Processing method for fermenting astragalus membranaceus by utilizing probiotics

Abstract

The invention provides a processing method for fermenting astragalus membranaceus by utilizing probiotics, and aims to solve the problems that the yield of total flavonoids, total saponins and total polysaccharides in astragalus membranaceus cannot be effectively improved and strains are difficult to play a role to the greatest extent in the prior art. The processing method comprises the following steps: culturing a freeze-dried probiotic strain in a solid culture medium at 37 DEG C for 24 hours by adopting a plate streaking method, transferring the freeze-dried probiotic strain into a liquid culture medium for culturing at 30 DEG C for 24 hours after microscopic examination, and continuously activating for three generations for later use; then, the astragalus membranaceus slices are placed in a fermentation culture medium, the fermentation culture medium is inoculated with the activated probiotics according to the inoculation amount of 3% for static light-shielding fermentation, stirring is conducted uniformly every 24 h during the fermentation, sampling is conducted from a fermentation tank regularly, the change of the content of active ingredients in the astragalus membranaceus is monitored through the HPLC technology, and the optimal fermentation technology is determined. The contents of total flavonoids, total polysaccharides and total saponins in the astragalus membranaceus fermented by the method disclosed by the invention are greatly increased.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation traditional Chinese medicine, and in particular to a method for preparing astragalus by fermenting it with probiotics. Background Art

[0002] Astragalus is a traditional tonic Chinese medicine. In recent years, studies have found that the main active ingredients in Astragalus are astragalus polysaccharides, astragalosides, bioflavonoids, as well as monosaccharides, amino acids and trace elements. They all have multiple biological activities, including anti-inflammatory, antiviral, antioxidant and immune-enhancing effects. They are widely used in humans, livestock and other livestock. At present, the extraction rate of the medicinal ingredients of Astragalus is low, and the utilization efficiency of Astragalus needs to be improved.

[0003] Compared with traditional Chinese medicine processing methods, the main advantages of microbial biotransformation technology for Chinese medicine are: (1) mild conditions. The water extraction temperature is generally around 100°C, while the fermentation processing temperature is generally not more than 40°C. Mild conditions have little damage to the medicinal properties of Chinese medicine; (2) increase the efficacy. Fermentation processing can also increase the efficacy of some Chinese medicines and produce some new effects. For example, after fermentation, Baiyao decoction can enhance the antitussive, expectorant, anti-inflammatory and antibacterial effects; (3) produce new metabolites. For example, the metabolites derived from fermentation of Chinese medicine have biological activity. These "post-metabolites" include fatty acids, polyamines, polyphosphates, proteins and peptides. (4) reduce toxicity. Some Chinese medicines are toxic and need to be processed before they can be used. For example, Pinellia ternata irritates the mucous membranes of animals. After fermentation processing, the toxicity will be reduced.

[0004] Probiotics are active microorganisms that can produce health benefits when consumed in sufficient amounts. Maintaining adequate levels of probiotics in the body has been shown to support immune function, aid digestive health, and even boost metabolism. Screening specific probiotics to act on astragalus in vitro, using the enzymes secreted by probiotics to break down the cell walls of traditional Chinese medicine, promote the release of active ingredients, and convert some macromolecules into small molecules, can significantly improve the utilization rate of astragalus.

[0005] In the existing method of using biological fermentation technology to improve the effective ingredients and antioxidant activity of Astragalus, due to the fermentation process and strain selection, it is impossible to effectively improve the yield of total flavonoids, total saponins and total polysaccharides in Astragalus, and it is difficult to maximize the role of the strain. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of the prior art. By fermenting astragalus with probiotics, the release and transformation of effective ingredients can be provided through biotransformation; secondly, the fermentation process parameters are optimized to maximize the effect of the probiotics.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing astragalus by fermenting probiotics. The method comprises the following steps: firstly culturing the probiotic freeze-dried strain in a solid culture at 37°C for 24 hours by a plate streak method, transferring the strain to a liquid culture medium after microscopic examination to culture it again at 30°C for 24 hours after no foreign bacteria are found, and then continuously activating it for three generations for standby use; then placing the astragalus slices in a fermentation culture medium, inoculating the activated probiotics into the fermentation culture medium at an inoculation rate of 3% for static fermentation in the dark, stirring the culture medium evenly every 24 hours, taking samples from a fermentation tank regularly, monitoring the changes in the content of active ingredients in the astragalus by HPLC technology, and determining the optimal fermentation process.

