Method for extracting chlorogenic acid from sunflower seed meal by using eutectic solvent

Abstract

The invention discloses a method for extracting chlorogenic acid from sunflower seed meal by using a deep eutectic solvent. The method comprises the following steps: detecting crude protein content of sunflower seed meal; crushing to obtain sunflower seed meal particles; uniformly mixing the sunflower seed meal particles with a deep eutectic solvent according to a mass-volume ratio of 1g: (5-5) mL, and soaking for 30-90 minutes; the selection of the eutectic solvent is determined according to the content of crude protein; when the content of the crude protein is 35-40%, the hydrogen bond acceptor and the hydrogen bond donor are mixed according to the molar ratio of (1.5-1.8): 1; when the concentration is 30-35%, the hydrogen bond acceptor and the hydrogen bond donor are mixed according to the molar ratio of (1.8-2): 1; when the content of the hydrogen bond acceptor is 40-45%, the hydrogen bond acceptor and the hydrogen bond donor are mixed according to the molar ratio of (1.0-1.5): 1, the hydrogen bond acceptor is L-proline or choline chloride, and the hydrogen bond donor is any one of urea, D-tartaric acid and acetamide; after soaking is completed, microwave treatment is carried out for 1-5 min under the microwave conditions of 30-70 DEG C and 500-900 W, filtering and purifying are carried out, and the chlorogenic acid extract is obtained. According to the method, under the assistance of the eutectic solvent and microwaves, the extraction rate of the chlorogenic acid can be increased, and the extraction time can be shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and more specifically, to a method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent. Background Art

[0002] Sunflower seed meal is the residue left after pressing or directly extracting oil from sunflower seeds. Sunflower seed meal contains 1.1~4.5% (the content depends on the shell content) of chlorogenic acid. Chlorogenic acid is an important biologically active dietary polyphenol with many important pharmacological effects such as antioxidant, antibacterial, hypolipidemic, hypotensive, and hypoglycemic effects. It has certain application value in the fields of food, medicine, and cosmetics. At present, compared with honeysuckle (chlorogenic acid content of 4.29~6.07%) and Eucommia ulmoides (chlorogenic acid content of 2~5%), the industrial production of chlorogenic acid mainly uses low-priced sunflower seed meal as raw material.

[0003] At present, the methods for extracting chlorogenic acid mainly include solvent extraction, ethanol reflux method, and enzymatic hydrolysis. Among them, the extractant of the solvent extraction method is water or organic solvent. Although the extraction process is simple, impurities such as polysaccharides and proteins in the raw materials will also dissolve during the extraction process, resulting in reduced purity of the product and too long extraction time; the ethanol reflux method uses 75% ethanol to reflux extraction at about 40°C, which also takes a long time to complete the extraction; Chen Shuisheng et al. used sunflower seed cake as raw material to study the effect of cellulase, protease and the combined action of cellulase and protease on the extraction of chlorogenic acid from dry sunflower seed cake, and the chlorogenic acid extraction rate reached 1.90%, while Wang Zhihua et al. used pectinase and cellulase to extract chlorogenic acid from sunflower seeds, with a content of 1.36%, and the extraction rate was low. Therefore, it is urgent to have a method for extracting chlorogenic acid from sunflower seed cake to obtain the beneficial effects of short extraction time and high extraction rate, which is worth pondering. Summary of the invention

[0004] One object of the present invention is to provide a method for extracting chlorogenic acid from sunflower seed meal with a deep eutectic solvent, comprising the following steps: S1. Detecting the crude protein content in sunflower seed meal; S2, crushing the sunflower seed meal to obtain sunflower seed meal particles; S3, mixing the sunflower seed meal particles and the low eutectic solvent at a mass volume ratio of 1 g: 5-25 mL, and soaking for 30-90 min; Wherein, when the crude protein content of the sunflower seed meal is 35-40%, the low eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1.5-1.8:1; When the crude protein content of the sunflower seed meal is 30-35%, the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1.8-2:1:1; When the crude protein content of the sunflower seed meal is 40-45%, the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1.0-1.5:1; The hydrogen bond acceptor is L-proline or choline chloride, and the hydrogen bond donor is any one of urea, D-tartaric acid, and acetamide; S4. After soaking, microwave treatment is performed at 30-70°C and 500-900W for 1-5 min. After filtration and purification, chlorogenic acid extract is obtained.

