Method for supercritical carbon dioxide extraction of glycine soja isoflavone and product

Abstract

The invention relates to the technical field of food processing, in particular to a method for supercritical carbon dioxide extraction of glycine soja isoflavone and a product. The method comprises the following steps: taking 30-40-mesh defatted soybean meal, putting the defatted soybean meal into an extraction kettle, putting 70% ethanol into an entrainer pump, and carrying out supercritical carbon dioxide extraction under the conditions that the extraction temperature is 50 DEG C, the extraction pressure is 35 mpa, the extraction time is 2 hours, the separation I temperature is 32 DEG C, the separation I pressure is 6 mpa, and the separation II temperature is 30 DEG C and the separation II pressure is 5 mpa; an extracted substance is yellow green liquid, and solanum nigrum isoflavone solid powder can be obtained through subsequent rotary evaporation and freeze-drying treatment. By exploring the interactive influence of the material-liquid ratio, the extraction pressure, the extraction temperature, the extraction time, the particle size and different extraction conditions on the extraction amount of the soybean isoflavone, the method for extracting the soybean isoflavone through the supercritical carbon dioxide is provided, and the extraction amount of the soybean isoflavone can be remarkably increased.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a method for extracting wild soybean isoflavones with supercritical carbon dioxide and a product thereof. Background Art

[0002] Wild soybean (Glycine soja Siebold & Zucc.) is an annual twining herb of the genus Glycine max in the legume family. It is also known as black bean, wild yellow bean, white-flowered broad-leafed creeping bean, etc. The total isoflavone content of wild soybean is about twice that of ordinary soybean. Wild soybean is mainly composed of daidzein, while ordinary soybean is mainly composed of malonyl genistin. Daidaidzein is hydrolyzed into daidzein, and malonyl genistin is hydrolyzed into genistein. Daidaidzein can be converted into equol in the intestine, and its physiological function tends to be estrogenic. It is targeted at women's health, protecting the nest and alleviating menopause. It is mild and non-irritating, and is more suitable for long-term use. The core function of genistein is a potent multi-target anti-cancer effect, especially for breast cancer and prostate cancer.

[0003] Patent publication number CN1570125A discloses a supercritical CO2 extraction process for high-purity soybean isoflavones. The process relates to a method for extracting natural extracts, particularly isoflavones from soybeans, and aims to simplify the process and improve purity. Crude soybean isoflavones are diluted five times with water, adjusted to a pH of 9.5, and ultrafiltered. 1-3% β-glucosidase is added to the filtrate, adjusting the pH to 6.5. The filtrate is incubated at 40°C for 5 hours, filtered, and the precipitate is washed three times with acidic water at pH 5. 30 kg of the precipitate is extracted with supercritical CO2 in a 100 L extraction kettle at a pressure of 8 MPa, a temperature of 40°C, and a CO2 flow rate of 10 kg / h. The resulting off-white powder is soybean isoflavones with a purity of over 90%. However, those skilled in the art have found in experiments that the extraction yield of wild soybean isoflavones using this method is low. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method and product for extracting wild soybean isoflavones with supercritical carbon dioxide, so as to increase the extraction amount of wild soybean isoflavones.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In one aspect, a method for extracting soybean isoflavones using supercritical carbon dioxide is provided, which specifically comprises the following steps:

[0007] Defatted soybean meal with a mesh size of 30-40 is placed in an extraction kettle, and 70% ethanol is placed in an entrainer pump. Supercritical carbon dioxide extraction is performed under the conditions of an extraction temperature of 50°C, an extraction pressure of 35 MPa, an extraction time of 2 hours, a separation I temperature of 32°C, a pressure of 6 MPa, and a separation II temperature of 30°C, a pressure of 5 MPa. The extracted product is a yellow-green liquid, which is subsequently subjected to rotary evaporation and freeze-drying to obtain a solid powder of wild soybean isoflavones.

[0008] Furthermore, the material-liquid ratio of the entrainer to the raw material is 1:4 g / mL.

[0009] In another aspect, a soy isoflavone soft capsule comprising wild soybean isoflavones is provided.

[0010] Furthermore, it also includes evening primrose oil, kudzu flavonoids, calcium citrate, and vitamin D3.

