Astragalus membranaceus residue fermentation method, coriolus versicolor fermented astragalus membranaceus residue and application thereof

Abstract

The invention belongs to the technical field of feed additives, and particularly relates to an astragalus membranaceus residue fermentation method, coriolus versicolor fermented astragalus membranaceus residues and application thereof. According to the method disclosed by the invention, the astragalus membranaceus dregs are fermented by utilizing Trametes versicolor, and the contents of astragaloside, general flavone and soluble sugar are obviously increased after fermentation. And secondary fermentation is further coupled, and mixed bacteria of lactobacillus plantarum, enterococcus faecium and saccharomyces cerevisiae are used for performing secondary fermentation on the mixture of the coriolus versicolor fermented astragalus membranaceus dregs, the corn flour and the soybean meal, so that the content of total protein and acid-soluble protein in the feed is increased, the growth of probiotics is promoted, and the feed is high in nutrition and has an important application value. According to the invention, the reutilization of the Chinese herbal medicine astragalus residue is realized, and the effective components in the medicine can be fully utilized. The fermentation substrate is simple in component, low in fermentation cost, simple and convenient in preparation process, high in nutrition and good in popularization and application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, and in particular to a method for fermenting astragalus root residues, astragalus root residues fermented from versicolor rich in astragaloside IV, flavonoids and soluble sugar, and applications thereof. Background Art

[0002] Astragalus is the root of Astragalus mongolica or Astragalus membranaceus, a plant of the genus Astragalus in the leguminous family. It has anti-inflammatory, antioxidant, and immune-regulating functions. Astragalus is a tonic medicine and a medicinal material that is both a medicine and a food. It is included in the "Catalogue of Feed Raw Materials" issued by the Ministry of Agriculture and Rural Affairs. Astragalus residues are obtained by water extraction or alcohol extraction of Astragalus medicinal materials, which still contain rich active ingredients remaining in the Astragalus residues, especially flavonoids, astragaloside IV, soluble sugars and other beneficial ingredients. The residues can be used directly or developed appropriately to replace feed antibiotics, improve animal production performance, and enhance antioxidant properties. However, the content of remaining beneficial ingredients in the residues is low. Therefore, the secondary development of Astragalus residues, especially the redevelopment of the active ingredients therein, has received more and more attention.

[0003] Using microorganisms for fermentation to release active ingredients for a second time is a feasible solution. For example, in the study "Experimental Study on the Production of Functional Feed by Mixed Fermentation of Astragalus Residues", Xie Yijun et al. used Aspergillus niger, Bacillus subtilis, and Candida utilis to ferment Astragalus residues, which can increase the crude protein, amino acids, total flavonoids, total saponins, and isoflavones content of Astragalus residues, and degrade crude fiber, so that the feeding value is effectively improved. Liu Chao et al. disclosed in "Research Progress on Astragalus Fermentation Strains and Fermentation Processes" that the pharmacological function of Astragalus is further enhanced after microbial fermentation, and analyzed the fermentation strains disclosed in recent years, mainly probiotics, Ganoderma lucidum, edible and medicinal fungi, as well as molds and streptococci. Comprehensive existing studies show that the active ingredients of Astragalus can change after microbial fermentation. Some produce new active substances, some promote bacterial growth, and some change the content of active ingredients. At the same time, the content of one or more active ingredients can be increased through process optimization. The commonly used fermentation bacteria for fermenting Astragalus residues are Trichoderma aculeatus, Paecilomyces chinensis, Aspergillus niger and Bacillus. There are also studies using edible and medicinal fungi Pleurotus ostreatus, Lentinus edodes, Ganoderma lucidum and Cordyceps sinensis to ferment Astragalus. After fermenting Astragalus with Pleurotus ostreatus, the content of Astragalus polysaccharides and free amino acids decreased, while the content of total flavonoids, total saponins and astragaloside A increased. Fu Xiaoxue fermented Astragalus with Lentinus edodes and found that some components of Astragalus were utilized and transformed by Lentinus edodes.

[0004] As a kind of white rot fungus, Coriolus versicolor belongs to Fungi, Basidiomycetes, Polyporaceae, Polyporaceae, and Coriolus genus in taxonomy. Its main active ingredients, Coriolus versicolor polysaccharide and Coriolus versicolor glycopeptide, not only have immunomodulatory and hepatoprotective and choleretic effects, but also have anti-tumor, antioxidant, anti-aging, lipid-lowering, qi-invigorating, detoxifying and analgesic effects. As an important active ingredient raw material, Coriolus versicolor polysaccharide is widely used in food, medicine and health care products. If the residue of Astragalus membranaceus can be fermented with Coriolus versicolor, it is expected that the effective ingredients of Astragalus membranaceus and Coriolus versicolor can be used at the same time to exert a comprehensive effect. However, there is no research report on the fermentation of Astragalus membranaceus residue by Coriolus versicolor, and it is still unknown what effect the fermentation of Astragalus membranaceus residue has on its effective ingredients.

