Method for extracting beta-carotene from broccoli stems by deep eutectic solvent method

Abstract

The invention provides a method for extracting beta-carotene from broccoli stems by a eutectic solvent method, which can directly extract beta-carotene from milled broccoli stems by a one-step method by adopting a eutectic solvent consisting of lauric acid and L-menthol (in a mass ratio of 1: 1.5), and optimizes the extraction process by adopting a single factor and response surface test. According to the method, the extraction yield of beta-carotene in the broccoli stems is remarkably improved, so that the broccoli stems which are usually cut and discarded by people before cooking are fully and efficiently utilized, the process is simple and efficient, and the consumption demand of modern people for pursuing green and healthy can be met.

Description

Technical Field

[0001] The invention belongs to the technical field of carotene extraction and relates to a method for extracting beta-carotene from broccoli stems using a low eutectic solvent method. Background Art

[0002] β-Carotene (C 40 H 56 ) belongs to carotenoids and is the most common and stable natural pigment in nature. Its pure product is a purple to dark red shiny crystalline powder, which is sensitive to light and easily oxidized. It is easily soluble in organic solutions such as chloroform, dichloromethane, acetone, n-hexane, carbon disulfide, but insoluble in water, propylene glycol, and glycerol. β-carotene can be converted into vitamin A after being ingested by humans, which can effectively prevent night blindness and also improve the skin. In addition, it has both antioxidant and anti-inflammatory effects, can prevent cataracts and cardiovascular diseases, enhance the connection and communication between human cell gaps, increase resistance, enhance the body's immune system's ability to resist cancer, help the body protect against free radical damage, and has a detoxification effect. It is an important nutrient that cannot be lacking in maintaining human health.

[0003] Many natural foods contain a lot of beta-carotene, such as green fruits and vegetables, sweet potatoes, spinach, carrots, papaya, wax gourd, mango, etc. Among them, broccoli ranks among the top in beta-carotene content. Broccoli has a comprehensive range of nutrients, rich in protein, vitamins and carotenoids, and a high mineral content. Therefore, it ranks among the world's similar vegetables and has the reputation of "the crown of vegetables". Broccoli originated in Italy along the Mediterranean coast of Europe and was introduced to China in the late 19th century. The high nutritional value of broccoli makes it popular with more consumers, and its cultivation area and production volume are gradually expanding. The total area of ​​broccoli cultivation in China is more than one million mu, and the planting area in Zhejiang Province accounts for about a quarter. However, since consumers mainly eat the green tender stems, flower buds, flower branches, etc. of broccoli, 50% to 70% of the broccoli stems and leaves are used as feed for livestock, but more are discarded in the fields, resulting in a waste of food resources and even environmental pollution.

[0004] At present, the extraction of β-carotene from pure natural food mainly adopts organic solvents for extraction. Commonly used organic extraction solvents are mainly methanol, petroleum ether, acetone, etc. Generally, organic solvents are widely used as extractants and are relatively cheap. However, with the large-scale production and use of some organic solvents and the difficulty in recycling and treatment, they will cause varying degrees of damage to the surrounding environment. The large-scale use of some organic solvent products may have an impact on human health and endanger health. Supercritical fluid CO2 is also used to extract β-carotene, but this method requires more production equipment, high cost, and still requires the assistance of organic solvents. It is not suitable for large-scale industrial production. The emergence of low eutectic solvents makes the extraction of β-carotene from natural plants possible with low cost, safer and more effective. Low eutectic solvents are low eutectic mixtures composed of hydrogen bond acceptors and hydrogen bond donors. They have the advantages of low cost, simple preparation, low toxicity, etc., and have good prospects in many fields.

[0005] CN115043766A provides a method for extracting beta-carotene from millet using a low eutectic solvent, wherein the low eutectic solvent is a mixture of N,N-dimethylcyclohexylamine and n-butanol. However, an antioxidant needs to be added during the extraction process to ensure the stability of the beta-carotene, and the yield of the beta-carotene is low. More importantly, the addition of N,N-dimethylcyclohexylamine, n-butanol, and an antioxidant may have a potential impact on the safety of the beta-carotene, and is not suitable for extracting beta-carotene from broccoli stems.

[0006] Wang Chengya's "Determination of β-carotene in tobacco leaves by low eutectic solvent microextraction-high performance liquid chromatography" discloses the use of a low eutectic solvent composed of menthol and lauric acid to microextract β-carotene from tobacco leaves. However, the low eutectic solvent cannot directly extract β-carotene from tobacco leaves, but requires a two-step extraction, namely, first an extraction with acetone, and then a second microextraction from the acetone extractant with a low eutectic solvent. At the same time, sodium chloride needs to be added to the low eutectic solvent to promote stratification, so that the extraction can be successful.

