Epimedium enzymolysis probiotic fermentation refined extract and preparation method thereof

Abstract

The invention relates to the technical field of functional food production through microbial fermentation, in particular to a herba epimedii enzymolysis probiotic fermentation refined extract and a preparation method thereof.The preparation method comprises the following steps that herba epimedii and beta-cyclodextrin are mixed, an obtained mixed solution is subjected to enzymolysis treatment through pectinase, cellulase and glycosidase and then serves as a solid-state fermentation substrate, and the herba epimedii enzymolysis probiotic fermentation refined extract is obtained; adding mixed bacteria of lactobacillus casei, bifidobacterium lactis and lactobacillus gasseri, performing solid-state fermentation, and performing vacuum freeze drying to obtain a herba epimedii probiotic fermentation refined extract; the herba epimedii probiotic fermentation refined extract is prepared by adopting an enzymolysis fermentation method in cooperation with a beta-cyclodextrin inclusion technology, the extraction efficiency, the solubility, the biological activity, the stability and other performance of effective components in herba epimedii are improved, the preparation method is simple, and operation is easy and convenient; the used materials are edible, green and safe; the process saves energy consumption, has good environmental benefits, greatly saves manpower and material resources, and can be applied to industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of functional food produced by microbial fermentation, and in particular to an epimedium enzymatic hydrolysis probiotic fermentation extract and a preparation method thereof. Background Art

[0002] Epimedium brevicornu Maxim. is a perennial herbaceous plant of the genus Epimedium in the family Berberidaceae. The rhizome is short and thick, dark brown. The complete leaves are trifoliate with long petioles. The leaflets are papery or thickly papery, with thorny edges. The flowers are white or light yellow. There are 68 species of the genus Berberidaceae in the world, 58 of which are distributed in China. China is the geographical distribution and variety center of Epimedium. The famous medical scientist Tao Hongjing learned that the frequency of mating and reproduction of male sheep increased significantly after eating a certain plant. He believed that this plant could enhance the energy of "Yang" and named it "Yin Yang Fire". Later, it was identified as an Epimedium plant. More than 15 species of Epimedium have been used in traditional Chinese medicine. Epimedium has a long history of medicinal use and is believed to have the properties of tonifying the kidney and tonifying Yang. A large number of studies have shown that the flavonoid compounds in Epimedium have significant therapeutic effects on symptoms such as kidney yang deficiency, low back pain, soreness of the waist and knees, cold body and limbs, lower limb weakness, frequent urination at night, etc. It plays the role of warming yang and tonifying the kidney, strengthening the muscles and bones, and can be used to treat primary osteoporosis.

[0003] As one of China's natural plant resources, epimedium contains a variety of active ingredients such as polysaccharides and flavonoids. Under the current market environment, the extraction of effective ingredients from epimedium faces many challenges, and efficient new technologies have not yet appeared. Traditional extraction methods are complex and have many disadvantages. Long-term heating not only consumes a lot of energy, pushes up production costs and is not conducive to environmental protection, but also easily destroys heat-sensitive active ingredients such as polysaccharides in epimedium. Microwave extraction has poor selectivity for ingredients. The simultaneous dissolution of multiple ingredients increases the difficulty of subsequent separation, and the equipment has major safety hazards. Uneven heating can easily cause local temperatures to be too high to destroy active ingredients, and the extraction quality is unstable due to fluctuations in multiple factors. Ultrasonic extraction has limited extraction capacity for certain ingredients that are closely bound to cells or located in special structures, and it is difficult to ensure the stability and consistency of the extraction quality. In addition, it is costly, requires professional operation and maintenance, and is not suitable for large-scale industrial production. Therefore, a new extraction method needs to be proposed. Summary of the invention

[0004] The object of the present invention is to provide an enzymatically hydrolyzed probiotic fermented extract of Epimedium and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing an extract of enzymatically hydrolyzed probiotic fermented epimedium, comprising the following steps: S1: Pretreatment: crushing the stems and leaves of Epimedium and sieving them to obtain Epimedium powder, mixing the Epimedium powder with a surfactant, adding a solvent, stirring and mixing them thoroughly to obtain a mixed solution; S2: Enzymatic hydrolysis: adding biological enzymes to the mixed solution, stirring the mixed solution for enzymatic hydrolysis under certain temperature and time conditions, and then sterilizing the mixed solution at high temperature to obtain the epimedium solid fermentation matrix; S3: fermentation: adding the lactic acid bacteria liquid to the epimedium solid fermentation matrix, fermenting under certain temperature and time conditions, and sterilizing at high temperature after the fermentation to obtain the epimedium solid fermentation liquid; S4: Preparation of epimedium extract: centrifugally separating the epimedium solid fermentation liquid to obtain a supernatant as the epimedium fermentation liquid, and vacuum freeze-drying the fermentation liquid to obtain the epimedium extract.

[0006] Preferably, the epimedium stems and leaves in S1 are crushed and then passed through a 60-mesh sieve, the surfactant is β-cyclodextrin, the solvent is a citric acid aqueous solution, the solid-liquid ratio of the epimedium micropowder to the citric acid aqueous solution is 1 g: (10~15) mL; and the content of β-cyclodextrin in the citric acid aqueous solution is 1%~10%.