[0008] As the preferred technical solution: Optionally, the freeze-dried probiotic strain is one or more of a freeze-dried strain of Bacillus subtilis, a freeze-dried strain of Saccharomyces cerevisiae or a freeze-dried strain of Lactobacillus plantarum, and the light-proof fermentation time is 5 to 7 days.

[0009] Optionally, the optimal fermentation process is: the best strain is Saccharomyces cerevisiae or a mixed strain of Bacillus subtilis and Saccharomyces cerevisiae, and the optimal fermentation time is 6 days; the total flavonoids content in Astragalus on the 6th day of fermentation reaches a maximum of 2.14 mg / g, an increase of 57.35% compared with unfermented Astragalus; the total polysaccharide content reaches a maximum of 90.94 mg / g, an increase of 11.84% compared with the polysaccharide content of unfermented Astragalus; the total saponin content reaches a maximum of 11.32 mg / g, an increase of 14.98% compared with the saponin content of unfermented Astragalus.

[0010] Optionally, the method specifically includes the following steps: S1. Bacteria Activation Take out the lyophilized probiotic strain from the strain collection center, inoculate the lyophilized probiotic strain into MRS or PDA solid culture medium by plate streaking method, culture at 37°C for 24 hours, transfer it to MRS or PDA liquid culture medium after no foreign bacteria are found by microscopic examination, and culture it again at 30°C for 24 hours; activate it for three generations continuously for standby use; S2. Fermentation Place the dried slices of Astragalus in a fermentation medium, inoculate the activated probiotics into the fermentation medium, set the fermentation temperature to 37°C, adjust the pH value to 6.5, control the ventilation volume to maintain a micro-aerobic environment, ferment statically in the dark, and stir evenly every 24 hours to ensure full contact between the strain and the Astragalus; S3. Fermentation process monitoring The temperature, pH value and dissolved oxygen parameters during the fermentation process were monitored in real time using sensors and automatically adjusted to maintain optimal fermentation conditions. Samples were taken from the fermentation tank regularly and the changes in the content of active ingredients in Astragalus were monitored using HPLC technology to determine the optimal fermentation process. S4. Draw the standard curve of the active ingredients of Astragalus The fermented astragalus samples were used to draw standard curves of total flavonoids, total polysaccharides and total saponins by ultraviolet detection method; S5. Optimization and Adjustment The fermentation conditions were optimized according to the standard curves of total flavonoids, total polysaccharides and total saponins.

[0011] Optionally, in step S2, Inoculating the activated probiotics into the fermentation medium means inoculating the activated Bacillus subtilis into the fermentation medium No. 1 and marking it as sample 1; Saccharomyces cerevisiae was inoculated into fermentation medium No. 2 and marked as sample 2; Lactobacillus plantarum was inoculated into fermentation medium No. 3 and marked as sample 3; A mixed strain of activated Bacillus subtilis and Saccharomyces cerevisiae in a mass ratio of 1:1 was inoculated into fermentation medium No. 4 and marked as sample 4; A mixed strain of activated Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1 was inoculated into fermentation medium No. 5 and marked as sample 5; A mixed strain of activated Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1 was inoculated into fermentation medium No. 6 and marked as sample 6; A mixed strain of Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1:1 was inoculated into fermentation medium No. 7 and marked as sample 7.

[0012] Optionally, in step S2, the stirring speed is set to 150 rpm and the stirring time is set to 30 min.

[0013] Optionally, in step S4, the method for processing the astragalus sample is to pre-treat the fermented astragalus after the fermentation is completed, dry it in an oven at 40° C. to constant weight, and grind it into powder.

[0014] Optionally, the pretreatment refers to adding enzymes to the fermented astragalus after fermentation, enzymatic hydrolysis and stabilization treatment, centrifuging, taking the supernatant, sterilizing or adding a preservative.

[0015] Optionally, the enzyme used for enzymatic hydrolysis is selected from one or more of cellulase, β-glucanase, pectinase, protease, amylase or lipase; the temperature of the enzymatic hydrolysis is 50-60°C, the time is 5-10h, and the temperature of the stabilization treatment is 90-100°C, the time is 10-15min.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The preparation method of probiotic fermented astragalus of the present invention has a simple process. Probiotic fermentation of astragalus is used, preferably activated brewer's yeast is used to ferment astragalus, which can greatly increase the content of active ingredients such as calycosin glucoside, calycosin isoflavones, formononetin and the like in astragalus.

