Method for fermenting astragalus by using strain and application of product in antiviral and immunity enhancing products

Abstract

The application belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a method for fermenting radix astragali by a strain and application of a product in antiviral and immunity-enhancing products. The preparation method comprises the following steps: (1) after extracting radix astragali powder and water under ultrasonic conditions, the pH is adjusted to 4.0-6.0 to obtain a pretreatment liquid; (2) enzyme preparation is added to the pretreatment liquid, and enzymolysis is carried out at a temperature of 40-50 DEG C for 20-40 min, and then the pH is adjusted to 6.5-7.0 to obtain an enzymolysis liquid; (3) the enzymolysis liquid is stirred and extracted at a temperature of 80-90 DEG C for 30-50 min to obtain a sterilization liquid; (4) a fermentation strain is added to the sterilization liquid, and fermentation is carried out at a temperature of 25-30 DEG C for 60 min-90 min to obtain a fermentation liquid, the fermentation liquid is separated into solid and liquid, and the upper layer is taken to obtain a fermentation supernatant; and (5) the fermentation supernatant is spray dried to obtain a radix astragali fermentation product, and the radix astragali fermentation product has a high content of astragaloside IV.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine fermentation technology, specifically relating to a method for fermenting Astragalus membranaceus with a bacterial strain and the application of the product in antiviral and immune-enhancing products. Background Technology

[0002] Astragalus root, the dried root of *Astragalus mongholicus* or *Astragalus membranaceus*, is a commonly used traditional Chinese medicine for strengthening the body and replenishing vital energy, making it both a food and a medicine. Besides its medicinal uses, astragalus root has also been developed into health products. Modern medical research has confirmed that astragalus root possesses pharmacological activities and health benefits such as enhancing immunity, protecting the liver and reducing inflammation, strengthening the kidneys and lowering blood pressure, and anti-tumor effects. It also has few toxic side effects and leaves no residue. It has now been developed into immune enhancers and other products, widely used in the prevention of human and animal diseases and the improvement of immune function, with satisfactory results.

[0003] The main chemical components of Astragalus membranaceus are saponins, flavonoids, polysaccharides, alkaloids, organic acids, amino acids, and trace elements. Among these, saponins and flavonoids are the most important chemical components. Structurally, Astragalus saponins are mainly tetracyclic triterpenoid saponins of the cycloatunane type. They are generally obtained by hydrolyzing the acetyl and glycosyl groups in total Astragalus saponins using microorganisms containing deacetylase and glycosidase to yield astragaloside A, with the chemical formula C. 41 H 68 O 14 Astragaloside A not only possesses the effects of astragalus polysaccharides, but also exhibits some effects unmatched by them. Its potency is more than twice that of conventional astragalus polysaccharides, and its antiviral activity is 30 times stronger. Due to its low content and high efficacy, it is even known as a "super astragalus polysaccharide." Cycloastragalol is the aglycone portion of astragaloside, with the chemical formula C. 30 H 50 O5.

[0004] Study on the effect of different extraction processes on the extraction of total saponins from Astragalus membranaceus [J]. Chinese Journal of Veterinary Drugs, 2016, 50(12) 24-28. The release amount of total saponins from Astragalus membranaceus can be extracted by fermentation. Chinese patent document CN102559828A discloses a method for preparing astragaloside A by microbial conversion of total saponins from Astragalus membranaceus, which includes the following steps: preparing microbial strains into bacterial suspensions or spore suspensions, inoculating them into seed culture medium, culturing at 28℃ and 220rpm for 24-48h to obtain seed liquid, then inoculating the seed liquid into fermentation culture medium, culturing at 28℃ and 220rpm for 24-48h, adding total saponins from Astragalus membranaceus, and converting under the same conditions for 48-96h to obtain conversion liquid; the conversion liquid is purified by organic solvent extraction and macroporous resin separation to obtain pure astragaloside A, wherein the microorganism is *Pterocarya stenoptera*. Chinese patent document CN106011213A discloses a method for the biotransformation and degradation of astragaloside A to prepare cycloastragalool. This method uses astragaloside A as a substrate and ferments it with the microbial strain *Absidia sp.* CGMCC3.2843 to obtain cycloastragalool. This demonstrates that different products can be obtained from the microbial fermentation of astragalus, and the choice of appropriate technology to achieve high-efficiency conversion of astragaloside A, while minimizing or eliminating the conversion of cycloastragalool, is particularly important. Summary of the Invention

