Preparation method of trifolium pratense isoflavone and polysaccharide

Abstract

The invention discloses a preparation method of trifolium pratense isoflavone and polysaccharide, and belongs to the field of natural product separation and extraction. The method comprises the following steps: performing ultrasonic extraction on isoflavone from red clover powder by using 50-100% ethanol, and performing freeze drying, extraction and refining to obtain an isoflavone product; and performing ultrasonic extraction on the polysaccharide from the extraction residues by using water, and performing freezing or spray drying to obtain a polysaccharide product. Wherein the polysaccharide product spray drying process is optimized, the optimal conditions are that the concentration multiple is 5 times, the inlet temperature is 151 DEG C and the like, and the yield is improved by 10% compared with that before optimization. According to the method, two products are prepared from one plant material, the activity of the prepared isoflavone product for removing DPPH free radicals is improved by 2 times compared with that of crude isoflavone, and the antioxidant capacity of a polysaccharide product is improved by 2-5 times compared with that of a direct water extraction method. The method realizes scientific and comprehensive utilization of red clover resources, greatly improves the industrial added value, and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of natural product separation and extraction, and particularly relates to a two-step extraction technology for red clover isoflavones and polysaccharides. BACKGROUND

[0002] With the pursuit of natural green and health, the overall plant extract industry in China is in a rapid development stage. According to the data of China Medical and Health Products Import and Export Chamber of Commerce, the total export volume of plant extracts in China reached 4.312 billion US dollars in 2022, with an increase of 8.07% year on year. It is estimated that the market size of China's plant extract industry will reach 7.8 billion US dollars in 2028.

[0003] Red clover (Trifolium pratense L.) is a perennial herbaceous plant widely cultivated in temperate regions, which is rich in bioactive components such as polysaccharides and isoflavones, and has important application value in food, medicine and cosmetics (Zhang et al., 2020; et al., 2024). Red clover isoflavones have a plant estrogen effect, and have significant clinical efficacy in treating women's menopausal syndrome. They can also be used as health products to enhance resistance and improve immune capacity, and are used for the prevention and treatment of breast cancer, cardiovascular disease, osteoporosis and other diseases. Previous studies by the project team have found that red clover polysaccharides have various antioxidant activities, and the activity of scavenging superoxide anion free radicals is significantly higher than that of vitamin C and vitamin E, which can be applied to repair and anti-aging cosmetics production.

[0004] However, current plant extraction is mainly targeted at single components, with low extraction efficiency and low utilization rate of plant resources. The present application utilizes the chemical properties of effective components to establish a method for sequentially extracting and preparing red clover isoflavones and polysaccharides, thereby improving the utilization of red clover plant resources and greatly increasing the added value of the industry. SUMMARY

[0005] The present application aims to provide a two-step extraction technology for red clover isoflavones and polysaccharides to fully improve the utilization of red clover plant resources and solve the problem of low resource utilization rate in current plant extraction which mainly targets single components.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] A preparation method of red clover isoflavones and polysaccharides, comprising the following steps:

[0008] (1) Extraction and purification of isoflavones: red clover powder is added to 10 times the volume of ethanol, and ultrasonic extraction is carried out at 30℃ for 30 minutes. After filtration, the supernatant is collected, concentrated under reduced pressure, and freeze-dried to obtain crude isoflavones. The crude isoflavones are dissolved in water, and repeated extraction is carried out with an equal volume of ethyl acetate for 3 times. The ethyl acetate phase and the water phase are separated, and the ethyl acetate phase is naturally volatilized and dried to obtain isoflavone products.

[0009] (2) Extraction and preparation of polysaccharides: the residue after extracting isoflavones in step (1) is added to 10 times the volume of water, and ultrasonic extraction is carried out at 30℃ for 30 minutes. The extraction is repeated twice, and the supernatant is collected after filtration. The supernatant is concentrated under reduced pressure, and freeze-dried or spray-dried to obtain polysaccharide products.

[0010] Further, the ethanol in step (1) is an ethanol solution with a volume fraction of 50% to 100%.

[0011] Further, the parameters for freeze-drying in steps (1) and (2) are: pressure 0.293hpa, condenser temperature -102℃, shelf temperature 35℃, and product temperature 37℃.

[0012] Further, the optimal conditions for spray drying in step (2) are: concentration ratio 5 times, inlet temperature 151℃, inlet air flow rate 36m 3 / h, feed flow rate 6mL / min, and spray gas flow rate 41mm.

[0013] Further, the total isoflavone content of the isoflavone product prepared in step (1) is 354.36mg / g, and the contents of daidzein, genistein, formononetin and biochanin A are 3-4 times that of the crude isoflavones.