[0005] Preferably, the particle size of the sunflower seed meal particles is 30-60 mesh.

[0006] Preferably, the low eutectic solvent further comprises 20-40% by mass of water.

[0007] Preferably, the method for preparing the deep eutectic solvent comprises the following steps: The hydrogen bond donor, the hydrogen bond acceptor and water are uniformly mixed and stirred at 90-100° C. until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent.

[0008] Preferably, when the crude protein content of the sunflower seed meal is 35-40%, the hydrogen bond acceptor is L-proline, and the hydrogen bond donor is urea; When the crude protein content of the sunflower seed meal is 30-35%, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is urea; When the crude protein content of the sunflower seed meal is 40-45%, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is acetamide.

[0009] Preferably, in step S4, a stabilizer is added to the mixture formed by the soaked sunflower seed meal particles and the low eutectic solvent, and the mixture is stirred evenly, and the stabilizer is citric acid or malic acid.

[0010] Preferably, the stabilizer is citric acid, and the volume of the citric acid is 1 / 15 to 2 / 15 of the volume of the deep eutectic solvent.

[0011] Preferably, the purification operation is as follows: adding the filtered filtrate to AB-8 resin, eluting once with deionized water, and then eluting with 50% ethanol aqueous solution, collecting the eluate obtained by eluting with ethanol aqueous solution, and subjecting the eluate to rotary evaporation and freeze-drying to obtain the chlorogenic acid extract.

[0012] Preferably, the mass volume ratio of the sunflower seed meal particles to the low eutectic solvent is 1 g:15 mL.

[0013] Preferably, the microwave temperature of the microwave treatment is 60-70° C., the microwave power is 600W-700W, and the microwave time is 3-4 min.

[0014] The present invention has at least the following beneficial effects: First, the low eutectic solvent of the present invention, under the assistance of microwave, has a higher extraction rate and a shorter extraction time for chlorogenic acid in sunflower seed meal with different crude protein contents. Among them, the low eutectic solvent composed of L-proline and uric acid has an extraction rate of chlorogenic acid of 2.59% for sunflower seed meal with a crude protein content in the range of 35-40%, which is 1.65 times that of the control group soaked in 75% ethanol + refluxed.

[0015] Second, the optimal process for extracting chlorogenic acid from sunflower seed meal with a crude protein content of 30-35% using the low eutectic solvent composed of choline chloride-urea of ​​the present invention under microwave assistance is: the water content of the low eutectic solvent is 30 wt%, the material-liquid ratio is 1:15, the microwave power is 700 W, the microwave temperature is 60°C, and the microwave time is 3 min. Under these conditions, the chlorogenic acid extraction rate is 2.63%.

[0016] Third, the present invention adds citric acid or malic acid to the low eutectic solvent system at a microwave temperature of 70°C, and the extraction rate of chlorogenic acid is more effectively improved under the assistance of microwave. Among them, when 1-2 mL of citric acid is added, that is, the volume of citric acid is 1 / 15-2 / 15 of the volume of the low eutectic solvent, the extraction rate of chlorogenic acid is the highest (2.68%). The addition of citric acid can overcome the problem of reduced extraction rate caused by excessively high microwave temperature, and can expand the applicable range of microwave temperature.