[0005] In view of this, the present invention combines traditional Chinese medicine fermentation technology to provide a method for preparing Astragalus membranaceus by fermentation of strains. The Astragalus membranaceus fermentation product obtained by this method has a high content of astragaloside A and has a good effect on enhancing immunity and antiviral activity.

[0006] The present invention achieves the above objectives using the following technical solutions.

[0007] A method for fermenting Astragalus membranaceus using a bacterial strain includes the following steps:

[0008] (1) Pretreatment: Astragalus powder and water were mixed and extracted under ultrasonic conditions, and then the pH was adjusted to 4.0-6.0 to obtain a pretreated solution;

[0009] (2) Enzymatic hydrolysis: Add enzyme preparation to the pretreatment solution, and enzymatically hydrolyze at a temperature of 40-50℃ for 20-40 min, and adjust the pH to 6.5-7.0 to obtain the enzymatic hydrolysate;

[0010] (3) Enzyme inactivation extraction: The enzyme hydrolysate is stirred and extracted at 80-90℃ for 30-50 min to obtain a sterile solution;

[0011] (4) Fermentation: Add the fermentation strain to the sterilization solution and ferment at 25-30℃ for 60-90 minutes to obtain the fermentation solution. Separate the solid and liquid of the fermentation solution and take the upper layer to obtain the fermentation clear liquid.

[0012] (5) Spray drying: The fermentation liquid is spray dried to obtain Astragalus fermentation product;

[0013] The enzyme preparation consists of cellulase and glucoamylase, and the fermentation strain consists of Bacillus subtilis and Leuconostoc mesenteroides subsp. milk fat.

[0014] Preferably, the weight ratio of the Astragalus powder to the fermentation strain is (400-500):1.

[0015] More preferably, the weight ratio of the Astragalus powder to the fermentation strain is 400:1.

[0016] Preferably, the weight ratio of Bacillus subtilis and Leuconostoc mesenteroides subsp. milk fat is 1:(1-2).

[0017] More preferably, the weight ratio of Bacillus subtilis and Leuconostoc mesenteroides subsp. milk fat is 1:1.

[0018] More preferably, the activity level of the Bacillus subtilis is 100-500 billion CFU / g, and the activity level of the Leuconostoc mesenteroides subsp. milk fat is 200-4000 billion CFU / g.

[0019] More preferably, the Bacillus subtilis has a bacterial activity of 200 billion CFU / g, and the Leuconostoc mesenteroides subsp. milk fat has a bacterial activity of 300 billion CFU / g.

[0020] Preferably, the weight ratio of the Astragalus powder to the enzyme preparation is (8-15):1.

[0021] More preferably, the weight ratio of the astragalus powder to the enzyme preparation is 13.3:1.

[0022] Preferably, the weight ratio of cellulase to glucoamylase is (1-3):(0.5-2).

[0023] More preferably, the weight ratio of cellulase to glucoamylase is 2:1.

[0024] Preferably, the cellulase has an activity of 10,000-20,000 U / g, and the glucoamylase has an activity of 50,000-70,000 U / g.

[0025] More preferably, the cellulase has an activity of 20,000 U / g and the glucoamylase has an activity of 60,000 U / g.

[0026] Preferably, the weight ratio of the astragalus powder to water is 1:(100-500).

[0027] More preferably, the temperature of the ultrasound in step (1) is 35-50℃ and the time is 30-60min.

[0028] More preferably, the temperature of the ultrasound in step (1) is 40°C and the time is 50 min.

[0029] Preferably, the astragalus powder is obtained by pulverizing astragalus and passing it through a 20-mesh sieve.