[0005] Chinese patent CN1663424A discloses a feed additive and its preparation process and application in the manufacture of feed for preventing avian influenza, which involves fermenting Coriolus versicolor in astragalus residue, corn cobs, taro residue and bran to obtain a feed additive, the function of which is to improve the immunity of animals against avian influenza; however, the study did not use Coriolus versicolor to ferment astragalus residue alone, and there is no research on the effect of fermentation on the effective ingredients in the astragalus residue. Chinese patent CN108653518A discloses a probiotic fermented Chinese medicine preparation and a preparation method thereof, which discloses using probiotics (including: Enterococcus faecium, Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Rhizopus oryzae) to ferment a Chinese medicine composition (including: Angelica sinensis, White Peony Root, Rehmannia Glutinosa, Chuanxiong, Astragalus, Codonopsis pilosula, Bupleurum, Cyperus rotundus, Vaccaria segetalis, Gleditsia sinensis, Smilax glabra, Leonurus japonicus and Taraxacum officinale) to increase the release of effective ingredients. These beneficial substances can improve the intestinal health of sows, thereby promoting food digestion and absorption in sows, and further improving the lactation output and quality of sows; however, in this study, Astragalus membranaceus was not fermented alone, and no research has been found on the effect of fermentation on the effective ingredients in Astragalus membranaceus; and this study used Chinese medicinal materials rather than Chinese medicinal residues, and the cost of using Chinese medicinal herbs as substrates for fermentation is relatively high.

[0006] Therefore, it is a research goal for those skilled in the art to provide a method with low fermentation cost and capable of fully utilizing the effective ingredients in the Astragalus residue, especially astragaloside IV, soluble sugars and flavonoids. Summary of the invention

[0007] The present invention aims to provide a method for fermenting astragalus residues, which uses astragalus residues as fermentation substrates and can fully improve the effective ingredients (especially astragaloside IV, soluble sugars and flavonoids) in astragalus residues. The method ferments astragalus residues with versicolor fungi, and the contents of astragaloside IV, total flavonoids and soluble sugars are significantly improved after fermentation; and the fermented versicolor fermented astragalus residues are mixed with corn flour and soybean meal and then secondary fermented with mixed bacteria (Lactobacillus plantarum, Enterococcus faecium and Saccharomyces cerevisiae) to effectively degrade neutral and acidic detergent fibers, and the total protein and acid-soluble protein contents in the obtained product are improved, and the number of lactic acid bacteria (including Lactobacillus plantarum and Enterococcus faecium) and live yeasts is significantly increased, which has very good application value in feed.

[0008] The invention aims to provide a method for fermenting astragalus residues for increasing the contents of astragaloside IV, flavonoids and soluble sugar.

[0009] Another object of the present invention is to provide the dregs of Astragalus membranaceus fermented from the above fermentation and the application thereof.

[0010] Another object of the present invention is to provide feed containing the fermented Astragalus membranaceus residue or fermented with the residue as raw material.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] After research and exploration, the present invention found that fermenting Astragalus membranaceus residue with Versicolor fungus can significantly increase the content of astragaloside IV, flavonoids and soluble sugar therein.

[0013] Therefore, the present invention provides a method for fermenting astragalus residues for increasing the content of astragaloside IV, flavonoids and soluble sugar. The method comprises fermenting the astragalus residues with versicolor fungus.

[0014] Preferably, the fermentation condition is 20-30° C. for 1-7 days.

[0015] More preferably, the fermentation condition is 20-30° C. for 3-7 days.

[0016] Most preferably, the fermentation condition is 25°C for 5 days.

[0017] Preferably, the inoculation amount of Coriolus versicolor is 0.5-3%.

[0018] Preferably, the water content of the Astragalus residue is adjusted to 40-80% before fermentation. More preferably, the water content is 60%.

[0019] Preferably, the astragalus residue is crushed into powder of 10-40 mesh before fermentation.

[0020] The fermented Astragalus residue obtained by the above fermentation is rich in astragaloside IV, flavonoids, and soluble sugars, and has a good application value in feed. Therefore, the obtained fermented Astragalus residue and its application in the preparation of feed should be within the protection scope of the present invention.

[0021] The invention also provides feed containing the dregs of Astragalus membranaceus fermented by Coriolus versicolor.

[0022] As a preferred embodiment, the feed comprises the fermented Astragalus root residue of Coriolus versicolor, corn flour and soybean meal. Preferably, the mass ratio of the fermented Astragalus root residue of Coriolus versicolor, corn flour and soybean meal is (1-5):(1-5):(1-5). More preferably, the mass ratio is 2:5:3.