[0007] Preferably, the biological enzymes in S2 are pectinase, cellulase and glycosidase, the added amount of enzymatic pectinase is 0.5%~1.5%, the added amount of cellulase is 1%~4%, and the added amount of glycosidase is 1%~4% (calculated based on the mass fraction of Epimedium).

[0008] Preferably, the enzymatic hydrolysis treatment in S2 refers to adding pectinase, cellulase and glycosidase to the citric acid aqueous solution of epimedium powder and β-cyclodextrin; the enzymatic hydrolysis conditions are pH 3.5-6.0, temperature 30°C-80°C, and enzymatic hydrolysis time 30 min-180 min.

[0009] Preferably, the lactic acid bacteria liquid in S3 is Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri, the inoculation amounts of Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri are 0.1% to 1% respectively, the fermentation temperature is 28° C. to 43° C., and the fermentation time is 12 h to 72 h.

[0010] Preferably, the high temperature sterilization condition temperature in S2 and S3 is 90°C to 120°C, and the sterilization time is 10 min to 30 min.

[0011] Preferably, the rotation speed for centrifugal separation of the solid-state fermentation liquid of Epimedium in S4 is 4000-8000 rpm; and the time for the centrifugal separation is 8-15 min.

[0012] Preferably, the vacuum freeze-drying conditions for the fermentation of epimedium in S4 are a temperature of 0°C to 5°C, a pressure of 0 Pa to 50 Pa, and a time of 24 h to 72 h to obtain an epimedium extract.

[0013] The present invention also provides the following technical solution: an enzymatically hydrolyzed and fermented probiotic extract of epimedium, which is prepared by the preparation method of the enzymatically hydrolyzed and fermented probiotic extract of epimedium.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The invention adopts an enzymatic fermentation method in coordination with a beta-cyclodextrin inclusion technology to prepare an epimedium probiotic fermentation extract, thereby improving the extraction efficiency, solubility, biological activity, stability and other properties of the effective components in the epimedium.

[0015] β-cyclodextrin has a hydrophobic inner cavity and a hydrophilic outer shell. β-cyclodextrin is used as the main molecule to encapsulate the hydrophobic guest molecule flavonoids in Epimedium, which has strong adsorption. During the enzymatic hydrolysis stage, the cell wall of Epimedium is decomposed, the cell membrane permeability increases, and various active ingredients in the cell (flavonoids and polysaccharides, etc.) are dissolved and contacted with β-cyclodextrin, and are targeted and dissolved in the solvent; during the fermentation stage, microorganisms will continue to grow and reproduce, and mycelium can penetrate the Epimedium plant cells and further destroy the cell structure. On the basis of the initial decomposition of the cell wall by enzymatic hydrolysis, the fermentation stage can further break up the Epimedium cells, so that the active ingredients in the cells can be fully released.

[0016] Microorganisms will also transform the active ingredients in Epimedium through their own metabolic enzyme systems, converting the flavonoid glycosides in Epimedium into flavonoid aglycones and producing a variety of secondary metabolites. These new products can synergize with the original ingredients in Epimedium to enhance the health and medicinal value of Epimedium, while introducing probiotics to improve the intestinal microecological environment.

[0017] The invention discloses a new production technology for preparing epimedium extract with high osteoblast proliferation activity, which provides guidance for the development of industrial green and healthy production of new epimedium extract.