[0007] Therefore, with regard to the broccoli stems that are usually cut off and discarded before cooking, it is urgent to find a more environmentally friendly, safe, simple and efficient method for extracting β-carotene from broccoli stems, thereby improving the utilization rate of broccoli stems and enhancing the extraction effect of β-carotene. Summary of the invention

[0008] To solve the above problems, the present invention provides a method for extracting β-carotene from broccoli stems by a low eutectic solvent method, wherein a low eutectic solvent composed of lauric acid and L-menthol (mass ratio 1:1.5) is used to directly extract β-carotene from broccoli stems that have been ground, and the extraction process is optimized by single factor and response surface experiments, thereby significantly improving the extraction rate of β-carotene in broccoli stems. The process is simple and efficient, and can meet the modern people's consumption needs for green and healthy living.

[0009] On the one hand, the present invention provides a method for extracting β-carotene from broccoli stems using a low eutectic solvent method, wherein the β-carotene is directly extracted from the broccoli stems using a low eutectic solvent, and the low eutectic solvent includes lauric acid and L-menthol.

[0010] Although there are related reports in the prior art on the use of low eutectic solvents to extract β-carotene, the low eutectic solvents used therein usually have safety hazards. The present application adopts a more green and environmentally friendly low eutectic solvent composed of lauric acid (hydrogen bond acceptor, HBA for short) and L-menthol (hydrogen bond donor, HBD for short), which are easily available raw materials, and can directly extract β-carotene from broccoli stems in one step, thereby improving the utilization rate of broccoli stems. Compared with traditional extraction methods, it has the characteristics of environmental protection and safety, simple operation, green and healthy, and higher extraction yield.

[0011] Furthermore, the broccoli stems need to be ground into powder with a particle size of less than 60 meshes.

[0012] In order to improve the extraction rate of β-carotene, the broccoli stems need to be sliced, dried, ground, and sieved multiple times with a 60-mesh sieve before extraction with a low eutectic solvent.

[0013] Furthermore, the mass ratio of lauric acid to L-menthol in the deep eutectic solvent is (1-3):(1-5).

[0014] The mass ratio of lauric acid and L-menthol in the low eutectic solvent has a great influence on the extraction effect of β-carotene in broccoli stems. If the mass ratio of lauric acid and L-menthol is not appropriate, it will directly lead to a very low extraction yield, and it will be difficult to extract from broccoli stems in one step. The extraction can only be successfully completed by first using other extraction reagents for extraction and then further microextraction.

[0015] Furthermore, the water content of the low eutectic solvent is 20-60%.

[0016] Furthermore, the solid-liquid ratio of the broccoli stem to the low eutectic solvent is (10-60):1.

[0017] Furthermore, the extraction time of the low eutectic solvent is 10 to 60 minutes.

[0018] Furthermore, the extraction time of the low eutectic solvent is 50 min, and the mass ratio of lauric acid to L-menthol in the low eutectic solvent is 1:1.5.

[0019] The present invention has been proved through research that only when the mass ratio of lauric acid and L-menthol in the low eutectic solvent is 1:1.5, can the one-step extraction of beta-carotene from broccoli stems be truly realized, and the extraction yield can be significantly improved, and the extraction yield can reach 89.8 μg / g.

[0020] Furthermore, the extraction temperature of the low eutectic solvent is 25-75°C.

[0021] Furthermore, the solid-liquid ratio of the broccoli stem to the low eutectic solvent is 42:1, the extraction temperature is 56° C., and the water content is 22%.

[0022] Response surface experiment analysis showed that the extraction yield of β-carotene in broccoli stems could be further improved when the solid-liquid ratio was 42:1, the extraction temperature was 56℃, and the water content was 22%.

[0023] In another aspect, the present invention provides a use of a deep eutectic solvent for preparing a preparation for improving the extraction yield of β-carotene in broccoli stems, wherein the deep eutectic solvent comprises lauric acid and L-menthol.