[0023] As a preferred embodiment, the feed is obtained by mixing the residue of Astragalus membranaceus fermented by Coriolus versicolor, corn flour and soybean meal, and then fermenting with mixed bacteria, wherein the mixed bacteria is a mixture of Lactobacillus plantarum, Enterococcus faecium and Saccharomyces cerevisiae.

[0024] Preferably, the inoculation amount of the mixed bacteria is 0.5-3%, and more preferably, the inoculation amount is 1%.

[0025] Preferably, the inoculation volume ratio of Lactobacillus plantarum, Enterococcus faecium and Saccharomyces cerevisiae is (1-2):(1-2):(1-2). More preferably, the volume ratio is 1:1:1.

[0026] Preferably, the mass ratio of the fermented Astragalus membranaceus residue from Coriolus versicolor, corn flour and soybean meal is (1-5):(1-5):(1-5). More preferably, the mass ratio is 2:5:3.

[0027] Preferably, the fermentation substrates, namely, the residue of fermented Astragalus membranaceus from Coriolus versicolor, corn flour and soybean meal, are mixed and the moisture content is adjusted to 30-70% before fermentation.

[0028] Preferably, the fermentation conditions of the mixed bacteria are: sealed fermentation at 20-30° C. for 5-7 days, and the fermentation is terminated when the pH drops to 3.0-5.0.

[0029] More preferably, the fermentation conditions of the mixed bacteria are: fermentation at 25° C. for 7 days, and fermentation is terminated when the pH drops to 4.0-4.5.

[0030] Specifically, as a preferred embodiment, the present invention provides a method for preparing a feed rich in astragaloside IV, flavonoids, and soluble sugars, comprising the following steps:

[0031] S1. Pretreatment of Astragalus residue: crush the Astragalus residue into 10-40 mesh and adjust the moisture content to 40-80%;

[0032] S2. Cultivation of Versicolor versicolor: inoculating Versicolor versicolor into a fermentation medium for cultivation to obtain Versicolor versicolor seed liquid;

[0033] S3. Fermenting the Astragalus residue with Coriolus versicolor: inoculating the pre-treated Astragalus residue with a volume ratio of 0.5 to 3% of the Coriolus versicolor seed liquid, fermenting at a temperature of 20 to 30° C. for 3 to 7 days, and sterilizing the Astragalus residue after the fermentation to obtain the Coriolus versicolor fermented Astragalus residue;

[0034] S4. Cultivation of mixed bacteria: three fermentation seed liquids of Lactobacillus plantarum, Enterococcus faecium and Saccharomyces cerevisiae were mixed in a volume ratio of (1-2):(1-2):(1-2) to obtain a mixed bacteria;

[0035] S5. Secondary fermentation of mixed bacteria: Mix the residue of Astragalus membranaceus fermented by Versicolor, corn flour and soybean meal in a mass ratio of (1-5):(1-5):(1-5), adjust the moisture content to 30-70%, the fermentation temperature to 20-30°C, and the fermentation time to 5-7 days to obtain a feed rich in astragaloside I, flavonoids and soluble sugars.

[0036] Preferably, the specific method of pre-treating the astragalus residue in step S1 is: first drying the astragalus residue to reduce its moisture content to below 10%, then crushing it to 10-40 meshes, and then adjusting the moisture content to 40-80%.

[0037] Preferably, the culture conditions of the versicolor fungus in step S2 are: fermentation at 20-30°C for 3-7 days. More preferably, the conditions are fermentation at 25°C for 5 days. More specifically, the culture conditions of the versicolor fungus are: liquid volume of 100 / 250mL, fermentation temperature of 25°C, and culture on a shaking table at 100rpm for 3 days, until the mycelium is spherical.

[0038] The feed prepared by the invention is not only rich in astragaloside IV, flavonoids and soluble sugar, but also has increased contents of total protein and acid-soluble protein, significantly increased numbers of lactic acid bacteria (including Lactobacillus plantarum and Enterococcus faecium) and live yeast, high in nutrition and important in application value.

[0039] The present invention has the following beneficial effects:

[0040] (1) The present invention ferments the astragalus residue with versicolor fungus, and after fermentation, the contents of astragaloside IV, total flavonoids, and soluble sugar are significantly increased, thereby achieving full utilization of the astragalus residue.

[0041] (2) The present invention couples two fermentations, further mixing the fermented Astragalus membranaceus residue with corn flour and soybean meal and then performing secondary fermentation with mixed bacteria (Lactobacillus plantarum, Enterococcus faecium and Saccharomyces cerevisiae), effectively degrading neutral and acidic detergent fibers, increasing the content of total protein and acid-soluble protein in the obtained product, and significantly increasing the number of live lactic acid bacteria (including Lactobacillus plantarum and Enterococcus faecium) and yeast. The total protein and acid-soluble protein content in the feed is increased, the growth of probiotics is promoted, the nutrition is high, and it has important application value.