[0014] Further, the polysaccharide product prepared in step (2) has an O2 ·- , DPPH and ABTS free radical scavenging ability 2-5 times higher than that of polysaccharides prepared by direct water extraction.

[0015] Further, the polysaccharide product prepared by spray drying in step (2) has a solubility of 83.05%, which is significantly higher than that of polysaccharide products prepared by freeze-drying.

[0016] Further, the monosaccharide composition of the polysaccharide product in step (2) includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the sum of the molar percentages of glucose, galactose and arabinose is about 83%.

[0017] The present application also provides a red clover isoflavone product prepared by the above method, and the product has a DPPH free radical scavenging ability 1.9 times that of crude isoflavones.

[0018] The application also provides a red clover polysaccharide product prepared by the method, wherein the product is spherical after being spray dried, has a particle size of 558.44±50.21 nm, has an O2 ·- radical scavenging rate of 15 times that of vitamin C.

[0019] Based on the technical scheme, the application has the following technical effects:

[0020] 1. The application sequentially extracts isoflavones and polysaccharides from the same red clover plant powder to prepare two functional products. The total isoflavone content of the prepared isoflavone product is 354.36 mg / g, and the isoflavone product exhibits enhanced DPPH and ABTS radical scavenging activity compared with the crude isoflavone.

[0021] 2. The two-step extraction method of the application can not only obtain isoflavones and other substances, but also significantly improve the antioxidant capacity of polysaccharides, and the O2 ·- , DPPH and ABTS radical scavenging capacity of the polysaccharides is higher than that of the direct water extraction method. Different drying methods have a significant effect on polysaccharides. The polysaccharides prepared by the freeze-drying method have high yield, the polysaccharide product powder prepared by the spray drying method has a spherical shape, small particle size and high solubility. There is no significant difference in the antioxidant capacity of the polysaccharide products prepared by the freeze-drying and spray drying methods.

[0022] 3. Based on the single factor test and response surface optimization design, the optimal conditions for spray drying of red clover polysaccharides are as follows: concentration multiple 5 times, inlet temperature 151℃, inlet air flow rate 36m 3 / h, feed flow rate 6mL / min, and spray gas flow rate 41mm. Under the conditions, the expected polysaccharide yield is 20.66%, which is 10% higher than that before optimization.

[0023] The application develops a process technology for preparing isoflavones and polysaccharides from the same red clover plant material, realizes in-depth development and comprehensive utilization of red clover resources, provides a new idea for the use of plant extracts, and has a wide market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Component analysis of the crude isoflavone sample, the isoflavone product and the polysaccharide sample in the crude isoflavone. Different small letters represent significant differences (P<0.05) in the components of different samples, and ** represents extremely significant differences (P<0.01), and the same below.

[0025] Figure 2 Antioxidant activity evaluation of the crude isoflavone and the isoflavone product. Figures A, B and C respectively represent O2 .- , DPPH and ABTS radical scavenging activity.

[0026] Figure 3Scanning electron microscope images of the freeze-dried and spray-dried polysaccharides. Figures A and B represent the electron microscope images of the freeze-dried and spray-dried polysaccharides, respectively. Among them, EW-F and EW-S represent the freeze-dried polysaccharide and the spray-dried polysaccharide, respectively, and the same applies hereinafter.

[0027] Figure 4 Polysaccharide and uronic acid contents in the freeze-dried and spray-dried polysaccharides.

[0028] Figure 5 Protein and isoflavone contents in the freeze-dried and spray-dried polysaccharides.

[0029] Figure 6 Single-factor results of the spray-drying of the polysaccharide. Among them, A, B, C, D and E represent the effects of the concentration multiple, the inlet temperature, the inlet air flow, the feed flow and the spray gas flow on the polysaccharide yield and the moisture content, respectively.

[0030] Figure 7 Two-factor interaction. Among them, Figures A, B and C represent the interaction of the inlet air flow with the inlet temperature, the inlet air flow with the spray gas flow and the inlet temperature with the spray gas flow, respectively. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0032] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels if not specifically stated.

[0033] The specific techniques or conditions not specified in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0034] The technical solutions of the present application will be described in detail below in combination with the embodiments, but the protection scope is not limited thereto.

[0035] The embodiments of the present application provide a preparation method of red clover isoflavones and polysaccharides. The embodiments are as follows:

[0036] Example 1: Preparation of red clover isoflavones

[0037] 1. Test materials

[0038] The red clover was collected from the above-ground part at the initial flowering stage, dried at 50°C to constant weight, crushed and used.

