Astragalus fermented bacteria, preparation method and use

Abstract

The invention discloses an astragalus fermentation substance, a preparation method and a use thereof. The astragalus fermentation substance is fermented by fermenting raw materials including the following weight components: 15 to 25 weight parts of astragalus, 4 to 12 weight parts of Poria cocos fermentation liquid and 5 to 25 weight parts of water. The astragalus fermentation substance of the invention has good antioxidant capacity.

Description

Technical Field

[0001] The invention relates to astragalus fermentation bacteria, a preparation method and application thereof. Background Art

[0002] Traditional Chinese medicine fermentation is the fermentation of traditional Chinese medicine under certain environmental conditions (such as humidity, temperature, etc.) with the help of microorganisms to change its original properties, enhance or produce new effects, thereby expanding the variety of medicines and meeting the needs of clinical medication. Modern medical research shows that traditional Chinese medicine fermentation can reduce the toxicity of traditional Chinese medicine, enhance or produce new effects, thereby broadening the types of traditional Chinese medicines to meet clinical needs, and can effectively improve the resource utilization rate of traditional Chinese medicines, so it has broad prospects and application value.

[0003] Astragalus is one of the oldest Chinese medicines for invigorating the middle and replenishing qi. It contains a variety of active ingredients such as saponins, flavonoids, polysaccharides, etc. Among them, the saponins include astragaloside I, astragaloside II, astragaloside III, astragaloside IV, etc., and the flavonoids include verbascoside, formononetin, verbascoside glycosides, formononetin, etc. It has anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory pharmacological effects.

[0004] CN 105193905 A discloses a traditional Chinese medicine processing technology of Ganoderma lucidum-Astragalus membranaceus bidirectional fermentation, which comprises astragalus membranaceus pretreatment, Ganoderma lucidum strain activation, bidirectional fermentation medium preparation, bidirectional fermentation and medicinal property change and fermentation bacterial substance processing.

[0005] CN 106880588 A discloses a method for preparing tangerine peel and tuckahoe oral liquid, wherein tuckahoe strains are picked from the inclined surface of a test tube and inoculated into a fermentation medium, and the tuckahoe fermentation liquid is obtained after culturing for 5-7 days. The tuckahoe fermentation liquid is boiled for 30-40 minutes, and then filtered, blended, filled, sealed, and sterilized to obtain a finished product.

[0006] Although the above methods all use fermentation to treat medicinal materials, which can improve the conversion rate of components to a certain extent and have certain antioxidant capacity, the preparation of raw materials is more complicated and the process is more complicated. Summary of the invention

[0007] In view of this, the first object of the present invention is to provide an Astragalus fermented fungus, which has significant antioxidant capacity.

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned astragalus fermentation bacteria, which can greatly improve the bioconversion rate, DPPH free radical scavenging rate and ABTS+ free radical scavenging rate of astragaloside IV in astragalus.

[0009] The third object of the present invention is to provide the use of the above-mentioned astragalus fermentation bacteria.

[0010] The fourth object of the present invention is to provide a use of Poria cocos.

[0011] The present invention adopts the following technical solutions to achieve the above-mentioned purpose.

[0012] In one aspect, the present invention provides an astragalus fermented fungus, which is fermented from raw materials including the following components by weight: 15 to 25 parts by weight of astragalus, 4 to 12 parts by weight of Poria cocos fermentation liquid and 5 to 25 parts by weight of water.

[0013] According to the Astragalus fermented fungus of the present invention, preferably, the Poria cocos fermented broth is prepared by the following method:

[0014] The method comprises the following steps: inoculating the Poria cocos on a potato glucose agar slant culture medium, activating and culturing the Poria cocos for 3 to 6 days at 24 to 29 DEG C and a relative humidity of 70 to 90%, so as to obtain activated Poria cocos; and fermenting the activated Poria cocos in a potato liquid culture medium for 2 to 5 days at a fermentation temperature of 24 to 29 DEG C and a shaking table rotation speed of 150 to 200 r / min, so as to obtain a Poria cocos fermentation liquid.

[0015] According to the astragalus fermented fungus of the present invention, preferably, the astragalus fermented fungus is prepared by the following method:

[0016] 15-25 parts by weight of astragalus, 4-12 parts by weight of Poria cocos fermentation liquid and 5-25 parts by weight of water are fermented at 24-29° C. for 6-12 days.