[0017] 2. On the one hand, the present invention establishes an ultraviolet detection method and standard curve for total flavonoids, total saponins and polysaccharides, and on the other hand, establishes an HPLC-UV detection method for four flavonoid components (calycosin glucoside, calycosin, formononetin, formononetin), and then dynamically detects the changes in the content of each component at different fermentation times of Astragalus.

[0018] 3. The in vitro antioxidant activity of Astragalus membranaceus fermented by the method of the present invention before and after fermentation was compared, and the antioxidant activity was reflected by DPPH scavenging rate, hydroxyl radical scavenging rate and total reducing power. The experimental results showed that the antioxidant activity of fermented Astragalus membranaceus was greatly improved compared with that of unfermented Astragalus membranaceus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is a flow chart of the preparation method of the present invention; FIG2 is a standard curve of the total flavonoids, total saponins and total polysaccharides content of the fermented Astragalus sample prepared by the method of the present invention, wherein (a) is the standard curve of total flavonoids, (b) is the standard curve of total polysaccharides, and (c) is the standard curve of total saponins; Figure 3 is a comparison of the yields of total flavonoids, total polysaccharides and total saponins of fermented Astragalus of samples 1 to 7, wherein (a) is a comparison of the content of total flavonoids at different fermentation times, (b) is a comparison of the content of total polysaccharides at different fermentation times, and (c) is a comparison of the content of total saponins at different fermentation times; Figure 4 This is the HPLC spectrum of Astragalus before and after fermentation; Figure 5 shows the in vitro DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and in vitro total reducing power (OD 700 ) at different concentrations. DETAILED DESCRIPTION

[0021] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0022] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0023] The present invention provides a method for preparing Astragalus membranaceus by fermenting probiotics, such as Figure 1 As shown, the specific steps are as follows: S1. Bacteria Activation Take out the lyophilized probiotic strain from the strain collection center, inoculate the lyophilized probiotic strain into MRS or PDA solid culture medium by plate streaking method, culture at 37°C for 24 hours, transfer it to MRS or PDA liquid culture medium after no foreign bacteria are found by microscopic examination, and culture it again at 30°C for 24 hours; activate it for three generations continuously for standby use; The probiotic freeze-dried strain is one or more of a freeze-dried strain of Bacillus subtilis, a freeze-dried strain of Saccharomyces cerevisiae or a freeze-dried strain of Lactobacillus plantarum. The strain of Bacillus subtilis is a commercial strain of Bacillus subtilis ATCC6633; the strain of Saccharomyces cerevisiae is a commercial strain of Saccharomyces cerevisiae ATCC9763; and the strain of Lactobacillus plantarum is a commercial strain of ATCC8014. S2. Fermentation The dried slices of Astragalus were placed in a fermentation medium, and activated Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum were inoculated into the fermentation medium at an inoculum size of 3%, respectively. The fermentation temperature was set to 37°C, the pH value was adjusted to 6.5, the ventilation volume was controlled to maintain a micro-aerobic environment, and the culture was fermented statically and in the dark for 5-7 days, and the culture was stirred evenly every 24 hours to ensure that the strains were in full contact with the Astragalus. The stirring speed was set to 150 rpm and the stirring time was 30 min during the stirring. S3. Fermentation process monitoring The temperature, pH value and dissolved oxygen parameters during the fermentation process were monitored in real time using sensors and automatically adjusted to maintain optimal fermentation conditions. Samples were taken from the fermentation tank regularly and the changes in the content of active ingredients in Astragalus were monitored using HPLC technology to determine the optimal fermentation process. S4. Draw the standard curve of the active ingredients of Astragalus The fermented astragalus samples were used to draw standard curves of total flavonoids, total polysaccharides and total saponins by ultraviolet detection method; The preparation method of the astragalus sample is to spray-dry the fermented astragalus after pretreatment and then package it into powder or granule form, specifically, add one or more of cellulase, β-glucanase, pectinase, protease, amylase or lipase to the fermented astragalus after fermentation, perform enzymolysis at 50-60°C for 5-10h, stabilize it at 90-100°C for 10-15min, centrifuge it, take the supernatant, sterilize it or add a preservative, dry it in an oven at 40°C to constant weight, and grind it into powder; S5. Optimization and Adjustment The fermentation conditions were optimized according to the standard curves of total flavonoids, total polysaccharides and total saponins.