[0018] The preparation method of the invention is simple and easy to operate; the materials used are all edible, green and safe; the process saves energy consumption, has good environmental benefits, greatly saves manpower and material resources, and can be applied to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The solutions are epimedium aqueous solution (a), epimedium enzymatic hydrolyzate (b), epimedium enzymatic hydrolyzate fermentation solution (c) and epimedium 50% ethanol solution (d); Figure 2 The effect of different β-cyclodextrin contents on the extraction rate of total polysaccharides from Epimedium. Figure 3 The effect of different enzyme types on the extraction rate of total polysaccharides from Epimedium; Figure 4The effect of different pH on the extraction rate of total polysaccharides from Epimedium; Figure 5 The effect of different enzymatic hydrolysis temperatures on the extraction rate of total polysaccharides from Epimedium; Figure 6 The effect of different enzymatic hydrolysis time on the extraction rate of total polysaccharides from Epimedium; Figure 7 The effect of different lactic acid bacteria species on the extraction rate of total polysaccharides from Epimedium. Figure 8 To study the effect of different lactic acid bacteria inoculation amounts on the extraction rate of total polysaccharides from Epimedium. Fig. 9 The effect of different fermentation temperatures on the extraction rate of total polysaccharides from Epimedium; Fig.10 The effect of different fermentation time on the extraction rate of total polysaccharides in Epimedium; Fig.11 The effect of different β-cyclodextrin contents on the extraction rate of total flavonoids from Epimedium. Fig.12 The effect of different enzyme types on the extraction rate of total flavonoids from Epimedium; Fig.13 The effect of different pH on the extraction rate of total flavonoids from Epimedium; Fig.14 The effect of different enzymatic hydrolysis temperatures on the extraction rate of total flavonoids from Epimedium; Fig.15 The effect of different enzymatic hydrolysis time on the extraction rate of total flavonoids in Epimedium; Fig.16 The effect of different lactic acid bacteria species on the extraction rate of total flavonoids from Epimedium; Fig.17 To study the effect of different lactic acid bacteria inoculation amounts on the extraction rate of total flavonoids from Epimedium. Fig.18 The effect of different fermentation temperatures on the extraction rate of total flavonoids from Epimedium; Fig.19 The effect of different fermentation time on the extraction rate of total flavonoids in Epimedium; Fig. 20 The HPLC chart of the standard products of four active ingredients in Epimedium (a. epimedin C, b. icariin, c. baculoside 1, d. icariin); Fig.21 This is the HPLC chart of four active ingredients extracted from Epimedium by traditional method; Fig. 22 This is the HPLC chart of the four active ingredients extracted from Epimedium by enzymatic hydrolysis and β-cyclodextrin inclusion technology; Fig.23This is the HPLC chart of the four active ingredients extracted from Epimedium by enzymatic fermentation method and β-cyclodextrin inclusion technology; Fig.24 This is a graph showing the effect of different concentrations of epimedium extract on the viability of mouse embryonic osteoblasts (MC3T3-E1) cells; Fig.25 This is a comparison of the effects of different concentrations of Epimedium extract on mouse embryonic osteoblast (MC3T3-E1) cell proliferation before and after. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The present invention provides a technical solution: an enzymatically hydrolyzed probiotic fermented extract of Epimedium and a preparation method thereof, the preparation method comprising the following steps: S1: Pretreatment: crush the stems and leaves of Epimedium with a grinder and pass through a 60-mesh sieve to obtain Epimedium powder, mix the Epimedium powder and β-cyclodextrin, add citric acid aqueous solution, stir and mix thoroughly to obtain a mixed solution; S2: Enzymatic hydrolysis: adding pectinase, cellulase and glycosidase to the mixed solution, stirring and enzymatic hydrolysis under certain temperature and time conditions, and then high-temperature sterilization to obtain the epimedium solid fermentation matrix; S3: fermentation: adding Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri bacterial liquid into the Epimedium solid fermentation matrix, fermenting under certain temperature and time conditions, and sterilizing at high temperature after fermentation to obtain the Epimedium solid fermentation liquid; S4: Preparation of epimedium extract: centrifugally separating the epimedium solid fermentation liquid to obtain a supernatant as the epimedium fermentation liquid, and vacuum freeze-drying the fermentation liquid to obtain the epimedium extract.

[0022] In S1, the ratio of the epimedium to the citric acid aqueous solution is preferably 1:(10-15), and more preferably 1:(13-15).

[0023] The content of the β-cyclodextrin is preferably 1wt%~10wt%, more preferably 5wt%~10wt%; the β-cyclodextrin can effectively include the active ingredients in Epimedium, and cooperate with the enzymatic hydrolysis process to make it quickly dissolve out of the cell, thereby achieving the purpose of efficient adsorption and release.

[0024] In S2, the enzymatic hydrolysis form is preferably a composite enzymatic hydrolysis form of pectinase + cellulase + glycosidase; the added amount of the pectinase is preferably 0.5~1.5%, more preferably 0.7~1%; the added amount of the cellulase is preferably 1~4%, more preferably 2~3%; the added amount of the glycosidase is preferably 1~4%, more preferably 2~3%; the enzymatic hydrolysis pH is preferably 3.5~6.0, more preferably 4.5~5.5; the enzymatic hydrolysis temperature is preferably 30~80°C, more preferably 40~60°C; the enzymatic hydrolysis time is preferably 30~180 min, more preferably 100~150 min.

[0025] The high temperature sterilization temperature is preferably 90-120° C., more preferably 100-110° C.; the high temperature sterilization time is preferably 10-30 min, more preferably 15-25 min.

[0026] S1 and S2 are the preparation of solid-state fermentation matrix of Epimedium.

[0027] In S3, the lactic acid bacteria fermentation form is preferably a composite bacteria fermentation form of Lactobacillus casei + Bifidobacterium lactis + Lactobacillus gasseri; the addition amount of Lactobacillus casei is preferably 0.1%~1%, more preferably 0.3%~0.5%; the addition amount of Bifidobacterium lactis is preferably 0.1%~1%, more preferably 0.3%~0.5%; the addition amount of Lactobacillus gasseri is preferably 0.1%~1%, more preferably 0.3%~0.5%; the fermentation temperature is preferably 28~43°C, more preferably 35~40°C; the fermentation time is preferably 12~72 h, more preferably 36~60 h.

[0028] The high temperature sterilization temperature is preferably 90-120° C., more preferably 100-110° C.; the high temperature sterilization time is preferably 10-30 min, more preferably 15-25 min.

[0029] In S4, the rotation speed of the fermentation liquid centrifugal separation is preferably 4000-8000 rpm, more preferably 5000-7000 rpm; the time of the centrifugal separation is preferably 8-15 min, more preferably 10 min.

[0030] The freeze-drying temperature is preferably 0-5°C, more preferably 0°C; the freeze-drying vacuum pressure is preferably 1-50 Pa, more preferably 10 Pa; the freeze-drying time is preferably 24-72 h, more preferably 48 h.

[0031] S3 and S4 are preparation methods for enzymatically hydrolyzed and fermented extracts of Epimedium.

[0032] In the epimedium enzymatic probiotic fermentation extract prepared by the present invention, the total flavonoid extraction rate is preferably 98.66%, and the total polysaccharide extraction rate is preferably 11.51%.