[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the standard curve of chlorogenic acid; Figure 2 It is a graph of extraction rates of different embodiments of one of the technical solutions of the invention and a control group; Figure 3 An extraction rate diagram of factors affecting the water content of a low eutectic solvent according to one of the technical solutions of the invention; Figure 4 An extraction rate diagram of factors affecting the solid-liquid ratio of sunflower seed meal and deep eutectic solvent according to one of the technical solutions of the invention; Figure 5 It is an extraction rate diagram of microwave power influencing factors of one of the technical solutions of the invention; Figure 6 The extraction rate diagram of microwave temperature influencing factors of one of the technical solutions of the invention; Figure 7 This is a diagram of the extraction rate of microwave time factors affecting one of the technical solutions of the invention. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0020] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0021] <Example 1> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 35-40%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After the soaking is completed, microwave treatment is performed at 60°C and 600W for 3 min, and the mixture is filtered through a polyester non-woven fabric to obtain a filtrate, and the filtrate is added to AB-8 resin, and the mixture is first eluted once with deionized water, and then eluted with a 50% ethanol aqueous solution, and the eluate obtained by eluting with the ethanol aqueous solution is collected, and the eluate is rotary evaporated at 45°C, and then freeze-dried to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: L-proline and urea are mixed in a molar ratio of 1.8:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0022] <Example 2> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After the soaking is completed, microwave treatment is performed at 60°C and 600W for 3 min, and the mixture is filtered through a polyester non-woven fabric to obtain a filtrate, and the filtrate is added to AB-8 resin, and the mixture is first eluted once with deionized water, and then eluted with a 50% ethanol aqueous solution, and the eluate obtained by eluting with the ethanol aqueous solution is collected, and the eluate is rotary evaporated at 45°C, and then freeze-dried to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0023] <Example 3> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 40-45%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After the soaking is completed, microwave treatment is performed at 60°C and 600W for 3 min, and the mixture is filtered through a polyester non-woven fabric to obtain a filtrate, and the filtrate is added to AB-8 resin, and the mixture is first eluted once with deionized water, and then eluted with a 50% ethanol aqueous solution, and the eluate obtained by eluting with the ethanol aqueous solution is collected, and the eluate is rotary evaporated at 45°C, and then freeze-dried to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and acetamide are mixed in a molar ratio of 1:1, water is added, heated to 90°C, and stirred continuously until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt% <Example 4> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 35-40%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After the soaking is completed, microwave treatment is performed at 60°C and 600W for 3 min, and the mixture is filtered through a polyester non-woven fabric to obtain a filtrate, and the filtrate is added to AB-8 resin, and the mixture is first eluted once with deionized water, and then eluted with a 50% ethanol aqueous solution, and the eluate obtained by eluting with the ethanol aqueous solution is collected. The eluate is rotary evaporated at 45°C and freeze-dried to obtain a chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: L-proline and urea are mixed in a molar ratio of 1:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0024] <Control group> The method for extracting chlorogenic acid from sunflower seed meal using an existing extraction method comprises the following steps: S1. Grind the dried sunflower seed meal, and pass it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S2, stir 1 g of sunflower seed meal particles with 25 mL of 75% ethanol to make them evenly mixed, and soak them for 80 min; S3. After soaking, reflux extraction was performed at 40°C in a water bath for 80 min, and the filtered solution was subjected to rotary evaporation at 45°C and then freeze-dried to obtain the purified chlorogenic acid extract.

[0025] <Chlorogenic acid extraction rate test> 1.1. Preparation of standard curve of chlorogenic acid The chlorogenic acid standard was dissolved in 50 wt% methanol solution and prepared into a series of standard concentrations (2, 10, 20, 40, 80 mg / L). The absorption peak area of ​​the chlorogenic acid standard solution of various concentrations was determined by high performance liquid chromatography. The standard curve was drawn with the chlorogenic acid concentration (mg / L) as the horizontal axis and the peak area as the vertical axis. The standard curve is shown in Figure 2. Figure 1 As shown, the regression equation of chlorogenic acid concentration and absorption peak area is: y=30695x-2099, and the correlation coefficient R 2 =0.99972, linear relationship in the range of 2~80 mg / L; The chromatographic conditions of high performance liquid chromatography are as follows: Chromatographic column: Pursuit 5 C18 column (150×4.6 mm), column temperature: 35°C, mobile phase: acetic acid (0.5wt%) and acetonitrile solution (volume ratio of 9:1), flow rate: 1.0 mL / min, injection volume: 10μL, detection wavelength: 327 nm.

[0026] 1.2. The filtrates obtained by filtering Examples 1 to 5 and the control group through the polyester non-woven fabric were diluted 100 times with water, and the absorption peak area was detected according to the above chromatographic conditions. The chlorogenic acid concentration was calculated by the regression equation, and then the extraction rate was calculated, wherein the chlorogenic acid extraction rate was calculated according to the following formula: Where: c is the concentration of chlorogenic acid (mg / L), N is the dilution factor, V is the volume of the extract (L), and W is the mass of the sunflower seed meal particles (mg).