[0030] More preferably, the inlet temperature of the spray drying in step (5) is 100-130℃ and the outlet temperature is 30-50℃.

[0031] The Astragalus fermentation product obtained by the above-mentioned strain fermentation method provided by the present invention can be used in the preparation of immune-enhancing or antiviral foods, health products, cosmetics, medical products and other products.

[0032] The technical solution of this invention has the following advantages:

[0033] 1. This invention provides a method for fermenting Astragalus membranaceus using microbial strains. Astragalus membranaceus powder is subjected to ultrasonic pretreatment, enzymatic hydrolysis, enzyme inactivation extraction, and fermentation steps in sequence. The enzyme preparations selected are cellulase and glucoamylase, and the fermentation strains are Bacillus subtilis and Leuconostoc mesenteroides subsp. lactis. The above method can improve the conversion rate of astragaloside A, and the fermented Astragalus membranaceus product has a high content of astragaloside A, while the content of cycloastragalool can be controlled to be less than 2.2%.

[0034] 2. The present invention provides a method for fermenting Astragalus membranaceus using strains. This method prepares astragaloside A through microbial transformation. The method is simple and suitable for industrial production.

[0035] 3 The Astragalus fermentation product obtained by the strain fermentation method provided by the present invention has a good effect on enhancing immunity and antiviral activity, and can be used for the development of products that enhance immunity and antiviral activity. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be described in further detail below. All other embodiments obtained by those skilled in the art based on the embodiments disclosed in this invention without inventive effort are within the scope of protection of this disclosure.

[0037] For any experimental steps or conditions not specified in the examples, the procedures or conditions described in the literature in this field can be followed. All reagents without specified manufacturers are commercially available standard reagent products.

[0038] In the following examples, the source information for some products is as follows:

[0039] Astragalus membranaceus was purchased from Bozhou Jinfang Qiancao Pharmaceutical Co., Ltd., with a diameter of 8mm-10mm from Gansu Province. Its moisture content was no higher than 10%, total ash content was no higher than 5%, and astragaloside A content was no less than 0.04%. Cellulase was purchased from Shandong Longkete Enzyme Preparation Co., Ltd.; glucoamylase was purchased from Nanjing Tongying Biotechnology Co., Ltd.; Bacillus subtilis was purchased from Junyao Runying Biotechnology (Shanghai) Co., Ltd., with Bacillus subtilis BS-GA28; Leuconostoc mesenteroides subsp. lactis was purchased from Unico (Shanghai) Life Science Co., Ltd., with product number YLK-J3274h.

[0040] Example 1

[0041] This embodiment provides a method for fermenting Astragalus membranaceus using a bacterial strain, including the following steps:

[0042] (1) Pretreatment: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 3000mL of water. Then place it in an ultrasonic extraction device at a temperature of 40℃ and sonicate for 50min. Adjust the pH to 5.0 to obtain the pretreated solution.

[0043] (2) Enzymatic hydrolysis: The pretreated solution was transferred to a fermenter, and then 0.5g of cellulase and 0.25g of glucoamylase were added. The temperature was raised to 40℃ and the mixture was stirred for 30 minutes. The pH was adjusted to 7.0 to obtain the hydrolysate.

[0044] (3) Enzyme inactivation extraction: Heat the enzyme hydrolysate to 90℃ and stir for 40 min to obtain sterilized solution;

[0045] (4) Fermentation: After the temperature of the sterilized liquid is reduced to 25°C, add 0.0125g of Bacillus subtilis and 0.0125g of Leuconostoc mesenteroides subsp. lactis, stir and ferment for 80 minutes to obtain fermentation liquid. Separate the solid and liquid of the fermentation liquid and take the upper layer to obtain fermentation clear liquid.

[0046] (5) Spray drying: The fermentation liquid is spray dried at an inlet temperature of 120°C and an outlet temperature of 40°C to obtain the fermentation product of Astragalus membranaceus.

