Soybean folic acid extract and preparation method thereof

Abstract

The invention relates to a soybean folic acid extract and a preparation method thereof, and the preparation method comprises the following steps: selecting sprouted soybeans as a raw material, spraying a culture solution containing Mg < 2 + >, Zn < 2 + > and serine in the soybean sprouting process, activating an endogenous enzyme system of sprouted seeds, and increasing the enrichment amount of soybean folic acid (5-CH3-THF); 5-CH3-THF is separated by adopting a supercritical CO2 extraction technology, preliminary purification of an AB-8 macroporous adsorption resin column and secondary purification of an ultrafiltration membrane; ascorbic acid and EDTA-2Na are added into the extracting solution, and the stability of soybean folic acid is further improved after freeze drying. According to the technical scheme, the characteristics of the germinated soybeans are deeply excavated and efficiently utilized, and a technical foundation is laid for production and popularization of natural folic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and in particular to a soybean folic acid extract and a preparation method thereof. Background Art

[0002] Folic acid is a general term for pteroylglutamic acid. It is inactive on its own and requires the catalysis of dihydrofolate reductase to produce tetrahydrofolate and its derivatives. As a carrier of one-carbon units, it participates in key physiological processes such as nucleic acid synthesis and amino acid metabolism, and is a core substance for cell proliferation and DNA synthesis. Folic acid deficiency can lead to anemia, fetal neural tube defects, cardiovascular and cerebrovascular diseases and other problems. Its activated form plays a pivotal role in the metabolic network connecting nucleic acid and amino acid metabolism, and is crucial for maintaining normal physiological functions of the body. Currently, the main way to improve the problem of insufficient folic acid intake in the human body is to add artificial folic acid to food or take it orally. However, the method of supplementing artificial folic acid has the limitation of masking the risk of vitamin B12 deficiency. Increasing the content of natural folic acid in the diet through biotechnology is a healthy, sustainable and low-cost solution.

[0003] Natural folate in food exists primarily as reduced tetrahydrofolate derivatives, including 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, and 10-formyltetrahydrofolate. The most predominant naturally occurring folate is 5-methyltetrahydrofolate (5-CH3-THF), which accounts for over 85% of the content in most foods and nearly 100% in fruits. Compared to synthetic oxidized folate, 5-CH3-THF has higher bioactivity and bioavailability and is the only form of folate that can directly participate in the methylation cycle. However, natural folate is a pteroylglutamate structure composed of pterin, para-aminobenzoic acid, and glutamate, resulting in poor stability. It is particularly susceptible to degradation under environmental conditions such as light, temperature, humidity, and oxidation, resulting in reduced bioactivity and application value.

[0004] Sprouted soybeans are one of the high-quality sources of natural folic acid. Improving the content and stability of soybean folic acid extracted using sprouted soybeans is of great significance to increasing the yield and economic benefits of natural folic acid. Summary of the Invention

[0005] The invention aims to improve the content and stability of soybean folic acid extracted from germinated soybeans.

[0006] To achieve the above objectives, this application is implemented through the following technical solutions: In one aspect, the present application provides a method for preparing a soybean folic acid extract, comprising the following steps: S1. Raw material processing: Soaking soybean seeds in clean water and germinating them to obtain germinated soybeans, and then washing, freeze-drying, and crushing the germinated soybeans to obtain germinated soybean powder; wherein: during the germination of soybean seeds, using a mixture containing Mg 2+ 、Zn 2+ Spraying the culture solution containing serine and germination soybeans; S2. Separation and extraction: Using supercritical CO2 extraction technology and ethanol as an entrainer, the germinated soybean powder was extracted. The extraction process was regulated by a gradient pressure method to finally produce an extract A rich in folic acid; S3, purification: After the extract A is adsorbed and purified using an AB-8 macroporous adsorption resin column, the AB-8 macroporous adsorption resin column is eluted with an ethanol solution, and the eluate is subjected to a secondary purification treatment using an ultrafiltration membrane to obtain a purified extract B rich in folic acid; S4. Stabilization treatment: After adding ascorbic acid and EDTA-2Na to the above extract B, a secondary freeze-drying treatment is performed to obtain a soybean folic acid extract.