[0027] The calculated chlorogenic acid extraction rate is as follows: Figure 2 The specific data are shown in Table 1 below: Table 1 Chlorogenic acid extraction rate of Examples 1 to 5 and the control group According to the data in Table 1, the extraction rates of Examples 1 to 4 are all higher than the extraction rate of the control group using 75% ethanol reflux extraction. In particular, the low eutectic solvent composed of L-proline and uric acid and the low eutectic solvent composed of choline chloride and urea have higher extraction rates for chlorogenic acid in sunflower seed meal. Among them, the extraction rate of the low eutectic solvent composed of L-proline and uric acid is 1.65 times that of the control group, and the extraction rate of the low eutectic solvent composed of choline chloride and urea is 1.61 times that of the control group. In addition, the extraction time of Examples 1 to 5 (immersion for 60 min + microwave for 3 min) is significantly lower than the extraction time of the control group 1 (immersion for 80 min + reflux for 80 min), indicating that the low eutectic solvent of the present application has a higher extraction rate and a shorter extraction time for extracting chlorogenic acid under the assistance of microwave.

[0028] <Comparative Example 1> The extraction method is the same as that in Example 1, except that L-proline and urea are replaced by choline chloride and urea (a1), choline chloride and D-tartaric acid (b1), choline chloride and urea (c1), and choline chloride and acetamide (d1), respectively.

[0029] <Comparative Example 2> The extraction method is the same as that in Example 1, except that choline chloride and urea are replaced by L-proline and urea (a2), choline chloride and D-tartaric acid (b2), choline chloride and urea (c2), and choline chloride and acetamide (d2), respectively.

[0030] <Comparative Example 3> The extraction method is the same as that in Example 1, except that choline chloride and acetamide are replaced by L-proline and urea (a3), choline chloride and D-tartaric acid (b3), choline chloride and urea (c3), and choline chloride and urea (d3), respectively.

[0031] The extraction rate of the filtrate obtained by extracting each group of Comparative Examples 1 to 3 and filtering through polyester non-woven fabric was calculated according to the method in "1.2", and compared with Examples 1 to 3. The results are shown in Table 2 below: Table 2 Effects of different deep eutectic solvent compositions on the extraction rate of chlorogenic acid in sunflower seed meal with different crude protein content ranges According to the data in Table 2, among the sunflower seed meal with different crude protein content ranges, the chlorogenic acid extraction rate of Example 1 (the low eutectic solvent is composed of L-proline and uric acid) with a crude protein content of 35-40% is the highest, reaching 2.59%, the chlorogenic acid extraction rate of Example 2 (the low eutectic solvent is composed of choline chloride and uric acid) with a crude protein content of 30-35% is the highest, reaching 2.52%, and the chlorogenic acid extraction rate of Example 3 (the low eutectic solvent is composed of choline chloride and acetamide) with a crude protein content of 40-45% is the highest, reaching 2.21%, indicating that when the crude protein content of sunflower seed meal is between 35 ~40%, it is more conducive to the extraction of chlorogenic acid with a low eutectic solvent. A higher or lower crude protein content range will lead to a decrease in the extraction rate of chlorogenic acid. By comparing the data of Example 1 and Comparative Example 1, the data of Example 2 and Comparative Example 2, and the data of Example 3 and Comparative Example 3, it can be seen that the specific low eutectic solvent composition used for sunflower seed meal with different crude protein content ranges has a better effect on the extraction of chlorogenic acid, and changing its composition will lead to a decrease in the extraction rate of chlorogenic acid.

[0032] Since the extraction rate of the low eutectic solvent composed of L-proline and uric acid is slightly different from that of the low eutectic solvent composed of choline chloride and urea, and the price of L-proline is significantly higher than that of choline chloride, based on extraction cost considerations, subsequent experiments all used low eutectic solvents composed of choline chloride and urea for research.

