Extraction method and application of broccoli seed extract
The sulforaphane in broccoli seeds is extracted by compounding organic acids and choline chloride, and nanoparticles are made into complex with Bifidobacterium longan, which solves the problems of environmental pollution and high cost of extracting sulforaphane in the prior art, achieves efficient extraction and stability improvement, and maintains intestinal health.
Patent Information
- Application Number
- CN202510137646.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-07
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art has the problems of solvent volatile, toxic and harmful to the environment when extracting sulforaphane from broccoli seeds. The enzymatic solution is costly and complicated by by-products. The analysis requirements of modern extraction technology instruments are high and difficult to promote on a large scale.
A certain weight ratio of organic acid and choline chloride are used as the main extracts, and are dispersed in water to form an aqueous extract. Broccoli seed extract is obtained by sonication and freeze-drying, and nanoparticles are made into complex with Bifidobacterium longan, and dispersed in normal saline.
The efficient extraction and stability of sulforaphane have been achieved. The nanoparticles have good sustained release in the body, and the half-life and bioavailability have been significantly improved. The sulforaphane content in feces has reached more than 62 wt%, maintaining intestinal health.
Smart Images
Figure SMS_1 
Figure SMS_2
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of plant extracts, and in particular to an extraction method and application of broccoli seed extract. Background Art
[0002] Sulforaphane is a natural active substance that is abundant in broccoli seeds. It has significant anti-cancer effects and many other health benefits, such as lowering blood pressure, protecting the heart, relieving diabetes, reducing weight, and improving mental illness. This makes the research on its preparation and purification extremely important, which can not only promote the development of the health industry, but also bring a wide range of medical application value. In recent years, as people's pursuit of a healthy lifestyle continues to deepen, the development of new deep-processed products using sulforaphane in broccoli seeds has become a research hotspot; however, the technical development in this field is still in the initial exploration stage, especially in terms of the development and utilization of its extracts.
[0003] At present, the extraction of sulforaphane is mainly based on solvent extraction, enzymatic hydrolysis and modern extraction technology. The commonly used extraction solvents for solvent extraction are dichloromethane, ethyl acetate, acetone, hot ethanol solution, etc. However, the existing extraction solvents are volatile, toxic, and harmful to the environment. In addition, the cost of waste liquid treatment is high; enzymatic hydrolysis is currently the main method for people to obtain natural sulforaphane from plants, but the activity of the enzyme is easily affected by the environment and the cost of the enzyme is high, and there are a large number of by-products after the enzymatic hydrolysis of glucosinolate, and the subsequent purification work is complicated; modern extraction technology mainly includes the application of instrumental analysis technologies such as supercritical fluid extraction, ultrasonic-assisted extraction and microwave-assisted extraction, which effectively shortens the extraction time of sulforaphane and improves the extraction efficiency, but the accuracy of instrumental analysis is high and it is difficult to achieve large-scale promotion. Therefore, how to extract a large amount of sulforaphane from broccoli seeds under the premise of being green, non-toxic and can be promoted on a large scale has become a difficult problem. Summary of the invention
[0004] In order to solve the above technical problems, the present application provides an extraction method and application of broccoli seed extract.
[0005] In a first aspect, the present application provides a method for extracting a broccoli seed extract, comprising the following steps: washing, drying, crushing, sieving, and then immersing the broccoli seeds in an aqueous extract, mixing, standing, ultrasonically treating, and then cooling to room temperature, centrifuging to obtain a supernatant, extracting with a resin, eluting to obtain an eluate, and freeze-drying to obtain a broccoli seed extract, wherein the aqueous extract comprises a main extract and water in a weight ratio of (1.25-1.85):1, and the main extract comprises an organic acid and choline chloride in a weight ratio of (3-3.5):1, and the organic acid comprises one or more of citric acid, lactic acid, and acetic acid.