[0042] (3) The fermentation substrate of the present invention has simple components, a simple production process, and low fermentation cost, and has a good prospect for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0044] Figure 1 This is the growth situation of Versicolor plate.

[0045] Figure 2 These are photos of Chinese medicinal residues before and after fermentation of Coriolus versicolor (Figure a is a photo of the Chinese medicinal residues before fermentation of Coriolus versicolor; Figure b is a photo of the Chinese medicinal residues after fermentation of Coriolus versicolor).

[0046] Figure 3 These are the changes in the content of important parameters during the fermentation process of Astragalus residue and Coriolus versicolor (Figure a shows the changes in the content of total soluble sugars during the fermentation process of Astragalus residue and Coriolus versicolor; Figure b shows the changes in the content of astragaloside I in Coriolus versicolor fermented with Astragalus residue; Figure c shows the changes in the content of total flavonoids during the fermentation process of Astragalus residue and Coriolus versicolor).

[0047] Figure 4 These are the changes in the contents of important parameters during the fermentation of Astragalus residue and Hericium erinaceus (Figure a shows the changes in the content of total soluble sugars during the fermentation of Astragalus residue; Figure b shows the changes in the content of astragaloside IV during the fermentation of Astragalus residue; Figure c shows the changes in the content of total flavonoids during the fermentation of Astragalus residue).

[0048] Figure 5 The pH and total number of live bacteria after fermentation with mixed bacteria at different inoculation ratios (Figure a shows the pH after fermentation with mixed bacteria at different inoculation ratios; Figure b shows the total number of live bacteria after fermentation with mixed bacteria at different inoculation ratios).

[0049] Figure 6 It is the pH after the fermentation of mixed bacteria with different inoculation amounts is completed (Figure a is the pH after the fermentation of mixed bacteria with different inoculation amounts is completed; Figure b is the total number of live bacteria after the fermentation of mixed bacteria with different inoculation amounts is completed).

[0050] Figure 7 Figure 3 shows the changes in pH and total acid content during the fermentation of Astragalus residues using mixed bacteria or unfermented Astragalus residues (Figure a shows the changes in pH during the fermentation of Astragalus residues using mixed bacteria; Figure b shows the changes in total acid content during the fermentation of Astragalus residues using mixed bacteria).

[0051] Figure 8Figure 1 shows the changes in the number of live bacteria of different strains during the fermentation of Astragalus residue by mixed bacteria (Figure a shows the changes in the number of live lactic acid bacteria during the fermentation of Astragalus residue by mixed bacteria; Figure b shows the changes in the number of live yeasts during the fermentation of Astragalus residue by mixed bacteria). DETAILED DESCRIPTION

[0052] The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.

[0053] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0055] In the following embodiments:

[0056] (1) The total viable count is:

[0057] The number of yeasts is determined in accordance with the National Food Safety Standard for Microbiological Examination of Foods - Count of Molds and Yeasts (GB4789.15-2016), and the number of lactic acid bacteria is determined in accordance with the National Food Safety Standard for Microbiological Examination of Foods - Lactic Acid Bacteria (GB4789.35-2016). Since both Lactobacillus plantarum and Enterococcus faecium are lactic acid bacteria, the total number of live bacteria = number of live yeasts + number of live lactic acid bacteria.

[0058] (2) The pH determination method is:

[0059] Take 5g of feed and add 10mL of deionized water, soak for 30min and ultrasonically treat for 30min, and measure the pH with a pH meter.

[0060] (3) The culture medium used was:

[0061] PDA medium (g / L): potato 200, glucose 20, agar powder 18.

[0062] Liquid seed and fermentation medium (g / L): glucose 30, peptone 4, MgSO4·7H2O 1, KH2PO4 2, corn steep liquor 15.

[0063] MRS medium: tryptone 10.0; beef extract 10; yeast powder 5; glucose 20g; diammonium hydrogen citrate 2; sodium acetate 5; K2HPO4·3H2O 2.6; MgSO4·7H2O 0.2; MnSO4·4H2O0.05; Tween 80 1. pH adjusted to 6.8±0.2.

[0064] MRS solid medium: before sterilization, add agar to the above MRS liquid medium so that its mass volume concentration in the liquid medium is 20g / L.

[0065] PDB medium (g / L): potato powder 6; glucose 20.

[0066] PDB solid medium: before sterilization, add agar to the above PDB liquid medium so that its mass volume concentration in the liquid medium is 20g / L.