[0024] The method for extracting β-carotene from broccoli stems by a deep eutectic solvent method provided by the present invention has the following beneficial effects:

[0025] 1. A greener and healthier low eutectic solvent consisting of lauric acid and L-menthol (mass ratio 1:1.5) was used to extract β-carotene from broccoli stems in one step;

[0026] 2. The extraction process was optimized by single factor and response surface test, which significantly improved the extraction rate of β-carotene in broccoli stems to 89.8μg / g;

[0027] 3. Turn waste into treasure, making more efficient use of broccoli stems that are usually cut and discarded before cooking;

[0028] 4. Simple preparation, convenient operation, low cost, and readily available raw materials;

[0029] 5. Greener and safer, beneficial to human health and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the β-carotene standard curve in Example 1;

[0031] Figure 2Schematic diagram of the yield of β-carotene directly extracted from broccoli stems by comparing the mass ratios of different hydrogen bond acceptors and hydrogen bond donors in Example 3;

[0032] Figure 3 This is a schematic diagram of the yield of β-carotene from broccoli stems under different water contents of deep eutectic solvents in Example 5;

[0033] Figure 4 Schematic diagram of the yield of β-carotene from broccoli stems at different liquid-to-solid ratios of deep eutectic solvents in Example 6;

[0034] Figure 5 This is a schematic diagram of the yield of β-carotene from broccoli stems at different extraction times in Example 7;

[0035] Figure 6 Schematic diagram of the yield of β-carotene from broccoli stems at different extraction temperatures in Example 8;

[0036] Figure 7 is the temperature and liquid-to-solid ratio interaction diagram in Example 9;

[0037] Figure 8 is the temperature and water content interaction diagram in Example 9;

[0038] Fig. 9 This is the interaction diagram between the solid-liquid ratio and the water content in Example 9. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be pointed out that the following embodiments are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0040] The broccoli used as the experimental research object in the following examples was selected from Yinzhou District, Ningbo City, Zhejiang Province.

[0041] The main reagents used:

[0042] Table 1. Main reagents

[0043]

[0044] Main equipment used:

[0045] Table 2. Main instruments and equipment

[0046]

[0047] Example 1: Method for extracting β-carotene from broccoli stems using a deep eutectic solvent method provided by the present invention

[0048] The method for extracting β-carotene from broccoli stems by a deep eutectic solvent method provided in this embodiment comprises the following steps:

[0049] (1) Preparation of deep eutectic solvent

[0050] The low eutectic solvent was prepared by heating method. Lauric acid and L-menthol were mixed in a clean and dry round-bottom flask at a mass ratio of 1:1.5, and a magnetic rotor was added to the bottom of the flask. After the working temperature of the constant temperature magnetic stirrer was set to 80°C, the flask was placed on the constant temperature magnetic stirrer for heating and stirring until a colorless and uniform liquid appeared in the flask, and then cooled to room temperature for subsequent experiments.

[0051] (2) Extraction of β-carotene from broccoli stems

[0052] After washing the broccoli with clean water, remove the flower head and keep the flower stem for later use. Take an appropriate amount of broccoli stems, slice them, put them in a vacuum drying oven for drying, and grind the dried broccoli stem slices into powder with a mortar after cooling. Sieve them several times with a 60-mesh sieve for later use.

[0053] Accurately weigh 0.5 g of the processed broccoli stem powder, put it into a clean and dry centrifuge tube, add 21 mL of a low eutectic solvent with a water content of 22%, and perform ultrasonic extraction at 56°C for 50 min. After extraction, centrifuge at 8000 r / min in a centrifuge for 15 min to obtain the supernatant, and then make the volume to 10 mL with the low eutectic solvent to obtain the β-carotene extract of the broccoli stem.

[0054] (3) Preparation of β-carotene standard curve

[0055] It is implemented in accordance with the method of GB 5009.83-2016 "National Food Safety Standard Determination of β-carotene in Infant Food and Dairy Products", see Table 3 for details.

[0056] Table 3. Preparation method of test solution

[0057]

[0058]

[0059] Measurement method: Take an appropriate amount of β-carotene standard working solution of different concentrations and put it into a 1 cm colorimetric tube, and measure its absorbance value A at a wavelength of 450 nm.

[0060] The concentration of the β-carotene standard solution is set as the x-axis variable (abscissa) of the standard curve, and the absorbance corresponding to the concentration of the β-carotene standard solution is set as the y-axis value (ordinate) of the standard curve to draw the standard curve (such as Figure 1 As shown), the linear regression equation of the standard solution curve is obtained:

[0061] Y=0.2432x+0.0034(R 2 =0.9995)

[0062] (4) Determination of β-carotene content in broccoli stems

[0063] Take an appropriate amount of sample β-carotene extract, and perform subsequent operations according to the method of determining the standard curve. According to the measured absorbance, substitute each data into the following formula, and the β-carotene yield of broccoli stems can be calculated according to the formula. The formula is as follows:

[0064] X=[(Ab)×V×10 -6 ] / (m×K)×100%

[0065] Wherein: X represents the yield of β-carotene in the test sample; A represents the absorbance of the test solution; b represents the vertical intercept of the β-carotene standard curve; V represents the volume of the test solution, in milliliters (mL); m represents the mass of the broccoli stem powder, in grams (g); K represents the slope of the β-carotene standard curve.