[0006] By adopting the above technical solution, the present application uses a certain weight ratio of organic acid and choline chloride as the main extract, disperses it in water to obtain a water extract, and the water extract has the properties of low melting point, green and harmless, and high extraction rate of sulforaphane. The extraction rate of sulforaphane in the broccoli seed extract finally obtained is not less than 1.28wt%, and the present application controls the weight ratio of the main extract and water to increase the extraction rate of sulforaphane as much as possible. If the proportion of the main extract is too large, the viscosity of the water extract will increase excessively, and the contact effect between the water extract and the broccoli seeds will be poor, resulting in a loss of extraction rate; if the proportion of the main extract is too small, the hydrogen bonds in the water extract will be destroyed, weakening the interaction between the water extract and the broccoli seeds, and also reducing the extraction rate.
[0007] Preferably, the aqueous extract comprises the main extract and water in a weight ratio of 1.45:1.
[0008] By adopting the above technical solution, the present application strictly controls the weight ratio of the main extract and water, thereby controlling the proportion of the main extract in the water extract. At this time, the extraction effect is more excellent. Experimental data show that the extraction rate of sulforaphane is significantly improved.
[0009] Preferably, the main extract comprises organic acid and choline chloride in a weight ratio of 3.2:1.
[0010] By adopting the above technical solution, the present application further controls the ratio of organic acid and choline chloride. Under this ratio, the proportion of organic acid and the extraction effect are at a high level. At the same time, choline chloride can also reasonably regulate the viscosity and surface tension of the water extract, increase the diffusion and mass transfer capacity of the target component, and further improve the extraction rate of sulforaphane.
[0011] Preferably, the organic acid comprises acetic acid and lactic acid in a weight ratio of 1:(4-5).
[0012] By adopting the above technical solution, the present application uses acetic acid and lactic acid as organic acids. The two can be well soluble in each other in the system. At the same time, acetic acid can ensure that the viscosity of the water extract is at a relatively low level, and lactic acid can achieve a very efficient extraction effect. The two can be mixed in a certain weight ratio to fully exert the synergistic effect, so that the viscosity and extraction effect of the organic acid are in a balanced state, and further the water extract as a whole has both low viscosity and high extraction capacity, thereby improving the extraction rate of sulforaphane.
[0013] Preferably, the weight ratio of acetic acid to lactic acid is 1:4.8.
[0014] By adopting the above technical solution, the present application controls the weight ratio of acetic acid to lactic acid to 1:4.8. At this time, the viscosity and extraction effect of the organic acid are in an optimal balance, which further makes the water extract as a whole have both low viscosity and higher extraction capacity, thereby improving the extraction rate of sulforaphane.
[0015] In a second aspect, the present application provides a broccoli seed extract obtained by the above extraction method, wherein the extraction rate of sulforaphane is not less than 1.28 wt %.
[0016] In a third aspect, the present application provides a dietary preparation, which is prepared by the following method: the nanoparticles prepared from the broccoli seed extract and Bifidobacterium longum are dispersed in physiological saline to obtain a nanoparticle concentration of 0.85 g / kg-1 g / kg and a viable count of Bifidobacterium longum of 3×10 9 CFU / mL-8×10 9 CFU / mL of dietary preparation.
[0017] Preferably, the method for preparing the nanoparticles comprises the following steps: mixing zein, propylene glycol alginate and water to obtain a binary solution, then adding anthocyanins and broccoli seed extract to the binary solution, mixing evenly and removing ethanol by rotary evaporation, then adding calcium chloride to the system, and vacuum freeze-drying to obtain nanoparticles, wherein the mass ratio of anthocyanins, broccoli seed extract, calcium chloride and the binary solution is (1-3):50:(1-3):950.
[0018] Preferably, the concentration of the nanoparticles is 0.95 g / kg, and the number of viable bacteria of Bifidobacterium longum is 5×10 9 CFU / mL.
[0019] Preferably, the mass ratio of anthocyanins, broccoli seed extract, calcium chloride and binary solution is 3:50:3:950.
[0020] Since the half-life of sulforaphane is relatively short, it will be rapidly metabolized and excreted in the body, which greatly limits its scope of application. Therefore, by adopting the above technical solution, the present application makes it into nanoparticles, uses zein and propylene glycol alginate as coating layers, and adds calcium chloride and anthocyanins to improve the overall structural stability, thermal stability and sulforaphane loading of the nanoparticles. Therefore, the nanoparticles prepared in the present application have good sustained release in the body, and the half-life and bioavailability are significantly improved; the present application strictly controls the mass ratio of anthocyanins, broccoli seed extract, calcium chloride and binary solution, and improves the stability and sustained release of the nanoparticles to the maximum. According to experimental detection, the half-life can be increased by 1h and the bioavailability can be increased by more than 35%.