[0067] (4) The strains used are:

[0068] Saccharomyces cerevisiae, Enterococcus faecium, Lactobacillus plantarum, Coriolus versicolor, and Hericium erinaceus were purchased from China Industrial Microorganism Collection Center. 1624, Enterococcus faecium 20537, Lactiplantibacillus plantarum 6026, Trametes versicolor 50001 and Hericium erinaceus 50081.

[0069] Example 1: Study on Astragalus membranaceus dregs fermented by Versicolor fungus

[0070] 1. Preparation of Versicolor seed solution

[0071] Inoculate the commercially purchased Versicolor fungus on a plate containing PDA medium, activate it 1-2 times, and observe whether the colony morphology of the plate is uniform. If the morphology is uniform, it indicates that the strain is relatively pure and further experiments can be carried out. Take the soybean-sized bacterial block after rejuvenation and purification and inoculate it in the center of the plate containing PDA medium. Culture it for 5-7 days. When it is almost full of the plate, use the punching method to inoculate, that is, use a metal puncher to obtain bacterial blocks of the same size (8mm in diameter) of different strains as seeds, inoculate them in the fermentation medium, and the liquid volume is 100 / 250mL. The fermentation temperature is 25℃, and the speed is 100rpm. Culture on a shaking table for 3 days. When the fungal hyphae appear spherical, the Versicolor seed liquid is obtained.

[0072] 2. Yunzhi fermented Astragalus residue

[0073] The versicolor seed liquid was inoculated into the astragalus residue at a ratio of 1:100, and the fermentation temperature was 25°C, and the fermentation was sealed with a breathable membrane for 7 days. During the fermentation process, samples of the unfermented (0th day), 1st, 3rd, 5th, and 7th day versicolor fermentation products of the astragalus residue were taken for detection and analysis.

[0074] 3. Determination of soluble sugar content

[0075] Take the Astragalus residue before fermentation with Versicolor and the Astragalus residue and Versicolor fermentation product after fermentation, keep their moisture content consistent, weigh 1g of each, add 10mL of distilled water, put it in a boiling water bath, cover it and boil it for 10min, filter it after cooling, collect the filtrate in a 100mL volumetric flask, make up to 100mL with distilled water, shake it well for later use, and determine the total sugar content by phenol-sulfuric acid method.

[0076] Total sugar content of Astragalus = (measured total sugar content / dry weight of Astragalus) × 100%

[0077] 4. Determination of Astragaloside IV Content

[0078] HPLC chromatographic conditions: mobile phase: acetonitrile: water = 32:68; flow rate: 1 mL / min; detection wavelength: 210 nm; column temperature: 30°C; injection volume: 10 μL.

[0079] Drawing of the astragaloside IV standard curve: take 0.1, 0.3, 0.5, 0.7, and 0.9 mL of the standard solution, respectively, dilute to 1 mL with methanol, take 10 μL of the diluted standard solution and inject it into the liquid chromatograph, and draw the standard curve with the peak area of ​​astragaloside IV as the horizontal axis and the concentration of astragaloside IV as the vertical axis.

[0080] Sample treatment: Take 5g of Astragalus residue before fermentation and 5g of Astragalus residue after fermentation, add them into Soxhlet extractor, extract with methanol, anhydrous ethanol and 95% ethanol respectively, heat and reflux until the extract is colorless, combine the extracts and concentrate to 10mL, extract with 40mL water-saturated n-butanol, extract 4 times continuously, mix the n-butanol solution extracted each time, and then wash with 40mL ammonia solution, wash twice, discard the ammonia solution, evaporate the combined n-butanol solution in an evaporating dish, add 5mL ultrapure water to dissolve the residue, pass the obtained solution through D101 macroporous adsorption resin column with inner diameter of 1.5cm and column height of 12cm, elute with 50mL pure water, discard the water, elute with 30mL 40% ethanol solution, discard the eluent, and continue eluting with 80mL 70% ethanol solution, retain the eluent, evaporate to dryness in the evaporating dish, dissolve the residue with methanol, and make up to volume with a 10mL volumetric flask as the test solution. Take 10 μL of the test sample and inject it to determine the peak area and calculate its astragaloside IV content.

[0081] 5. Determination of total flavonoids content

[0082] Preparation of flavonoid standard curve: Weigh 20 mg of rutin standard, accurately weigh, prepare into 1 mg / mL stock solution, dilute, shake well to obtain rutin standard solution with mass concentration of 0.1 mg / mL. Accurately aspirate 0, 40, 80, 120, 160, 200 μL of rutin standard solution into 1.5 mL EP tubes, add 70% ethanol solution to 200 μL, add 40 μL of 5% NaNO2 solution, shake well and place for 6 minutes, add 40 μL of 10% Al(NO3)3 solution, shake well, place for another 6 minutes, add 400 μL of 4% NaOH solution, shake well and place for 15 minutes, make up to 1 mL with 70% ethanol, let stand for 10 minutes, and measure absorbance at a wavelength of 510 nm. Draw the total flavonoid standard curve with rutin concentration as the ordinate and solution absorbance as the abscissa.