[0066] Example 2: Preliminary exploration of different extraction methods

[0067] This example initially uses a two-step extraction method to extract β-carotene from broccoli stems, and by comparing the two-step extraction method with the one-step extraction method, it is hoped that a more suitable low eutectic solvent can be found to achieve a one-step method for efficient extraction of β-carotene from broccoli stems. This example uses the following three extraction methods:

[0068] 1. First, β-carotene in 0.5 g broccoli stem powder was extracted with 21 mL of methanol, ultrasonically extracted for 50 min at room temperature, filtered, and the filtrate was diluted to 10 mL with methanol; then lauric acid and L-menthol (mass ratio 1:2) were used as low eutectic solvents, and ultrasonically extracted for 30 min under the conditions of 50% water content, 40:1 liquid-to-solid ratio, and extraction temperature of 40°C, centrifuged to obtain the supernatant, and diluted to 10 mL with the low eutectic solvent;

[0069] 2. First, β-carotene in 0.5 g of broccoli stem powder was extracted with 21 mL of acetone, and ultrasonic extraction was performed at room temperature for 50 min. The filtrate was filtered and the volume of the filtrate was adjusted to 10 mL with acetone. Then, lauric acid and L-menthol (mass ratio 1:2) were used as low eutectic solvents. Ultrasonic extraction was performed for 30 min under the conditions of 50% water content, 40:1 liquid-to-solid ratio, and extraction temperature of 40°C. The supernatant was obtained by centrifugation and the volume was adjusted to 10 mL with the low eutectic solvent.

[0070] 3. Directly use lauric acid and L-menthol (mass ratio 1:2) as a low eutectic solvent 21 mL, ultrasonically extract for 30 min under the conditions of a water content of 50%, a liquid-to-solid ratio of 40:1, and an extraction temperature of 40°C, centrifuge to obtain the supernatant, and dilute to 10 mL with a low eutectic solvent;

[0071] The yield of β-carotene was tested for the samples obtained by the three extraction methods according to the method provided in Example 1. The results are shown in Table 4.

[0072] Table 4. Comparison of the effects of different extraction methods

[0073] Serial number Extraction method β-carotene yield (μg / g) 1 1. Methanol; 2. Low eutectic solvent 35.5 2 1. Acetone; 2. Low eutectic solvent 44.6 3 Deep eutectic solvents 18.9

[0074] It can be seen from Table 4 that compared with the two-step extraction method, the β-carotene yield is relatively low when the deep eutectic solvent is directly used for extraction, which can only reach 18.9 μg / g. Therefore, it is necessary to further explore suitable deep eutectic solvents and specific process parameters in the hope of improving the one-step extraction rate of β-carotene from broccoli stems.

[0075] Example 3: Effect of different hydrogen bond acceptor and hydrogen bond donor mass ratios on the direct extraction of β-carotene from broccoli stems

[0076] In this example, lauric acid and L-menthol are selected as the components of the low eutectic solvent for extracting β-carotene from broccoli stems. Under the conditions of 50% water content of the low eutectic solvent, 40:1 liquid-to-solid ratio, 30 minutes of extraction time, and 40°C of extraction temperature, β-carotene from broccoli stems is directly extracted. By changing the mass ratio of lauric acid to L-menthol, the yield of β-carotene from broccoli stems when the mass ratio is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3 is investigated. The results are as follows: Figure 2 shown.

[0077] Depend on Figure 2It can be seen that different mass ratios of lauric acid and L-menthol have a very significant effect on the yield of β-carotene in broccoli stems. When the mass ratio of lauric acid to L-menthol is 1:1.5, the yield of β-carotene in broccoli stems is particularly high, reaching 48.5μg / g, while when the ratio is not appropriate, the yield of β-carotene decreases significantly, making it difficult to achieve one-step extraction with a low eutectic solvent. It can be seen that when the mass ratio of lauric acid to L-menthol in a low eutectic solvent is 1:1.5, there is hope for one-step extraction of β-carotene from broccoli stems.

