Technology for extracting total flavonoids from fermented soybean by using ultrasonic-assisted deep-eutectic solvent

Abstract

The invention belongs to the field of fermented soybean general flavone extraction, and particularly relates to a process for extracting fermented soybean general flavone through an ultrasonic-assisted deep-eutectic solvent, which comprises the following steps: degreasing fermented soybean to obtain degreased fermented soybean, uniformly mixing the degreased fermented soybean with the deep-eutectic solvent, performing ultrasonic-assisted extraction, cooling to room temperature after the extraction is completed, and centrifuging to obtain the fermented soybean general flavone. And taking the supernate, namely the fermented soybean total flavone extracting solution. The DES technology and the ultrasonic technology are combined, so that the total flavonoids in the fermented soybean can be obviously promoted to be dissolved out, and the extraction effect is good. The optimal optimization conditions are as follows: the liquid-material ratio is 36: 1mL / g, the water content of the fermented soybean is 32%, the extraction temperature is 73 DEG C, and finally the fermented soybean total flavone extracting solution with the fermented soybean total flavone content of 6.13 mg / g can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of extracting total flavonoids from fermented black beans, and particularly relates to a process for extracting total flavonoids from fermented black beans using an ultrasound-assisted deep eutectic solvent. Background Art

[0002] Light fermented black beans, also known as "fragrant fermented black beans," "douchi," and "fermented black beans," are fermented from the dried, mature seeds (black beans) of the legume plant Glycine max (L.) Merr. It is used to treat colds, fever, headaches, chest tightness, and insomnia. Research has shown that the main components of light fermented black beans are flavonoids, organic acids, and polysaccharides. Flavonoids are the most important active substances in light fermented black beans, exhibiting pharmacological effects such as anti-cancer, anti-osteoporosis, antipyretic, antibacterial, anti-tumor, anti-radiation, blood pressure lowering, and anti-atherosclerosis.

[0003] Total flavonoids are a class of natural compounds widely found in plants and are a general term for flavonoids. Fermented black beans contain a variety of flavonoids, mainly including daidzein, daidzin, genistin, and genistein. Among them, free aglycones (such as daidzein) and bound glycosides (such as genistin) are its main components. Studies have shown that bound glycosides are converted into free aglycones during the fermentation process, thereby enhancing their biological activity.

[0004] The solvents used to extract flavonoids from traditional Chinese medicine (TCM) are primarily water and organic solvents, which pose challenges such as high impurities, difficulty in purification and separation, and environmental pollution. Therefore, exploring new extraction methods to improve resource utilization and reduce environmental pollution is a key issue that needs to be addressed. Summary of the Invention

[0005] The purpose of the present invention is to provide an ultrasound-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans to solve the above problems.

[0006] The ultrasound-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans comprises the following steps:

[0007] The light fermented black beans are defatted to obtain defatted light fermented black beans, and the defatted light fermented black beans are uniformly mixed with a low eutectic solvent and then subjected to ultrasound-assisted extraction. After the extraction is completed, the mixture is cooled to room temperature and centrifuged, and the resulting supernatant is the light fermented black bean total flavonoid extract.

[0008] As a further improvement, the deep eutectic solvent is prepared by mixing choline chloride and urea in a molar ratio of 1:2.

[0009] As a further improvement, the liquid-to-solid ratio of defatted light fermented black beans to the low eutectic solvent is 36:1 mL / g.

[0010] As a further improvement, the water content of the light fermented black beans is 32%.

[0011] As a further improvement, the extraction temperature is 73±1℃.

[0012] The total flavonoids extract of light fermented soya bean is used as an antioxidant food additive or antioxidant drug. The concentration of the total flavonoids of light fermented soya bean in the total flavonoids extract of light fermented soya bean is 50 μg / ml.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The present invention adopts DES in combination with ultrasonic technology, which can significantly promote the dissolution of total flavonoids in light fermented black beans, has a good extraction effect, and provides a theoretical basis for the development and utilization of light fermented black beans.