[0039] 2. Test method

[0040] 2.1. Crude isoflavone preparation method

[0041] Weigh 570.17 g of red clover powder, add 10 times the amount of ethanol, and extract at 30°C for 30 min. Then filter and collect the supernatant. Concentrate the supernatant under reduced pressure, and dry the ethanol extract using a freeze dryer (CoolSafe 110-4, Gene Co., Ltd.). Repeat the process three times for each group. The freeze-drying parameters are: pressure (0.293 hpa), condenser temperature (-102°C), shelf temperature (35°C), and product temperature (37°C).

[0042] 2.2. Isoflavone purification

[0043] According to the principle of "like dissolves like", we use extraction technology to separate the fat-soluble and water-soluble components in the crude isoflavone. First, dissolve the crude isoflavone in an appropriate amount of water, then extract it with an equal volume of ethyl acetate three times. After separating the ethyl acetate phase and the water phase, concentrate them under reduced pressure. The ethyl acetate phase is dried naturally to obtain the isoflavone product, while the water phase is freeze-dried to obtain the polysaccharide component in the crude isoflavone.

[0044] 2.3. Isoflavone content determination

[0045] The total isoflavone content is determined using a microplate reader (Synergy Neo2, Bio Tek, USA). The method is based on the reference Li YS et al. (2019) "Effects of different wavelengths and standard products on the determination of red clover isoflavone content". The contents of four isoflavones, daidzein, genistein, formononetin, and biochanin A, are determined using a high-performance liquid chromatograph (Agilent 1260 II, Agilent Technologies). The method is based on the reference Li YT et al. (2024) "Effects of red and blue light on red clover (Trifolium pratense L.) growth and secondary metabolism".

[0046] 2.4. Polysaccharide content determination

[0047] The polysaccharide content is determined using the phenol-sulfuric acid method. The method is based on the reference Shi WJ. (2020) "Study on the antioxidant activity of polysaccharides and flavonoids in red clover and red clover". The absorbance value is measured at 490 nm, and the polysaccharide content is calculated.

[0048] Polysaccharide content (mg / g) = polysaccharide concentration of the sample to be tested (mg / mL) x extraction volume (mL) / red clover sample mass (g)

[0049] The standard curve is Y=5.1498X+0.0335, R 2 =0.9992.

[0050] 2.5. Antioxidant activity determination

[0051] The radical scavenging activity was determined using a microplate reader. O2 ·- The radical scavenging activity was determined according to the method described in Li Y et al. (2024) “Biomass fractionation techniques impact on the structure and antioxidant properties of isolated lignins”. The DPPH radical scavenging activity was determined according to the method described in Shen SA et al. (2014) “Effects of extraction methods on antioxidant activities of polysaccharides from camellia seed cake”. The ABTS radical scavenging activity was determined according to the method described in Zheng MJ et al. (2024) “Effects of ultra-high pressure assisted extraction on the structure, antioxidant and hypolipidemic activities of Porphyra Haitanensis polysaccharides”.

[0052] 3. Results and analysis

[0053] The isoflavone yield of the crude isoflavone prepared by the present application was 3.33%.

[0054] 3.1. Chemical composition of isoflavone product

[0055] The polysaccharide content in the prepared crude isoflavone was 3.7 times higher than the isoflavone content Figure 1 A). The impurity polysaccharide in the crude isoflavone was removed by extraction, improving the purity of the isoflavone, and the isoflavone product was prepared. The total isoflavone content in the isoflavone product was 1.4 times higher than that of the crude isoflavone Figure 1 A). The daidzein, genistein, formononetin and biochanin A contents in the isoflavone product were significantly higher than those in the crude isoflavone (P<0.05), which were 3-4 times higher than those in the crude isoflavone Figure 1) But the absolute mass of isoflavones did not change much, 24.36 mg in the crude isoflavones and 24.29 mg in the extracted isoflavones, indicating that the extraction process caused little loss of isoflavones. Although there was still a small amount of polysaccharides in the isoflavone product, the content was within 5% (25.12-40.26 mg / g), which was significantly lower than that of the crude isoflavones (P < 0.05).

[0056] 3.2. Antioxidant activity of isoflavone product

[0057] It can be seen that the ability of the isoflavone product to scavenge DPPH free radicals was significantly improved compared with the crude isoflavones (P < 0.05) after extraction, and the activity was 1.9 times that of the crude isoflavones (B). Figure 2 Figure 2 The IC50 value of the isoflavone product for scavenging ABTS free radicals decreased by 31.89% compared with the crude isoflavones (B), but there was no significant difference in the IC50 value between the isoflavone product and the crude isoflavones (P > 0.05, C). 50 50 Figure 2 The results showed that the purity of the isoflavone product was significantly improved by extraction and purification, and the ability to scavenge DPPH and ABTS free radicals was enhanced.