[0078] The molar ratio of hydrogen bond donors and hydrogen bond acceptors is a key factor affecting the properties of low eutectic solvents. The viscosity and number of hydrogen bonds of low eutectic solvents prepared with different mass ratios are different, which will lead to different extraction effects of low eutectic solvents on β-carotene in broccoli stems. When analyzing the results, in addition to comparing the extraction effects, it is also necessary to take into account the actual situation and compare the convenience and feasibility of experimental operation of low eutectic solvents with different mass ratios of hydrogen bond donors and hydrogen bond acceptors. This embodiment also investigates a variety of other mass ratio relationships. During the experiment, it was found that when the mass ratio of lauric acid to L-menthol was 1:1, 1:0.5 (2:1) and 3:1, the above three groups of low eutectic solvents were very easy to solidify at room temperature, which was not convenient for subsequent experimental operations and had a greater impact on the absorbance measurement results. When the mass ratio of lauric acid to L-menthol was 1:1.5, the low eutectic solvent was still in a liquid state at room temperature, which was convenient for subsequent experimental operations, and the viscosity was relatively suitable for the dissolution of β-carotene in broccoli stems. Therefore, the mass ratio of lauric acid to L-menthol was determined to be 1:1.5 as the optimal condition for the single factor mass ratio of hydrogen bond acceptor and hydrogen bond donor.

[0079] Example 4: Effect of different extraction methods on the extraction of β-carotene from broccoli stems

[0080] This embodiment compares the following four extraction methods:

[0081] (1) First, β-carotene in 0.5 g of broccoli stem powder was extracted with 21 mL of acetone, and ultrasonic extraction was performed at room temperature for 50 min. The filtrate was filtered and the volume of the filtrate was adjusted to 10 mL with acetone. Then, lauric acid and L-menthol (mass ratio 1:2) were used as low eutectic solvents. Ultrasonic extraction was performed for 30 min under the conditions of 50% water content, 40:1 liquid-to-solid ratio, and extraction temperature of 40°C. The supernatant was obtained by centrifugation and the volume was adjusted to 10 mL with the low eutectic solvent.

[0082] (2) First, β-carotene in 0.5 g of broccoli stem powder was extracted with 21 mL of acetone, and ultrasonic extraction was performed at room temperature for 50 min. The filtrate was filtered and the volume of the filtrate was adjusted to 10 mL with acetone. Then, lauric acid and L-menthol (mass ratio 1:1.5) were used as low eutectic solvents. Ultrasonic extraction was performed for 30 min under the conditions of 50% water content, 40:1 liquid-to-solid ratio, and extraction temperature of 40°C. The supernatant was obtained by centrifugation and the volume was adjusted to 10 mL with the low eutectic solvent.

[0083] (3) directly using lauric acid and L-menthol (mass ratio 1:2) as a low eutectic solvent 21 mL, ultrasonic extraction for 30 min under the conditions of a water content of 50%, a liquid-to-solid ratio of 40:1, and an extraction temperature of 40°C, centrifuging to obtain the supernatant, and diluting to 10 mL with a low eutectic solvent;

[0084] (4) directly using lauric acid and L-menthol (mass ratio 1:1.5) as a low eutectic solvent 21 mL, ultrasonic extraction for 30 min under the conditions of a water content of 50%, a liquid-to-solid ratio of 40:1, and an extraction temperature of 40°C, centrifuging to obtain the supernatant, and diluting to 10 mL with a low eutectic solvent;

[0085] The yield of β-carotene was tested for the samples obtained by the four extraction methods according to the method provided in Example 1. The results are shown in Table 5.

[0086] Table 5. Comparison of the effects of different extraction methods

[0087]

[0088] It can be seen from Table 5 that when the mass ratio of lauric acid to L-menthol is 1:2, the two-step extraction can significantly improve the extraction rate of β-carotene in broccoli stems; and when the mass ratio of lauric acid to L-menthol is 1:1.5, the effect of two-step extraction is very close to that of one-step direct extraction, and even the one-step extraction rate is slightly higher, and the two-step method will also introduce more chemical reagent components. Therefore, it is preferred to use a one-step extraction with a mass ratio of lauric acid to L-menthol of 1:1.5, which is simpler to operate, and more green, healthy and safe. The subsequent examples all use a mass ratio of lauric acid to L-menthol of 1:1.5 for extraction.

[0089] Example 5: Effect of different eutectic solvent water contents on β-carotene yield

[0090] A fixed mass ratio of 1:1.5 of lauric acid and L-menthol was used as the components of the eutectic solution for extracting β-carotene from broccoli stems. Under the conditions of a liquid-to-solid ratio of 40:1, an extraction time of 30 min, and an extraction temperature of 40°C, the water content of the eutectic solvent was changed to investigate the yield of β-carotene from broccoli stems when the water content was 10%, 20%, 30%, 40%, 50%, and 60%. The results are as follows: Figure 3 shown.