[0015] 2. By screening different deep eutectic solvents, the present invention determined that choline chloride-urea had the highest extraction yield. The effects of water content, material-liquid ratio, and temperature on extraction efficiency were investigated, and the optimal extraction conditions were identified using response surface methodology. The antioxidant activity of total flavonoids from fermented black beans was evaluated based on their ability to scavenge DPPH and ABTS free radicals. The optimal conditions were a liquid-to-liquid ratio of 36:1 mL / g, a fermented black bean water content of 32%, and an extraction temperature of 73°C. The resulting fermented black bean total flavonoid extract contained 6.13 mg / g of total flavonoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the standard curve of rutin;

[0017] Figure 2 The extraction yield of total flavonoids from fermented soya bean curd with different DESs solvents;

[0018] Figure 3 The effect of DESs with different water contents on the yield of total flavonoids;

[0019] Figure 4 Effect of different liquid-to-solid ratios on the yield of total flavonoids

[0020] Figure 5 The effect of different extraction temperatures on the yield of total flavonoids;

[0021] Figure 6 The effect of different extraction times on the yield of total flavonoids;

[0022] Figure 7 Response surface analysis of the effects of water content, liquid-to-solid ratio, and extraction temperature on the extraction efficiency of total flavonoids from fermented black beans Figure 1 ;

[0023] Figure 8 Response surface analysis of the effects of water content, liquid-to-solid ratio, and extraction temperature on the extraction efficiency of total flavonoids from fermented black beans Figure 2 ;

[0024] Figure 9Response surface analysis of the effects of water content, liquid-to-solid ratio, and extraction temperature on the extraction efficiency of total flavonoids from fermented black beans Figure 3 ;

[0025] Figure 10 Response surface analysis of the effects of water content, liquid-to-solid ratio, and extraction temperature on the extraction efficiency of total flavonoids from fermented black beans Figure 4 ;

[0026] Figure 11 Response surface analysis of the effects of water content, liquid-to-solid ratio, and extraction temperature on the extraction efficiency of total flavonoids from fermented black beans Figure 5 ;

[0027] Figure 12 Response surface analysis of the effects of water content, liquid-to-solid ratio, and extraction temperature on the extraction efficiency of total flavonoids from fermented black beans Figure 6 ;

[0028] Figure 13 The DPPH free radical scavenging ability of total flavonoids extract of fermented black beans;

[0029] Figure 14 The ABTS free radical scavenging ability of total flavonoids extract from fermented black beans. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0031] Example 1

[0032] 1. Raw materials and reagents

[0033] Light fermented black beans were purchased from Anhui Hejitang Chinese Medicine Pieces Co., Ltd.

[0034] Rutin (Shanghai Yuanye Biotechnology Co., Ltd.), anhydrous ethanol, petroleum ether, acetic acid, glycerol, citric acid, lactic acid, urea, ethylene glycol (all analytical grade, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.), sodium nitrite, aluminum nitrate, sodium hydroxide (Tianjin Damao Chemical Reagent Factory), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), L-ascorbic acid, and AB-8 macroporous adsorption resin.

[0035] 2. Equipment and instruments

[0036] Microplate reader, centrifuge, electronic analytical balance, ultrasonic cleaning machine, magnetic stirrer, rotary evaporator.

[0037] 3. Experimental methods

[0038] 3.1 DES screening

[0039] A hydrogen bond acceptor (choline chloride) and different hydrogen bond donors were mixed in different molar ratios, and water was added. The mixture was heated in a water bath at 80°C with magnetic stirring until a stable, uniform, transparent liquid was formed. The mixture was then cooled to room temperature to obtain a deep eutectic solvent. Six DESs were obtained, with 60% ethanol used as the traditional extraction solvent as a control. The specific configurations are shown in Table 1.

[0040] Table 1 Different types of DES solvents

[0041]

[0042] 3.2 Sample pretreatment

[0043] Take light fermented black bean powder, add petroleum ether, seal and stir for 3 hours, defatted, filter out petroleum ether, take powder, dry and set aside.

[0044] 3.3 Process optimization experiment

[0045] 3.3.1 Effect of water content on the extraction rate of total flavonoids from fermented black beans

[0046] The liquid-to-solid ratio was controlled at 30:1, the extraction temperature was 30°C, and the extraction time was 30 min. The effects of the water content of light fermented black beans (10%, 20%, 30%, 40%, 50%) on the extraction rate of total flavonoids from light fermented black beans were investigated, and the process was repeated three times.

[0047] 3.3.2 Effect of liquid-to-solid ratio on the extraction rate of total flavonoids from fermented black beans

[0048] The water content was controlled at 30%, the extraction temperature was 30℃, and the extraction time was 30min. The effects of liquid-to-solid ratios (10:1, 20:1, 30:1, 40:1, and 50:1) on the extraction rate of total flavonoids from fermented black beans were investigated, and the process was repeated three times.

[0049] 3.3.3 Effect of extraction temperature on the extraction rate of total flavonoids from fermented black beans

[0050] The water content was controlled at 30%, the liquid-to-solid ratio was 40:1, the extraction time was 30 min, and the effects of temperature (15°C, 30°C, 45°C, 60°C, 75°C) on the extraction rate of total flavonoids from fermented black beans were investigated, and repeated three times.