[0058] Example 2: Preparation of red clover polysaccharides

[0059] 1. Test method

[0060] 1.1. Polysaccharide preparation method

[0061] 285 g of the extraction residue was weighed, 30 ℃ water was added at 10 times the amount, and ultrasonic extraction was performed for 30 min, twice. The supernatant was collected by filtration, and the supernatant was concentrated under reduced pressure and spray dried to obtain the polysaccharide product. In order to compare with the direct water extraction method commonly reported, the same method was used to extract and prepare polysaccharides from red clover powder with water. The direct water extract was dried by a spray dryer to obtain polysaccharides prepared by direct extraction.

[0062] Spray drying conditions: air inlet flow 100% (40 m 3 / h), inlet temperature 160 ℃, feed flow 20% (3 mL / min), spray gas flow 40 mm, outlet temperature 90 ℃. Freeze-drying parameters: pressure (0.293 hpa), condenser temperature (-102 ℃), shelf temperature (35 ℃), product temperature (37 ℃).

[0063] 1.2. Determination of yield, solubility, pH, density and porosity of polysaccharide product

[0064] Yield (%): determined by the ratio between the mass of the polysaccharide product collected after drying and the mass of the red clover residue.

[0065] ​​​Solubility (%): After the polysaccharide product of mass M1 was prepared into a 1% (w / v) sample solution with ultrapure water, it was placed in a 30°C water bath for 30 min. Then the solution was centrifuged at 800 x g for 30 min, the precipitate was transferred to a pre-weighed aluminum box, the mass of the aluminum box was recorded as M2, and dried at 120°C to a constant weight M3. Solubility = [M1-(M3-M2)] / M1 x 100.

[0066] pH: A certain mass of frozen and spray-dried sample was prepared into a 2.0 mg / mL solution with ultrapure water, and the pH was recorded after the pH meter was stabilized.

[0067] Bulk density (g / cm 3 ): 0.5 g of sample was placed in a 5 mL graduated glass cylinder. Bulk density = sample mass / total volume.

[0068] Tap density (g / cm 3 ): 0.5 g of sample was placed in a 5 mL graduated glass cylinder, and the cylinder was tapped 100 times on a soft rubber pad 15 cm from the ground. Tap density = sample mass / total volume.

[0069] Particle density (g / cm 3 ): 0.5 g of sample was placed in a 5 mL graduated glass cylinder, and 2.5 mL of petroleum ether was added and shaken well. Then 1 mL of petroleum ether was added along the wall of the cylinder. The total volume was recorded. Particle density = sample mass / (total volume-3.5).

[0070] Porosity (%) = (Particle density - Tap density) / Particle density

[0071] 1.3. Determination of polysaccharide content, uronic acid content, protein content and isoflavone content

[0072] The polysaccharide content and isoflavone content determination method is shown in Example 1. The protein content was determined by the Coomassie Brilliant Blue method, and the uronic acid content was determined by the carbazole sulfate method.

[0073] 1.4 Scanning electron microscopy and monosaccharide composition determination of polysaccharide product

[0074] The appearance and morphology of the polysaccharide product were observed using a desktop scanning electron microscope (JSM-IT200 LA, Japan Corporation). The polysaccharide product was fixed and gold-coated, and its surface structure was observed under high vacuum conditions at an acceleration potential of 15 kV.

[0075] The sample was treated by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method, and the monosaccharide composition in the polysaccharide was analyzed by high performance liquid chromatography. The linear regression equation of the monosaccharide standard is shown in Table 1.

[0076] Table 1 Linear regression equation of monosaccharide standard

[0077]

[0078] 1.5. Determination of molecular weight

[0079] The molecular weight and its distribution of polysaccharide were determined by high performance gel permeation chromatography (Nexera LC-40D XR, Shimadzu Corporation, Japan) equipped with a differential refractive index detector (RID-20A, Shimadzu Corporation, Japan).

[0080] 1.6. Determination of antioxidant activity

[0081] O2 ·- The method for determining the scavenging ability of O2, DPPH and ABTS free radicals is shown in Example 1.

[0082] 2. Results and analysis

[0083] 2.1. Evaluation of antioxidant activity of polysaccharides prepared by different extraction methods