[0033] In the enzymatically hydrolyzed and fermented extract of epimedium prepared by the present invention, the effective ingredients are preferably epimedin C, icariin, baculoside 1 and icariin; and the contents thereof are preferably 5.11 mg / g, 6.08 mg / g, 1.62 mg / g and 0.44 mg / g, respectively.

[0034] The enzymatically hydrolyzed probiotic fermented extract of Epimedium prepared by the present invention has no effect on cell viability at medium concentrations (52.50 μg / mL and 25.25 μg / mL) and high concentrations (210 μg / mL and 105 μg / mL), has good biocompatibility, and has a significant promoting effect on the proliferation of osteoblasts (MC3T3-E1).

[0035] The preparation method provided by the invention is simple, has mild reaction, and the materials and solvents used in the preparation are non-toxic, has good environmental benefits, and can be applied to factory production.

[0036] The present invention also provides an epimedium enzymatic probiotic fermentation extract, which includes an epimedium extract and a marker active ingredient in the extract. The epimedium extract and the marker active ingredient in the extract include the epimedium extract described in any one of the above technical solutions or the epimedium extract prepared by the preparation method described in any one of the above technical solutions.

[0037] In the present invention, the auxiliary materials for enzymatic fermentation of epimedium are auxiliary materials acceptable in the food industry. The present invention has no special limitation on the types of auxiliary materials acceptable in the food industry, and auxiliary materials well known to those skilled in the art can be used.

[0038] The enzymatic probiotic fermentation extract of epimedium of the present invention is a novel product prepared by combining epimedium with modern biotechnology, such as molecular adsorption technology, enzymolysis, and probiotic fermentation. Through enzymolysis and fermentation technology, the macromolecular substances in epimedium can be decomposed into small molecular components, and after targeted adsorption by beta-cyclodextrin, its solubility in aqueous medium can be significantly improved, thereby improving the absorption of the effective components of epimedium by human body, and enhancing the health care effect of the product. During the fermentation process, the metabolism of microorganisms can produce some new bioactive substances, which synergize with the effective components of epimedium, further enhance the biological activity, and make it have more significant effects in improving human body functions. At the same time, probiotics multiply in large quantities during the fermentation process, help regulate the balance of human intestinal microecology, promote the growth of beneficial bacteria, inhibit the reproduction of harmful bacteria, enhance intestinal barrier function, improve intestinal digestion and absorption function, thereby improving the absorption capacity of human body to nutrients, and also have a positive impact on human health status, realizing the organic integration of traditional Chinese medicine and modern technology. Due to its good absorbability, biological activity and safety, the enzymatically hydrolyzed and fermented extract of Epimedium should have broad market development prospects. Example 1

[0039] 1. The stems and leaves of Epimedium were crushed and passed through a 60-mesh sieve to obtain Epimedium powder. The Epimedium powder and β-cyclodextrin (the content of β-cyclodextrin in the solvent was 1%) were fully mixed, and then citric acid aqueous solution was added at a solid-liquid ratio of 1:10. After stirring evenly, 0.5% pectinase, 1% cellulase and 1% glycosidase (corresponding to the actual amount of Epimedium added) were added. After enzymolysis at pH 3.5 and temperature of 30°C for 30 min, the mixture was sterilized at high temperature at 90°C for 10 min to obtain the Epimedium solid fermentation matrix.

[0040] 2. Activated Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri were added to the Epimedium solid fermentation matrix at a ratio of 0.1%, stirred and mixed, and placed in an incubator at 30°C. After fermentation for 12 hours, high-temperature sterilization was performed at 90°C for 10 minutes to obtain the Epimedium solid fermentation liquid.

[0041] 3. After centrifuging the solid fermentation liquid of Epimedium at 4000 rpm for 8 min, the precipitate was discarded to obtain the Epimedium fermentation liquid, and the Epimedium fermentation liquid was vacuum freeze-dried at 5°C and a pressure of 50 Pa for 24 h to obtain the Epimedium extract.

[0042] 4. The content of active ingredients (total flavonoids and total polysaccharides) in the epimedium extract was detected by ultraviolet colorimetry, and the extraction rate of epimedium was calculated. The total polysaccharide extraction rate was 9.12%, and the total flavonoid extraction rate was 85.32%. Example 2

[0043] 1. The stems and leaves of Epimedium were crushed and passed through a 60-mesh sieve to obtain Epimedium powder. The Epimedium powder and β-cyclodextrin (the content of β-cyclodextrin in the solvent was 2%) were fully mixed, and then citric acid aqueous solution was added at a solid-liquid ratio of 1:11. After stirring evenly, 0.7% pectinase, 2% cellulase and 2% glycosidase (corresponding to the actual amount of Epimedium added) were added. After enzymolysis at pH 4.0 and temperature of 40°C for 60 min, the mixture was sterilized at high temperature at 95°C for 15 min to obtain the Epimedium solid fermentation matrix.

[0044] 2. Activated Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri were added to the Epimedium solid fermentation matrix at a ratio of 0.3%, stirred and mixed, and placed in an incubator at 35°C. After fermentation for 36 hours, high-temperature sterilization was performed at 95°C for 15 minutes to obtain the Epimedium solid fermentation liquid.

[0045] 3. After centrifuging the solid fermentation liquid of Epimedium at 4500 rpm for 12 min, the precipitate was discarded to obtain the Epimedium fermentation liquid, and the Epimedium fermentation liquid was vacuum freeze-dried at 3°C ​​and a pressure of 40 Pa for 36 h to obtain the Epimedium extract.