[0007] As a further improvement of the present application, in step S1, the soybean seeds are soaked in clean water for 8 to 12 hours, and germinated at a temperature of 22 to 23°C and a relative humidity of 85 to 90% to obtain germinated soybeans with a sprout length of 3 to 5 cm; the germinated soybeans are sequentially washed, freeze-dried, and crushed to obtain germinated soybean powder with a particle size of 160 to 190 mesh; wherein: during the germination of the soybean seeds, the germinated soybeans are sprayed with a culture solution containing MgSO4, ZnSO4 and serine.

[0008] As a further improvement of the present application, during the germination of soybean seeds, the concentration of MgSO4 added to the culture medium is 0.5-0.9 mmol / L, the concentration of ZnSO4 is 0.1-0.2 mmol / L, and the concentration of serine is 0.5-1.0 mmol / L; the optimized MgSO4 concentration is 0.7 mmol / L, the ZnSO4 concentration is 0.15 mmol / L, and the serine concentration is 0.75 mmol / L.

[0009] As a further improvement of the present application, in step S2, the volume fraction of ethanol is 6-9%, the extraction temperature is 35-45°C, the gradient pressure range is 25-28 MPa, and the extraction time lasts 75-105 min.

[0010] As a further improvement of the present application, in step S2, in the supercritical CO2 extraction technology: the volume fraction of ethanol is 8%, the extraction temperature is 38°C to 40°C, the extraction time is 100 minutes, the pressure is maintained at 25 MPa for the first 30 minutes, and then gradually increased to 28 MPa.

[0011] As a further improvement of the present application, in step S3, during the purification treatment, the extract A is passed through the AB-8 macroporous adsorption resin column at a preset flow rate, where the preset flow rate can be 0.5BV / h, 1BV / h, 1.5BV / h, etc. After rinsing with deionized water, it is eluted with an ethanol solution with a volume fraction of 6-10% and the eluate is collected. The eluate is secondary purified using an ultrafiltration membrane with a molecular weight cutoff of 600-1000Da to obtain a purified extract B rich in folic acid.

[0012] As a further improvement of the present application, in step S3, the extract A is passed through an AB-8 macroporous adsorption resin column at a flow rate of 1BV / h, eluted with a 9% volume fraction ethanol solution, and the eluate is secondary purified using an ultrafiltration membrane with a molecular weight cutoff of 800Da to obtain a purified extract B rich in folic acid.

[0013] As a further improvement of the present application, in step S4, 0.15% to 0.25% by mass of ascorbic acid and 0.05% to 0.08% by mass of EDTA-2Na are added to the extract B, stirred evenly, and subjected to secondary freeze-drying to obtain a soybean folic acid extract.

[0014] As a further improvement of the present application, in step S4, 0.20% by mass of ascorbic acid and 0.075% by mass of EDTA-2Na are added to the extract B, stirred evenly, and subjected to secondary freeze-drying to obtain a soybean folic acid extract.

[0015] In order to further optimize the preparation method of soybean folic acid extract, the present invention optimizes the components and concentrations of the germinated soybean culture solution using the accumulation of folic acid (5-CH3-THF) in germinated soybeans as an indicator. In the orthogonal experiment, the experimental conditions were fixed: the soybean germination time was selected as 4 days, the germination temperature was 22-23°C, the relative humidity was 85%-90%, and the Mg content was set to 0. 2+ 、Zn 2+ and serine, three influencing factors, were selected at three levels, among which Mg 2+ The concentrations were 0.5mmol / L, 0.7mmol / L, 0.9mmol / L, Zn 2+ The concentrations were 0.10mmol / L, 0.15mmol / L, and 0.20mmol / L, and the amount of serine added was 0.50mmol / L, 0.75mmol / L, and 1.00mmol / L, respectively. A three-factor three-level orthogonal experiment L9 (3 3 ), to obtain the optimal culture medium composition Mg 2+ 、Zn 2+ and serine concentrations, and the experimental results are shown in Table 1.

[0016] Table 1 Orthogonal experiment results and analysis

[0017] From the range R analysis in Table 1, we can see that: Mg in the culture medium 2+ 、Zn 2+ The order of influence of three factors, serine concentration, on the enrichment of 5-CH3-THF by soybean germination is: serine concentration>Mg 2+ Concentration>Zn 2+ From the K value, we can know that the concentration of the most suitable culture medium components for soybean germination to enrich 5-CH3-THF is: Mg 2+ 0.7mmol / L, Zn 2+ Further validation test results showed that under the optimal combination conditions, the initial folic acid (5-CH3-THF) content in germinated soybeans reached 1346±37µg / 100g DW.