[0024] The following is a detailed description of the embodiments: Example

[0025] A method for preparing astragalus by fermenting probiotics, the specific steps are as follows: (1) Activation of bacterial strains The frozen Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum were cultured in solid culture and liquid culture respectively by plate streak method, specifically, Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum were first inoculated into MRS / PDA solid culture medium, cultured at 37°C for 24 hours, and then transferred to MRS / PDA liquid culture medium after microscopic examination without foreign bacteria, and cultured again at 30°C for 24 hours; activated three generations continuously for standby use; (2) Fermentation The activated Bacillus subtilis was inoculated into fermentation medium No. 1 and marked as sample 1; The activated Saccharomyces cerevisiae was inoculated into fermentation medium No. 2 and marked as sample 2; The activated Lactobacillus plantarum was inoculated into fermentation medium No. 3 and marked as sample 3; A mixed strain of activated Bacillus subtilis and Saccharomyces cerevisiae in a mass ratio of 1:1 was inoculated into fermentation medium No. 4 and marked as sample 4; A mixed strain of activated Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1 was inoculated into fermentation medium No. 5 and marked as sample 5; A mixed strain of activated Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1 was inoculated into fermentation medium No. 6 and marked as sample 6; A mixed strain of Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1:1 was inoculated into fermentation medium No. 7 and marked as sample 7.

[0026] The samples 1 to 7 and the astragalus slices were fermented in a fermentation medium in a static dark environment for 5 to 7 days, and stirred evenly every 24 hours to ensure that the strain and the astragalus were fully in contact. During the fermentation, the temperature in the fermentation tank was maintained at 37°C, the pH value was adjusted to 6.5, and the ventilation volume was controlled to maintain a micro-oxygen environment. During this period, the fermented astragalus in the fermentation tanks of samples 1 to 7 was sampled every day; (3) Pretreatment of each group of samples Take 200 ml of fermented astragalus, add 5 ml of cellulase, perform enzymolysis at 50°C for 8 hours, stabilize at 100°C for 10 minutes, centrifuge, take the supernatant, sterilize or add preservatives, dry in an oven at 40°C to constant weight, and grind; (4) Total flavonoids extraction and standard curve drawing, the method is as follows: Weigh 1g of fermented Astragalus sample powder (passed through a 40-mesh sieve) accurately, place it in a stoppered conical flask, add 10mL of 75% ethanol, and perform ultrasonic extraction at 75°C for 30min, repeat 3 times. Centrifuge, collect the supernatant, and dilute to 30mL. Accurately pipette 0.0, 0.25, 0.50, 1.00, 2.00, 3.00, 4.00, and 6.00 mL of 0.2mg / mL rutin standard solution, place them in 25mL colorimetric tubes, fill them up to 5mL with 60% ethanol solution, add 1mL of sodium nitrite, shake well, let stand for 6min, add 1.5mL of aluminum nitrate, shake well, let stand for 6min, add 4mL of sodium hydroxide, dilute to the mark with ethanol, shake well, and let stand for 15min. Zero with blank (without aluminum nitrate), and measure the absorbance at a wavelength of 510nm.

[0027] With absorbance Y as the ordinate and total rutin concentration X as the abscissa, a standard curve was drawn and linear fitting was performed using the least squares method to obtain the linear regression equation and correlation coefficient R between X and Y. 2 .

[0028] The standard curve was drawn based on the absorbance value corresponding to the content as follows: Y = 0.009640*X + 3.151e-005, R 2 =0.9996; The standard curve of total flavonoids represented by rutin in the active ingredients of fermented Astragalus is shown in Figure 2(a).

[0029] (5) Total polysaccharide extraction and standard curve drawing are as follows: Take about 1g of fermented astragalus powder (passed through a 40-mesh sieve), weigh it accurately, place it in a stoppered conical flask, add 10mL of purified water accurately, weigh the mass, extract it by ultrasonic at 60℃ for 30min, repeat 3 times, centrifuge, and collect the supernatant. Add anhydrous ethanol to the supernatant to a final concentration of 80%, let it stand at room temperature overnight, and centrifuge. Add water to the precipitate, heat to dissolve, and make up to 50mL. Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of 0.1mg / mL standard glucose solution, and make up to 1.0 mL with distilled water; first add 1 mL of 5% phenol solution, and quickly add 5 mL of sulfuric acid vertically, shake it immediately and let it stand for 10 min; heat it in a boiling water bath for 15min, quickly cool it with cold water, and let it stand at room temperature for 20min, and measure its absorbance at 490nm.