[0021] The present application compounded the nanoparticles with good stability with Bifidobacterium longum and dispersed them in physiological saline to obtain a dietary preparation that can promote the conversion of glucoraphanin existing in the human body into sulforaphane. After taking it, the level of sulforaphane in the intestine can be effectively regulated, thereby maintaining intestinal health. Experiments have shown that the dietary preparation of the present application can make the content of sulforaphane in the feces of living organisms reach more than 62wt% of the total content of sulforaphane and glucoraphanin. The present application strictly controls the concentration of nanoparticles in the dietary preparation to 0.95g / kg and the number of live bacteria of Bifidobacterium longum to 5×10 9 CFU / mL, so that the total content of sulforaphane and glucoraphanin reached 64.8wt%.
[0022] In summary, this application has the following beneficial technical effects: 1. The present application uses a certain weight ratio of organic acid and choline chloride as the main extract, disperses it in water to obtain a water extract, which has the properties of low melting point, green and harmless, and high extraction rate of sulforaphane. The extraction rate of sulforaphane in the broccoli seed extract finally obtained is not less than 1.28wt%; 2. The present application uses acetic acid and lactic acid as organic acids. The two are well soluble in each other in the system. At the same time, acetic acid can ensure that the viscosity of the water extract is at a relatively low level, and lactic acid can achieve a very efficient extraction effect. The two are mixed in a certain weight ratio to fully exert their synergistic effect, so that the viscosity and extraction effect of the organic acid are in a balanced state, and further the water extract as a whole has both low viscosity and high extraction capacity, thereby improving the extraction rate of sulforaphane; 3. The present application compounds the nanoparticles with good stability with Bifidobacterium longum and disperses them in physiological saline to obtain a dietary preparation that can promote the conversion of glucoraphanin existing in the human body into sulforaphane. After taking the dietary preparation, the level of sulforaphane in the intestine can be effectively regulated, thereby maintaining intestinal health and making the content of sulforaphane in the feces of the living organism reach more than 62wt% of the total content of sulforaphane and glucoraphanin. DETAILED DESCRIPTION
[0023] Material Source Unless otherwise specified, the raw materials used in this application are all commercially available products, specifically: Citric acid was purchased from Guangzhou Chemical Reagent Factory; Acetic acid was purchased from Guangzhou Chemical Reagent Factory; Lactic acid was purchased from Guangzhou Chemical Reagent Factory; Choline chloride was purchased from Guangzhou Chemical Reagent Factory; Macroporous separation resin XAD-2 was purchased from Tianjin Haiguang Chemical Co., Ltd., and was soaked in 95% by volume ethanol for 24 h and then washed with ultrapure water until there was no alcohol smell, thereby obtaining the treated macroporous separation resin XAD-2 for later use; Zein powder was purchased from Hefei Bomei Biotechnology Co., Ltd., food grade; Propylene glycol alginate was purchased from Huazhong Biotechnology Company, food grade; Anthocyanins were purchased from Chenguang Biotechnology Group Co., Ltd., food grade; Bifidobacterium longum, with the deposit number CCFM1206, was activated for 3 generations in MRS medium and then centrifuged for 15 minutes. The supernatant was taken and washed 2-3 times with physiological saline. The bacterial sludge was obtained by centrifugation and resuspended in a sterilized 30% glycerol solution. After counting, it was stored at -80°C for use.
[0024] The present application is further described in detail below with reference to embodiments and comparative examples.