[0017] On the other hand, the present invention provides a method for preparing the above-mentioned fermented astragalus, comprising the following steps:

[0018] 15-25 parts by weight of astragalus, 4-12 parts by weight of Poria cocos fermentation liquid and 5-25 parts by weight of water are fermented at 24-29° C. for 6-12 days to obtain astragalus fermentation fungus.

[0019] According to the preparation method of the present invention, preferably, astragalus and water are mixed and then sterilized to obtain a first mixture; the Poria cocos fermentation liquid is inoculated into the first mixture and fermented to obtain astragalus fermentation fungus.

[0020] The preparation method according to the present invention preferably further comprises the following steps:

[0021] The method comprises the following steps: inoculating the Poria cocos on a potato glucose agar slant culture medium, activating and culturing the Poria cocos for 3 to 6 days at 24 to 29 DEG C and a relative humidity of 70 to 90%, so as to obtain activated Poria cocos; and fermenting the activated Poria cocos in a potato liquid culture medium for 2 to 5 days at a fermentation temperature of 24 to 29 DEG C and a shaking table rotation speed of 150 to 200 r / min, so as to obtain a Poria cocos fermentation liquid.

[0022] According to the preparation method of the present invention, preferably, the weight ratio of astragalus to water is 0.5 to 4:1.

[0023] According to the preparation method of the present invention, preferably, the liquid-to-solid ratio of the Poria cocos fermentation broth to the Astragalus membranaceus is 1:2-5.

[0024] In another aspect, the present invention provides a use of the above-mentioned Astragalus fermentation bacteria in the preparation of antioxidant drugs.

[0025] In another aspect, the present invention provides a use of Poria cocos in improving the conversion rate of astragaloside IV during the fermentation of Astragalus.

[0026] The fermented astragalus of the present invention has significant antioxidant capacity. The preparation method of the fermented astragalus of the present invention can greatly improve the bioconversion rate of astragaloside IV in astragalus and the DPPH free radical scavenging rate and ABTS+ free radical scavenging rate. The tuckahoe of the present invention can improve the conversion rate of astragaloside IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison chart of the DPPH radical scavenging ability of the Astragalus fermented fungus in Experimental Example 1 and the control sample.

[0028] Figure 2 This is a comparison chart of the ABTS free radical scavenging abilities of the Astragalus fermented bacteria in Experimental Example 1 and the control sample. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0030] <Astragalus Fermented Bacteria>

[0031] The astragalus fermented fungus substance of the invention is obtained by fermenting raw materials including the following components: astragalus, fermented bacteria and water.

[0032] In the present invention, water can be any drinking water, preferably pure water, more preferably ultrapure water. Astragalus can be 15 to 25 parts by weight, preferably 16 to 24 parts by weight, more preferably 18 to 22 parts by weight. Poria fermentation broth can be 4 to 12 parts by weight, preferably 6 to 10 parts by weight, more preferably 7 to 9 parts by weight. Water can be 5 to 25 parts by weight, preferably 7 to 20 parts by weight, more preferably 8 to 15 parts by weight. The use of the above-mentioned proportions of the components makes the fermented Astragalus have significant antioxidant capacity.

[0033] <Method for preparing fermented astragalus>

[0034] The astragalus root and water are mixed and then sterilized to obtain a first mixture; the Poria cocos fermentation liquid is inoculated into the first mixture and fermented to obtain the astragalus fermentation fungus substance.

[0035] In the present invention, preferably, the astragalus is astragalus powder; the particle size of the astragalus powder can be 50-80 mesh, preferably 55-75 mesh, and more preferably 60-65 mesh. This can increase the surface area, which is conducive to the full contact between the medicinal material and the bacteria. At the same time, the effective ingredients in the medicinal material in powder form are easier to leach, greatly improving the extraction rate.

[0036] In the present invention, the weight ratio of astragalus powder to water can be 0.5 to 4: 1, preferably 1 to 4: 1, and more preferably 1.5 to 4: 1. This is conducive to the subsequent fermentation bacteria fully contacting with the astragalus, achieving better fermentation effect, higher growth rate of fermentation bacteria and higher yield of astragalus fermentation bacteria.

[0037] In the present invention, preferably, high pressure sterilization is adopted, and the sterilization temperature may be 110-150°C; preferably 110-130°C; more preferably 115-125°C.

[0038] In the present invention, the sterilization time may be 15 to 30 min, preferably 20 to 30 min, and more preferably 25 to 30 min.

[0039] In the present invention, preferably, the Poria cocos fermentation liquid is obtained by culturing and activating Poria cocos. Poria cocos is a fungus of the Polyporaceae family, Poria cocos (Schw.) Wolf., which can be purchased on the market.