[0046] 4. The content of active ingredients (total flavonoids and total polysaccharides) in the epimedium extract was detected by ultraviolet colorimetry, and the extraction rate of epimedium was calculated. The total polysaccharide extraction rate was 9.45%, and the total flavonoid extraction rate was 88.32%. Example 3

[0047] 1. The stems and leaves of Epimedium were crushed and passed through a 60-mesh sieve to obtain Epimedium powder. The Epimedium powder and β-cyclodextrin (the content of β-cyclodextrin in the solvent was 6%) were fully mixed, and then citric acid aqueous solution was added at a solid-liquid ratio of 1:12. After stirring evenly, 0.9% pectinase, 4% cellulase and 4% glycosidase (corresponding to the actual amount of Epimedium added) were added. After enzymolysis at pH 5.5 and temperature of 60°C for 90 min, the solid-state fermentation matrix of Epimedium was obtained by high-temperature sterilization at 100°C for 25 min.

[0048] 2. Activated Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri were added to the Epimedium solid fermentation matrix at a ratio of 0.5%, stirred and mixed, and placed in an incubator at 40°C. After fermentation for 36 hours, high-temperature sterilization was performed at 100°C for 25 minutes to obtain the Epimedium solid fermentation liquid.

[0049] 3. After centrifuging the solid fermentation liquid of Epimedium at 5000 rpm for 12 min, the precipitate was discarded to obtain the Epimedium fermentation liquid, and the Epimedium fermentation liquid was vacuum freeze-dried at 2°C and a pressure of 20 Pa for 60 h to obtain the Epimedium extract.

[0050] 4. The content of active ingredients (total flavonoids and total polysaccharides) in the epimedium extract was detected by ultraviolet colorimetry, and the extraction rate of epimedium was calculated. The total polysaccharide extraction rate was 10.83%, and the total flavonoid extraction rate was 91.22%. Example 4

[0051] 1. The stems and leaves of Epimedium were crushed and passed through a 60-mesh sieve to obtain Epimedium powder. The Epimedium powder and β-cyclodextrin (the content of β-cyclodextrin in the solvent was 8%) were fully mixed, and then citric acid aqueous solution was added at a solid-liquid ratio of 1:15. After stirring evenly, 1.5% pectinase, 3% cellulase and 3% glycosidase (corresponding to the actual amount of Epimedium added) were added. After enzymolysis at pH 6.0 and temperature of 80°C for 180 min, the mixture was sterilized at high temperature at 120°C for 30 min to obtain the Epimedium solid fermentation matrix.

[0052] 2. Activated Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri were added to the Epimedium solid fermentation matrix at a ratio of 1.0%, stirred and mixed, and placed in an incubator at 45°C. After fermentation for 72 hours, high-temperature sterilization was performed at 120°C for 30 minutes to obtain the Epimedium solid fermentation liquid.

[0053] 3. After centrifuging the solid fermentation liquid of Epimedium at 8000 rpm for 15 min, the precipitate was discarded to obtain the Epimedium fermentation liquid, and the Epimedium fermentation liquid was vacuum freeze-dried at 1°C and 0 Pa for 72 h to obtain the Epimedium extract.

[0054] 4. The content of active ingredients (total flavonoids and total polysaccharides) in the epimedium extract was detected by ultraviolet colorimetry, and the extraction rate of epimedium was calculated. The total polysaccharide extraction rate was 11.12%, and the total flavonoid extraction rate was 96.21%. Example 5

[0055] 1. The stems and leaves of Epimedium were crushed and passed through a 60-mesh sieve to obtain Epimedium powder. The Epimedium powder and β-cyclodextrin (the content of β-cyclodextrin in the solvent was 10%) were fully mixed, and then citric acid aqueous solution was added at a solid-liquid ratio of 1:13. After stirring evenly, 1% pectinase, 3% cellulase and 3% glycosidase (corresponding to the actual amount of Epimedium added) were added. After enzymolysis at pH 5.0 and temperature of 50°C for 120 min, the mixture was sterilized at high temperature at 110°C for 20 min to obtain the Epimedium solid fermentation matrix.

[0056] 2. Activated Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri were added to the Epimedium solid fermentation matrix at a ratio of 0.3%, stirred and mixed, and placed in an incubator at 37°C. After fermentation for 48 hours, high-temperature sterilization was performed at 110°C for 20 minutes to obtain the Epimedium solid fermentation liquid.

[0057] 3. After centrifuging the solid fermentation liquid of Epimedium at 6000 rpm for 10 min, the precipitate was discarded to obtain the Epimedium fermentation liquid, and the Epimedium fermentation liquid was vacuum freeze-dried at 0°C and a pressure of 10 Pa for 48 h to obtain the Epimedium extract.

[0058] 4. The content of active ingredients (total flavonoids and total polysaccharides) in the epimedium extract was detected by ultraviolet colorimetry, and the extraction rate of epimedium was calculated. The total polysaccharide extraction rate was 11.51%, and the total flavonoid extraction rate was 98.66%.