[0033] <Study on the Extraction Process of Chlorogenic Acid> 2.1 Effect of water content of deep eutectic solvent on the extraction rate of chlorogenic acid Referring to the extraction method of Example 2, the effect of different water contents (20-50 wt%) of the low eutectic solvent on the extraction rate of chlorogenic acid was investigated. The filtrate obtained by extraction with different water contents (other conditions were the same as those in Example 2) and filtration through polyester non-woven fabric was calculated according to the method in "1.2". The results are as follows Figure 3 The specific data are shown in Table 3 below: Table 3 Effect of water content of deep eutectic solvent on the extraction rate of chlorogenic acid According to the data in Table 3, the water content of the low eutectic solvent composed of choline chloride and urea has a significant effect on the extraction rate of chlorogenic acid in sunflower seed meal. When the water content of the low eutectic solvent is 30 wt%, the extraction rate of chlorogenic acid reaches the highest, which is 2.52%. As the water content continues to increase, the extraction rate of chlorogenic acid shows a downward trend. Therefore, in the process of extracting chlorogenic acid from sunflower seed meal using a low eutectic solvent composed of choline chloride and urea, controlling the water content of the low eutectic solvent to 30wt% helps to obtain a higher extraction rate of chlorogenic acid.

[0034] 2.2 Effect of solid-liquid ratio on chlorogenic acid extraction rate Referring to the extraction method of Implementation 2, the effect of the solid-liquid ratio (1 g: 5-25 mL) on the extraction rate of chlorogenic acid was investigated. The extraction rate of the filtrate obtained by extraction with different solid-liquid ratios (other conditions were the same as those in Example 2) and filtration through polyester non-woven fabric was calculated according to the method in "1.2". The results are as follows: Figure 4 The specific data are shown in Table 4 below: Table 4 Effect of solid-liquid ratio on chlorogenic acid extraction rate According to the data in Table 4, the chlorogenic acid extraction rate showed significant differences under different solid-liquid ratios. When the solid-liquid ratio was 1:15, the chlorogenic acid extraction rate reached a peak of 2.52%. In the process of gradually increasing the solid-liquid ratio from 1:5 to 1:15, the chlorogenic acid extraction rate showed an upward trend, indicating that appropriately increasing the amount of low eutectic solvent composed of choline chloride and urea can help improve the extraction effect. However, when the solid-liquid ratio continued to increase from 1:15 to 1:20 and 1:25, the chlorogenic acid extraction rate began to decrease, falling to 2.42% and 2.40%, respectively. This means that excessive use of low eutectic solvents will not continue to improve the extraction efficiency, but may lead to a decrease in the extraction rate due to factors such as dilution. Therefore, in the process of extracting chlorogenic acid from sunflower seed meal using a low eutectic solvent composed of choline chloride and urea, a solid-liquid ratio of 1:15 is a relatively good choice, which can achieve a higher chlorogenic acid extraction rate.

[0035] 2.3 Effect of microwave power ratio on chlorogenic acid extraction rate Referring to the extraction method of Example 2, the effect of microwave power (500-900 W) on the extraction rate of chlorogenic acid was investigated. The extraction rate of the filtrate obtained by extraction with different microwave powers (other conditions were the same as those in Example 2) and filtration through polyester non-woven fabric was calculated according to the method in "1.2". The results are as follows: Figure 5 The specific data are shown in Table 5 below: Table 5 Effect of microwave power ratio on chlorogenic acid extraction rate According to the data in Table 5, the change of microwave power has a significant effect on the extraction rate of chlorogenic acid. When the microwave power is 700 W, the extraction rate of chlorogenic acid reaches the maximum value of 2.63%. In the process of increasing the power from 500 W to 700 W, the extraction rate of chlorogenic acid shows an upward trend, which indicates that within a certain range, increasing the microwave power can enhance the extraction process and release more chlorogenic acid from sunflower seed meal. However, when the microwave power is further increased to 800 W and 900 W, the extraction rate decreases significantly, to 2.37% and 1.78%, respectively. This may be due to the destruction of the chlorogenic acid structure caused by excessive microwave power, or other side reactions that are not conducive to extraction. Therefore, in the process of extracting chlorogenic acid from sunflower seed meal using a low eutectic solvent, a microwave power of 700 W is a better choice, which can effectively improve the extraction rate of chlorogenic acid.