[0083] Determination of total flavonoids content: Take the astragalus residue before fermentation and the fermented astragalus residue after fermentation, keep the water content consistent, add 5 times the mass of 70% ethanol solution, ultrasonically treat for 30 minutes, centrifuge at 5000rpm for 10 minutes, take the supernatant and prepare the test solution according to the standard curve sample preparation method, and also measure the absorbance at a wavelength of 510nm. Calculate the total flavonoids content according to the standard curve.

[0084] Combination Figure 3 As shown in Figure a, the soluble total sugar content of Astragalus residue increased significantly after fermentation with Versicolor, from 4.29% on the 0th day before fermentation to the highest soluble total sugar content of 11.35% on the 5th day of fermentation, and the increase was particularly obvious in the first 3 days of fermentation.

[0085] Combination Figure 3 As shown in Figure b, since the sample used was the residue of Astragalus membranaceus after extraction, the initial astragaloside content was low. The astragaloside content of the sample on day 0 was only 0.1 mg / g. As the fermentation progressed, its content increased significantly, reaching the highest content of 5.2 mg / g on day 5 of fermentation.

[0086] Combination Figure 3 As shown in Figure c, the total flavonoids content showed a significant increasing trend after fermentation with Coriolus versicolor. There was no significant difference in the total flavonoids content after 5 days of fermentation and 7 days of fermentation, and its content increased by 2.9 times compared with the 0th day.

[0087] In addition, further research on fermentation conditions showed that the inoculation amount of Yunzhi seed liquid can be 0.5-3%. The fermentation temperature can be preferably 20-30°C, and the air-permeable membrane sealed fermentation time can be preferably 3-7 days, and most preferably 5 days. Comparative Example 1: Research on Hericium erinaceus fermentation of Astragalus medicated residue

[0088] 1. Preparation of Hericium erinaceus seed solution

[0089] Inoculate the commercially purchased Hericium erinaceus on a plate containing PDA culture medium, activate it 1 to 2 times, and observe whether the colony morphology of the plate is uniform. If the morphology is uniform, it indicates that the strain is relatively pure and further experiments can be carried out. Take the soybean-sized fungus block after rejuvenation and purification and inoculate it in the center of the plate containing PDA culture medium. Culture it for 5 to 7 days. When it is almost full of the plate, use the punching method to inoculate, that is, use a metal puncher to obtain fungus blocks of the same size (8mm in diameter) of different strains as seeds, inoculate them in the fermentation medium, and the liquid volume is 100 / 250mL. The fermentation temperature is 25℃, and the speed is 100rpm. Culture on a shaking table for 3 days. When the fungal hyphae appear spherical, the Hericium erinaceus seed liquid is obtained.

[0090] 2. Hericium erinaceus fermented Astragalus dregs

[0091] The Hericium erinaceus seed liquid was inoculated into the Astragalus residue at a ratio of 1:100, and the fermentation temperature was 25°C, and the fermentation was sealed with a breathable membrane for 7 days. During the fermentation process, samples of unfermented (0th day), 1st, 3rd, 5th, and 7th days were taken for detection and analysis.

[0092] 3. Determination of soluble sugar content

[0093] The detection method is the same as in Example 1.

[0094] 4. Determination of Astragaloside IV Content

[0095] The detection method is the same as in Example 1.

[0096] 5. Determination of total flavonoids content

[0097] The detection method is the same as in Example 1.

[0098] Combination Figure 4 As shown in Figures a, b and c, the soluble total sugar content, astragaloside IV content and total flavonoids content of Astragalus residue after fermentation with Hericium erinaceus increased slightly. Except for the soluble total sugar content reaching the highest content at 3 days of fermentation, the astragaloside IV content and total flavonoids content both reached the highest content at 5 days of fermentation. However, the highest contents of the three were not as high as those of Astragalus residue after fermentation with Versicolor.

[0099] Example 2: Preparation and optimization of mixed bacterial solution

[0100] 1. Preparation of fermented seed liquid

[0101] The commercially available Lactobacillus plantarum and Enterococcus faecium strains were inoculated into MRS medium for activation culture and cultured at 37°C for 24 h. After activation, they were re-inoculated into MRS medium at a 3% inoculum volume and cultured under the same conditions for 18 h. At this time, the number of viable Lactobacillus plantarum was about 4×10 9 CFU / mL, the number of viable Enterococcus faecium is about 2×10 9 CFU / mL.