[0091] Depend on Figure 3 It can be seen that when the water content rises to 20%, the yield of β-carotene is the highest, which is 61.5μg / g. When the water content starts from 30%, as the water content continues to rise, the yield begins to fluctuate and decline. This may be because compared with conventional organic solvents, the viscosity of the low eutectic solvent itself is higher, which is not conducive to the exudation of the target product, and has a greater impact on the overall extraction rate. This is also the main inconvenience factor for choosing a low eutectic solvent as an extractant. Therefore, when preparing a low eutectic solvent, we need to reduce the viscosity of the low eutectic solvent by adding an appropriate amount of water. In addition, adding an appropriate amount of water to the low eutectic solvent also helps to increase the polarity of the system. With the increase of water, the viscosity of the low eutectic solvent decreases, the fluidity gradually increases, and the conductivity increases, which all contribute to the extraction of the target product. However, after reaching a certain range, as the polarity increases, it may cause the hydrogen bonds between molecules to break, and the inherent molecular structure of the low eutectic solvent is broken, resulting in a decrease in the conductivity of the low eutectic solvent, affecting the extraction effect of β-carotene. Therefore, adding excessive water to the deep eutectic solvent may affect the breaking of hydrogen bonds between hydrogen bond donors and hydrogen bond acceptors, thereby changing the molecular structure of the deep eutectic solvent, thereby weakening the molecular interaction between the deep eutectic solvent and β-carotene, resulting in a decrease in the yield of β-carotene. Therefore, the water content of the deep eutectic solvent was set at 20% as the optimal condition for the single factor of water content.

[0092] Example 6: Effect of different eutectic solvent liquid-to-solid ratios on β-carotene yield

[0093] A fixed mass ratio of lauric acid to L-menthol of 1:1.5 was used as a component of the eutectic solution for extracting β-carotene from broccoli stems. Under the conditions of a water content of 20% for the eutectic solvent, an extraction time of 30 min, and an extraction temperature of 40°C, the liquid-to-solid ratio of the eutectic solvent was changed to investigate the yield of β-carotene when the liquid-to-solid ratio was 10:1, 20:1, 30:1, 40:1, 50:1, and 60:1. The results are as follows: Figure 4 shown.

[0094] Depend on Figure 4It can be seen that as the liquid-to-solid ratio gradually increases, the yield of β-carotene also gradually increases. When the liquid-to-solid ratio is 40:1, the yield of β-carotene is the highest, reaching 80.6μg / g. When the liquid-to-solid ratio exceeds 40:1 and the liquid-to-solid ratio of the low eutectic solvent is increased, the yield of β-carotene decreases instead. Changes in the liquid-to-solid ratio will affect the degree of contact between the sample and the low eutectic solvent. Properly increasing the volume of the low eutectic solvent can increase the contact area between the solvent and the solute, increase the dispersion, increase the solubility, and promote the overflow of β-carotene, which has a driving effect.

[16] . Too low a liquid-to-solid ratio will result in insufficient contact between broccoli powder and the low eutectic solvent, slow dispersion of the sample in the low eutectic solvent, and even the heat transfer effect will be affected to a certain extent, resulting in insufficient extraction of β-carotene. When the liquid-to-solid ratio is too high, the yield of β-carotene is reduced. This may be because the low eutectic solvent diffuses into the plant cells for too long, dissolving more impurities and reducing the yield of β-carotene. At the same time, from an economic perspective, an excessively high liquid-to-solid ratio will increase the extraction cost. Therefore, the liquid-to-solid ratio of the low eutectic solvent was set at 40:1 as the optimal condition for the single factor of the liquid-to-solid ratio.

[0095] Example 7: Effect of different extraction times on β-carotene yield

[0096] A fixed mass ratio of 1:1.5 of lauric acid and L-menthol was used as the components of the eutectic solution for extracting β-carotene from broccoli stems. Under the conditions of 20% water content of the eutectic solvent, 40:1 liquid-to-solid ratio and 40°C extraction temperature, the extraction time of the eutectic solvent was changed to investigate the yield of β-carotene when the extraction time was 10min, 20min, 30min, 40min, 59min and 60min. The results are as follows: Figure 5 shown.

[0097] Depend on Figure 5It can be seen that in the range of 10min to 50min, the yield of β-carotene gradually increases. When the extraction time increases to 50min, the yield of β-carotene is the highest, reaching 81.9μg / g. When the extraction time continues to increase, the yield of β-carotene begins to decrease. This may be because the β-carotene in the early broccoli powder will continue to infiltrate the low eutectic solvent with the increase of time, so that the yield of β-carotene continues to increase. Therefore, a shorter extraction time may cause incomplete extraction of the target component, resulting in the target component that could have been extracted not being extracted. From an economic point of view, it also causes a certain degree of cost waste of the sample. However, too long extraction time is also undesirable. If the extraction time is too long, the target component that can be extracted has been extracted, which will cause a waste of time and reduce the overall operation rate. At the same time, too long extraction time may cause more serious oxidation of the target component β-carotene, and the structure of β-carotene may also be destroyed as a result. As the extraction time increases, the various mechanical effects and thermal effects of ultrasound will make the degradation of β-carotene in the extraction system more serious, resulting in a decrease in the extraction rate as the extraction time increases. In addition, in addition to the substances to be extracted, other impurities may be dissolved, affecting the final yield. Therefore, the time for extracting β-carotene from broccoli stems with a low eutectic solvent was finally set at 50 minutes as the optimal condition for this single factor.