[0047] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0048] Example 2

[0049] This embodiment provides a method for fermenting Astragalus membranaceus using a bacterial strain, including the following steps:

[0050] (1) Pretreatment: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 1000mL of water. Then place it in an ultrasonic extraction device at a temperature of 35℃ and sonicate for 60min. Adjust the pH to 6.0 to obtain the pretreated solution.

[0051] (2) Enzymatic hydrolysis: The pretreated solution was transferred to a fermenter, and then 0.75 g of cellulase and 0.5 g of glucoamylase were added. The temperature was raised to 50°C and the mixture was stirred for 30 min. The pH was adjusted to 7.0 to obtain the hydrolysate.

[0052] (3) Enzyme inactivation extraction: Heat the enzyme hydrolysate to 80℃ and stir for 30 min to obtain sterilized solution;

[0053] (4) Fermentation: After the temperature of the sterilized liquid is reduced to 30℃, add 0.011g of Bacillus subtilis and 0.011g of Leuconostoc mesenteroides subsp. lactis, stir and ferment for 60min to obtain fermentation liquid. Separate the solid and liquid of the fermentation liquid and take the upper layer to obtain fermentation clear liquid.

[0054] (5) Spray drying: The fermentation liquid is spray dried at an inlet temperature of 130°C and an outlet temperature of 30°C to obtain the fermentation product of Astragalus membranaceus.

[0055] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0056] Example 3

[0057] This embodiment provides a method for fermenting Astragalus membranaceus using a bacterial strain, including the following steps:

[0058] (1) Pretreatment: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 5000mL of water. Then place it in an ultrasonic extraction device at a temperature of 50℃ and sonicate for 30min. Adjust the pH to 5.0 to obtain the pretreated solution.

[0059] (2) Enzymatic hydrolysis: The pretreated solution was transferred to a fermenter, and then 0.5 g of cellulase and 0.17 g of glucoamylase were added. The temperature was raised to 45°C and the mixture was stirred for 20 min. The pH was adjusted to 6.5 to obtain the hydrolysate.

[0060] (3) Enzyme inactivation extraction: Heat the enzyme hydrolysate to 85℃ and stir for 50 min to obtain sterilized solution;

[0061] (4) Fermentation: After the temperature of the sterilized liquid is reduced to 25°C, add 0.007g of Bacillus subtilis and 0.013g of Leuconostoc mesenteroides subsp. lactis, stir and ferment for 90 minutes to obtain fermentation liquid. Separate the solid and liquid of the fermentation liquid and take the upper layer to obtain fermentation clear liquid.

[0062] (5) Spray drying: The fermentation liquid is spray dried at an inlet temperature of 100°C and an outlet temperature of 50°C to obtain the fermentation product of Astragalus membranaceus.

[0063] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0064] Example 4

[0065] This embodiment provides a method for fermenting Astragalus membranaceus using a bacterial strain, including the following steps:

[0066] (1) Pretreatment: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 4000mL of water. Then put it into an ultrasonic extraction device at a temperature of 45℃ and sonicate for 45min. Adjust the pH to 5.0 to obtain the pretreated solution.

[0067] (2) Enzymatic hydrolysis: The pretreated solution was transferred to a fermenter, and then 0.28 g of cellulase and 0.55 g of glucoamylase were added. The temperature was raised to 40°C and the mixture was stirred for 25 min. The pH was adjusted to 7.0 to obtain the hydrolysate.

[0068] (3) Enzyme inactivation extraction: Heat the enzyme hydrolysate to 83℃ and stir for 35 min to obtain sterile solution;

[0069] (4) Fermentation: After the temperature of the sterilized liquid is reduced to 27°C, add 0.01g of Bacillus subtilis and 0.01g of Leuconostoc mesenteroides subsp. lactis, stir and ferment for 70 minutes to obtain fermentation liquid. Separate the solid and liquid of the fermentation liquid and take the upper layer to obtain fermentation clear liquid.

[0070] (5) Spray drying: The fermentation liquid is spray dried at an inlet temperature of 120°C and an outlet temperature of 40°C to obtain the fermentation product of Astragalus membranaceus.

