular flask was tied with eight layers of gauze, and cultured at 30°C for 3 days to obtain fermented Atractylodes macrocephala powder.

(3) Add 3000 mL of deionized water to all the Atractylodes macrocephala powder obtained in step (2), stir evenly, place in a 90°C constant temperature water bath, extract for 5 hours, and shake it by hand once every 15 minutes. Afterwards, the flask was transferred to an ultrasonic cleaning machine with a water temperature of 80°C, and 200W ultrasonic extraction was performed for 30 minutes. After the ultrasonic water extraction was completed, the Buchner funnel was suction-filtered while it was hot, and the filtrate was concentrated to 600 mL under reduced pressure at 65° C. to obtain a concentrated solution.

(4) Add 2400 mL of 95% ethanol to the entire concentrated solution obtained in step (3), and after standing for precipitation at room temperature for 16 hours, suction filter through a Buchner funnel, collect the polysaccharide precipitate, and vacuum-dry at 60° C. for 10 hours to obtain the crude polysaccharide of Rhizoma Atractylodes Rhizome. Determination of polysaccharide content in crude polysaccharide by phenol-sulfuric acid method.

According to the above steps, 22.2 g of Atractylodes Rhizoma Atractylodes Polysaccharides was obtained, the polysaccharide content was 80.6%, that is, 17.9 g of Atractylodes Rhizoma Polysaccharides was obtained, the extraction yield was 17.9%, and the product was off-white powder.

If the fermentation process of step (2) of this embodiment is not carried out, other steps are according to the method of this embodiment, 100g of Atractylodes Rhizome powder can be extracted to obtain 14.4g of Atractylodes Rhizome polysaccharide, the polysaccharide content is 81.7%, that is, 11.8g of Atractylodes Rhizoma Atractylodes polysaccharide is obtained, and the extraction yield The rate was 11.8%. It can be seen that in this embodiment, the fermentation pretreatment of Rhizopus oryzae CH5 is added in advance of the ultrasonic water of Atractylodes macrocephala polysaccharide, and the extraction yield of Atractylodes macrocephala polysaccharide can be increased by 51.7% compared with the conventional method without fermentation pretreatment.

sequence listing

<110> Zhejiang Shuren College (Zhejiang Shuren University)

<120> A method of extracting polysaccharides from Atractylodes macrocephala by microbial fermentation

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Claims (6)

1. A method for extracting Atractylodes atractylodes polysaccharides by microbial fermentation, characterized in that the method is: inoculate Rhizopus oryzae ( Rhizopus oryzae ) GDMCC No: 60990 spores in Atractylodes atractylodes powder containing sterile water, and in 28-30 Fermented at ℃ for 2-3 days, the fermented product was concentrated by ultrasonic water extraction to obtain a concentrate; the concentrate was subjected to alcohol precipitation and vacuum drying to obtain Atractylodes macrocephala crude polysaccharide. 2. The method according to claim 1, characterized in that the volumetric dosage of the sterile water is 2-3mL/g based on the weight of Atractylodes macrocephala powder. 3. The method according to claim 1, characterized in that the inoculum amount of the Rhizopus oryzae GDMCC No: 60990 spores is 2-5×10 ⁷ pcs/g by weight of Atractylodes macrocephala powder. 4. the method for claim 1 is characterized in that described rhizopus oryzae GDMCC No: 60990 spore adds with the form of spore liquid, and the preparation method of described spore liquid is: the rhizopus oryzae GDMCC No of cryopreservation: 60990 Inoculate on the PDA plate medium, after 2-3 days of constant temperature cultivation at 28-30°C, add sterile water, stir with an inoculation loop to suspend the spores, and obtain the spore liquid; the final concentration of the PDA plate medium is composed of: potato 200 g/L, 20 g/L sucrose, 20 g/L agar, tap water as solvent, natural pH. 5. The method according to claim 1, characterized in that the preparation method of the concentrate is as follows: adding deionized water to the fermented product, stirring evenly, placing it in a constant temperature water bath at 80-90°C for 3-5 hours, Transfer to an ultrasonic cleaning machine with a water temperature of 80°C, and perform ultrasonic extraction at 100–200 W for 20–30 min; after ultrasonic water extraction, filter while hot, and concentrate the filtrate under reduced pressure at 65°C to 1/4–1/5 of the original volume , to obtain a concentrate; the added volume of deionized water is 20-30mL/g based on the weight of Atractylodes macrocephala powder before fermentation. 6. The method according to claim 1, characterized in that the preparation method of Atractylodes macrocephala polysaccharide is: add 3-4 times the volume of 95% ethanol to the concentrate, leave it at room temperature for precipitation for 10-16 h, and then Filter the funnel, collect the polysaccharide precipitate, and dry it in vacuum at 60°C to constant weight to obtain the crude polysaccharide of Atractylodes macrocephala.

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