[0025] Example 1.1 A method for extracting broccoli seed extract comprises the following steps: I. Preparation of aqueous extract: 1.25 kg of the main extract (0.97 kg of citric acid and 0.28 kg of choline chloride) was dispersed in 1 L of water and stirred at 80°C until a uniform, transparent liquid was formed to obtain an aqueous extract; II. Wash the broccoli seeds with deionized water, dry them at 35°C, crush them, pass them through a 60-mesh sieve, and then immerse them in the water extract obtained in step I at a solid-liquid ratio of 20:1, mix them, let them stand for 2 hours, perform ultrasonic treatment at a temperature of 35°C and a power of 350 W, then cool them to room temperature, centrifuge them at a speed of 8000 r / min for 15 minutes to obtain a supernatant, and then add macroporous separation resin XAD-2 to the supernatant in an amount of 1 g / mL, place it in a shaking incubator at a temperature of 30°C and a speed of 180 r / min, wash the macroporous separation resin XAD-2 after adsorption equilibrium with ultrapure water for multiple times, and then elute them with ethanol with a volume fraction of 95%, shake them at 30°C for 12 hours to obtain an eluate, and freeze-dry them at a temperature of -50°C to obtain a broccoli seed extract.
[0026] Example 1.2 A method for extracting broccoli seed extract comprises the following steps: I. Preparation of aqueous extract: 1.85 kg of the main extract (1.3875 kg of acetic acid and 0.4625 kg of choline chloride) was dispersed in 1 L of water and stirred at 80°C until a uniform, transparent liquid was formed to obtain an aqueous extract; II. Wash the broccoli seeds with deionized water, dry them at 35°C, crush them, pass them through a 60-mesh sieve, and then immerse them in the water extract obtained in step I at a solid-liquid ratio of 20:1, mix them, let them stand for 2 hours, perform ultrasonic treatment at a temperature of 35°C and a power of 350 W, then cool them to room temperature, centrifuge them at a speed of 8000 r / min for 15 minutes to obtain a supernatant, and then add macroporous separation resin XAD-2 to the supernatant in an amount of 1 g / mL, place it in a shaking incubator at a temperature of 30°C and a speed of 180 r / min, wash the macroporous separation resin XAD-2 after adsorption equilibrium with ultrapure water for multiple times, and then elute them with ethanol with a volume fraction of 95%, shake them at 30°C for 12 hours to obtain an eluate, and freeze-dry them at a temperature of -50°C to obtain a broccoli seed extract.
[0027] Example 1.3 A method for extracting broccoli seed extract comprises the following steps: I. Preparation of aqueous extract: 1.55 kg of the main extract (1.2 kg of lactic acid and 0.35 kg of choline chloride) was dispersed in 1 L of water and stirred at 80°C until a uniform, transparent liquid was formed to obtain an aqueous extract; II. Wash the broccoli seeds with deionized water, dry them at 35°C, crush them, pass them through a 60-mesh sieve, and then immerse them in the water extract obtained in step I at a solid-liquid ratio of 20:1, mix them, let them stand for 2 hours, perform ultrasonic treatment at a temperature of 35°C and a power of 350 W, then cool them to room temperature, centrifuge them at a speed of 8000 r / min for 15 minutes to obtain a supernatant, and then add macroporous separation resin XAD-2 to the supernatant in an amount of 1 g / mL, place it in a shaking incubator at a temperature of 30°C and a speed of 180 r / min, wash the macroporous separation resin XAD-2 after adsorption equilibrium with ultrapure water for multiple times, and then elute them with ethanol with a volume fraction of 95%, shake them at 30°C for 12 hours to obtain an eluate, and freeze-dry them at a temperature of -50°C to obtain a broccoli seed extract.
[0028] Example 1.4 A method for extracting broccoli seed extract, which is different from Example 1.1 in that the amount of main extract used is 1.45 kg, the amount of citric acid used is 1.13 kg, the amount of choline chloride used is 0.32 kg, and the rest is the same as Example 1.1.
[0029] Example 2 A method for extracting broccoli seed extract, which is different from Example 1.4 in that the amount of main extract used is 1.45 kg, the amount of citric acid used is 1.1 kg, the amount of choline chloride used is 0.35 kg, and the rest is the same as Example 1.4.
[0030] Example 3.1 A method for extracting broccoli seed extract is different from that of Example 2 in that citric acid is replaced by acetic acid, and the rest is the same as that of Example 2.