[0018] The optimized Mg 2+ 、Zn 2+ , serine concentration can be used as the optimal concentration of germinated soybean culture solution in the preparation method of soybean folic acid extract of the present invention.

[0019] On the other hand, the present application also provides a soybean folic acid extract, which is prepared by any of the above-mentioned methods for preparing a soybean folic acid extract, wherein: The folic acid (5-CH3-THF) content in the soybean folic acid extract reaches a maximum of 1300µg / 100g DW; After being placed under conditions of a temperature of 25° C. and a relative humidity of 60% for 180 days, the purity of folic acid in the soybean folic acid extract is greater than 91%; After being placed under the conditions of a temperature of 40° C. and a relative humidity of 75% for 7 days, the purity of folic acid in the soybean folic acid extract is above 89%.

[0020] The beneficial effects of this application are as follows: This application makes full use of the characteristics of soybean raw materials, and with the help of the synergistic effect of the active ingredients of the germinated soybeans themselves and exogenous additives, increases the initial folic acid content in the germinated soybeans, and realizes the in-depth exploration and efficient utilization of the characteristics of the germinated soybeans.

[0021] This application adopts supercritical CO2 extraction technology. The extraction process does not use traditional organic solvents, which reduces environmental pollution and solvent residue problems. All additives are safe and non-toxic substances.

[0022] The present application also adds ascorbic acid and EDTA-2Na to the extract, and the two act synergistically to improve the stability of folic acid in the soybean folic acid extract. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions of the present application. Obviously, the embodiments described are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0024] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below in conjunction with specific implementation methods.

[0025] In order to improve the content and stability of soybean folic acid extracted from germinated soybeans, the present application provides a method for preparing soybean folic acid extract, which is as follows: During the germination of soybean seeds, Mg 2+ 、Zn 2+ Spraying the germinated soybeans with a culture solution containing serine activates the endogenous enzyme system of the germinating seeds and increases the enrichment of soybean folic acid (including 5-CH3-THF, etc.); Supercritical CO2 extraction technology was used to extract germinated soybean powder with ethanol as the entrainer. The extraction process was regulated by the gradient pressure method, and finally, extract A rich in folic acid was obtained. Extract A is adsorbed and purified using an AB-8 macroporous adsorption resin column, the AB-8 macroporous adsorption resin column is eluted with an ethanol solution, and the eluate is subjected to a secondary purification treatment using an ultrafiltration membrane to obtain a purified extract B rich in folic acid; After adding ascorbic acid and EDTA-2Na to the extract B, the extract was freeze-dried twice to obtain a soybean folic acid extract.

[0026] Example 1 S1. Raw material processing: Soybean seeds with high germination rate were selected, soaked in clean water for 10 hours, transferred to a constant temperature and humidity chamber, and germinated at a temperature of 22°C and a relative humidity of 85%. During the germination of soybean seeds, the Mg-containing 2+ 、Zn 2+ Spraying the germinated soybeans with a culture solution containing 0.7 mmol / L MgSO4, 0.15 mmol / L ZnSO4 and 0.75 mmol / L serine, wherein the culture solution is added with 0.7 mmol / L MgSO4, 0.15 mmol / L ZnSO4 and 0.75 mmol / L serine, and harvesting the germinated soybeans when the bean sprouts are 4.5 cm in length; After cleaning the germinated soybeans, place them in a freezing device with a vacuum degree of 20Pa and a cold trap temperature of -65℃. Spread the washed germinated soybeans on the tray of the freezing device with a thickness of 2.5cm. Pre-freeze them at -45℃ for 2.5h, then heat them to -20℃ at a rate of 0.8℃ / min under a vacuum degree of 20Pa, maintain them for 4h for the first drying, then heat them to 20℃ at a rate of 0.4℃ / min, maintain them for 2.5h to complete the second drying, and finally grind them into 170 mesh fine powder.

[0027] S2. Separation and extraction: The above-mentioned fine powder was loaded into a CO2 extraction device, CO2 was used as the extraction agent, and 8% by volume of ethanol was added as the entrainer. Supercritical CO2 extraction was carried out at a pressure of 25-28 MPa and a temperature of 38-40°C. The pressure was maintained at 25 MPa for the first 30 minutes and then gradually increased to 28 MPa. The entire extraction time was 100 minutes, and finally, an extract A rich in folic acid was obtained.