[0011] The beneficial effects of the present invention are:

[0012] This study explores the interactive effects of different extraction conditions, including material-liquid ratio, extraction pressure, temperature, time, particle size, and soy isoflavone extraction, to provide a method for extracting wild soybean isoflavones using supercritical carbon dioxide. This method significantly increases the yield of wild soybean isoflavones. The extracted wild soybean isoflavones can be used to prepare soy isoflavone soft capsules for menopausal relief, hot flashes, and osteoporosis prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the analysis results of the effect of the material-liquid ratio on isoflavone extraction in Example 1;

[0014] Figure 2 Schematic diagram of the analysis results of the effect of extraction pressure on isoflavone extraction in Example 1;

[0015] Figure 3 Schematic diagram of the analysis results of the effect of extraction temperature on isoflavone extraction in Example 1;

[0016] Figure 4 Schematic diagram of the analysis results of the effect of extraction time on isoflavone extraction in Example 1;

[0017] Figure 5 Schematic diagram of the analysis results of the effect of particle size on isoflavone extraction in Example 1;

[0018] Figures 6A-6FSchematic diagram of the analysis results of the interactive effects of different extraction conditions on the extraction amount of soy isoflavones in Example 1; wherein, 6A is a response surface and contour diagram of the effects of extraction temperature and time on the extraction amount of soy isoflavones at a constant particle size of 30 mesh; 6B is a partial enlarged view of 6A; 6C is a response surface and contour diagram of the effects of extraction temperature and particle size on the extraction amount of soy isoflavones at a constant extraction time of 2 h; 6D is a partial enlarged view of 6C; 6E is a response surface and contour diagram of the effects of extraction time and particle size on the extraction amount of soy isoflavones at a constant extraction temperature of 50°C; 6F is a partial enlarged view of 6E. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0020] Example 1

[0021] 1. Implementation Methods

[0022] 100g of defatted soybean meal (30-40 mesh) was placed in an extraction kettle, along with 400ml of 70% ethanol in an entrainer pump. Supercritical carbon dioxide extraction was performed at a temperature of 50°C, a pressure of 35 MPa, and a time of 2 hours. Separation I was performed at a temperature of 32°C and a pressure of 6 MPa, and Separation II was performed at a temperature of 30°C and a pressure of 5 MPa. The product was a yellow-green liquid. Subsequent rotary evaporation and freeze-drying were performed to obtain a solid powder of wild soybean isoflavones.

[0023] 2. Single-factor experimental results

[0024] (1) Effect of solid-liquid ratio on isoflavone extraction

[0025] The analysis results of the effects of different entrainer to raw material ratios (1:1 g / mL, 1:2 g / mL, 1:3 g / mL, 1:4 g / mL) on supercritical extraction of isoflavones are shown in Figure 2. Figure 1 The total isoflavone extraction amount showed a trend of first decreasing and then increasing, and the extraction amount (116μg / g) was the highest when the ratio of entrainer to raw material was 1:4g / mL. The proportion of 12 monomers of isoflavones extracted by supercritical fluid under different material-liquid ratios was as follows: Figure 1The results show that when the solid-liquid ratio is 1:1 g / mL and 1:4 g / mL, the proportion of daidzein is the highest, at 41% and 37% respectively. When the solid-liquid ratio is 1:2 g / mL and 1:3 g / mL, the proportion of genistein is the highest, at 40% and 41% respectively. The extraction content of acetyl daidzein (1.04ug / g) was the highest when the solid-liquid ratio was 1:1g / mL; the extraction content of acetyl glycitin (0.96ug / g), acetyl genistin (1.43ug / g), and malonyl genistin (0.83ug / g) was the highest when the solid-liquid ratio was 1:2g / mL; the extraction content of glycitin (1.85ug / g), malonyl daidzein (2.91ug / g), malonyl daidzein (0.47ug / g), and genistein (28.56ug / g) was the highest when the solid-liquid ratio was 1:3g / mL; the extraction content of daidzein (42.89ug / g), glycitein (12.32ug / g), genistin (2.91ug / g), and daidzein (8.57ug / g) was the highest when the solid-liquid ratio was 1:4g / mL.