[0071] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0072] Comparative Example 1

[0073] This comparative example provides a method for fermenting Astragalus membranaceus using a bacterial strain, which differs from Example 1 in that it does not include a pretreatment step. Specifically, it includes the following steps:

[0074] (1) Enzymatic hydrolysis: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 3000mL of water to adjust the pH to 5.0 and then transfer it to a fermenter. Then add 0.5g of cellulase and 0.25g of glucoamylase, raise the temperature to 40℃ and stir for 30min, and adjust the pH to 7.0 to obtain the enzymatic hydrolysate.

[0075] (2) Enzyme inactivation extraction: Heat the enzyme hydrolysate to 90℃ and stir for 40 min to obtain sterilized solution;

[0076] (3) Fermentation: After the temperature of the sterilized liquid is reduced to 25°C, add 0.0125g of Bacillus subtilis and 0.0125g of Leuconostoc mesenteroides subsp. lactis, stir and ferment for 80 minutes to obtain fermentation liquid. Separate the solid and liquid of the fermentation liquid and take the upper layer to obtain fermentation clear liquid.

[0077] (4) Spray drying: The fermentation liquid is spray dried at an inlet temperature of 120°C and an outlet temperature of 40°C to obtain the Astragalus fermentation product.

[0078] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0079] Comparative Example 2

[0080] This comparative example provides a method for fermenting Astragalus membranaceus using a bacterial strain. The difference between this method and Example 1 is that it does not include an enzymatic hydrolysis step. Specifically, it includes the following steps:

[0081] (1) Pretreatment: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 3000mL of water. Then put it into an ultrasonic extraction device at a temperature of 40℃ and sonicate for 50min. Adjust the pH to 5.0 to obtain the pretreated solution.

[0082] (2) Enzyme inactivation extraction: The pretreated solution was heated to 90℃ and stirred for 40 min to obtain a sterilized solution;

[0083] (3) Fermentation: After the temperature of the sterilized liquid is reduced to 25°C, add 0.0125g of Bacillus subtilis and 0.0125g of Leuconostoc mesenteroides subsp. lactis, stir and ferment for 80 minutes to obtain fermentation liquid. Separate the solid and liquid of the fermentation liquid and take the upper layer to obtain fermentation clear liquid.

[0084] Spray drying: The fermentation liquid is spray dried at an inlet temperature of 120°C and an outlet temperature of 40°C to obtain the Astragalus fermentation product.

[0085] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0086] Comparative Example 3

[0087] This comparative example provides a method for fermenting Astragalus membranaceus with a strain. The only difference from Example 1 is that the enzyme preparation includes only 0.75g of cellulase, while the rest is the same as in Example 1.

[0088] Comparative Example 4

[0089] This comparative example provides a method for fermenting Astragalus membranaceus with a strain. The only difference from Example 1 is that the enzyme preparation includes only 0.75g of glucoamylase, while the rest is the same as in Example 1.

[0090] Comparative Example 5

[0091] This comparative example provides a method for fermenting Astragalus membranaceus with a strain. The only difference from Example 1 is that the strain includes only 0.025g of Bacillus subtilis, and the rest is the same as in Example 1.

[0092] Comparative Example 6

[0093] This comparative example provides a method for fermenting Astragalus membranaceus with a strain. The only difference from Example 1 is that the strain includes only 0.025g of Leuconostoc mesenteroides subsp. lactis, and the rest is the same as in Example 1.

[0094] Comparative Example 7

[0095] This comparative example provides a method for fermenting Astragalus membranaceus using a bacterial strain. The difference between this method and Example 1 is that it does not include the enzyme inactivation and extraction step. Specifically, it includes the following steps:

[0096] (1) Pretreatment: After pulverizing Astragalus membranaceus and passing it through a 20-mesh sieve, take 10g of Astragalus membranaceus powder and add it to 1000mL of water. Then place it in an ultrasonic extraction device at a temperature of 35℃ and sonicate for 60min. Adjust the pH to 6.0 to obtain the pretreated solution.