[0028] S3. Purification treatment: First, the extract A is passed through an AB-8 macroporous adsorption resin column at a flow rate of 1 BV / h; secondly, the AB-8 macroporous adsorption resin column is rinsed with deionized water until it becomes colorless; thirdly, the AB-8 macroporous adsorption resin column is eluted with a 9% by volume ethanol solution and the eluate is collected; finally, the eluate is subjected to a secondary purification using an ultrafiltration membrane with a molecular weight cutoff of 800 Da to obtain a purified extract B rich in folic acid.

[0029] S4. Stabilization treatment: After adding 0.20% by mass of ascorbic acid and 0.075% by mass of EDTA-2Na to the extract B, stir evenly to obtain a mixed solution, and place the mixed solution on a tray with a thickness of 1.5 cm in a freeze-drying device with a vacuum degree of 20 Pa and a cold trap temperature of -65°C. First, pre-freeze it at -45°C for 1.5 hours, and then slowly heat it to -20°C at a heating rate of 0.4°C / min under a vacuum environment of 20 Pa, maintain it for 2.5 hours for primary drying, and then heat it to 10°C at a heating rate of 0.25°C / min, maintain it for 1.5 hours to complete secondary drying, and obtain soybean folic acid extract.

[0030] Example 2 S1. Raw material processing: Soybean seeds with high germination rate were selected, soaked in clean water for 8 hours, transferred to a constant temperature and humidity chamber, and germinated at a temperature of 23°C and a relative humidity of 90%. During the germination of soybean seeds, the Mg-containing 2+ 、Zn 2+Spraying the germinated soybeans with a culture solution containing 0.7 mmol / L MgSO4, 0.15 mmol / L ZnSO4 and 0.75 mmol / L serine, wherein the culture solution is added with 0.7 mmol / L MgSO4, 0.15 mmol / L ZnSO4 and 0.75 mmol / L serine, and harvesting the germinated soybeans when the bean sprouts are 3 cm in length; After cleaning the germinated soybeans, place them in a freezing device with a vacuum degree of 20Pa and a cold trap temperature of -65℃. Spread the washed germinated soybeans on the tray of the freezing device with a thickness of 2.5cm. Pre-freeze them at -45℃ for 2.5h, then heat them to -20℃ at a rate of 0.8℃ / min under a vacuum degree of 20Pa, maintain them for 4h for the first drying, then heat them to 20℃ at a rate of 0.4℃ / min, maintain them for 2.5h to complete the second drying, and finally grind them into 160 mesh fine powder.

[0031] S2. Separation and extraction: The above-mentioned fine powder was loaded into a CO2 extraction device, CO2 was used as the extraction agent, and 6% by volume of ethanol was added as the entrainer. Supercritical CO2 extraction was carried out at a pressure of 25-28 MPa and a temperature of 35°C. The pressure was maintained at 25 MPa for the first 30 minutes and then gradually increased to 28 MPa. The whole extraction time was 75 minutes, and finally, an extract A rich in folic acid was obtained.

[0032] S3. Purification treatment: First, the extract A is passed through an AB-8 macroporous adsorption resin column at a flow rate of 1 BV / h; secondly, the AB-8 macroporous adsorption resin column is rinsed with deionized water until it becomes colorless; thirdly, the AB-8 macroporous adsorption resin column is eluted with a 6% by volume ethanol solution and the eluate is collected; finally, the eluate is subjected to a secondary purification using an ultrafiltration membrane with a molecular weight cutoff of 600 Da to obtain a purified extract B rich in folic acid.

[0033] S4. Stabilization treatment: After adding 0.15% by mass of ascorbic acid and 0.05% by mass of EDTA-2Na to the extract B, stir evenly to obtain a mixed solution, and place the mixed solution on a tray with a thickness of 1.5 cm in a freeze-drying device with a vacuum degree of 20 Pa and a cold trap temperature of -65°C. First, pre-freeze it at -45°C for 1.5 hours, and then slowly heat it to -20°C at a heating rate of 0.4°C / min under a vacuum environment of 20 Pa, maintain it for 2.5 hours for primary drying, and then heat it to 10°C at a heating rate of 0.25°C / min, maintain it for 1.5 hours to complete secondary drying, and obtain soybean folic acid extract.