[0036] 2.4 Effect of microwave temperature ratio on chlorogenic acid extraction rate Referring to the extraction method of Example 2, the effect of microwave temperature (30-70°C) on the extraction rate of chlorogenic acid was investigated. The extraction rate of the filtrate obtained by extraction at different microwave temperatures (other conditions were the same as those in Example 2) and filtration through polyester non-woven fabric was calculated according to the method in "1.2". The results are as follows: Figure 6 The specific data are shown in Table 6 below: Table 6 Effect of microwave temperature ratio on chlorogenic acid extraction rate According to the data in Table 6, the change of microwave temperature significantly affects the extraction rate of chlorogenic acid. Within the set microwave temperature range (30~70℃), when the microwave temperature is 60℃, the extraction rate of chlorogenic acid reaches a peak of 2.63%. As the microwave temperature increases from 30℃ to 60℃, the extraction rate of chlorogenic acid gradually increases, indicating that in this temperature range, appropriately increasing the temperature helps to enhance the extraction effect of low eutectic solvent on chlorogenic acid, which may be because the increase in temperature promotes the diffusion of chlorogenic acid from sunflower seed meal to the low eutectic solvent. However, when the microwave temperature is further increased to 70℃, the extraction rate of chlorogenic acid drops to 2.26%, perhaps because the high temperature causes the decomposition of chlorogenic acid or other chemical reactions that are not conducive to extraction. Therefore, in the process of extracting chlorogenic acid from sunflower seed meal using low eutectic solvent, a microwave temperature of 60℃ is a more suitable condition, which can effectively improve the extraction rate of chlorogenic acid.

[0037] 2.5 Effect of microwave time ratio on chlorogenic acid extraction rate Referring to the extraction method of Example 2, the effect of microwave time (1-5 min) on the extraction rate of chlorogenic acid was investigated. The extraction rate of the filtrate obtained by extraction with different microwave times (other conditions were the same as those in Example 2) and filtration through polyester non-woven fabric was calculated according to the method in "1.2". The results are as follows: Figure 7 The specific data are shown in Table 7 below: Table 7 Effect of microwave time ratio on chlorogenic acid extraction rate According to the data in Table 7, the change of microwave time has a certain effect on the extraction rate of chlorogenic acid. Within the set microwave time range of 1-5 min, when the microwave time is 3 min, the extraction rate of chlorogenic acid reaches the maximum value of 2.63%. Within 1-3 min, as the microwave time increases, the extraction rate of chlorogenic acid shows an upward trend, which indicates that appropriately extending the microwave treatment time helps chlorogenic acid to be more fully dissolved from sunflower seed meal into the low eutectic solvent, thereby improving the extraction rate. However, when the microwave time exceeds 3 min, such as extending to 4 min and 5 min, the extraction rate fluctuates, but the overall improvement is not obvious, and even decreases compared to 3 min. This shows that if the microwave treatment time is too long, it may not further significantly improve the extraction efficiency of chlorogenic acid. On the contrary, it may cause some side reactions that are not conducive to extraction due to long-term microwave action, or the chlorogenic acid has been basically extracted completely, and it is not meaningful to continue to extend the time. Therefore, in the process of extracting chlorogenic acid from sunflower seed meal using a low eutectic solvent, a microwave treatment time of 3 min is a more suitable choice, which can obtain a higher chlorogenic acid extraction rate.

[0038] In summary, the optimal extraction process conditions for chlorogenic acid in sunflower seed meal were as follows: 30 wt% water content of the low eutectic solvent, 1:15 solid-liquid ratio, 700 W microwave power, 60 °C microwave temperature, and 3 min microwave time. Under these conditions, the extraction rate of chlorogenic acid was 2.63%.

[0039] <Comparative Example 4> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, 1 g of sunflower seed meal particles was stirred with 15 mL of low eutectic solvent to make them evenly mixed, then soaked for 63 min, filtered through polyester non-woven fabric to obtain a filtrate, added the filtrate to AB-8 resin, first eluted once with deionized water, then eluted with 50% ethanol aqueous solution, collected the eluate obtained by eluting with ethanol aqueous solution, and the eluate was rotary evaporated at 45 ° C and freeze-dried to obtain chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0040] <Comparative Example 5> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stir 1 g of sunflower seed meal particles with 15 mL of 75% ethanol to mix evenly, and soak for 60 min; S4. After soaking, microwave treatment was performed at 60°C and 700 W for 3 min, and the mixture was filtered through polyester non-woven fabric to obtain a filtrate. The filtrate was added to AB-8 resin, and the mixture was first eluted once with deionized water, and then eluted with 50% ethanol aqueous solution. The eluate obtained by eluting with ethanol aqueous solution was collected, and the eluate was rotary evaporated at 45°C and freeze-dried to obtain the chlorogenic acid extract.