[0030] With absorbance Y as the ordinate and glucose concentration X as the abscissa, a standard curve was drawn and linear fitting was performed using the least squares method to obtain the linear regression equation and correlation coefficient R between X and Y. 2 .

[0031] Draw the standard curve as follows: Y = 0.01501*X + 0.03360, R 2 =0.9992; The standard curve of total polysaccharides represented by glucose in the active ingredients of fermented Astragalus is shown in Figure 2(b).

[0032] (6) Total saponin extraction and standard curve drawing, the method is as follows: Take about 1g of fermented astragalus powder (passed through a 40-mesh sieve), weigh it accurately, place it in a stoppered conical flask, add 10mL of methanol accurately, and extract it by ultrasonic at 75℃ for 30min. Repeat 3 times. Centrifuge, collect the supernatant, and make up to 30mL. Recover methanol by reflux at 65℃ under reduced pressure, add 30mL of water to the residue, dissolve it in a 70℃ water bath, extract it with chloroform twice to remove chlorophyll and anthraquinone substances, and then shake and extract it with water-saturated n-butanol 3 times, 30mL each time. Combine the n-butanol solution, wash it with 40mL of 10% ammonia water twice, discard the ammonia solution, and evaporate it to dryness under reduced pressure at 60℃. Dissolve the residue in methanol and dilute it to an appropriate concentration. According to the vanillin-perchloric acid method for color development test, accurately weigh 5mg of astragaloside IV reference substance, add methanol to make up to 25mL. Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL into a test tube, evaporate the solvent in a 60 ℃ water bath, add 0.2 mL of freshly prepared 5% vanillin glacial acetic acid solution and 0.8 mL of perchloric acid, incubate in a 60 ℃ water bath for 15 min, cool in an ice-water bath, add 5 mL of glacial acetic acid, shake well, and use the reagent as a blank control. Scan in the wavelength range of 400 to 700 nm on a double-beam UV-visible spectrophotometer, and the screening measurement wavelength is 543 nm.

[0033] The standard curve was drawn with the absorbance Y at 543 nm as the ordinate and the mass of astragaloside IV as the abscissa. The linear regression equation and correlation coefficient R between X and Y were obtained by the least squares method. 2 .

[0034] Draw the standard curve as follows: Y = 0.0001360*X + 0.006349, R 2 =0.9963.

[0035] The standard curve of total saponins represented by astragaloside IV in the active ingredients of fermented Astragalus is shown in Figure 2(c). Example

[0036] The total flavonoids, total polysaccharides and total saponins were extracted from each fermented astragalus sample at different fermentation stages of samples 1 to 7 using the method of steps (4) to (6) of Example 1. The content of total flavonoids, total polysaccharides and total saponins in each sample was then determined, and the yields of total flavonoids, total polysaccharides and total saponins of astragalus fermented by single bacteria and mixed bacteria were calculated, as shown in Figures 3 (a) to (c).

[0037] According to the test results, when single bacteria is cultured, the best strain is Saccharomyces cerevisiae, and the best fermentation time is 6 days; the total flavonoids content in the fermented astragalus sample on the 6th day of fermentation (i.e. fermented sample 4 inoculated with Bacillus subtilis and Saccharomyces cerevisiae) reached a maximum of 2.14 mg / g, which was 57.35% higher than that of the unfermented astragalus; the total polysaccharide content in the fermented astragalus sample on the 6th day of fermentation (i.e. fermented sample 2 inoculated with Saccharomyces cerevisiae) reached a maximum of 90.94 mg / g, which was 11.84% higher than that of the unfermented astragalus; the total saponin contents of samples 1, 2, 3 and 4 were relatively large on the 6th day of fermentation, which were 9.55 mg / g, 10.88 mg / g, 9.99 mg / g and 11.32 mg / g respectively. The total saponin content of sample 4 (i.e. fermented astragalus inoculated with Bacillus subtilis and Saccharomyces cerevisiae) reached the maximum, which was 14.98% higher than that of the unfermented astragalus. Therefore, the contents of astragalus polysaccharides, saponins and flavonoids were used as evaluation indicators, and Saccharomyces cerevisiae or a mixture of Bacillus subtilis and Saccharomyces cerevisiae was the best strain. Example