[0059] The following test example is described by taking the analysis of the extract of Epimedium enzymatic fermentation prepared in Example 5 as an example: Test Example 1

[0060] The total polysaccharide content of the enzymatic fermentation extract of Epimedium prepared in Example 5 was tested by the following method: Drawing of glucose standard curve: Take the glucose standard and place it in a 55℃ vacuum drying oven to dry to constant weight. Accurately weigh 5 mg of glucose standard, transfer it to a 50 ml volumetric flask, and add distilled water to make up to volume. Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of glucose standard solution into stoppered test tubes, and then add water to 1 mL respectively. Add 1 mL of 5% phenol solution to each test tube, shake well, and then slowly add 5 mL of concentrated sulfuric acid along the wall of the tube and shake well. After standing at room temperature for 30 min, measure the absorbance value at a wavelength of 490nm. Use the concentration of glucose solution as the horizontal axis and the absorbance value as the vertical axis to draw a standard curve and calculate the linear regression equation.

[0061] Detection of total polysaccharide content in enzymatic hydrolysis and fermentation extract of Epimedium: 0.5 g of Epimedium powder was reacted under different enzymatic hydrolysis and fermentation factors. After completion, the extract was diluted 10 times with water, 1.0 mL of sample was placed in a centrifuge tube, 1.0 mL of 8% phenol solution was added to each test tube, and then 5.0 mL of concentrated sulfuric acid was slowly added along the tube wall and shaken. After standing at room temperature for 30 minutes, the absorbance value was measured at a wavelength of 490 nm, and the total polysaccharide content was calculated by substituting it into the glucose standard curve.

[0062] Figure 1 The solutions are epimedium aqueous solution (a), epimedium enzymatic hydrolyzate (b), epimedium enzymatic hydrolyzate fermentation solution (c) and epimedium 50% ethanol solution (d).

[0063] Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6The effect of different enzymatic hydrolysis conditions on the extraction rate of total polysaccharides in Epimedium was studied. When the β-cyclodextrin content was 8%, the enzyme type was pectinase + cellulase + saccharifying enzyme complex, the enzymatic pH was in the range of 4.5-5.5, the enzymatic temperature was 50℃, and the enzymatic time was 90-150 min, the extraction rate was ideal, indicating that the enzymatic reaction was sufficient and stable during this period. After the cell wall was decomposed, more channels were formed inside and outside the cell, which was conducive to the solvent entering the cell and fully contacting with the polysaccharide, thereby increasing the extraction of total polysaccharides. The efficient extraction of total polysaccharides in Epimedium was achieved, and the total polysaccharide extraction rate was 10.88%.

[0064] Figure 7 , Figure 8 , Fig. 9 and Fig.10 The effect of different fermentation conditions on the extraction rate of total polysaccharides in Epimedium was studied. When the lactic acid bacteria were a composite species of Lactobacillus casei + Bifidobacterium lactis + Lactobacillus gasseri, the inoculation amount was 0.5%~0.9%, the fermentation temperature was 37℃, and the fermentation time was 48 h, the microorganisms would produce a variety of substances that could decompose the cell walls of plant cells, releasing the cell wall polysaccharides that were originally difficult to extract, thereby increasing the content of total polysaccharides. The total polysaccharide extraction rate was 11.51%. Test Example 2

[0065] The total flavonoid content of the enzymatic fermentation extract of Epimedium prepared in Example 5 was tested by the following method: Standard curve drawing of rutin: Accurately weigh 5.0 mg of rutin standard, place it in a 10 mL volumetric flask, and dilute to the scale with 70% ethanol to obtain a rutin standard solution with a concentration of 500 μg / mL. The half-dilution method was used, and the dilution concentrations were 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, and 7.8125 μg / mL, respectively. 1.0 mL of rutin standard solution of different concentrations was pipetted, and 0.3 mL of 5% NaNO2 solution was added, mixed evenly, and then allowed to stand for 6 min; then 0.3 mL of 10% Al(NO3)3 solution was added, mixed evenly, and allowed to stand for 6 min; then 4 mL of 4% NaOH solution was added, and allowed to stand for 15 min, and the volume was fixed to the mark with 70% ethanol; the same volume of 70% ethanol was used to replace the rutin standard solution as a blank, and the absorbance at 510 nm was measured in the same way. The standard curve was drawn with rutin concentration as the horizontal axis and absorbance as the vertical axis, and the linear regression equation was calculated.

[0066] Detection of total flavonoids content in enzymatic hydrolysis and fermentation extract of Epimedium: 0.5 g of Epimedium powder was reacted under different enzymatic hydrolysis and fermentation factors, and the extract was diluted 10 times with 70% ethanol. 1.0 mL of sample was placed in a centrifuge tube, 0.3 mL of 5% NaNO2 solution was added, shaken, and allowed to stand for 6 min; then 0.3 mL of 10% Al(NO3)3 solution was added, shaken, and allowed to stand for 6 min; then 4 mL of 4% NaOH solution was added, allowed to stand for 15 min, and the volume was made up to the scale with 70% ethanol; the same volume of 70% ethanol was used to replace the test sample as a blank, and the same method was used to measure the absorbance at 510 nm, and the total flavonoids content was calculated by substituting it into the rutin standard curve.

[0067] Fig.11 , Fig.12 , Fig.13 , Fig.14 and Fig.15 The effect of different enzymatic hydrolysis conditions on the extraction rate of total flavonoids in Epimedium was studied. When the β-cyclodextrin content was 10%, the enzyme type was pectinase + cellulase + saccharifying enzyme complex, the enzymatic pH was 4.0, the enzymatic temperature was 50℃, and the enzymatic time was 120 min, the extraction rate was ideal, and the extraction rate of total flavonoids was 92.44%. Enzymatic hydrolysis may make the plant cell wall structure loose and reduce adhesion, destroy the cell wall barrier, and make the encapsulated active ingredients easier to release.