[0097] (2) Enzymatic hydrolysis: The pretreated solution was transferred to a fermenter, and then 0.5g of cellulase and 0.25g of glucoamylase were added. The temperature was raised to 40℃ and the mixture was stirred for 30 minutes. The pH was adjusted to 7.0 to obtain the hydrolysate.

[0098] (3) Fermentation: After the temperature of the enzymatic hydrolysate is lowered to 25°C, 0.0125 g of Bacillus subtilis and 0.0125 g of Leuconostoc mesenteroides subsp. lactis are added and stirred for 80 min to obtain fermentation liquid. The fermentation liquid is then separated into solid and liquid phases, and the upper layer is taken to obtain fermentation clear liquid.

[0099] (4) Spray drying: The fermentation liquid is spray dried at an inlet temperature of 120°C and an outlet temperature of 40°C to obtain the Astragalus fermentation product.

[0100] The bacterial activity of Bacillus subtilis was 200 billion CFU / g, the bacterial activity of Leuconostoc mesenteroides subsp. lactis was 300 billion CFU / g, the enzyme activity of cellulase was 20,000 U / g, and the enzyme activity of glucoamylase was 60,000 U / g.

[0101] Experimental Example 1: Detection of Active Ingredients

[0102] 1. Determination of Astragaloside A: The astragaloside A was determined according to the high performance liquid chromatography method (General Rule 0512) described in the Chinese Pharmacopoeia (2020 edition). The difference lies in the preparation of the test solution. In this invention, 4g each of the Astragalus fermentation products obtained in Examples 1-4 and Comparative Examples 1-7, and three parallel samples of each, were accurately weighed and placed in round-bottom flasks.

[0103] The release rate of astragaloside A was used as an example, and its calculation formula was: Astragaloside A release rate = Astragaloside A content / Mass of Astragalus fermentation product * 100%. The specific results are shown in Table 1.

[0104] 2. Determination of Cycloastragalol: HPLC-ELSD method was used: chromatographic column: Phenomenex C18 (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A) - 0.3% formic acid water (B); flow rate: 0.5 mL / min; column temperature: 35℃; injection volume: 5 μL; gradient elution (0-10 min, 50%-60% A; 10-20 min, 60%-70% A; 20-30 min, 70%-75% A; 30-35 min, 75%-90% A; 35-40 min, 90%-100% A).

[0105] The linear regression equation for cycloastragalool is Y = 1.217X + 7.2638 (R²). 2 =0.9981).

[0106] The release rate of cycloastragalool is expressed as follows: cycloastragalool release rate = cycloastragalool content / Astragalus fermentation product * 100%. The specific results are shown in Table 1.

[0107] The specific results are shown in Table 1.

[0108] Table 1

[0109]

[0110] Results analysis:

[0111] As can be seen from the overall analysis of the table above, the astragaloside A content in the fermented products of Astragalus membranaceus obtained in Examples 1-4 of this invention can reach more than 60%, and the cycloastragalool content can be controlled within 2.5%, indicating that the preparation method of Astragalus membranaceus fermented by the strain provided in this application can be used to prepare Astragalus membranaceus products with a high content of astragaloside A.

[0112] The difference between Example 1 and Comparative Example 1 is that Comparative Example 1 did not have an ultrasonic extraction step. As can be seen from the results in Table 1, the content of astragaloside A in Comparative Example 1 is much lower than that in Example 1. The applicant speculates that the ultrasonic extraction improved the dissolution of some components in Astragalus membranaceus, providing a higher content of relatively small molecular weight components for subsequent enzymatic hydrolysis and fermentation, which is more conducive to the release of astragaloside A.

[0113] Comparative analysis of the astragaloside A content in Example 1 and Comparative Examples 2, 3, and 4 shows that enzymatic hydrolysis is beneficial for the release of astragaloside A, and the combined action of cellulase and glucoamylase is necessary to achieve the maximum effect.