[0038] The fermented Astragalus sample in sample 4 on the 6th day of fermentation was selected as the test sample by the method of Example 1, and the changes of the active ingredients in Astragalus before and after fermentation were monitored by HPLC method, specifically: (1) Selecting acetonitrile, calycosin glucoside, formononetin, calycosin and formononetin as reagents; (2) Chromatographic conditions: Chromatographic column: Ultimate XB-Phenyl C18 (4.6 mm × 250 mm, 5 µm); Mobile phase: acetonitrile as mobile phase A, 0.2% phosphoric acid aqueous solution as mobile phase B, elution according to the following elution order and elution ratio, detection wavelength 210~400nm, column temperature 30℃. The elution process is shown in Table 1: Table 1

[0039] (3) Preparation of reference substances Take appropriate amount of four reference substances, namely, calycosin glucoside, formononetin, calycosin and formononetin, and prepare standard stock solutions with concentrations of 0.500 mg / mL, 0.500 mg / mL, 0.500 mg / mL, and 0.800 mg / mL, respectively. Accurately pipette the mother solutions and add methanol to prepare mixed reference substance solutions with concentrations of 0.250 mg / mL, 0.250 mg / mL, 0.250 mg / mL, and 0.400 mg / mL.

[0040] (4) Experimental results 4.1 Fingerprint The fingerprints of fermented astragalus samples and unfermented astragalus (i.e. blank astragalus) were obtained by HPLC method. Figure 4 The specific values ​​are shown in Table 2 below: Table 2

[0041] As shown in Table 2, there are 11 peaks after fermentation of Astragalus, of which 6 peaks have increased peak area, 4 peaks have decreased peak area, and the peak at 5.145min disappears after fermentation, and a new peak is added at 22.81min. The peak area before and after is not much different, which is speculated to be the transformation of substances during the fermentation process. In general, the total peak area after fermentation is higher than that before fermentation, which corresponds to the increase in total flavonoid content after fermentation.

[0042] The fingerprints of the four groups of reference substances were obtained by HPLC method, and the specific values ​​are shown in Table 3 below: Table 3

[0043] As shown in Table 3, after yeast fermentation of Astragalus, the content of formononetin decreased, which was presumably converted into other flavonoid components or degraded, and the contents of calycosin glucoside, calycosin and formononetin increased. It can be seen that the processing method of the present invention can effectively increase the active ingredients of Astragalus. Example

[0044] The method of Example 1 was used to select the fermented astragalus sample in sample 4 on the 6th day of fermentation as the test sample, and the in vitro antioxidant activity of the fermented astragalus sample was monitored, specifically: (1) DPPH, salicylic acid, FeSO4, H2O2, anhydrous ethanol, PBS buffer (concentration 0.01 mol / L, pH 7.2-7.4), trichloroacetic acid, potassium ferrocyanide, FeCl3 and distilled water were selected as reagents; (2) Determination of the ability of fermented Astragalus to scavenge DPPH free radicals in vitro Weigh 1 g of fermented Astragalus sample powder (passed through a 40-mesh sieve) accurately, place in a stoppered conical flask, add 10 mL of 75% ethanol, perform ultrasonic extraction at 75°C for 30 min, repeat 3 times, centrifuge, collect the supernatant, and use it as the sample solution for later use; Take 1 mL of the sample solution, mix it with 1 mL of DPPH solution and shake it evenly, record it as sample A, place it at room temperature away from light for 30 minutes, and measure the absorbance at 517 nm; 1 mL of the sample solution was mixed with 1 mL of anhydrous ethanol and shaken well, which was recorded as blank sample A. After being placed in the dark at room temperature for 30 min under the same conditions, the absorbance was measured at 517 nm. Mix 1 mL of anhydrous ethanol and 1 mL of DPPH solution evenly, record it as A control sample, place it at room temperature and away from light for 30 minutes under the same conditions, and measure the absorbance at 517 nm; The calculation formula of DPPH clearance rate (%) is as follows: ; in, A x It indicates the absorbance value of sample A at 517nm; A xo Indicates the absorbance value of blank sample A at 517nm; A 0 It represents the absorbance value of A control sample at 517nm.