[0068] Fig.16 , Fig.17 , Fig.18 and Fig.19 The effect of different fermentation conditions on the extraction rate of total flavonoids in Epimedium was studied. When the lactic acid bacteria were a composite species of Lactobacillus casei + Bifidobacterium lactis + Lactobacillus gasseri, the inoculation amount was 0.3-0.5%, the fermentation temperature was 37°C, and the fermentation time was 48 h, the fermentation process could improve the metabolic activity of microorganisms, promote the release and conversion of flavonoids, and the extraction rate of total flavonoids was 98.66%. Test Example 3

[0069] The content of characteristic components of the enzymatic fermentation extract of Epimedium prepared in Example 5 was tested by the following method: Standard curves of epimedin C, icariin, baohuoside 1, and icariin were drawn: 10 mg of epimedin C, icariin, baohuoside 1, and icariin standard were accurately weighed and placed in a 10 mL volumetric flask, and the volume was adjusted to the mark with methanol to obtain a standard solution with a concentration of 1 mg / mL. The diluted concentrations were 1 mg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL, respectively, and HPLC was tested. HPLC detection conditions were: 0-25 min, 25% acetonitrile (A) and 75% water (B); 25-33 min, 30% acetonitrile (A) and 70% water (B); 33-43 min, 52% acetonitrile (A) and 48% water (B), flow rate of 0.7 mL / min, column oven temperature of 30 °C, UV detection wavelength of 270 nm, injection volume of 10 μL. The standard curve was drawn with the concentration of the standard as the abscissa and the absorbance as the ordinate, and the linear regression equation was calculated.

[0070] Detection of the content of characteristic components in the enzymatic hydrolysis and fermentation extract of Epimedium: 0.5 g of Epimedium powder was reacted under different enzymatic hydrolysis and fermentation factors. After completion, the extract was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. After passing through a 0.22 μm organic filter membrane, 10 μL was taken for HPLC testing and the content of epimedin C, icariin, baculoside 1, and icariin was calculated by substituting the standard curve.

[0071] Fig. 20 The HPLC chart of the standard products of four active ingredients in Epimedium (a. epimedin C, b. icariin, c. baculoside 1, d. icariin); Fig.21 , Fig. 22 and Fig.23The HPLC graphs of the four active ingredients (a. epimedin C, b. icariin, c. baculoside 1, d. icariin) before and after the traditional extraction method and enzymatic fermentation are shown respectively. The solubility of the characteristic ingredients of traditional water extraction of epimedium is limited, and it is easily affected by factors such as temperature and pH during the extraction process, and a variety of impurities are extracted. The impurities interact with the characteristic ingredients, and the stability is poor. Chemical changes are prone to occur, resulting in a low content of the characteristic ingredients. Compared with the traditional water extraction method, after enzymatic hydrolysis of epimedium, cellulase and pectinase can decompose the cell wall of epimedium, making it easier for the effective ingredients to be released from the cells. At the same time, glycosidase can convert the flavonoid glycosides in epimedium into more active flavonoid aglycones. Combined with the fermentation process, microbial metabolism can change the pH value and improve the solubility and stability of the effective ingredients. Finally, the content of the characteristic ingredients in the enzymatic fermentation extract of epimedium is higher than that of the traditional extraction method. After enzymatic hydrolysis and fermentation, the contents of epimedin C, icariin, baculoside 1 and icariin were 5.11 mg / g, 6.08 mg / g, 1.62 mg / g and 0.44 mg / g, respectively, which were significantly increased compared with the traditional extraction method of 0.58 mg / g, 0.17 mg / g, 0.02 mg / g and 0.05 mg / g, providing strong data support for the subsequent more efficient development and utilization of epimedium. Test Example 4

[0072] The activity test of epimedium extract on mouse embryonic osteoblast (MC3T3-E1) cells is as follows: Mouse MC3T3-E1 cells were used as a model to evaluate the cytotoxicity of epimedium extract. MC3T3-E1 was cultured in 10% fetal bovine serum + 1% penicillin-streptomycin + 90% DMEM high-glucose culture medium. After digestion with trypsin containing 0.25% EDTA and centrifugation at 1500 r / min for 10 min, passage or suspension preparation was performed. The MTT method was used to determine the toxic effect of epimedium extract on mouse MC3T3-E1 cells. The above centrifuged cells were added to complete fresh culture medium at 5×10 3 / well density, volume of 100 μL / well, put into 96-well plate, incubate at 37℃, 5% CO2 for 12 h. The extract of Epimedium essence was prepared into different concentrations (13.13 μg / mL, 26.25 μg / mL, 52.50 μg / mL, 105 μg / mL, 210 μg / mL) with blank culture medium, and 100 μL of the samples of each concentration were added to the 96-well plate, replacing the original culture medium, repeated 3 times, with blank culture medium as control. Then, 10 μL MTT solution was added to each well, and 100 μL Formazan solution was added to each well, mixed appropriately, and incubated in the incubator for another 3-4 h. Until the Formazan was completely dissolved under an ordinary optical microscope. The absorbance was measured with an enzyme marker at a wavelength of 570 nm. The changes before and after cell proliferation were observed under an optical microscope. The formula for cell survival rate is as follows:

[0073] ODe is the average absorbance of the experimental group, and ODc is the average absorbance of the control group.