[0031] Example 3.2 A method for extracting broccoli seed extract is different from that of Example 2 in that citric acid is replaced by lactic acid, and the rest is the same as that of Example 2.
[0032] Example 3.3 A method for extracting broccoli seed extract is different from that of Example 2 in that 1.1 kg of citric acid is replaced by 0.22 kg of acetic acid and 0.88 kg of lactic acid, and the rest is the same as that of Example 2.
[0033] Example 3.4 A method for extracting broccoli seed extract is different from that of Example 2 in that 1.1 kg of citric acid is replaced by 0.18 kg of acetic acid and 0.92 kg of lactic acid, and the rest is the same as that of Example 2.
[0034] Example 3.5 A method for extracting broccoli seed extract is different from that of Example 2 in that 1.1 kg of citric acid is replaced by 0.22 kg of acetic acid and 0.88 kg of citric acid, and the rest is the same as that of Example 2.
[0035] Example 3.6 A method for extracting broccoli seed extract is different from that of Example 2 in that 1.1 kg of citric acid is replaced by 0.22 kg of citric acid and 0.88 kg of lactic acid, and the rest is the same as that of Example 2.
[0036] Example 3.7 A method for extracting broccoli seed extract is different from that of Example 2 in that 1.1 kg of citric acid is replaced by 0.19 kg of citric acid and 0.91 kg of lactic acid, and the rest is the same as that of Example 2.
[0037] Comparative Example 1.1 The difference from Example 1.1 is that the amount of main extract used is 1 kg, the amount of citric acid used is 0.78 kg, the amount of choline chloride used is 0.22 kg, and the rest is the same as Example 1.1.
[0038] Comparative Example 1.2 The difference from Example 1.1 is that the amount of main extract used is 2 kg, the amount of citric acid used is 1.56 kg, the amount of choline chloride used is 0.44 kg, and the rest is the same as Example 1.1.
[0039] Comparative Example 2.1 The difference from Example 1.1 is that the amount of citric acid used is 0.5 kg, the amount of choline chloride used is 0.6 kg, and the rest is the same as Example 1.1.
[0040] Comparative Example 2.2 The difference from Example 1.1 is that the amount of citric acid used is 1 kg, the amount of choline chloride used is 0.1 kg, and the rest is the same as Example 1.1.
[0041] Performance Testing The extraction rates of sulforaphane in the broccoli seed extracts obtained in the examples and comparative examples were measured and calculated using high performance liquid chromatography, and the results are recorded in Table 1.
[0042] Table 1 Extraction rate of sulforaphane
[0043] Data Analysis: As can be seen from Table 1, the sulforaphane extraction rate of Examples 1.1-1.3 is 1.28-1.29 wt %, which proves that the aqueous extract obtained by using a certain weight ratio of organic acid and choline chloride as the main extract and dispersing it in water has the properties of low melting point, green and harmless, and high extraction rate of sulforaphane, and can obtain a large amount of sulforaphane; The sulforaphane extraction rate of Example 1.4 is higher than that of Example 1.1, which proves that the present application controls the proportion of the main extract in the water extract by strictly controlling the weight ratio of the main extract and water, thereby further improving the extraction effect of the present application; The sulforaphane extraction rate of Example 2 is higher than that of Example 1.4, which proves that the present application further controls the ratio of organic acid and choline chloride. Under this ratio, the proportion of organic acid and the extraction effect are at a high level. At the same time, choline chloride can also reasonably regulate the viscosity and surface tension of the water extract, increase the diffusion and mass transfer capacity of the target component, and further improve the extraction rate of sulforaphane. In Examples 3.1-3.6, the sulforaphane extraction rate of Examples 3.3-3.4 is higher than that of other Examples, which proves that the present application uses acetic acid and lactic acid as organic acids, and the two can be well soluble in the system. At the same time, acetic acid can ensure that the viscosity of the water extract is at a relatively low level, and lactic acid can achieve a very efficient extraction effect. The two can be mixed in a certain weight ratio to give full play to the synergistic effect, so that the viscosity and extraction effect of the organic acid are in a balanced state, and further make the water extract as a whole have both low viscosity and high extraction capacity, thereby improving the extraction rate of sulforaphane; The sulforaphane extraction rate of Example 3.7 is higher than that of Examples 3.3-3.4, which proves that the weight ratio of acetic acid to lactic acid is controlled to 1:4.8 in the present application. At this time, the viscosity and extraction effect of the organic acid are in an optimal balance, and the overall water extract has both low viscosity and higher extraction capacity, thereby improving the extraction rate of sulforaphane.