[0045] (3) Determination of the ability of fermented astragalus to scavenge hydroxyl radicals in vitro Weigh 1 g of fermented Astragalus sample powder (passed through a 40-mesh sieve) accurately, place in a stoppered conical flask, add 10 mL of 75% ethanol, perform ultrasonic extraction at 75°C for 30 min, repeat 3 times, centrifuge, collect the supernatant, and use it as the sample solution for later use; In a stoppered conical flask, add 1 mL of salicylic acid, 1 mL of FeSO4 solution, 1 mL of sample solution and 8 mL of deionized water in sequence, and finally add 1 mL of H2O2 solution. Use it as sample D, start the reaction, react in a 37°C water bath for 15 min, and measure the absorbance at 510 nm.

[0046] In a stoppered conical flask, add 1 mL of salicylic acid, 1 mL of FeSO4 solution, 12 mL of deionized water, and finally 1 mL of H2O2 solution, i.e., add no sample solution. Use it as the D control sample, start the reaction, react in a 37°C water bath for 15 min, and measure the absorbance at 510 nm.

[0047] In a stoppered conical flask, add 1 mL of salicylic acid, 1 mL of FeSO4 solution, 1 ml of sample solution and 9 mL of deionized water in sequence. Do not add H2O2 solution. Use it as D blank sample. Start the reaction. React in a 37°C water bath for 15 min. Measure the absorbance at 510 nm.

[0048] The calculation formula of hydroxyl radical scavenging rate (%) is as follows: ; in, D x It represents the absorbance value of sample D at 510nm; D xo D represents the absorbance value of blank sample at 510nm; D 0 It represents the absorbance value of D control sample at 510nm.

[0049] (4) Determination of the total reducing power of fermented Astragalus in vitro Add 1.0mL of sample solution to a stoppered conical flask, then add 2.5mL of PBS buffer and 5.0mL of 1.0% potassium ferricyanide solution in sequence, shake well; react at a constant temperature of 50℃ in a water bath for 20min, and add 5.0mL of 10% trichloroacetic acid to terminate the reaction. Centrifuge the reaction solution at 3000r / min for 10min, take 2.5mL of supernatant, add 2.5mL of distilled water and 0.5mL of 10% FeCl3 solution and mix thoroughly, measure the absorbance at 700nm, the reducing power of the sample is positively correlated with the absorbance of the solution being tested, and the absorbance of the sample solution is used to evaluate its reducing power.

[0050] (5) Test results As shown in Figure 5 (a) to (c), compared with blank astragalus (i.e., unfermented astragalus), the DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, and total reducing power values ​​of the fermented astragalus sample extracted by 75% ethanol were higher than those of the unfermented astragalus within a certain concentration range. This indicates that yeast fermentation of astragalus can significantly enhance the antioxidant activity.

[0051] It can be seen that after the processing method of the present invention is used to ferment the astragalus with probiotics, the content of active ingredients is significantly improved, and the yield of total flavonoids, total polysaccharides and total saponins is significantly improved, making brewer's yeast as the best strain to maximize the effect of the effective ingredients of astragalus. In addition, the antioxidant activity of the fermented astragalus of the method of the present invention is greatly improved compared with that of the unfermented astragalus.

[0052] The above describes the specific embodiments of the present invention. The present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments may be combined with each other at will.

Claims

1. A method for preparing astragalus by fermenting with probiotics, characterized in that: The preparation method comprises the following steps: first culturing the freeze-dried probiotic strain at 37° C. for 24 hours in a solid culture medium by a plate streak method, transferring the strain to a liquid culture medium and culturing it again at 30° C. for 24 hours after microscopic examination for the presence of foreign bacteria, and continuously activating the strain for three generations for standby use; then placing the astragalus slices in a fermentation medium, inoculating the activated probiotics into the fermentation medium at an inoculation rate of 3% for static fermentation in the dark, stirring the strain evenly every 24 hours, taking samples from the fermentation tank regularly, monitoring the changes in the content of active ingredients in the astragalus by HPLC, and determining the optimal fermentation process.

2. The method for preparing astragalus by fermenting with probiotics according to claim 1, characterized in that: The probiotic freeze-dried strain is one or more of a freeze-dried strain of Bacillus subtilis, a freeze-dried strain of Saccharomyces cerevisiae or a freeze-dried strain of Lactobacillus plantarum, and the light-proof fermentation time is 5 to 7 days.