[0041] The extraction rates of Comparative Example 4, Comparative Example 5 and the best process were calculated by filtering the filtrate obtained by polyester non-woven fabric according to the method in "1.2". The results are shown in Table 8: Table 8 Extraction rate of chlorogenic acid in each group According to the data in Table 8, the extraction rate of chlorogenic acid in Comparative Example 4 using only a low eutectic solvent and the extraction rate of chlorogenic acid in Comparative Example 5 using 75% ethanol immersion combined with microwave treatment are significantly lower than the extraction rate of chlorogenic acid under the optimal process, indicating that when extracting chlorogenic acid from sunflower seed meal, the extraction effect is not ideal when simply using a low eutectic solvent or ethanol combined with microwave treatment. In the optimal process, there is an obvious synergistic effect between microwaves and low eutectic solvents, and this synergistic effect significantly improves the extraction efficiency of chlorogenic acid.

[0042] <Example 5> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After the soaking is completed, microwave treatment is performed at 70°C and 700W for 3 min, and the mixture is filtered through a polyester non-woven fabric to obtain a filtrate, and the filtrate is added to AB-8 resin, and the mixture is first eluted once with deionized water, and then eluted with a 50% ethanol aqueous solution, and the eluate obtained by eluting with the ethanol aqueous solution is collected, and the eluate is rotary evaporated at 45°C, and then freeze-dried to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0043] <Example 6> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After soaking, add 2 mL of citric acid, stir evenly, and microwave for 3 min at 70°C and 700 W. Filter through polyester non-woven fabric to obtain a filtrate, add the filtrate to AB-8 resin, elute once with deionized water, and then elute with 50% ethanol aqueous solution, collect the eluate obtained by eluting with ethanol aqueous solution, rotary evaporate the eluate at 45°C, and then freeze-dry to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0044] <Example 7> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After soaking, add 2 mL of malic acid, stir evenly, and microwave for 3 min at 70°C and 700 W. Filter through polyester non-woven fabric to obtain a filtrate, add the filtrate to AB-8 resin, elute once with deionized water, and then elute with 50% ethanol aqueous solution, collect the eluate obtained by eluting with ethanol aqueous solution, rotary evaporate the eluate at 45°C, and then freeze-dry to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0045] <Example 8> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After soaking, add different volumes of citric acid, stir evenly, microwave treat at 70°C and 700W for 3 min, filter through polyester non-woven fabric to obtain a filtrate, add the filtrate to AB-8 resin, elute once with deionized water, and then elute with 50% ethanol aqueous solution, collect the eluate obtained by eluting with ethanol aqueous solution, rotary evaporate the eluate at 45°C, and then freeze-dry to obtain the chlorogenic acid extract; The volume of citric acid is set to: A: 1 mL; B: 3 mL; C: 4 mL; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0046] <Comparative Example 6> The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent comprises the following steps: S1. Detect crude protein content in sunflower seed meal using Dumas nitrogen analyzer; S2, grinding the dried sunflower seed meal with a crude protein content of 30-35%, and passing it through a 60-mesh sieve and a 30-mesh sieve to obtain sunflower seed meal particles with a particle size of 30-60 mesh; S3, stirring 1 g of sunflower seed meal particles with 15 mL of deep eutectic solvent to make them evenly mixed, and then soaking for 60 min; S4. After the soaking is completed, 2 mL of low eutectic solvent is added, and after stirring evenly, microwave treatment is performed at 70°C and 700 W for 3 min, and filtered through a polyester non-woven fabric to obtain a filtrate, and the filtrate is added to AB-8 resin, and the mixture is first eluted once with deionized water, and then eluted with a 50% ethanol aqueous solution, and the eluate obtained by eluting with the ethanol aqueous solution is collected, and the eluate is rotary evaporated at 45°C, and then freeze-dried to obtain the chlorogenic acid extract; Wherein, the preparation method of the deep eutectic solvent comprises the following steps: Choline chloride and urea are mixed in a molar ratio of 2:1, water is added, the mixture is heated to 90° C., and the mixture is continuously stirred until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent, wherein the water content of the low eutectic solvent is 30 wt %.