[0044] Application Example 1 A method for preparing a dietary preparation comprises the following steps: The zein powder was dissolved in 70% ethanol aqueous solution to prepare a zein solution with a concentration of 2 wt%. 1 L of the zein solution was added to 250 mL of 2.5 wt% propylene glycol alginate solution, and magnetically stirred at 1000 rpm overnight to obtain a binary solution. 950 g of the binary solution was added thereto with 3 g of anthocyanidins and 50 g of the broccoli seed extract extracted in Example 3.7, and magnetically stirred at room temperature for 4 h. The ethanol was then removed by rotary evaporation. 1 g of calcium chloride was added to the system, and then vacuum freeze-dried at a vacuum degree of 20 Pa and a temperature of -45 ° C to obtain nanoparticles. The nanoparticles and the cultured Bifidobacterium longum were then dispersed in physiological saline to obtain nanoparticles with a concentration of 1 g / kg and a viable count of 3 × 10-6 bifidobacterium longum. 9 CFU / mL of dietary preparation.
[0045] Application Example 2 A method for preparing a dietary preparation comprises the following steps: Zein powder was dissolved in 70% ethanol aqueous solution to prepare a 2wt% zein solution. 1L of zein solution was added to 250mL of 2.5wt% propylene glycol alginate solution, and magnetically stirred at 1000rpm overnight to obtain a binary solution. 950g of the binary solution was added with 3g of anthocyanidins and 50g of broccoli seed extract extracted in Example 3.7, and magnetically stirred at room temperature for 4h. The ethanol was then removed by rotary evaporation. 1g of calcium chloride was added to the system, and then vacuum freeze-dried at a vacuum degree of 20Pa and a temperature of -45°C to obtain nanoparticles. The nanoparticles and the cultured Bifidobacterium longum were then dispersed in physiological saline to obtain nanoparticles with a concentration of 0.85g / kg and a viable count of 8×10-6 bifidobacterium longum. 9 CFU / mL of dietary preparation.
[0046] Application Example 3 A method for preparing a dietary preparation comprises the following steps: Zein powder was dissolved in 70% ethanol aqueous solution to prepare a 2wt% zein solution. 1L of zein solution was added to 250mL of 2.5wt% propylene glycol alginate solution, and magnetically stirred at 1000rpm overnight to obtain a binary solution. 950g of the binary solution was added with 3g of anthocyanidins and 50g of broccoli seed extract extracted in Example 3.7, and magnetically stirred at room temperature for 4h. The ethanol was then removed by rotary evaporation. 1g of calcium chloride was added to the system, and then vacuum freeze-dried at a vacuum degree of 20Pa and a temperature of -45°C to obtain nanoparticles. The nanoparticles and the cultured Bifidobacterium longum were then dispersed in physiological saline to obtain nanoparticles with a concentration of 0.95g / kg and a viable count of 5×10-60. 9 CFU / mL of dietary preparation.
[0047] Application Example 4 A method for preparing a dietary preparation comprises the following steps: The zein powder was dissolved in 70% ethanol aqueous solution to prepare a zein solution with a concentration of 2 wt%. 1 L of the zein solution was added to 250 mL of 2.5 wt% propylene glycol alginate solution, and magnetically stirred at a speed of 1000 rpm overnight to obtain a binary solution. 950 g of the binary solution was added thereto with 1 g of anthocyanin and 50 g of the broccoli seed extract extracted in Example 3.7, and magnetically stirred at room temperature for 4 h. The ethanol was then removed by rotary evaporation. 3 g of calcium chloride was added to the system, and then vacuum freeze-dried at a vacuum degree of 20 Pa and a temperature of -45 ° C to obtain nanoparticles. The nanoparticles and the cultured Bifidobacterium longum were then dispersed in physiological saline to obtain nanoparticles with a concentration of 0.95 g / kg and a viable count of 5 × 10 9 CFU / mL of dietary preparation.