[0034] Example 3 S1. Raw material processing: Soybean seeds with high germination rate were selected, soaked in clean water for 12 hours, transferred to a constant temperature and humidity chamber, and germinated at a temperature of 22°C and a relative humidity of 85%. During the germination of soybean seeds, the Mg-containing 2+、Zn 2+ Spraying the germinated soybeans with a culture solution containing 0.7 mmol / L MgSO4, 0.15 mmol / L ZnSO4 and 0.75 mmol / L serine, wherein the culture solution is added with 0.7 mmol / L MgSO4, 0.15 mmol / L ZnSO4 and 0.75 mmol / L serine, and harvesting the germinated soybeans when the bean sprouts are 5 cm in length; After cleaning the germinated soybeans, place them in a freezing device with a vacuum degree of 20Pa and a cold trap temperature of -65℃. Spread the washed germinated soybeans on the tray of the freezing device with a thickness of 2.5cm. Pre-freeze them at -45℃ for 2.5h, then heat them to -20℃ at a rate of 0.8℃ / min under a vacuum degree of 20Pa, maintain them for 4h for the first drying, then heat them to 20℃ at a rate of 0.4℃ / min, maintain them for 2.5h to complete the second drying, and finally grind them into 190 mesh fine powder.

[0035] S2. Separation and extraction: The above-mentioned fine powder was loaded into a CO2 extraction device, CO2 was used as the extraction agent, and 9% by volume of ethanol was added as the entrainer. Supercritical CO2 extraction was carried out at a pressure of 25-28 MPa and a temperature of 45°C. The pressure was maintained at 25 MPa for the first 30 minutes and then gradually increased to 28 MPa. The whole extraction time was 105 minutes, and finally, an extract A rich in folic acid was obtained.

[0036] S3. Purification treatment: First, the extract A is passed through an AB-8 macroporous adsorption resin column at a flow rate of 1 BV / h; secondly, the AB-8 macroporous adsorption resin column is rinsed with deionized water until it becomes colorless; thirdly, the AB-8 macroporous adsorption resin column is eluted with a 10% by volume ethanol solution and the eluate is collected; finally, the eluate is subjected to a secondary purification using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain a purified extract B rich in folic acid.

[0037] S4. Stabilization treatment: After adding 0.25% by mass of ascorbic acid and 0.08% by mass of EDTA-2Na to the extract B, stir evenly to obtain a mixed solution, and place the mixed solution on a tray with a thickness of 1.5 cm in a freeze-drying device with a vacuum degree of 20 Pa and a cold trap temperature of -65°C. First, pre-freeze it at -45°C for 1.5 hours, and then slowly heat it to -20°C at a heating rate of 0.4°C / min under a vacuum environment of 20 Pa, maintain it for 2.5 hours for primary drying, and then heat it to 10°C at a heating rate of 0.25°C / min, maintain it for 1.5 hours to complete secondary drying, and obtain soybean folic acid extract.

[0038] Comparative Example 1 The difference between this comparative example and Example 1 is that, in step S1, pure water is used instead of culture solution for spraying during the germination of soybean seeds.

[0039] Comparative Example 2 The difference between this comparative example and Example 1 is that in step S1, ungerminated soybean seeds are directly crushed into 170 mesh fine powder.

[0040] Comparative Example 3 This comparative example differs from Example 1 in that, in step S2, the separation and extraction steps are as follows: using methanol as the solvent for extraction, soybean powder and methanol are mixed in a ratio of 1:10, and refluxed at 50°C for 3 hours. The extract is filtered and collected, and the methanol is then removed by reduced pressure distillation to produce extract A. Subsequent extraction and stabilization treatments are the same as in Example 1.

[0041] Comparative Example 4 The difference between this comparative example and Example 1 is that in step S4, during the stabilization treatment, only 0.2% by mass of ascorbic acid was added to the extract B without adding EDTA-2Na, and the mixture was stirred evenly and then freeze-dried to obtain a soybean folic acid extract.

[0042] Performance testing: (1) Folic acid content detection method High-performance liquid chromatography (HPLC) was used to determine the folic acid content in germinated soybeans and their extracts. Folic acid refers to tetrahydrofolate (THF) and its derivatives. 5-methyltetrahydrofolate (5-CH3-THF) is the most physiologically active form of folic acid and can be directly utilized by organisms. 5-CH3-THF accounts for the largest proportion of folic acid, and the 5-CH3-THF content is often used to represent the content of natural folic acid.