[0026] (2) Effect of extraction pressure on isoflavone extraction

[0027] The analysis results of the effects of different extraction pressures (20 MPa, 25 MPa, 30 MPa, 35 MPa) on supercritical extraction of isoflavones are as follows: Figure 2 The total isoflavone extraction amount showed an upward trend, and the extraction amount (126μg / g) was the highest when the extraction pressure was 35mpa. The proportion of 12 monomers of isoflavones extracted by supercritical fluid under different extraction pressure conditions Figure 2 The results show that genistein accounts for the highest proportions at extraction pressures of 20 MPa, 25 MPa, and 30 MPa, at 35%, 36%, and 41%, respectively. Daidzein accounts for the highest proportion at 35 MPa, at 34%. The extraction content of all 12 isoflavone monomers is highest at 35 MPa.

[0028] (3) Effect of extraction temperature on isoflavone extraction

[0029] The analysis results of the effects of different extraction temperatures (40℃, 45℃, 50℃, 55℃) on supercritical extraction of isoflavones are as follows: Figure 3 The total isoflavone extraction amount showed a trend of increasing first and then decreasing, with the highest extraction amount (83 μg / g) at an extraction temperature of 50°C. The proportion of 12 monomers of isoflavones extracted by supercritical extraction under different extraction temperature conditions Figure 3The results show that the highest proportions of genistein were 38%, 37%, 36% and 41% at extraction temperatures of 40, 45, 50 and 55°C, respectively. The highest contents of malonyl glycitin (2.97 μg / g) and acetyl glycitin (1 μg / g) were found at 45°C, while the highest contents of daidzein (29 μg / g), glycitein (8.92 μg / g), genistein (29.63 μg / g), daidzin (5.3 μg / g), malonyl daidzin (2.92 μg / g), genistin (1.51 μg / g), malonyl genistin (0.97 μg / g), acetyl daidzin (0.85 μg / g) and acetyl genistin (1.3 μg / g) were found at 50°C, and the highest content of glycitin was found at 55°C.

[0030] (4) Effect of extraction time on isoflavone extraction

[0031] The analysis results of the effects of different extraction times (1h, 2h, 3h, 4h) on supercritical extraction of isoflavones are as follows: Figure 4 The total isoflavone extraction amount showed a trend of first increasing and then decreasing, with the highest extraction amount (105.6μg / g) at an extraction time of 2h. The proportion of 12 monomers of isoflavones extracted by supercritical extraction under different extraction time conditions Figure 4 The results show that the content of genistein is the highest at 36%, 39%, 41% and 36% when the extraction time is 1h, 2h, 3h and 4h respectively. The content of daidzein (37.72μg / g), glycitein (9.67μg / g), genistein (41.29μg / g), daidzin (5.85μg / g), genistin (2.07μg / g), malonylgenistin (0.94μg / g), acetyldaidzin (0.99μg / g), acetyl glycitein (0.97μg / g) and acetylgenistin (1.52μg / g) is the highest when the extraction time is 2h, while the content of glycitein (1.85μg / g), malonyldaidzin (2.91μg / g) and malonylglycitein (0.47μg / g) is the highest when the extraction time is 3h.

[0032] (5) Effect of particle size on isoflavone extraction

[0033] The results of the analysis of the effects of different particle sizes (20-30 mesh, 30-40 mesh, 40-50 mesh, 50-60 mesh) on supercritical extraction of isoflavones are as follows: Figure 5 The total isoflavone extraction amount showed a trend of first increasing and then decreasing, and the extraction amount (83.4μg / g) was the highest when the particle size was 30-40 mesh. The proportion of 12 monomers of isoflavones extracted by supercritical fluid under different particle size conditions Figure 5It shows that when the particle size is 20-30 mesh, 40-50 mesh, and 50-60 mesh, the proportion of genistein is the highest at 35%, 41%, and 38%, respectively, and when the particle size is 30-40 mesh, the proportion of daidzein is the highest at 35%. The extraction content of malonyl glycitin (0.97μg / g), malonyl genistin (0.95μg / g), and acetyl glycitin (1μg / g) was the highest when the particle size was 20-30 mesh; the extraction content of daidzein (29.03μg / g), glycitein (8.01μg / g), daidzein (9.35μg / g), malonyl daidzein (3.45μg / g), genistin (3.07μg / g), acetyl daidzein (0.95μg / g), and acetyl genistin (1.54μg / g) was the highest when the particle size was 30-40 mesh; the extraction content of genistein (28.56μg / g) and glycitin (1.85μg / g) was the highest when the particle size was 40-50 mesh.