[0098] Example 8: Effect of different extraction temperatures on β-carotene yield

[0099] A fixed mass ratio of 1:1.5 of lauric acid and L-menthol was used as the components of the eutectic solution for extracting β-carotene from broccoli stems. Under the conditions of 20% water content of the eutectic solvent, 40:1 liquid-to-solid ratio, and 50 min extraction time, the extraction temperature of the eutectic solvent was changed. The yield of β-carotene at extraction temperatures of 25°C, 35°C, 45°C, 55°C, 65°C, and 75°C was investigated. The results were observed and recorded, such as Figure 6 shown.

[0100] Depend on Figure 6It can be seen that within the range of 25°C to 55°C, as the temperature increases, the yield of β-carotene also gradually increases. When the temperature rises to 55°C, the yield of β-carotene is the highest, reaching 89.5±1.9μg / g, and as the temperature continues to rise, its yield decreases instead. The viscosity of the low eutectic solvent will decrease significantly with the increase in temperature, which may be because the change in temperature will affect the van der Waals force and hydrogen bond. When the temperature rises, the van der Waals force between molecules weakens, the molecular movement rate accelerates, the resistance during ion movement decreases, the viscosity drops rapidly, and at the same time, the hydrogen bond effect is strengthened, and the conductivity increases accordingly, which promotes the overflow of β-carotene in broccoli stem powder. When the temperature is too low, the viscosity of the low eutectic solvent is high and the conductivity is low, the overflow of β-carotene is hindered, and its yield cannot increase. However, since the temperature will have a certain impact on the stability of the active substance, this leads to the fact that the extraction temperature is too high and is not conducive to the extraction of β-carotene. β-carotene is very sensitive to high temperatures. If the extraction temperature of β-carotene is too high, it will not be conducive to ensuring the relative stability of β-carotene, and may cause the molecular structure of β-carotene to be directly destroyed and unable to be further extracted. In addition, the content of non-pigment components extracted may also increase with the increase of temperature. Although the overall extraction results increase from the results, in fact, only the extraction ratio of impurities increases, which may have a certain impact on the accuracy of the final measurement results. Therefore, the temperature for extracting β-carotene from broccoli stems with a low eutectic solvent was finally set at 55°C as the optimal condition for this single factor.

[0101] Example 9, response surface experiment

[0102] Based on the results of the single factor test, the lauric acid-L-menthol deep eutectic solvent (with a mass ratio of 1:1.5 and an extraction time of 50 min) was used as the extractant, the β-carotene yield of broccoli stems was used as the response value, and the temperature of the deep eutectic solvent for extracting β-carotene, the liquid-to-solid ratio of the deep eutectic solvent, and the water content were set as the three main response factors (as shown in Table 6). The response surface design was performed using the software Design Expert 10 (as shown in Table 7) and the results were compared and analyzed in order to obtain the optimal process conditions for extracting β-carotene from broccoli stems with the deep eutectic solvent. The results are shown in Tables 8 and 9.

[0103] Table 6. Factors and levels

[0104]

[0105] The response surface design was performed using the software Design Expert 10 (as shown in Table 7) and the results were compared and analyzed in order to obtain the optimal process conditions for extracting β-carotene from broccoli stems using a low eutectic solvent. The results are detailed in Tables 8 and 9.

[0106] Table 7. Response surface design and results

[0107]

[0108] The response surface and contour lines of the regression results were made using the software Design Expert 10. The results are shown in Figures 7 to 9 , the shapes of contour lines and response surfaces can reflect the interactions among various factors.

[0109] Depend on Figure 7 It can be seen that when the water content of the low eutectic solvent (C) remains unchanged, with the increase of extraction temperature (A) and the liquid-to-solid ratio of the low eutectic solvent (B), the yield of β-carotene first increases and then decreases, and its contour line profile is close to a circle. The interaction between the two factors is not significant (P>0.05), which is consistent with the results of variance analysis.