[0074] Fig. 20 It can be seen that after the cells were co-incubated with the epimedium extract, the cell viability did not decrease significantly with the increase in the concentration of the epimedium extract. Even when the concentration reached 210 μg / mL, it did not show cytotoxicity, and the survival rate of MC3T3-E1 cells was still above 60%. Fig.21 It can be seen that different concentrations of epimedium extract have a significant effect on cell proliferation activity. Under medium concentration (52.50 μg / mL and 25.25 μg / mL) and high concentration (210 μg / mL and 105 μg / mL) treatment, the cell proliferation effect is most significant. After proliferation, the cell density increases significantly, and the cell morphology becomes irregular and overlaps with each other; under low concentration (12.13 μg / mL) treatment, the cell proliferation effect is relatively weak, the cell density does not increase significantly, and the cell morphology is still relatively regular. Although the control group (NC) has a small amount of proliferation under natural conditions, its proliferation amplitude is significantly smaller than that of the experimental group, which fully shows that the high concentration of epimedium extract has a more prominent effect on promoting cell proliferation. The above results collectively show that the epimedium extract has no obvious cytotoxicity to normal cells, has a high degree of biocompatibility, and has a significant promoting effect on osteoblast proliferation activity.

[0075] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all changes within the meaning and range of equivalents of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an extract of Epimedium enzymatic hydrolysis and probiotic fermentation, characterized in that: The following steps are involved: S1： Pretreatment: crushing the stems and leaves of Epimedium and sieving them to obtain Epimedium powder, mixing the Epimedium powder with a surfactant, adding a solvent, stirring and mixing them thoroughly to obtain a mixed solution; S2: Enzymatic hydrolysis: adding biological enzymes to the mixed solution, stirring the mixed solution for enzymatic hydrolysis under certain temperature and time conditions, and then sterilizing the mixed solution at high temperature to obtain the epimedium solid fermentation matrix; S3: fermentation: adding the lactic acid bacteria liquid to the epimedium solid fermentation matrix, fermenting under certain temperature and time conditions, and sterilizing at high temperature after the fermentation to obtain the epimedium solid fermentation liquid; S4: Preparation of epimedium extract: centrifugally separating the epimedium solid fermentation liquid to obtain a supernatant as the epimedium fermentation liquid, and vacuum freeze-drying the fermentation liquid to obtain the epimedium extract.

2. The method for preparing the enzymatically hydrolyzed and fermented extract of Epimedium according to claim 1, characterized in that: In S1, the stems and leaves of epimedium are crushed and passed through a 60-mesh sieve, the surfactant is β-cyclodextrin, the solvent is a citric acid aqueous solution, the solid-liquid ratio of epimedium micropowder to citric acid aqueous solution is 1 g: (10~15) mL; the content of β-cyclodextrin in the citric acid aqueous solution is 1%~10%.

3. The method for preparing the enzymatically hydrolyzed and fermented extract of Epimedium according to claim 1, characterized in that: The biological enzymes in S2 are pectinase, cellulase and glycosidase. The addition amount of pectinase for enzymatic hydrolysis is 0.5%~1.5%, the addition amount of cellulase is 1%~4%, and the addition amount of glycosidase is 1%~4% (calculated based on the mass fraction of Epimedium).

4. The method for preparing the extract of Epimedium enzymatic hydrolysis and probiotic fermentation according to claim 1, characterized in that: The enzymatic hydrolysis treatment in S2 refers to adding pectinase, cellulase and glycosidase to the citric acid aqueous solution of epimedium powder and β-cyclodextrin; the enzymatic hydrolysis conditions are pH 3.5-6.0, temperature 30°C-80°C, and enzymatic hydrolysis time 30 min-180 min.

5. The method for preparing the enzymatically hydrolyzed and fermented extract of Epimedium according to claim 1, characterized in that: The lactic acid bacteria liquid in S3 is Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri, the inoculation amounts of Lactobacillus casei, Bifidobacterium lactis and Lactobacillus gasseri are 0.1%-1% respectively, the fermentation temperature is 28°C-43°C, and the fermentation time is 12 h-72 h.

6. The method for preparing the enzymatically hydrolyzed and fermented extract of Epimedium according to claim 1, characterized in that: The high temperature sterilization conditions in S2 and S3 are 90℃~120℃, and the sterilization time is 10 min~30 min.

7. The method for preparing the enzymatically hydrolyzed and fermented extract of Epimedium according to claim 1, characterized in that: The rotation speed of the centrifugal separation of the solid-state fermentation liquid of Epimedium in S4 is 4000-8000 rpm; and the time of the centrifugal separation is 8-15 min.

8. The method for preparing the enzymatically hydrolyzed and fermented extract of Epimedium according to claim 1, characterized in that: The vacuum freeze-drying conditions for the fermentation of epimedium in S4 are 0℃~5℃, 0 Pa~50 Pa, and 24 h~72 h to obtain the epimedium extract.

9. An enzymatically hydrolyzed and fermented extract of Epimedium, characterized in that: The extract is prepared by the method for preparing the enzymatically hydrolyzed and fermented probiotic extract of Epimedium as described in any one of claims 1 to 8.

Images