[0034] Since the extraction pressure and the material-liquid ratio showed a linear upward trend, the subsequent response surface optimization experiment selected three factors for optimization: extraction temperature (45°C, 50°C, 55°C), extraction time (1h, 2h, 3h), and particle size (20 mesh, 30 mesh, 40 mesh).

[0035] 3. Response surface experiment results

[0036] Table 1 Optimization of soybean isoflavone extraction conditions by response surface methodology

[0037]

[0038]

[0039] Table 2 Variance analysis of regression model for soybean isoflavone extraction conditions using response surface methodology

[0040]

[0041] The independent variables were extraction temperatures of 45, 50, and 55°C, extraction times of 1, 2, and 3 hours, and particle sizes of 20, 30, and 40 mesh, with the soy isoflavone extraction yield as the response value. The response surface design and results are shown in Table 1. Using Design-expert V13.0.1 analysis software, a multiple regression fit was performed on the experimental data in Table 1, yielding the multiple regression fit equation for the soy isoflavone extraction yield Y versus A (extraction temperature), B (extraction time), and C (particle size):

[0042] Y=1525.26-111.94*A-108.87*B-219.81C-152.07*AB+164.83AC+24.42BC-217.77*A 2-257.04B 2 -940.41*C 2 .

[0043] To verify the reliability of the regression equation and determine the impact of various factors on soy isoflavone extraction, an analysis of variance (ANOVA) was performed on the regression equation. Table 2 shows the ANOVA results. The p-value for the regression model was 0.0001, < 0.05, indicating that the regression model was highly significant. The fitting error was 0.1771, > 0.05, indicating that the fitting error was not significant and that the regression model fitted the actual situation well. Based on the F-value, the influence of various factors on soy isoflavone extraction was C > A > B, i.e., particle size > extraction temperature > extraction time.

[0044] To more intuitively demonstrate the effects of the three factors on soy isoflavone extraction, response surface plots and contour plots were created between each factor and its corresponding value, as shown in Figure 6. Figure 6A shows the response surface plot and contour plot of the effects of extraction temperature and time on soy isoflavone extraction at a constant particle size of 30 mesh. As extraction temperature and time increase, the soy isoflavone extraction rate shows a trend from low to high. That is, at a specific extraction temperature and time, the soy isoflavone extraction rate reaches a maximum of 1581.22 μg / g, located at the top of the response surface plot. The shape of the contours reveals the strength of the interaction effect. When the contour lines are slanted ellipses or saddle-shaped (hyperbolic), an interaction is present. The contour lines in the right panel of Figure 6B show that the contour line is a slanted ellipse, indicating a significant interaction between the two factors, extraction temperature and time. Figure 6C shows the response surface plot and contour plot of the effects of extraction temperature and particle size on soy isoflavone extraction at a constant extraction time of 2 hours. As extraction temperature and particle size increase, the soy isoflavone extraction yield shows a trend from low to high. Specifically, at a specific extraction temperature and particle size, the soy isoflavone extraction yield reaches a maximum of 1581.22 μg / g, located at the top of the response surface plot. The contour lines in Figure 6D show that the plot is a tilted ellipse, indicating a significant interaction between extraction temperature and particle size. Figure 6E shows a response surface and contour plot of the effects of extraction time and particle size on soy isoflavone extraction yield at a constant extraction temperature of 50°C. As extraction time and particle size increase, the soy isoflavone extraction yield shows a trend from low to high. Specifically, at a specific extraction time and particle size, the soy isoflavone extraction yield reaches a maximum of 1581.22 μg / g, located at the top of the response surface plot. The contour lines in the right panel of Figure 6F show that the plot is a symmetrical ellipse, indicating that the interaction between extraction temperature and time is not significant.