[0102] Commercially available brewer's yeast (Angel Yeast) was streaked onto PDA solid medium and cultured on a PDB plate at 30°C for 48 h. After colonies grew out, a single colony was selected and inoculated into PDA medium for 24 h. The inoculation volume was then continued to be inoculated into PDA medium for 24 h at a rate of 3% to obtain a yeast seed solution with a live bacterial count of about 1 × 10 9 CFU / mL.

[0103] The three cultured bacterial liquids were uniformly mixed according to a volume ratio to prepare a mixed bacterial liquid.

[0104] 2. Optimization of the proportion of probiotics and inoculation amount in the mixed bacterial solution

[0105] The astragalus residue after fermentation in the above Example 1 was boiled and sterilized, and after cooling, the fermented astragalus residue: corn flour: soybean meal = 2:5:3 were mixed evenly, the moisture content was adjusted to 50%, and the mixture was placed in a fermentation bag with an air release valve. The seed solution with a volume ratio of Lactobacillus plantarum: Enterococcus faecium: Saccharomyces cerevisiae of 1:1:0.5, 1:1:1, 1:1:2, 2:1:1, and 1:2:1 was prepared respectively; the fermentation was carried out under the same conditions for 7 days, and the pH and the total number of viable bacteria after the fermentation were detected.

[0106] After determining the inoculation ratio of probiotics in the seed liquid, the same fermentation method was used, with inoculation amounts of 0.5%, 1%, 2%, and 3%, respectively. The fermentation was carried out under the same conditions for 7 days, and the pH value was tested, and the total number of live bacteria after the fermentation was completed.

[0107] 3. Results

[0108] like Figure 5 Figures a and b show that from the results of different bacterial species ratios in the seed solution, it can be seen that the pH of the two groups of Lactobacillus plantarum: Enterococcus faecium: Saccharomyces cerevisiae = 1:1:1 and 2:1:1 is the lowest, and the number of live bacteria in the two groups of Lactobacillus plantarum: Enterococcus faecium: Saccharomyces cerevisiae = 1:1:1 and 1:2:1 is the highest. Combined with the experimental results, it is judged that Lactobacillus plantarum: Enterococcus faecium: Saccharomyces cerevisiae = 1:1:1 is the best bacterial species ratio.

[0109] like Figure 6 Figures a and b show that the pH of fermented feed decreases with the increase of inoculation amount. When the inoculation amount is greater than 1%, the pH no longer changes significantly. However, the number of live bacteria increases with the increase of inoculation amount, reaching the maximum value at 1%, and continues to increase to 3%, and the number of live bacteria decreases slightly. Therefore, 1% is selected as the optimal inoculation concentration.

[0110] In addition, further research on the fermentation conditions of the mixed bacteria showed that the fermentation conditions can be sealed and fermented at 20-30°C for 5-7 days, and the fermentation can be terminated when the pH drops to 3.0-5.0.

[0111] The mass ratio of the fermentation substrate, the residue of Astragalus membranaceus fermented by Coriolus versicolor, corn flour and soybean meal, can be within the range of (1-5):(1-5):(1-5).

[0112] The fermentation substrate, the residue of Astragalus membranaceus fermented by Coriolus versicolor, corn flour and soybean meal, is mixed and the moisture content is adjusted to 30-70% before fermentation.

[0113] Example 3: Preparation of the mixture and the effect of the mixture in feed additives

[0114] 1. Preparation of the mixture: Boil and sterilize the fermented astragalus residue described in Example 1, and after cooling, mix them evenly according to the mass ratio of astragalus residue: corn flour: soybean meal = 2:5:3, adjust the moisture content to 50%, add them into a fermentation bag with a mass ratio of 1% and a ratio of Lactobacillus plantarum: Enterococcus faecium: Saccharomyces cerevisiae = 1:1:1, and ferment at room temperature for 7 days. The unfermented astragalus residue was fermented under the same conditions as the control group, and samples were taken at 0, 3, 5, and 7 days for testing.

[0115] 2. Determination of total protein and acid-soluble protein content

[0116] Total protein content: determined by Kjeldahl method according to GB / T 6432-2018;

[0117] Acid-soluble protein content: Refer to NY / T 3801-2020, feed 150g·L -1 The extract was extracted with trichloroacetic acid solution and the nitrogen was determined by Kjeldahl method after centrifugation.

[0118] 3. Determination of neutral and acid detergent fiber content

[0119] The neutral detergent fiber content in the sample was determined according to GB / T 20806-2022, and the acid detergent fiber content in the sample was determined according to NY / T 1459-2007.