[0043] 1. Preparation of Standards: Weigh 0.0100 g of 5-CH3-THF standard. In the dark, dilute to a 50 mL brown volumetric flask with a small amount of 0.1 M phosphate buffer (pH 6.1, containing 1% ascorbic acid and 0.1% 2-mercaptoethanol) as the extractant to prepare a 200 µg / mL 5-CH3-THF standard stock solution. Aliquot and store in a -20°C refrigerator. Upon use, gradually dilute the 5-CH3-THF standard stock solution to prepare test standard solutions of 400 ng / mL, 200 ng / mL, 100 ng / mL, 40 ng / mL, and 4 ng / mL.

[0044] 2. Sample preparation: 0.3 g of the germinated soybean powder freeze-dried in step S1 of each example and comparative example was taken, 7 mL of phosphoric acid extract and 1 mL of amylase-protease solution were added, nitrogen was added, vortexed, and enzymatically hydrolyzed at 37°C for 2 h, then in a boiling water bath for 10 min, rapidly cooled on ice, and centrifuged (12000 r / min, 20 min). 3 mL of the supernatant was taken, 200 μL of rat serum was added, nitrogen was added, vortexed, and enzymatically hydrolyzed at 37°C for 2 h, then in a boiling water bath for 5 min, rapidly cooled on ice, and centrifuged (12000 r / min, 20 min). The resulting sample solution was filtered through a membrane with a pore size of 0.45 μm and placed in a brown injection bottle for testing.

[0045] 3. Liquid phase determination conditions Mobile phase: acetonitrile, 30 mM sodium phosphate buffer (pH 2.3); chromatographic column: Zorbax SB, C18, 250*4.6 mm, 5 μm; column temperature: 35°C; flow rate: 0.4 mL / min; injection volume: 20 μL; detector: fluorescence detector at excitation wavelength / emission wavelength of 290 / 360 nm.

[0046] The elution program is shown in Table 2: Table 2 Gradient elution program

[0047] The 5-CH3-THF content of the sample was obtained from the standard curve.

[0048] The calculation formula is as follows:

[0049] Where: X—5-CH3-THF content in the sample, unit is μg / 100g C—5-CH3-THF concentration obtained from the standard curve, in ng / mL V—sample volume, in mL m—sample mass, in g (2) Test results and analysis 1. Initial folic acid content in germinated soybeans under different treatment conditions Table 3 Comparison of initial 5-CH3-THF content in germinated soybeans under different treatment conditions

[0050] From the comparative analysis of Comparative Example 1 and Examples 1-3 in Table 3, it can be seen that during the germination of soybeans, the Mg-containing 2+ 、Zn 2+ and serine culture medium, significantly increased the folic acid content in soybean folic acid extract, this is because the Mg 2+、Zn 2+ and serine in the culture medium is beneficial to the formation and accumulation of soybean folic acid (5-CH3-THF) in germinated soybeans.

[0051] A comparative analysis of Comparative Example 2 and Examples 1-3 shows that the use of germinated soybeans to prepare soybean folic acid extract significantly increases the folic acid content in the soybean folic acid extract. This is because the endogenous enzyme system of the seeds is activated during the soybean germination process, effectively increasing the amount of soybean folic acid (5-CH3-THF) formed. The folic acid (5-CH3-THF) content in the soybean folic acid extract obtained in Example 2 is increased by 53.83% compared with that in Comparative Example 2.

[0052] In addition, according to the comparison between Examples 1-3 and Comparative Examples 1 and 2, it can be seen that the soybean folic acid extract is prepared by selecting germinated soybeans, and at the same time, the Mg-containing solution is sprayed on the germinated soybeans during the germination process. 2+ 、Zn 2+ The folic acid content in the soybean folic acid extract obtained by adding culture medium containing 1% glutamic acid and 1% serine is much higher than that in the soybean folic acid extracts prepared in Comparative Example 1 and Comparative Example 2, reflecting the synergistic effect of the endogenous enzyme system and the additives.

[0053] Although the 5-CH3-THF content in the conventional parameter treatment groups (Examples 2 and 3) was lower than that in Example 1, it was still 30.63% higher than that in Comparative Example 2. Furthermore, according to Examples 1-3, the folic acid (5-CH3-THF) content in the soybean folic acid extract prepared using the technical solution of the present application reached 1114-1346 μg / 100 g DW.

[0054] 2. Results of folic acid stability test in soybean folic acid extract 1) Stability test results of folic acid in soybean folic acid extract under room temperature storage conditions After the soybean folic acid extracts prepared in Examples 1-3 and Comparative Examples 1-4 were stored at room temperature (25° C.) and a relative humidity of 60% for 180 days, the folic acid (5-CH 3 -THF) content in the soybean folic acid extracts of each Example and each Comparative Example was tested. The results are shown in Table 4.