[0051] 3.3.4 Effect of extraction time on the extraction rate of total flavonoids from fermented black beans

[0052] The water content was controlled at 30%, the liquid-to-solid ratio was 40:1, and the extraction temperature was 60°C. The effects of extraction time (10 min, 30 min, 50 min, 70 min, 90 min) on the extraction rate of total flavonoids from fermented black beans were investigated and repeated three times.

[0053] 3.4 Response surface optimization experiment

[0054] Based on the experimental results, water content (A), liquid-to-solid ratio (B), and extraction temperature (C) were the main factors affecting the total flavonoid yield of fermented black beans. Based on the Box-Benhnken central composite experimental design principle, the experimental design was optimized with the coding levels set to -1, 0, and 1, and the total flavonoid yield of fermented black beans (W) as the response value. The response surface experimental factors and levels are shown in Table 2.

[0055] Table 2 Experimental design of response surface analysis

[0056]

[0057] 3.5 Preparation of test solution

[0058] According to the test results, 1.00 g of light fermented black bean powder was accurately weighed and evenly mixed with the low eutectic solvent. The water content, liquid-to-solid ratio, temperature and other conditions were controlled for ultrasonic-assisted extraction. After the extraction was completed, the mixture was cooled and transferred to a centrifuge tube. It was centrifuged at 10,000 r / min for 10 min, and the supernatant was taken for later use.

[0059] 3.6. Drawing of the standard curve

[0060] Weigh 10.00 mg of rutin standard, dissolve it in methanol solution and dilute to a 10 mL volumetric flask, shake well to obtain a 1.00 mg / mL rutin standard solution. Transfer 0.00, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, and 0.70 mL respectively into 8 10.00 mL volumetric flasks, add 0.3 mL of 5% NaNO2 solution in turn, shake evenly and let it stand for 6 minutes; then add 0.3 mL of 10% Al(NO3)3 solution, mix well and let it stand for 6 minutes; add 4 mL of NaOH test solution, and finally dilute to the scale with methanol, shake evenly and let it stand for 15 minutes. Use ID3 enzyme reader to measure the absorbance value of rutin standard solution with different mass concentrations at 510 nm after the reaction. Draw a standard curve with the mass concentration of the standard as the horizontal axis (X) and the absorbance as the vertical axis (Y), as shown below. Figure 1 The linear regression equation is: y = 8.7057x - 0.0099, R 2 =0.9993.

[0061] 3.7 Determination of total flavonoids in fermented black beans

[0062] Accurately measure 1 mL of the total flavonoids extract solution of light fermented soybeans into a 10 mL volumetric flask, measure the absorbance according to the above determination method, and calculate the total flavonoids content of light fermented soybeans based on the obtained rutin standard curve.

[0063] 3.8 Antioxidant activity test

[0064] The total flavonoids from fermented black beans were extracted, and the supernatant was collected after centrifugation and enriched and purified by AB-8 macroporous adsorption resin chromatography column. The filtrate was then concentrated by a rotary evaporator to prepare solutions of different concentrations for later use.

[0065] 3.8.1 Determination of DPPH free radical scavenging rate

[0066] Accurately weigh 0.007886 g of DPPH and dissolve it in anhydrous ethanol by ultrasonication. The volume is adjusted to 100 mL. Pipette 0.5 mL of the various concentrations of the total flavonoid extracts from fermented black beans prepared in 1.3.8 into a 3 mL EP tube and add 1.0 mL of a 0.2 mmol / l DDPPH-ethanol solution. Incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm using ascorbic acid as a control. Calculate the DPPH free radical scavenging rate according to the formula.

[0067]

[0068] Where: A sample is the absorbance of the sample solution and DPPH working solution, A blank is the absorbance of the sample solution and distilled water, and A control is the absorbance of the DPPH working solution and distilled water.

[0069] 3.8.2 Determination of ABTS free radical scavenging rate

[0070] Preparation of ABTS working solution: Prepare 7.0 m mol / L ABTS solution and 2.45 m mol / L potassium persulfate solution in a 1:1 volume ratio, mix well, and store in the dark for 13 h. Dilute with ethanol to an absorbance of 0.70 ± 0.07 at 734 nm and set aside.

[0071] Pipette 0.1 ml of the prepared total flavonoid extract of various concentrations of fermented black beans into a 3 ml EP tube. Add 1.6 ml of ABTS working solution, mix thoroughly, and incubate at room temperature in the dark for 5 minutes. Measure the absorbance at 734 nm using ascorbic acid as a control. Calculate the ABTS free radical scavenging rate according to the formula.