[0040] In the present invention, the culture method of the Poria cocos fermentation liquid can be: inoculate the Poria cocos on a potato dextrose agar slant medium and activate and culture for 3 to 6 days under the conditions of a temperature of 24 to 29° C. and a relative humidity of 70 to 90% to obtain the activated Poria cocos; liquid ferment the activated Poria cocos in a potato liquid medium under the conditions of a fermentation temperature of 24 to 29° C. and a rotation speed of 150 to 200 r / min for 2 to 5 days to obtain the Poria cocos fermentation liquid. Preferably, the Poria cocos is inoculated on a potato dextrose agar slant medium and activated and cultured for 3.5 to 5.5 days under the conditions of a temperature of 25 to 28° C. and a relative humidity of 75 to 85% to obtain the activated Poria cocos; the activated Poria cocos is cultured in a potato liquid medium under the conditions of a shaking table at 25 to 28° C. and a rotation speed of 160 to 190 r / min for 2.5 to 4.5 days to obtain the Poria cocos fermentation liquid. More preferably, the Poria cocos is inoculated on a potato glucose agar slant culture medium and activated and cultured for 4 to 5 days at a temperature of 26 to 27° C. and a relative humidity of 78 to 81% to obtain activated Poria cocos; the activated Poria cocos is cultured in a potato liquid culture medium at 26 to 27° C. and a shaking table at a rotation speed of 170 to 185 r / min for 3 to 4 days to obtain a Poria cocos fermentation liquid.

[0041] In the present invention, the liquid-solid ratio of the Poria cocos fermentation liquid and the Astragalus powder can be 1:2 to 5, preferably 1:2 to 4, and more preferably 1:2 to 3. In this way, the Astragalus medicinal material can be effectively fermented, the strain waste can be avoided, and a better fermentation effect can be obtained.

[0042] In the present invention, preferably, the Astragalus membranaceus is fermented by solid culture; the fermentation time can be 6 to 12 days, preferably 8 to 11 days, and more preferably 9 to 10 days. This can ensure the activity of Poria cocos, give full play to the fermentation effect, and make the fermentation product more effective and active.

[0043] In the present invention, the fermentation temperature may be 24-29° C., preferably 25-28° C., and more preferably 26-28° C. This can ensure the activity of Poria cocos, give full play to the fermentation effect, and make the fermentation product have higher effective ingredients and activity.

[0044] <Application of Astragalus Fermented Bacteria>

[0045] The astragalus fermented bacteria of the present invention has significant antioxidant capacity. The antioxidant capacity is measured by DPPH free radical scavenging rate and ABTS+ free radical scavenging rate. Therefore, the present invention provides the use of the astragalus fermented bacteria in the preparation of antioxidant drugs.

[0046] <Application of Poria cocos fermented liquid>

[0047] Astragaloside IV is one of the main active ingredients in Astragalus and is the main criterion for evaluating the quality of Astragalus. The conversion rate of Astragaloside IV in Astragalus fermented with Poria fermentation liquid is significantly improved. Therefore, the present invention provides a use of Poria in improving the conversion rate of Astragaloside IV in the fermentation process of Astragalus.

[0048] The test method is described below. The components in the fermented astragalus obtained by the above method were determined by high performance liquid chromatography, and the test conditions were as follows:

[0049] Flavonoids determination

[0050] Phenomenex C18 column (4.60×250 mm, 5 μm) was used to quantitatively analyze the calycosin, calycosin glycosides, genistin, formononetin and formononetin in the sample. Liquid phase conditions: mobile phase: A is 0.05% formic acid aqueous solution, B is acetonitrile; gradient elution program was used for elution, gradient conditions: 0-5min, 0-5% B; 5-10min, 5-20% B; 10-25min, 20-25% B; 25-35min, 25-30% B; 35-40min, 30-35% B; 40-54min, 35-40% B; 54-55min, 40-0% B; flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 10μL; detection wavelength: 254nm.

[0051] Saponin determination

[0052] Phenomenex C18 column (4.60×250 mm, 5 μm) was used to quantitatively analyze astragaloside IV and II in the sample. Liquid phase conditions: mobile phase: A is acetonitrile, B is ultrapure water; gradient elution program was used for elution, gradient conditions: 0-20 min, 96%-80% (B); 20-40 min, 80%-70% (B); 40-65 min, 70%-40% (B); 65-70 min, 40-96% (B); 70-80 min, 96% (B); flow rate: 1.0 mL / min; column temperature: 25°C; injection volume: 20 μL; the drift tube temperature of ELSD was set to 100°C, and the carrier gas flow rate was 2.5 L / min.