[0055] Table 4 Retention rate of 5-CH3-THF in soybean folic acid extract under room temperature storage conditions

[0056] As shown in Table 4, after 180 days of storage at room temperature (25°C) and a relative humidity of 60%, the 5-CH3-THF in Examples 1 to 3 exhibited high stability, with retention rates much higher than those in Comparative Examples 1 to 4. The soybean folic acid extract prepared using the technical solution of the present application exhibited a folic acid retention rate exceeding 91% after 180 days of storage at 25°C and a relative humidity of 60%. This demonstrates that the preparation or storage methods of the Examples can effectively slow the natural degradation of 5-CH3-THF, and that stable protection can be achieved by optimizing the antioxidant system or inhibiting enzyme activity. In contrast, the comparative examples exhibited a high 5-CH3-THF loss rate due to insufficient processes or conditions.

[0057] 2) Stability test results of folic acid in soybean folic acid extract under high temperature storage conditions The soybean folic acid extracts prepared in Examples 1-3 and Comparative Examples 1-4 were placed at a temperature of 40° C. and a relative humidity of 75% for 7 days, and the folic acid (5-CH 3 -THF) content in the soybean folic acid extracts of each Example and each Comparative Example was tested. The results are shown in Table 5.

[0058] Table 5 Retention rate of 5-CH3-THF in soybean folic acid extract under high temperature storage conditions

[0059] As shown in Table 5, after 7 days of storage at 40°C and 75% relative humidity, the 5-CH3-THF retention rates in the soybean folic acid extracts of Examples 1-3 decreased, but they still maintained relatively high stability, with folic acid retention rates exceeding 88%. In Comparative Examples 1-4, the 5-CH3-THF retention rates were generally lower than those of the Examples, ranging from only 50.77% to 83.16%. This indicates that the synergistic effect of the germinated soybean folic acid extract and the various process conditions, and the synergistic effect of the active ingredients of the germinated soybeans and the exogenous additives during the stabilization treatment, significantly reduced the damage to 5-CH3-THF caused by the humid and hot environment. However, due to the lack of targeted protective measures in the comparative examples, the degradation rate of 5-CH3-THF was accelerated.

[0060] 3) Stability test results of folic acid in soybean folic acid extract under simulated sunlight conditions The soybean folic acid extracts prepared in Examples 1-3 and Comparative Examples 1-4 were placed under simulated sunlight for 5 days, and the folic acid (5-CH3-THF) content in the soybean folic acid extracts in each Example and each Comparative Example was tested. The results are shown in Table 6.

[0061] Table 6 Decrease rate of 5-CH3-THF content in soybean folic acid extract under light conditions

[0062] As shown in Table 6, after the soybean folic acid extracts were placed under simulated sunlight for 5 days, the 5-CH3-THF content in the soybean folic acid extracts of Examples 1-3 exhibited good stability under these conditions. In Comparative Examples 1-4, the 5-CH3-THF content decreased significantly compared to the Examples, with the decrease in Comparative Example 3 reaching a high of 30.41%. This indicates that the soybean folic acid extracts of the Examples better maintain the 5-CH3-THF content under light conditions, while light has a greater impact on the stability of 5-CH3-THF in the soybean folic acid extracts of the Comparative Examples.

[0063] Under the above three soybean folic acid extract 5-CH3-THF stability test conditions, the examples all significantly improved the stability of 5-CH3-THF through process optimization (such as formula design and stabilizer addition), verifying the key role of exogenous intervention in the protection of 5-CH3-THF and providing reliable technical support for industrial storage.

[0064] Although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0065] The series of detailed descriptions listed above are only specific descriptions of the feasible implementation methods of this application and are not intended to limit the scope of protection of this application. Any equivalent implementation methods or changes that do not deviate from the technical spirit of this application should be included in the scope of protection of this application.