[0114] Comparative analysis of the astragaloside A content in Example 1 and Comparative Examples 5 and 6 showed that the astragaloside content obtained in Comparative Examples 5 and 6 was lower, indicating that the choice of strain has a significant impact on the astragaloside A content. This means that fermentation is a relatively important step in the preparation method of astragaloside by strain fermentation, and that the combined use of Bacillus subtilis and Leuconostoc mesenteroides subsp. lactis is necessary to achieve better release of astragaloside A.

[0115] The difference between Example 1 and Comparative Example 7 is that Comparative Example 7 did not sterilize and extract the enzyme activity but carried out fermentation directly. In this case, the enzyme preparation and the strain were in the same system. The comparative analysis of the contents of astragaloside A and cycloastragalol showed that the content of cycloastragalol increased while the content of astragaloside A decreased significantly, indicating that astragaloside A was partially converted into cycloastragalol.

[0116] Experiment Example 2: Immunity Enhancement Experiment

[0117] Experimental Design

[0118] Eighty 6-week-old male SD rats weighing 110-130g were selected and, after one week of acclimatization, randomly divided into four groups: a blank control group, a model control group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, control group 1, and control group 2, with 10 mice in each group. The model control group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, control group 1, and control group 2 were intraperitoneally injected with cyclophosphamide at 80 mg / kg body weight daily, while the blank control group was intraperitoneally injected with physiological saline. These injections were continued for 3 days.

[0119] Three days later, experimental groups 1, 2, 3, and 4, control group 1, and control group 2 were administered the Astragalus fermentation products obtained from Examples 1, 2, 3, 4, and Comparative Examples 3 and 5 by gavage, respectively. The dosage for mice was determined to be 0.1 g / (d.kg.BW) based on a daily dose of 6.0 g per person (equivalent to 0.1 g / (d.kg.BW) for a 60 kg body weight). This dosage was 1 g / (d.kg.BW) per day for each group of mice, determined to be 10 times the recommended human dose. This was administered by gavage for 30 consecutive days. On day 31, before the morning feeding, the mice were sacrificed, and serum was collected to detect the levels of immunoglobulins (A, M) and interleukin-2, interleukin-6, and interleukin-10. The detection methods were as described in the kit instructions, which were manufactured by Nanjing Jiancheng.

[0120] Data Statistics and Analysis

[0121] SPSS 19.0 software was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation (X ± s). If the data of each group conformed to a normal distribution and homogeneity of variance, one-way ANOVA was performed. The LSD test was used for comparisons between groups. If the variances were unequal, nonparametric tests (Kruskal-Wallis one-way ANOVA) were used. P < 0.05 was considered statistically significant.

[0122] Results and Discussion

[0123] The results for each group of mice are shown in Table 2.

[0124] Table 2. Detection results of immunoglobulins and interleukins in each group of rats (X±s)

[0125]

[0126] Data Results Analysis

[0127] Compared with the control group, the levels of immunoglobulin M and immunoglobulin A in Examples 1-4 were significantly different, indicating statistical differences. This demonstrates that the Astragalus fermentation product obtained in this invention can effectively increase the levels of immunoglobulin A and immunoglobulin M in the body. Control groups 1 and 2 showed significant differences compared with group 1 of Example 1, indicating that the enzyme preparation and strain selected in this invention can better increase the levels of immunoglobulin A and immunoglobulin M in the body.

[0128] Compared with the control group, the levels of interleukin-2, interleukin-6, and interleukin-10 in Examples 1-4 showed statistically significant differences, indicating that the Astragalus fermentation product provided by this invention can effectively increase the levels of interleukin-2 and interleukin-10 in the body and significantly reduce the level of interleukin-6. Control groups 1 and 2 showed significant differences compared with the Example 1 group, indicating that the enzyme preparation and strain selected in this invention can better increase the number of interleukin-2 and interleukin-10 in the body and reduce the level of interleukin-6. In summary, the Astragalus fermentation product provided by this invention has the effect of improving immunity and can be used in the development of immune-enhancing products.