[0110] Depend on Figure 8 It can be seen that when the liquid-to-solid ratio (B) of the low eutectic solvent remains unchanged, with the increase of extraction temperature (A) and the water content of the low eutectic solvent (C), the yield of β-carotene first increases and then decreases. Its contour line profile presents an obvious ellipse and the response surface is steep, indicating that the two factors interact significantly and have a significant effect on the yield of β-carotene (P < 0.05), which is consistent with the results of variance analysis.

[0111] Depend on Fig. 9 It can be seen that when the extraction temperature (A) remains unchanged, with the increase of the liquid-to-solid ratio (B) and the water content (C) of the low eutectic solvent, the yield of β-carotene first increases and then decreases, and its contour line profile tends to be elliptical, and the response surface is steep, but in contrast Figure 8 The response surface of was relatively flat, indicating that the interaction between the two factors was more obvious, and the effect on the yield of β-carotene reached a significant level (P < 0.05), which was consistent with the results of variance analysis.

[0112] By analyzing the application software Design Expert 10, the optimal extraction process of β-carotene from broccoli stems is extraction temperature of 56.20℃, low eutectic solvent liquid-to-solid ratio of 41.68:1 and water content of 22.52%. Under this condition, the model predicts that the yield can reach 90.1μg / g. In order to verify the results and obtain the optimal extraction conditions of β-carotene, considering the practical operability, the extraction process of β-carotene from broccoli stems was adjusted to: lauric acid-L-menthol low eutectic solvent molar ratio of 1:2, extraction time of 50min, extraction temperature of 56℃, low eutectic solvent liquid-to-solid ratio of 42:1 and water content of 22%. Three parallel verification experiments were carried out under this condition, and the average extraction rate of β-carotene from broccoli stems was 89.8μg / g, which was only 0.3μg / g different from the predicted value, indicating that the extraction process optimized by response surface methodology is feasible.

[0113] The best extraction solvent for β-carotene from broccoli stems used in this example is the low eutectic solvent lauric acid-L-menthol. On the basis of the single factor experiment, the regression model is designed in combination with Design Expert 10 software. The liquid-to-solid ratio, water content and extraction temperature of the low eutectic solvent are used as response factors, and the yield of β-carotene is used as the response value. The optimization method of the extraction process of β-carotene from broccoli stems is discussed. The final results show that the designed regression model is significant in the experiment and has a good fit. The influence of the three factors on the yield of β-carotene extraction from broccoli stems is in the order of water content of the low eutectic solvent > extraction temperature of β-carotene > liquid-to-solid ratio of the low eutectic solvent. Taking into account the actual operability and combined with the optimal extraction conditions obtained by software analysis, the extraction process of β-carotene from broccoli stems was adjusted to: extraction temperature 56°C, low eutectic solvent liquid-to-solid ratio 42:1 and water content 22%. The yield of β-carotene can reach 89.8μg / g, which is close to the predicted value of 90.1μg / g. This shows that the application of single factor experiment and response surface experiment can optimize the process method for extracting β-carotene from broccoli stems, open up new process methods for the extraction of β-carotene in pure natural foods and propose new research directions for the utilization of broccoli stems.

[0114] The application of the present invention is not limited thereto. For example, the scope of application of the present invention can be expanded according to the environmental protection aspect. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for extracting β-carotene from broccoli stems using a deep eutectic solvent method, characterized in that: β-carotene is directly extracted from broccoli stems by using a deep eutectic solvent, wherein the deep eutectic solvent comprises lauric acid and L-menthol.

2. The method according to claim 1, characterized in that The broccoli stems need to be ground into powder with a particle size of less than 60 meshes.

3. The method according to claim 1, characterized in that The mass ratio of lauric acid to L-menthol in the deep eutectic solvent is (1-3):(1-5).

4. The method according to claim 3, characterized in that The water content of the deep eutectic solvent is 20-60%.

5. The method according to claim 4, characterized in that The solid-liquid ratio of the broccoli stem to the low eutectic solvent is (10-60):

1.

6. The method according to claim 5, characterized in that The extraction time of the deep eutectic solvent is 10 to 60 minutes.

7. The method according to claim 5, characterized in that The extraction time of the low eutectic solvent is 50 minutes, and the mass ratio of lauric acid to L-menthol in the low eutectic solvent is 1:1.

5.

8. The method according to claim 6, characterized in that The extraction temperature of the deep eutectic solvent is 25-75°C.

9. The method according to claim 7, characterized in that The solid-liquid ratio of the broccoli stem to the low eutectic solvent is 42:1, the extraction temperature is 56° C., and the water content is 22%.

10. Use of a deep eutectic solvent for preparing a preparation for improving the extraction yield of β-carotene in broccoli stems, characterized in that: The deep eutectic solvent includes lauric acid and L-menthol.

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