3. The method for preparing astragalus by fermenting with probiotics according to claim 2, characterized in that: The optimal fermentation process is as follows: the optimal strain is Saccharomyces cerevisiae or a mixed strain of Bacillus subtilis and Saccharomyces cerevisiae, and the optimal fermentation time is 6 days; the total flavonoids content in the astragalus on the 6th day of fermentation reaches a maximum of 2.14 mg / g, which is 57.35% higher than that of the unfermented astragalus; the total polysaccharide content reaches a maximum of 90.94 mg / g, which is 11.84% higher than that of the unfermented astragalus; the total saponin content reaches a maximum of 11.32 mg / g, which is 14.98% higher than that of the unfermented astragalus.

4. The method for preparing astragalus by fermenting with probiotics according to claim 1, characterized in that: The specific steps include: S1. Bacteria Activation Take out the lyophilized probiotic strain from the strain collection center, inoculate the lyophilized probiotic strain into MRS or PDA solid culture medium by plate streaking method, culture at 37°C for 24 hours, transfer it to MRS or PDA liquid culture medium after no foreign bacteria are found by microscopic examination, and culture it again at 30°C for 24 hours; activate it for three generations continuously for standby use; S2. Fermentation Place the dried slices of Astragalus in a fermentation medium, inoculate the activated probiotics into the fermentation medium, set the fermentation temperature to 37°C, adjust the pH value to 6.5, control the ventilation volume to maintain a micro-aerobic environment, ferment statically in the dark, and stir evenly every 24 hours to ensure full contact between the strain and the Astragalus; S3. Fermentation process monitoring The temperature, pH value and dissolved oxygen parameters during the fermentation process were monitored in real time using sensors and automatically adjusted to maintain optimal fermentation conditions. Samples were taken from the fermentation tank regularly and the changes in the content of active ingredients in Astragalus were monitored using HPLC technology to determine the optimal fermentation process. S4. Draw the standard curve of the active ingredients of Astragalus The fermented astragalus samples were used to draw standard curves of total flavonoids, total polysaccharides and total saponins by HPLC ultraviolet detection method; S5. Optimization and Adjustment The fermentation conditions were optimized according to the standard curves of total flavonoids, total polysaccharides and total saponins.

5. The method for preparing Astragalus membranaceus by fermenting it with probiotics according to claim 4, characterized in that: In step S2, Inoculating the activated probiotics into the fermentation medium means inoculating the activated Bacillus subtilis into the fermentation medium No. 1 and marking it as sample 1; Saccharomyces cerevisiae was inoculated into fermentation medium No. 2 and marked as sample 2; Lactobacillus plantarum was inoculated into fermentation medium No. 3 and marked as sample 3; A mixed strain of activated Bacillus subtilis and Saccharomyces cerevisiae in a mass ratio of 1:1 was inoculated into fermentation medium No. 4 and marked as sample 4; A mixed strain of activated Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1 was inoculated into fermentation medium No. 5 and marked as sample 5; A mixed strain of activated Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1 was inoculated into fermentation medium No. 6 and marked as sample 6; A mixed strain of Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1:1 was inoculated into fermentation medium No. 7 and marked as sample 7.

6. The method for preparing astragalus by fermenting with probiotics according to claim 4, characterized in that: In step S2, the stirring speed is set to 150 rpm and the stirring time is set to 30 min.

7. The method for preparing astragalus by fermenting with probiotics according to claim 4, characterized in that: In step S4, the method for processing the astragalus sample is to pre-treat the fermented astragalus after the fermentation is completed, dry it in an oven at 40° C. to constant weight, and grind it into powder.

8. The method for preparing Astragalus membranaceus by fermenting it with probiotics according to claim 4, characterized in that: The pretreatment refers to adding enzymes to the fermented astragalus after fermentation, enzymolysis and stabilization treatment, centrifugation, taking the supernatant, sterilization or adding preservatives.

9. A method for preparing astragalus by fermenting with probiotics according to claim 8, characterized in that: The enzyme used for enzymolysis is selected from one or more of cellulase, β-glucanase, pectinase, protease, amylase or lipase; the temperature of the enzymolysis is 50-60°C, the time is 5-10h, and the temperature of the stabilization treatment is 90-100°C, the time is 10-15min.

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