[0129] Experiment Example 3: Antiviral Experiment

[0130] Six-week-old SPF female BALB / c mice, weighing 18–20 g, were randomly divided into six groups of 15 mice each: a normal control group, a virus model group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4. After mild anesthesia with ether, the mice in the virus model group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4 were intranasally infected with 10 [unspecified substances]. -3 A / PR / 8 / 34 (H1N1) subtype influenza virus (PR8) dilution at concentration (the original virus solution was diluted with phosphate buffer to a concentration of 10). -3 The concentration of PR8 (purchased from the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences) was 30 μL, while the normal control group was given an equal volume of physiological saline, 30 μL per animal.

[0131] The mice were administered the drug subcutaneously one day prior to infection. Groups 1, 2, 3, and 4 received the Astragalus fermentation product solution prepared in Examples 1-4 (prepared by dissolving the Astragalus fermentation product in physiological saline to a concentration of 0.3 g / mL). The normal control group and the virus model group received 0.2 mL of physiological saline once daily for 7 consecutive days. The morbidity and mortality of the mice were observed daily. After 21 days of observation, the survival rate and average survival time of each group were calculated based on the results.

[0132] The experimental results are as follows:

[0133] In the virus control group, mice began to show symptoms on day 4 post-infection, including rough fur and decreased appetite. By day 5, most mice were infected, and the symptoms worsened, including tremors, lethargy, rapid weight loss, and eventually death. In contrast, groups 1-4 significantly increased the number of surviving mice and their average survival time, indicating that the Astragalus extract provided by this invention has a high in vivo antiviral activity against influenza A virus.

[0134] Table 3. Mouse survival status

[0135]

[0136] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A method for fermenting Astragalus membranaceus using a bacterial strain, characterized in that, Includes the following steps: (1) Pretreatment: Astragalus powder and water were mixed and extracted under ultrasonic conditions, and then the pH was adjusted to 4.0-6.0 to obtain a pretreated solution; the ultrasonic temperature was 35-50℃ and the time was 30-60min. (2) Enzymatic hydrolysis: Add enzyme preparation to the pretreatment solution, and enzymatically hydrolyze at a temperature of 40-50℃ for 20-40 min, and adjust the pH to 6.5-7.0 to obtain the enzymatic hydrolysate; (3) Enzyme inactivation extraction: The enzyme hydrolysate is stirred and extracted at 80-90℃ for 30-50 min to obtain a sterile solution; (4) Fermentation: Add the fermentation strain to the sterilization solution and ferment at 25-30℃ for 60-90 minutes to obtain the fermentation solution. Separate the solid and liquid of the fermentation solution and take the upper layer to obtain the fermentation clear liquid. (5) Spray drying: The fermentation liquid is spray dried to obtain Astragalus fermentation product; The enzyme preparation is composed of cellulase and glucoamylase, and the fermentation strain is composed of Bacillus subtilis and Leuconostoc mesenteroides subsp. lactis. The weight ratio of Astragalus powder to fermentation strain is (400-500):1; the weight ratio of Bacillus subtilis and Leuconostoc mesenteroides subsp. milk fat is 1:(1-2). The weight ratio of Astragalus powder to enzyme preparation is (8-15):1, and the weight ratio of cellulase to glucoamylase is (1-3):(0.5-2).

2. The method according to claim 1, characterized in that, The activity level of Bacillus subtilis is 100-500 billion CFU / g, and the activity level of Leuconostoc mesenteroides subsp. milk fat is 200-4000 billion CFU / g.

3. The method according to claim 1, characterized in that, The cellulase has an activity of 10,000-20,000 U / g, and the glucoamylase has an activity of 50,000-70,000 U / g.

4. The method according to claim 1, characterized in that, The weight ratio of Astragalus powder to water is 1:(100-500).

5. The method according to claim 1, characterized in that, The Astragalus powder is obtained by crushing Astragalus and passing it through a 20-mesh sieve; the inlet temperature of the spray drying in step (5) is 100-130℃ and the outlet temperature is 30-50℃.

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