[0072]

[0073] Where: A sample is the absorbance of the sample solution and ABTS working solution; A blank is the absorbance of the sample solution and distilled water; A control is the absorbance of the DPPH working solution and distilled water.

[0074]

[0075] Where: B 样品 is the absorbance of the sample solution and ABTS working solution; B 空白 is the absorbance of the sample solution and distilled water; B 对照is the absorbance of DPPH working solution and distilled water.

[0076] 4 Results and Analysis

[0077] 4.1 DES screening results

[0078] like Figure 2 As shown in the results, the yields of total flavonoids from F. douchi (Zhi Dou Chi) using different DESs were significantly different. Choline chloride-urea yielded the highest yield of 4.85 mg / g, which was superior to the 4.32 mg / g yield obtained using the traditional extraction method (60% methanol). Therefore, choline chloride-urea was selected as the extraction solvent for total flavonoids from F. douchi (Zhi Dou Chi).

[0079] 4.2 Experimental Results

[0080] 4.2.1 Effect of DESs water content on total flavonoid extraction rate

[0081] like Figure 3 As shown in the results, as the moisture content increases, the total flavonoid content gradually increases from 10% to 30% moisture content, reaching a maximum value (4.81 mg / g) at 30%, and the extractable content decreases from 30% to 50% moisture content. Therefore, the moisture content of the choline chloride-urea deep eutectic solvent component was determined to be 30%.

[0082] 4.2.2 Effect of liquid-to-solid ratio on total flavonoid extraction rate

[0083] Depend on Figure 4 It can be seen that when the liquid-to-solid ratio increases from 10:1 to 40:1, the extraction rate of total flavonoids from light fermented black beans continues to increase; when the liquid-to-solid ratio increases from 40:1 to 50:1, the extraction rate of total flavonoids decreases. Therefore, the optimal liquid-to-solid ratio for extracting total flavonoids is determined to be 40:1.

[0084] 4.2.3 Effect of extraction temperature on total flavonoid extraction rate

[0085] Depend on Figure 5 It can be seen that when the temperature rises from 15℃ to 60℃, the extraction rate increases significantly; when the temperature exceeds 60℃, the extraction rate decreases. Therefore, the optimal extraction temperature is set at 60℃.

[0086] 4.2.4 Effect of extraction time on total flavonoid extraction rate

[0087] Depend on Figure 6 It can be seen that with the increase of ultrasonic time, the extraction rate of total flavonoids from light fermented black beans showed an increasing trend, reaching the highest at 70 minutes, and the extraction rate at 90 minutes was slightly lower than that at 70 minutes. Therefore, 70 minutes was selected as the optimal extraction time.

[0088] 4.3 Response surface experiments

[0089] 4.3.1 Response surface test results

[0090] According to the selected factors and levels, a response surface experimental design was performed. The data in Table 3 were analyzed and processed using Design expert13 software to obtain the corresponding quadratic polynomial regression equation. The polynomial regression model of the total flavonoids yield y (mg / g) of light fermented black beans and the three factors of water content (A,%), liquid-to-solid ratio (B, mL / g), and extraction temperature (C, ℃) was: Y = 6.01 + 0.51A - 0.17B + 0.23C - 0.0025AB - 0.0250AC + 0.0200BC - 1.15A 2 -0.35B 2 -0.17C 2 .

[0091] Table 3 Response surface analysis scheme and experimental results

[0092]

[0093] 4.3.2 Analysis of variance for response surface experiments

[0094] According to the analysis results in Table 4, the P value of this model is less than 0.01, which is extremely significant. 2 =0.9939, R 2 adj =0.9860, indicating that the model has no significant error, and the model is reliable and accurate, and can be used for subsequent analysis. Combining the F value and P value of the variance analysis results of each factor, the influence of each factor on the extraction rate of total flavonoids from light fermented black beans is in the following order: water content (A) > ultrasonic power (C) > material-liquid ratio (B).

[0095] Table 4 Analysis of variance of regression model

[0096]

[0097] Note: Significant difference, *(P<0.05); extremely significant difference, **(P<0.01).