[0047] The extraction rates of the filtrates obtained by extracting the filtrates obtained in Examples 5 to 7, each group of Example 8 and Comparative Example 6 and filtering them through polyester non-woven fabric were calculated according to the method in "1.2". The results are shown in Table 9: Table 9 Extraction rate of chlorogenic acid in each group According to the data in Table 9, compared with Example 5, the extraction rate of chlorogenic acid in Example 6 and Example 7 is significantly improved, indicating that in the process of extracting chlorogenic acid from sunflower seed meal with a low eutectic solvent, under the microwave conditions of 70°C, 700 W, and 3 min, the addition of citric acid and malic acid can more effectively improve the extraction rate of chlorogenic acid than the simple use of a low eutectic solvent, and can overcome the problem of reduced extraction rate caused by excessively high microwave temperature. Among them, citric acid has a better effect on improving the extraction rate (2.68%), which is higher than the extraction rate (2.63%) of the optimal process (60°C, 700 W, 3 min); and compared with Comparative Example 6, the extraction rate of chlorogenic acid in Example 6 and Example 7 is significantly improved, indicating that when citric acid and malic acid are used in combination with a low eutectic solvent, respectively, the extraction rate is higher than that of Comparative Example 6 in which only the amount of the low eutectic solvent is increased, and the synergistic effect between citric acid and malic acid and the low eutectic solvent can effectively improve the extraction rate of chlorogenic acid; compared with the extraction rates of each group in Example 6 and Example 8, when 1-2 When mL of citric acid, i.e., the volume of citric acid is 1 / 15 to 2 / 15 of the volume of the low eutectic solvent, the extraction rate of chlorogenic acid is the highest, but adding too much citric acid will reduce the extraction rate of chlorogenic acid.

[0048] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent, characterized in that: The following steps are involved: S1. Detecting the crude protein content in sunflower seed meal; S2, crushing the sunflower seed meal to obtain sunflower seed meal particles; S3, mixing the sunflower seed meal particles and the low eutectic solvent at a mass volume ratio of 1 g: 5-25 mL, and soaking for 30-90 min; Wherein, when the crude protein content of the sunflower seed meal is 35-40%, the low eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1.5-1.8:1; When the crude protein content of the sunflower seed meal is 30-35%, the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1.8-2:1:1; When the crude protein content of the sunflower seed meal is 40-45%, the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1.0-1.5:1; The hydrogen bond acceptor is L-proline or choline chloride, and the hydrogen bond donor is any one of urea, D-tartaric acid, and acetamide; S4. After soaking, microwave treatment is performed at 30-70°C and 500-900W for 1-5 min. After filtration and purification, chlorogenic acid extract is obtained.

2. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: The particle size of the sunflower seed meal particles is 30-60 meshes.

3. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: The low eutectic solvent also includes water in a mass fraction of 20-40%.

4. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 3, characterized in that: The preparation method of the deep eutectic solvent comprises the following steps: The hydrogen bond donor, the hydrogen bond acceptor and water are uniformly mixed and stirred at 90-100° C. until a uniform, stable, clear and transparent solution is formed to obtain the low eutectic solvent.

5. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: When the crude protein content of the sunflower seed meal is 35-40%, the hydrogen bond acceptor is L-proline, and the hydrogen bond donor is urea; When the crude protein content of the sunflower seed meal is 30-35%, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is urea; When the crude protein content of the sunflower seed meal is 40-45%, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is acetamide.

6. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: In step S4, a stabilizer is added to the mixture formed by the soaked sunflower seed meal particles and the low eutectic solvent, and the mixture is stirred evenly. The stabilizer is citric acid or malic acid.

7. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 6, characterized in that: The stabilizer is citric acid, and the volume of the citric acid is 1 / 15 to 2 / 15 of the volume of the low eutectic solvent.

8. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: The purification operation is as follows: the filtered filtrate is added to AB-8 resin, first eluted once with deionized water, then eluted with 50% ethanol aqueous solution, the eluate obtained by eluting with ethanol aqueous solution is collected, and the eluate is subjected to rotary evaporation and freeze-drying to obtain the chlorogenic acid extract.

9. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: The mass volume ratio of the sunflower seed meal particles to the low eutectic solvent is 1 g:15 mL.

10. The method for extracting chlorogenic acid from sunflower seed meal using a deep eutectic solvent according to claim 1, characterized in that: The microwave temperature of the microwave treatment is 60-70° C., the microwave power is 600W-700W, and the microwave time is 3-4 min.

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