[0084] The polysaccharide product prepared by the present application has a stronger ability to scavenge O2 ·- , DPPH and ABTS free radicals than the polysaccharide prepared by direct extraction, with an activity 2-5 times higher (Table 2). The two-step extraction method not only obtains isoflavones and other substances, but also significantly improves the antioxidant capacity of polysaccharides. The polysaccharide sample in the crude isoflavones in Table 2 is derived from Example 1 and is the polysaccharide component contained in the crude isoflavones, which is separated during the refining process of isoflavone products. The polysaccharide sample in the crude isoflavones has a significantly weaker ability to scavenge O2 ·- , DPPH and ABTS free radicals than the polysaccharide product and the polysaccharide prepared by direct extraction. As shown in Table 3, the polysaccharide product and the polysaccharide sample in the crude isoflavones are both composed of 8 monosaccharides, namely mannose (Man), rhamnose (Rha), glucuronic acid (GluA), galacturonic acid (GalA), glucose (Glu), galactose (Gal), xylose (Xyl) and arabinose (Ara). The polysaccharide product and the polysaccharide sample in the crude isoflavones are both mainly composed of glucose. The glucose in the polysaccharide sample in the crude isoflavones accounts for about 92% of the monosaccharide components, with a significantly higher molar percentage than the polysaccharide product, while the molar percentages of the remaining seven monosaccharides are significantly lower than those of the polysaccharide product (P<0.05). The polysaccharide product has a relatively high proportion of glucose, galactose and arabinose, which account for about 83% of the monosaccharide components. The results show that the polysaccharide component obtained during the extraction of isoflavones in Example 1 is significantly different from the polysaccharide product and is a low-activity component, further demonstrating the scientificity of the polysaccharide preparation method of the present application.

[0085] Table 2 Free radical scavenging activity of polysaccharides prepared by different extraction methods

[0086]

[0087] Note: Different lower case letters indicate significant differences (P < 0.05) between different extraction samples.

[0088] Table 3 Monosaccharide composition of polysaccharide samples in polysaccharide products and crude isoflavones

[0089]

[0090] Note: Different lower case letters indicate significant differences (P < 0.05) in monosaccharide composition among different samples.

[0091] 2.2. Effects of different drying methods on polysaccharide products

[0092] The residue of red clover was weighed at 568.59 g, and 10 times water was added for ultrasonic extraction at 30 °C for 30 min, twice. The supernatant was collected after filtration, and the water extract was divided into two equal parts after appropriate concentration under reduced pressure. The water extract was dried by freeze-drying and spray-drying methods to obtain polysaccharide products, namely freeze-dried polysaccharide (EW-F) and spray-dried polysaccharide (EW-S), respectively, with three replicates in each group. The physicochemical properties, chemical composition, and antioxidant activity of the polysaccharide products were studied.

[0093] As shown in Table 4, the yield of freeze-dried polysaccharide (EW-F) was 21.88%, which was significantly higher than that of spray-dried polysaccharide (EW-S) (P < 0.05), and it was 1.19 times that of the latter. The solubility of spray-dried polysaccharide in water was 83.05% (Table 4), which was significantly higher than that of freeze-dried polysaccharide (P < 0.05), and this characteristic made it have higher potential application as a stabilizer and emulsifier in the food and pharmaceutical industries. In addition, there were differences in the pH values of the polysaccharides prepared by the two drying methods: freeze-dried polysaccharide was neutral, while spray-dried polysaccharide showed weak alkalinity (Table 4).

[0094] The results showed that the bulk and tap densities of spray-dried polysaccharide were significantly smaller than those of freeze-dried polysaccharide (Table 4, P < 0.05), and this difference might be due to the smaller particle size, higher uniformity, and stronger cohesive properties of the powder formed during spray-drying. Particle density and porosity are key factors affecting the stability and storage performance of powder products, and generally, lower porosity and higher particle density are more conducive to maintaining product stability and prolonging shelf life. However, it was found that there was no significant difference in the particle density and porosity of freeze-dried and spray-dried polysaccharides (Table 4, P > 0.05). Freeze-dried polysaccharide showed irregular crumb-like morphology with smooth surface, while spray-dried polysaccharide showed regular spherical shape with smoother surface texture. Figure 3

[0095] ​Table 4. Effect of freeze and spray drying methods on polysaccharide yield and physicochemical properties

[0096]

[0097] Note: Different lower case letters indicate significant difference (P < 0.05) between different dried samples.

[0098] The molecular weight was expressed as "pullulan equivalent" and the weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity index (Mw / Mn) were calculated. As shown in Table 5, the different polysaccharide components and molecular weight might be caused by the difference between the drying methods, which affected the physicochemical properties of the polysaccharide products. The weight average molecular weight of component 1 was the highest in the polysaccharide products prepared by freeze drying. The polysaccharide components 3 in the polysaccharide products prepared by freeze and spray drying methods, and components 2 and 4 in the polysaccharide products prepared by spray drying had a polydispersity index closer to 1, and the molecular weight was more uniform, which was beneficial to the separation and purification.