[0098] 4.3.3 Interaction of factors in response surface experiments

[0099] Through the response surface experiment, a 3D surface plot and contour map were obtained. The 3D surface plot can intuitively reflect the relationship between the two factors and the response variable. The more curved the surface shape, the stronger the interaction between the factors, and vice versa, the flatter it is, the less significant it is. And the redder the surface color, the higher the yield of flavonoids from light fermented soya bean curd. Figure 7As can be seen, the 3D surface plots between water content (A) and material-liquid ratio (B), and water content (A) and extraction temperature (C) are relatively curved, indicating that the AB and AC interactions have a strong influence on the total flavonoid yield of light fermented soya bean curd. The 3D surface plot between material-liquid ratio (B) and extraction temperature (C) is relatively flat, indicating that the BC interaction has no significant effect on the yield of light fermented soya bean curd ketones. Contour plots can show how the combination of two independent variables affects the response variable, while the other variables are fixed. The shape and spacing of the contour plots provide information about the strength of the interaction between factors, with the greater the ellipse eccentricity, the more significant the interaction. The contour lines for water content (A) and material-liquid ratio (B), and water content (A) and extraction time (C) in the figure are flat ellipses with a relatively dense distribution of contour lines, indicating that these two factors have a significant influence on the total flavonoid yield of light fermented soya bean curd. The contour lines formed by material-liquid ratio (B) and extraction time (C) have a slightly smaller eccentricity, indicating that the interaction is less significant than the previous two. The above analysis is consistent with the variance results.

[0100] 4.4 Antioxidant test results

[0101] 4.4.1 DPPH free radical scavenging ability

[0102] Depend on Figure 13 As shown, the DPPH free radical scavenging ability of the total flavonoids from Fructus leucoderma increased with increasing concentration. Within the 10-55 μg / mL range, the scavenging rate increased from 23.72% to 71.71%. At the same concentration, the DPPH free radical scavenging rate of the total flavonoids from Fructus leucoderma was lower than that of ascorbic acid. At a concentration of 50 μg / mL, the DPPH free radical scavenging rate reached 71.71%. The IC50 value for DPPH free radical scavenging activity calculated using GraphPad Prism 9 was 30.06 μg / mL, demonstrating that the total flavonoids from Fructus leucoderma possesses excellent DPPH free radical scavenging ability.

[0103] 4.4.2 ABTS free radical scavenging ability

[0104] Depend on Figure 14 The results show that the ABTS free radical scavenging activities of the total flavonoids extract from zushii and ascorbic acid showed similar growth trends, and the ABTS scavenging activity of the total flavonoids extract from zushii was positively correlated with concentration. Within the 10-50 μg / mL range, the ABTS free radical scavenging rate of the total flavonoids from zushii increased with increasing concentration. At a concentration of 50 μg / mL, the ABTS free radical scavenging rate of the total flavonoids from zushii was 86.47%. The IC50 of the total flavonoids from zushii was calculated using GraphPad Prism 9 to be 29.38 μg / mL, indicating that the total flavonoids from zushii have good ABTS scavenging activity.

[0105] 5 Conclusion

[0106] The results showed that the DES prepared with a molar ratio of choline chloride to urea of ​​1:2 had a higher flavonoid extraction rate than the traditional extraction method. When the liquid-to-solid ratio was 36:1 mL / g, the water content was 32%, and the extraction temperature was 73°C, the yield of soybean total flavonoids was the highest, with a content of 6.13 mg / g. The antioxidant results showed that the total flavonoids of light fermented soy beans had a scavenging capacity of 71.71% and 86.47% for DPPH free radicals and ABTS free radicals at 50 μg / ml, and the IC 50 They were 30.06μg / mL and 29.38μg / mL respectively, indicating that the total flavonoids of fermented black beans had good antioxidant effect.

[0107] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Ultrasonic-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans, characterized by: The following steps are involved: The light fermented black beans are defatted to obtain defatted light fermented black beans, and the defatted light fermented black beans are uniformly mixed with a low eutectic solvent and then subjected to ultrasound-assisted extraction. After the extraction is completed, the mixture is cooled to room temperature and centrifuged, and the resulting supernatant is the light fermented black bean total flavonoid extract.

2. The ultrasound-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans according to claim 1, characterized in that: The deep eutectic solvent is prepared by mixing choline chloride and urea in a molar ratio of 1:

2.

3. The ultrasound-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans according to claim 1, characterized in that: The liquid-to-solid ratio of defatted light fermented black beans to low eutectic solvent is 36:1 mL / g.

4. The ultrasound-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans according to claim 1, characterized in that: The water content of light fermented black beans is 32%.

5. The ultrasound-assisted deep eutectic solvent extraction process for total flavonoids from fermented black beans according to claim 1, characterized in that: The extraction temperature was 73±1℃.

6. The use of the extract of total flavonoids from fermented soya beans obtained by the ultrasound-assisted deep eutectic solvent extraction process of total flavonoids from fermented soya beans as an antioxidant food additive or antioxidant drug, characterized in that: The concentration of total flavonoids from fermented soya beans in the total flavonoids from fermented soya beans extract is 50 μg / ml.

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