[0045] Therefore, the optimal extraction conditions were as follows: extraction pressure 35 MPa, solid-liquid ratio 1:4 g / mL, extraction temperature 50°C, extraction time 2 h, and particle size 30-40 mesh. The maximum extraction amount of soybean isoflavones was 1581.22 ug / g.

[0046] Specifically, the difference from single-factor extraction is explained: When conducting single-factor experiments, the fixed extraction conditions were: extraction temperature 50°C, extraction pressure 30 MPa, solid-liquid ratio 1:3 g / mL, extraction time 3 h, and raw material particle size 40-50 mesh. Compared with the final optimal extraction conditions, it can be found that, except for the extraction temperature, the remaining conditions are inconsistent with the optimal conditions. In the response surface method optimization of soybean isoflavone extraction conditions in Table 1, the extraction yield obtained in the seventh group of experiments was the lowest, about 33 times lower than the optimal group (1581.22 ug / g). The response surface experiment shows that when the response surface experiment was conducted under the optimal conditions of extraction pressure (35 MPa) and solid-liquid ratio (1:4 g / mL), the extraction temperature and particle size, as well as the extraction temperature and extraction time, had extremely significant interactions. Therefore, under the conditions of multiple factors interacting with each other, the isoflavone extraction yield was significantly improved.

[0047] Comparative Example 1

[0048] The wild soybean isoflavones were extracted by the method disclosed in the patent with publication number CN1570125A.

[0049] 100 g of defatted soybean meal with a mesh size of 30-40 was diluted with water, adjusted to a pH of 9.5, and ultrafiltered. 1-3% β-glucosidase was added to the filtrate, and the pH was adjusted to 6.5. The mixture was kept at 40° C. for 5 hours, filtered, and the precipitate was washed three times with acidic water at pH 5. 30 kg of the precipitate was extracted with supercritical CO2 in a 100 L extraction kettle at a pressure of 8 MPa, a temperature of 40° C., and a CO2 flow rate of 10 kg / h. The extracted off-white powder was wild soybean isoflavones (extraction amount was 96.3 μg / g).

[0050] Example 2

[0051] A soybean isoflavone soft capsule comprises the wild soybean isoflavones prepared in Example 1.

[0052] Efficacy: Regulates female menopause, relieves menopausal hot flashes, and prevents osteoporosis.

[0053] Dosage: Take 2 capsules once a day, preferably with warm water half an hour after a meal.

[0054] Contraindications: Not recommended for minors, pregnant women, breastfeeding women, and those during menstruation.

[0055] Element:

[0056]

[0057] In summary, this invention provides a method for extracting wild soybean isoflavones using supercritical carbon dioxide by exploring the interactive effects of different extraction conditions on the yield of soy isoflavones, including the solid-liquid ratio, extraction pressure, temperature, time, particle size, and soy isoflavone extraction. This method significantly increases the yield of wild soybean isoflavones. The extracted wild soybean isoflavones can be used to prepare soy isoflavone soft capsules to regulate menopause in women, alleviate menopausal hot flashes, and prevent osteoporosis.

[0058] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0059] In addition, it should be understood that although this specification is described in terms of 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 embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for extracting soybean isoflavones using supercritical carbon dioxide, characterized in that: The specific steps include: Defatted soybean meal with a mesh size of 30-40 is placed in an extraction kettle, and 70% ethanol is placed in an entrainer pump. Supercritical carbon dioxide extraction is performed under the conditions of an extraction temperature of 50°C, an extraction pressure of 35 MPa, an extraction time of 2 hours, a separation I temperature of 32°C, a pressure of 6 MPa, and a separation II temperature of 30°C, a pressure of 5 MPa. The extracted product is a yellow-green liquid, which is subsequently subjected to rotary evaporation and freeze-drying to obtain a solid powder of wild soybean isoflavones.

2. The method for extracting soybean isoflavones with supercritical carbon dioxide according to claim 1, wherein The material-liquid ratio of entrainer to raw material is 1:4 g / mL.

3. Wild soybean isoflavones prepared by the method according to any one of claims 1-2.

4. A soy isoflavone soft capsule comprising the wild soybean isoflavones according to claim 3.

5. The soy isoflavone soft capsule according to claim 4, characterized in that Also includes evening primrose oil, kudzu flavonoids, calcium citrate, and vitamin D3.

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