[0099] Table 5. Effect of freeze and spray drying methods on polysaccharide molecular weight

[0100]

[0101] From Table 6, it can be seen that the polysaccharide content and uronic acid content in the freeze and spray dried polysaccharides had no significant difference (P > 0.05). From Table 6, it can be seen that the polysaccharide content and uronic acid content in the freeze and spray dried polysaccharides had no significant difference (P > 0.05). Figure 4 From Table 6, it can be seen that there were small amounts of protein and isoflavone substances in the polysaccharide products, and the protein and total isoflavone contents in the spray dried polysaccharides were significantly higher than those in the freeze dried polysaccharides (A and B, P < 0.05). From Table 6, it can be seen that the isoflavone content in the polysaccharide products from high to low was formononetin, biochanin A, genistein and daidzein. The contents of genistein, formononetin and biochanin A in the spray dried polysaccharides were high. Figure 5 Figure 5 From Table 6, it can be seen that there were small amounts of protein and isoflavone substances in the polysaccharide products, and the protein and total isoflavone contents in the spray dried polysaccharides were significantly higher than those in the freeze dried polysaccharides (A and B, P < 0.05). From Table 6, it can be seen that the isoflavone content in the polysaccharide products from high to low was formononetin, biochanin A, genistein and daidzein. The contents of genistein, formononetin and biochanin A in the spray dried polysaccharides were high. Figure 5 Figure 5 From Table 6, it can be seen that the isoflavone content in the polysaccharide products from high to low was formononetin, biochanin A, genistein and daidzein. The contents of genistein, formononetin and biochanin A in the spray dried polysaccharides were high.

[0102] From Table 6, it can be seen that the polysaccharide content and uronic acid content in the freeze and spray dried polysaccharides had no significant difference (P > 0.05). From Table 6, it can be seen that the polysaccharide content and uronic acid content in the freeze and spray dried polysaccharides had no significant difference (P > 0.05). ·- The O2 free radical scavenging activity of the polysaccharides prepared by freeze and spray drying methods had no significant difference (P > 0.05).

[0103] Table 6. Free radical scavenging activity of freeze and spray dried samples

[0104]

[0105] Note: Different lower case letters indicate significant difference (P < 0.05) between different dried samples. 50 values of different dried samples.

[0106] Example 3: Optimization of polysaccharide spray drying process ​​

[0107] The research on the physicochemical properties and antioxidant activity of polysaccharide products prepared by freezing and spray drying methods shows that the polysaccharide prepared by spray drying has high content and solubility, regular spherical surface morphology, small particle size and good antioxidant capacity. In order to improve the yield of polysaccharide, the spray drying conditions of polysaccharide are further optimized.

[0108] 1. Single factor test

[0109] 1.1. Effect of concentration multiple on polysaccharide yield and moisture content

[0110] As shown in Figure 6 A, under the conditions of inlet temperature 160℃, air flow rate 40m 3 / h, feed flow rate 6mL / min and spray gas flow rate 40mm, with the increase of concentration multiple, the polysaccharide yield shows a trend of first increasing and then decreasing, and the moisture content shows a downward trend. When the concentration multiple is 5, the polysaccharide yield reaches the highest value 19.03%, but the moisture content does not reach the minimum value. The increase of concentration multiple means the increase of time and energy consumption. Considering the extraction efficiency, time cost and polysaccharide yield, 5 times is the best concentration multiple.

[0111] 1.2. Effect of inlet temperature on polysaccharide yield and moisture content

[0112] As shown in Figure 6 B, under the conditions of concentration multiple 5 times, air flow rate, feed flow rate and spray gas flow rate being constant, with the increase of inlet temperature, the polysaccharide yield shows a trend of first increasing and then decreasing, and the moisture content shows a trend of first decreasing and then increasing. When the inlet temperature is 150℃, the polysaccharide yield reaches the maximum value 19.70%, but the polysaccharide yield at 160℃ has no significant difference (P>0.05) with it. When the inlet temperature is 170℃, the moisture content reaches the minimum value 8.07%, but the inlet temperature at this time has no significant difference (P>0.05) in moisture content with the sample at 160℃. Considering the polysaccharide yield and moisture content comprehensively, 160℃ is the best inlet temperature.

[0113] 1.3. Effect of air flow rate on polysaccharide yield and moisture content

[0114] As shown in Figure 6 C, under the conditions of concentration multiple 5 times, inlet temperature 160℃, feed flow rate and spray gas flow rate being constant, with the increase of air flow rate, the polysaccharide yield shows a trend of first increasing and then decreasing, and the moisture content shows a downward trend. When the air flow rate is 36m 3 / h, the polysaccharide yield reaches the maximum value 19.43%; when the air flow rate is 40m 3 / h, the moisture content reaches the minimum value 9.13%, and has no significant difference (P>0.05) in moisture content with the air flow rate being 36m3 There was no significant difference in moisture content at / h (P>0.05). Therefore, 36m 3 / h represents the optimal intake airflow rate.