[0053] Determination of DPPH free radical scavenging rate

[0054] (1) Dry the sample to a solid state;

[0055] (2) Accurately weigh 8 mg of DPPH reagent in a dark place, dissolve it in anhydrous ethanol, transfer it to a 100 mL volumetric flask, add anhydrous ethanol to the mark, and shake well to obtain a 0.08 mg / mL DPPH anhydrous ethanol solution;

[0056] (3) Accurately weigh 8 mg of dry sample into a centrifuge tube, add 8 mL of anhydrous ethanol to dissolve, and obtain a 1 mg / mL sample stock solution. Dilute the sample stock solution to the required concentration. Accurately pipette 100 μL of the sample solution into a centrifuge tube, add 1 mL of DPPH anhydrous ethanol solution, make up to 4 mL with anhydrous ethanol, shake well, let stand for 30 min, and detect the absorbance at 517 nm, which is recorded as A1. Accurately pipette 100 μL of the sample solution into a centrifuge tube, make up to 4 mL with anhydrous ethanol, and detect the absorbance at 517 nm, which is recorded as A2. Pipette 1 mL of DPPH anhydrous ethanol solution, add 3 mL of anhydrous ethanol, and detect the absorbance at 517 nm, which is recorded as A3. Use Vc as a positive control. Calculate the DPPH free radical scavenging rate of the sample according to the following formula.

[0057] DPPH free radical scavenging rate (%) = 100 × (A3 + A2 – A1) / A3

[0058] Determination of ABTS free radical scavenging rate

[0059] (1) Dry the sample to a solid state;

[0060] (2) Accurately weigh 0.3841g of 2,2-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt into a beaker and add 100mL of water. Accurately weigh 0.0662g of potassium persulfate into a beaker and add 100mL of water. Mix the two, shake well, and react in the dark for 12 to 16 hours to obtain ABTS+ free radical solution. Dilute it with water so that its absorbance A value at 734nm is 0.700±0.02. This solution is used as ABTS+ working solution.

[0061] (3) Accurately weigh 12 mg of the dry sample into a centrifuge tube and add 1 mL of water to obtain a 12 mg / mL sample stock solution. Dilute the sample stock solution to the required concentration. Pipette 0.1 mL of sample solutions of different concentrations into a centrifuge tube, add 4 mL of ABTS+ working solution, shake well, let stand for 6 minutes, and detect the absorbance at 734 nm, which is recorded as A. Pipette 0.1 mL of water, add 4 mL of ABTS+ working solution, shake well, let stand for 6 minutes, and detect the absorbance at 734 nm, which is recorded as A0. Pipette sample solutions of different concentrations, detect the absorbance at 734 nm, and record it as B. Use Vc as a positive control. Calculate the ABTS+ free radical scavenging rate of the sample according to the following formula.

[0062] ABTS + free radical scavenging rate (%) = [A0-(AB)] / A0×100

[0063] Here are the raw materials:

[0064] Poria cocos: Poria cocos bio-08656, purchased from Beijing Biobo Biotechnology Co., Ltd., Room 432, 4th Floor, Building 3, No. 16, Xinzhong North Street, Shunyi District, Beijing.

[0065] Preparation Example

[0066] The Poria cocos was inoculated on a potato dextrose agar (PDA) slant culture medium, and placed in a constant temperature and humidity chamber at 27°C and a relative humidity of 80% for activation culture for 5 days to obtain activated Poria cocos; then the activated Poria cocos was picked up with an inoculation loop and placed in a potato liquid culture medium, and cultured at 27°C and 180 r / min shaking conditions for 4 days to obtain Poria cocos fermentation liquid.

[0067] Example 1

[0068] 20 parts by weight of astragalus powder with a particle size of 60 meshes were weighed and placed in a solid fermentation bag, and then 10 parts by weight of water were added to the solid fermentation bag, and the bag was sterilized by high pressure at 121° C. for 25 minutes to obtain a first mixture.

[0069] 8 parts by weight of the Poria cocos fermentation liquid obtained in the preparation example were inoculated into the first mixture in a clean bench according to the sterile requirements, and solid-state cultured at 27° C. in a constant temperature incubator for 10 days to obtain the Astragalus fermentation fungus.