[0120] 4. Total acid determination

[0121] Take 5g of feed and add 10mL of deionized water, soak for 30min and ultrasonically treat for 30min, filter with neutral filter paper, take 1mL of filtrate, add 40-60mL of water and 0.2mL of 1% phenolphthalein indicator, and use 0.1mol·L -1 Sodium hydroxide standard titration solution is titrated until it turns slightly red and does not fade for 30 seconds. Record the consumption of 0.1 mol·L -1 The milliliters of sodium hydroxide standard titration solution (V1). The same sample to be tested must be tested three times. Replace the test solution with water and follow the analysis steps below. Record the consumption of 0.1 mol·L -1The number of milliliters of sodium hydroxide standard titration solution (V2). The total acid content is expressed as mass fraction X, and the value is expressed in grams per kilogram (g·kg -1 ) is expressed as, and the total acid is calculated as shown in formula (1).

[0122] X=(c×(V1-V2)×F×K) / m×1000 (1)

[0123] Where:

[0124] c: Concentration of sodium hydroxide standard titration solution (mol·L -1 );

[0125] V1: Volume of sodium hydroxide standard titration solution consumed when titrating the test solution (mL);

[0126] V2: Volume of sodium hydroxide standard titration solution consumed during blank test (mL);

[0127] F: dilution factor of the sample;

[0128] m: sample volume (g);

[0129] K: Acid conversion factor, lactic acid is 0.090.

[0130] As shown in Table 1, the fermented Astragalus residues were subjected to secondary feed fermentation, which was more conducive to the fermentation process. The total protein content did not change much during the fermentation process, but the experimental group (fermented Astragalus residues) was higher than the control group (unfermented Astragalus residues). The acid-soluble protein content gradually increased with the fermentation process, and the experimental group was higher than the control group during the fermentation process. Neutral and acidic detergent fiber gradually decreased with the fermentation process, and the experimental group was lower than the control group during the fermentation process.

[0131] Table 1 Test results of feed nutritional indicators

[0132]

[0133] like Figure 7 As shown in Figures a and b, after the fermentation of the experimental group, the total acid content was 10.62 g / kg, and the pH dropped to 4.13; while the total acid accumulation of the control group was 8.95 and the pH was 4.57. The fermented Astragalus residue was added to the feed for secondary fermentation, and the total acid content was significantly higher than that of the unfermented Astragalus residue, and the pH drop accumulation rate was also significantly increased. The reason may be that after Astragalus is fermented with Versicolor, the sugar content available for microbial growth increases, but the subsequent secondary fermentation produces more acid.

[0134] like Figure 8As shown in Figures a and b, the number of live bacteria in the feed fermentation process showed a trend of first increasing and then decreasing. However, the number of live bacteria in the experimental group was generally higher during the fermentation process. Both groups of lactic acid bacteria reached the highest number of live bacteria on the 5th day of fermentation, and the highest number of live bacteria in the control group was 5.25×10 8 CFU / g, the control group was 7.15×10 8 CFU / g, it may be that the nutrients in the fermented astragalus are more conducive to microbial utilization, resulting in faster microbial growth and higher biomass. The yeast in both groups reached the highest on the third day and then declined rapidly in the later period.

[0135] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A fermentation method for improving the content of astragaloside IV, flavonoids and soluble sugar in astragalus residues, characterized in that: The Astragalus residue was fermented by Trametes versicolor.

2. The fermentation method of astragalus root residues according to claim 1, characterized in that: The fermentation conditions are 20-30°C for 1-7 days.

3. The fermentation method of astragalus root residues according to claim 1, characterized in that: The inoculation amount of Versicolor versicolor is 0.5-3%.

4. The fermentation method of astragalus root residues according to claim 1, characterized in that: The water content of the astragalus root residue is 40-80%.

5. The fermentation method of astragalus root residues according to claim 1 or 2, characterized in that: The astragalus root residue is crushed into powder of 10-40 meshes and then fermented.

6. The drug residue of Astragalus membranaceus fermented from Versicolor prepared by the method according to any one of claims 1 to 5.

7. Use of the residues of Astragalus membranaceus fermented from Coriolus versicolor according to claim 6 in preparing feed.

8. A feed, characterized in that: The invention comprises the fermented Astragalus membranaceus residue of Versicolor as described in claim 6.

9. The feed according to claim 8, characterized in that The method comprises the fermented Astragalus membranaceus residues of Versicolor as claimed in claim 6, corn flour and soybean meal.

10. The feed according to claim 8, characterized in that The product is obtained by mixing the fermented Astragalus membranaceus residues of Coriolus versicolor as claimed in claim 6, corn flour and soybean meal, and then fermenting with mixed bacteria, wherein the mixed bacteria is a mixed bacteria of Lactobacillus plantarum, Enterococcus faecium and Saccharomyces cerevisiae.

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