[0115] 1.4. Effect of feed flow rate on polysaccharide yield and moisture content

[0116] like Figure 6 As shown in D, the concentration factor is 5 times, the inlet temperature is 160℃, and the inlet air flow rate is 36m³. 3 Under the condition of constant feed flow rate and spray gas flow rate, the polysaccharide yield first increases and then decreases with increasing feed flow rate, while the moisture content first decreases and then increases with increasing feed flow rate. When the feed flow rate is 6 mL / min, the polysaccharide yield reaches its maximum value of 18.43%, and the moisture content reaches its minimum value of 10.33%. Therefore, 6 mL / min is the optimal feed flow rate.

[0117] 1.5. Effect of spray gas flow rate on polysaccharide yield and moisture content

[0118] like Figure 6 As shown in E, with a concentration factor of 5 times, an inlet temperature of 160℃, and an inlet air flow rate of 36m³, 3 Under the condition of constant feed flow rate of 6 mL / min and spray gas flow rate, the polysaccharide yield first increases and then decreases with increasing spray gas flow rate, while the moisture content shows the opposite trend. When the spray gas flow rate is 40 mm, the polysaccharide yield reaches its maximum value of 19.70%, and the moisture content reaches its minimum value of 9.46%. Therefore, 40 mm is the optimal spray gas flow rate.

[0119] 2. Response surface design optimization

[0120] Based on the results of the single-factor experiments, three factors—inlet air flow rate, inlet temperature, and spray gas flow rate—were selected as independent variables from the five variables, with polysaccharide yield as the dependent variable. A three-factor, three-level response surface optimization experiment was designed. The experimental factors and levels are shown in Table 7.

[0121] Table 7 Box-Behnken Design Factor Levels

[0122]

[0123] The experimental design and results of the spray drying process optimization are shown in Table 8.

[0124] Using Design-Expert 13 software, a multiple regression model was fitted, yielding the regression equation: Y = 20.60 + 0.24A + 0.26B + 0.30C + 0.38AB + 0.35AC - 0.55BC - 1.94A 2 -1.34B 2 -0.61C2 .

[0125] The results of variance analysis are shown in Table 9. The significant level P of the model is less than 0.0001, indicating that the regression model fitting is extremely significant; the determination coefficient R 2 = 0.9848, the adjusted determination coefficient R 2 adj = 0.9288, the lack of fit P = 0.3779 > 0.05 is not significant, indicating that the equation has high reliability, the model fitting degree is good, and the model can be used for prediction and analysis of the spray drying process optimization results of red clover polysaccharide. The influence degree of each factor on the response value can be evaluated by comparing the size of F value. The larger the F value, the more significant the influence of the factor on the response value. By comparing the F value, it can be known that the arrangement order of the influence degree of each factor on the observation index is: the spray gas flow (C) > the inlet temperature (B) > the inlet air flow (A).

[0126] Table 8 Box-Behnken design scheme and results

[0127] Serial number A B C Yield (%) 1 1 0 1 18.70 2 -1 1 0 16.80 3 -1 0 -1 18.10 4 0 0 0 20.30 5 0 0 0 20.70 6 1 0 -1 17.60 7 0 -1 1 19.30 8 -1 0 1 17.80 9 0 0 0 20.40 10 1 -1 0 17.10 11 0 -1 -1 17.40 12 1 1 0 18.30 13 0 0 0 20.70 14 0 1 1 18.80 15 0 0 0 20.90 16 -1 -1 0 17.10 17 0 1 -1 19.10

[0128] Table 9 Regression analysis of Box-Behnken test results

[0129]

[0130]

[0131] Note: ** indicates extremely significant difference P < 0.01; * indicates significant difference P < 0.05.

[0132] The interaction between each factor was analyzed by Design-Expert 13 and Origin 2024b software. Figure 7 As shown in the response surface and contour plots, the polysaccharide yield increases first and then decreases with the increase of each factor value. The steepness of the response surface is positively correlated with the influence degree of each factor on the polysaccharide yield; if the response surface is steep, it indicates that the factor has a greater influence on the response value, and if the response surface is flat, the situation is the opposite. Figure 7 A1and Figure 7 B1response surface, the influence degree of polysaccharide yield on inlet temperature and spray gas flow is higher than that of inlet air flow, and the interaction between inlet air flow and spray gas flow is stronger than that between inlet air flow and inlet temperature. Figure 7 A1, Figure 7 B1and Figure 7The slope of the C1 response surface indicates that the influence of the spray gas flow on the polysaccharide yield is higher than that of the inlet temperature. When the contour is circular, it means that the interaction between the two factors is not significant, while the elliptical shape means that the interaction is significant. By observing Figure 7 A2、 Figure 7 B2and Figure 7 C2, it is found that the shapes of the three groups of contour maps are all elliptical, indicating that there is a significant interaction between the inlet flow and the inlet temperature, the inlet flow and the spray gas flow, and the inlet temperature and the spray gas flow.