[0070] Example 2

[0071] Except for the raw material ratio of the fermented astragalus, the rest of the preparation process is exactly the same as that of Example 1:

[0072] The fermented astragalus is prepared from the following raw materials: 20 parts by weight of astragalus, 9 parts by weight of Poria cocos fermentation liquid and 25 parts by weight of water.

[0073] Comparative Example 1

[0074] The solid state culture time was 15 days, and the other conditions were exactly the same as in Example 1.

[0075] Comparative Example 2

[0076] The remaining conditions were exactly the same as those in Example 1 except that the Tricholoma acuminata was replaced by Trametes chinensis.

[0077] Comparative Example 3

[0078] Except that the Astragalus was not fermented, the other conditions were exactly the same as those in Example 1.

[0079] The active ingredients of the fermented astragalus of Examples 1-2 and Comparative Examples 1-3 were determined using astragaloside IV as a standard indicator. The results are shown in Table 1.

[0080] Table 1

[0081]

[0082] Experimental Example 1 Evaluation of the efficacy of fermented Astragalus

[0083] The antioxidant capacity of the fermented Astragalus obtained in Example 1 was tested and compared with that of the unfermented Astragalus. Taking Vc as a reference, the antioxidant capacities of DPPH and ABTS were as follows: Figure 1 and Figure 2 shown.

[0084] The above experiments show that the fermented astragalus of the present invention has good antioxidant capacity as a whole. The DPPH free radical scavenging rate and ABTS+ free radical scavenging rate of the fermented astragalus are 63.81% and 98.60% respectively, which are 24% and 43% higher than those of the unfermented astragalus.

[0085] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. A fermented astragalus membranaceus, characterized in that: The astragalus fermentation bacteria are prepared by the following method: 15-25 parts by weight of astragalus, 4-12 parts by weight of Poria cocos fermentation liquid and 5-25 parts by weight of water are fermented at 24-29° C. for 6-12 days.

2. The astragalus fermentation bacteria according to claim 1, characterized in that The Poria cocos fermentation broth is prepared by the following method: The method comprises the following steps: inoculating the Poria cocos on a potato glucose agar slant culture medium, activating and culturing the Poria cocos for 3 to 6 days at 24 to 29 DEG C and a relative humidity of 70 to 90%, so as to obtain activated Poria cocos; and fermenting the activated Poria cocos in a potato liquid culture medium for 2 to 5 days at a fermentation temperature of 24 to 29 DEG C and a rotation speed of 150 to 200 r / min on a shaking table, so as to obtain a Poria cocos fermentation liquid.

3. A method for preparing astragalus fermentation bacteria, characterized in that: The steps include: 15-25 parts by weight of astragalus, 4-12 parts by weight of Poria cocos fermentation liquid and 5-25 parts by weight of water are fermented at 24-29° C. for 6-12 days to obtain astragalus fermentation fungus.

4. The preparation method according to claim 3, characterized in that: The astragalus root and water are mixed and then sterilized to obtain a first mixture; the Poria cocos fermentation liquid is inoculated into the first mixture and fermented to obtain the astragalus fermentation fungus substance.

5. The preparation method according to claim 4, characterized in that: The following steps are also included: The method comprises the following steps: inoculating the Poria cocos on a potato glucose agar slant culture medium, activating and culturing the Poria cocos for 3 to 6 days under the conditions of a temperature of 24 to 29 DEG C and a relative humidity of 70 to 90%, so as to obtain activated Poria cocos; and fermenting the activated Poria cocos in a potato liquid culture medium for 2 to 5 days under the conditions of a fermentation temperature of 24 to 29 DEG C and a rotation speed of 150 to 200 r / min on a shaking table, so as to obtain a Poria cocos fermentation liquid.

6. The preparation method according to claim 5, characterized in that: The weight ratio of astragalus to water is 1 to 4:

1.

7. The preparation method according to claim 5, characterized in that: The liquid-to-solid ratio of the Poria cocos fermentation liquid and Astragalus membranaceus is 1:2-5.

8. Use of the Astragalus fermentation microbiota according to any one of claims 1 to 2 in the preparation of antioxidant drugs.

9. A use of Poria cocos to increase the content of astragaloside IV in the fermentation process of Astragalus, characterized in that: 15-25 parts by weight of astragalus, 4-12 parts by weight of Poria cocos fermentation liquid and 5-25 parts by weight of water are fermented at 24-29° C. for 6-12 days to obtain astragalus fermentation fungus substance.

Images