Claims

1. A method for preparing soybean folic acid extract, characterized in that: The steps include: S1. Raw material processing: Soaking soybean seeds in clean water and germinating them to obtain germinated soybeans, and then washing, freeze-drying, and crushing the germinated soybeans to obtain germinated soybean powder; wherein: during the germination of soybean seeds, 2+ 、Zn 2+ Spraying the culture solution containing serine and germination soybeans; S2. Separation and extraction: Using supercritical CO2 extraction technology and ethanol as an entrainer, the germinated soybean powder was extracted. The extraction process was regulated by a gradient pressure method to finally produce an extract A rich in folic acid; S3, purification: After the extract A is adsorbed and purified using an AB-8 macroporous adsorption resin column, the AB-8 macroporous adsorption resin column is eluted with an ethanol solution, and the eluate is subjected to a secondary purification treatment using an ultrafiltration membrane to obtain a purified extract B rich in folic acid; S4. Stabilization treatment: After adding ascorbic acid and EDTA-2Na to the above extract B, a secondary freeze-drying treatment is performed to obtain a soybean folic acid extract.

2. The method for preparing the soybean folic acid extract according to claim 1, wherein In step S1, soybean seeds are soaked in clean water for 8 to 12 hours and germinated at a temperature of 22 to 23° C. and a relative humidity of 85 to 90% to obtain germinated soybeans with a sprout length of 3 to 5 cm; the germinated soybeans are washed, freeze-dried, and crushed in sequence to obtain germinated soybean powder with a particle size of 160 to 190 mesh; wherein: during the germination of the soybean seeds, the germinated soybeans are sprayed with a culture solution containing MgSO4, ZnSO4 and serine.

3. The method for preparing the soybean folic acid extract according to claim 2, wherein: During the germination of soybean seeds, the concentration of MgSO4 added to the culture medium is 0.5-0.9 mmol / L, the concentration of ZnSO4 is 0.1-0.2 mmol / L, and the concentration of serine is 0.5-1.0 mmol / L; preferably, the concentration of MgSO4 added to the culture medium is 0.7 mmol / L, the concentration of ZnSO4 is 0.15 mmol / L, and the concentration of serine is 0.75 mmol / L.

4. The method for preparing the soybean folic acid extract according to claim 1, wherein In step S2, the volume fraction of ethanol is 6-9%, the extraction temperature is 35-45° C., the gradient pressure ranges from 25-28 MPa, and the extraction time lasts for 75-105 min.

5. The method for preparing the soybean folic acid extract according to claim 4, characterized in that: In step S2, in the supercritical CO2 extraction technology: the volume fraction of ethanol is 8%, the extraction temperature is 38°C to 40°C, the extraction time is 100 minutes, the pressure is maintained at 25 MPa for the first 30 minutes, and then gradually increased to 28 MPa.

6. The method for preparing the soybean folic acid extract according to claim 1, characterized in that: In step S3, during the purification treatment, the extract A is passed through an AB-8 macroporous adsorption resin column at a preset flow rate, rinsed with deionized water, eluted with an ethanol solution with a volume fraction of 6 to 10%, and the eluate is collected. The eluate is subjected to secondary purification using an ultrafiltration membrane with a molecular weight cutoff of 600 to 1000 Da to obtain a purified extract B rich in folic acid.

7. The method for preparing the soybean folic acid extract according to claim 6, characterized in that: In step S3, the extract A is passed through an AB-8 macroporous adsorption resin column at a flow rate of 1 BV / h, eluted with a 9% volume fraction ethanol solution, and the eluate is secondary purified using an ultrafiltration membrane with a molecular weight cutoff of 800 Da to obtain a purified extract B rich in folic acid.

8. The method for preparing the soybean folic acid extract according to claim 1, wherein In step S4, 0.15% to 0.25% by mass of ascorbic acid and 0.05% to 0.08% by mass of EDTA-2Na are added to the extract B, stirred evenly, and subjected to secondary freeze-drying to obtain a soybean folic acid extract.

9. The method for preparing the soybean folic acid extract according to claim 8, characterized in that: In step S4, 0.20% by mass of ascorbic acid and 0.075% by mass of EDTA-2Na are added to the extract B, stirred evenly, and subjected to secondary freeze-drying to obtain a soybean folic acid extract.

10. A soybean folic acid extract, characterized in that It is prepared by the preparation method of the soybean folic acid extract according to any one of claims 1 to 9, wherein: The folic acid (5-CH3-THF) content in the soybean folic acid extract reaches a maximum of 1300µg / 100g DW; After being placed under conditions of a temperature of 25° C. and a relative humidity of 60% for 180 days, the retention rate of folic acid content in the soybean folic acid extract is above 91%; After being placed under conditions of a temperature of 40° C. and a relative humidity of 75% for 7 days, the retention rate of the folic acid content in the soybean folic acid extract is above 89%.

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