[0133] 3. Verification of the optimal drying process conditions

[0134] Through single-factor experiments and response surface regression equation model optimization, the optimal process conditions for spray drying are obtained: concentration ratio 5 times, inlet temperature 150.61℃, inlet flow rate 36.36m 3 / h, feed flow rate 6.00mL / min, and spray gas flow rate 41.21mm. Under these process conditions, the polysaccharide yield is the highest, with a predicted value of 20.66%. According to the spray process conditions, a verification test is carried out, and combined with the actual operation, the concentration ratio is selected as 5 times, the inlet temperature is 151℃, the inlet flow rate is 36m 3 / h, the feed flow rate is 6mL / min, and the spray gas flow rate is 41mm. The verification experiment is repeated three times, and the polysaccharide yield obtained is 20.20±0.07%. The relative error between the predicted value and the actual result is 2.24%, indicating that the conditions optimized by the response model have high reliability.

[0135] The present application first uses red clover plants to prepare two products from the same material. The prepared isoflavone product has a DPPH free radical scavenging activity that is 2 times higher than that of the crude isoflavone, and the polysaccharide product has an antioxidant capacity that is 2-5 times higher than that of the direct water extraction method. The plant resources are fully utilized, the industrial added value is greatly increased, the enthusiasm of farmers for planting is improved, the industrial upgrading and transformation are promoted, and a new idea is provided for the deep processing and scientific development of forage grass industry.

[0136] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all embodiments. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method for the preparation of red clover isoflavones and polysaccharides, characterized in that, The method comprises the following steps: (1) extraction and purification of isoflavones: adding red clover powder into 10 times volume of ethanol, and extracting at 30℃ for 30 minutes under ultrasonic, collecting supernatant after filtration, and obtaining crude isoflavones by freeze-drying after concentration under reduced pressure; dissolving the crude isoflavones in water, and repeatedly extracting with equal volume of ethyl acetate for 3 times, separating ethyl acetate phase and water phase, and obtaining isoflavone product by natural evaporation drying of ethyl acetate phase, and obtaining polysaccharide component in crude isoflavones by freeze-drying of water phase; (2) extraction and preparation of polysaccharides: taking the residue after extraction of isoflavones in step (1), adding 10 times volume of water, and extracting at 30℃ for 30 minutes under ultrasonic, repeating extraction for 2 times, collecting supernatant after filtration, and obtaining polysaccharide product by freeze-drying or spray-drying after concentration under reduced pressure.

2. The production method according to claim 1, characterized by, The ethanol in step (1) is an ethanol solution with a volume fraction of 50% to 100%.

3. The preparation method according to claim 1, characterized in that, The parameters of freeze-drying in step (1) and step (2) are: pressure 0.293hpa, condenser temperature -102℃, shelf temperature 35℃, and product temperature 37℃.

4. The method of claim 1, wherein, The conditions for spray drying in step (2) were: concentration factor 5 times, inlet temperature 151°C, inlet air flow 36 m 3 / h, feed flow 6 mL / min, spray gas flow 41 mm.

5. The preparation method according to claim 1, characterized in that, The total isoflavone content in the isoflavone product prepared in step (1) is 354.36mg / g, and the contents of daidzein, genistein, formononetin and biochanin A are 3-4 times of the content of crude isoflavones. The DPPH free radical scavenging activity of isoflavone product is improved by 2 times compared with that of crude isoflavones.

6. The method of claim 1, wherein, The polysaccharide product prepared in step (2) scavenges O2 ·- The ability to scavenge DPPH and ABTS radicals is 2-5 times higher than polysaccharides prepared by direct water extraction method.

7. The preparation method according to claim 1, characterized in that, The solubility of polysaccharide product prepared by spray-drying in step (2) is 83.05%.

8. The method of claim 1, wherein, The monosaccharide composition of polysaccharide product in step (2) includes one or more of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose.

9. A red clover isoflavone product prepared by the method according to any one of claims 1-5, characterized in that, The product has the ability to scavenge DPPH free radicals 1.9 times that of the crude isoflavones, and the IC50 value for scavenging ABTS free radicals is 31.89% lower than that of the crude isoflavones. 50 the product has the ability to scavenge DPPH free radicals 1.9 times that of the crude isoflavones, and the IC50 value for scavenging ABTS free radicals is 10. A red clover polysaccharide product prepared by the method of any one of claims 1 to 4, 6 to 8, characterised in that, The product is spherical after spray drying, with a particle size of 558.44 ± 50.21 nm, and can remove O2 ·- The activity of free radicals is 15 times that of vitamin C.

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