[0048] Application Example 5 A method for preparing a dietary preparation comprises the following steps: The zein powder was dissolved in 70% ethanol aqueous solution to prepare a zein solution with a concentration of 2 wt%. 1 L of the zein solution was added to 250 mL of 2.5 wt% propylene glycol alginate solution, and magnetically stirred at a speed of 1000 rpm overnight to obtain a binary solution. 950 g of the binary solution was added thereto with 1 g of anthocyanin and 50 g of the broccoli seed extract extracted in Example 3.7, and magnetically stirred at room temperature for 4 h. The ethanol was then removed by rotary evaporation. 1 g of calcium chloride was added to the system, and then vacuum freeze-dried at a vacuum degree of 20 Pa and a temperature of -45 ° C to obtain nanoparticles. The nanoparticles and the cultured Bifidobacterium longum were then dispersed in physiological saline to obtain nanoparticles with a concentration of 0.95 g / kg and a viable count of 5 × 10-6 bifidobacterium longum. 9 CFU / mL of dietary preparation.
[0049] Application Example 6 A method for preparing a dietary preparation comprises the following steps: The zein powder was dissolved in 70% ethanol aqueous solution to prepare a zein solution with a concentration of 2 wt%. 1 L of the zein solution was added to 250 mL of 2.5 wt% propylene glycol alginate solution, and magnetically stirred at a speed of 1000 rpm overnight to obtain a binary solution. 950 g of the binary solution was added thereto with 3 g of anthocyanidins and 50 g of the broccoli seed extract extracted in Example 3.7, and magnetically stirred at room temperature for 4 h. The ethanol was then removed by rotary evaporation. 3 g of calcium chloride was added to the system, and then vacuum freeze-dried at a vacuum degree of 20 Pa and a temperature of -45 ° C to obtain nanoparticles. The nanoparticles and the cultured Bifidobacterium longum were then dispersed in physiological saline to obtain nanoparticles with a concentration of 0.95 g / kg and a viable count of 5 × 10 9 CFU / mL of dietary preparation.
[0050] Performance Testing Half-life and bioavailability test: Several male SD rats (180-220 g) were taken and fed adaptively for 7 days at a temperature of 25±2°C and a relative humidity of 50±10%. The experiment was then carried out. The nanoparticles obtained in Application Examples 1-6 were made into a 20 mg / kg aqueous solution and the rats were gavaged (a blank control group of pure sulforaphane was also set up). Water was given overnight, and blood was collected through the eye sockets at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 12 and 24 hours after administration to obtain 300 µL blood samples, which were centrifuged at 4°C and 10,000 rpm for 10 minutes, and the supernatant was collected and stored at -80°C. The half-life and bioavailability of sulforaphane in rat blood were determined by chromatography and mass spectrometry, and the results are recorded in Table 2. Several male SD mice (18-20 g) were taken and adaptively fed for 7 days at a temperature of 25±2°C and a relative humidity of 50±10% before conducting the experiment. During the experiment, the mice could freely drink the dietary preparation prepared in Application Example 1-6. In addition, each mouse could eat freely. After 2 weeks of intervention, the mice were killed on the 15th day, and the feces of the mice were collected. The content of sulforaphane in the feces was determined to determine the ratio of the total content of sulforaphane and glucoraphanin. The results are recorded in Table 2.
[0051] Table 2 Performance test table
[0052] Data Analysis: As can be seen from Table 2, the half-life of sulforaphane in Application Example 1-2 is 7.72h, and the bioavailability is 77.86-77.89%, which are 1.01h and 37.32-37.35wt% higher than those of the blank group, respectively. This proves that the present application prepares broccoli seed extract into nanoparticles, uses zein and propylene glycol alginate as coating layers, and adds calcium chloride and anthocyanins to improve the overall structural stability, thermal stability and sulforaphane loading of the nanoparticles. Therefore, the nanoparticles prepared in the present application have good sustained release in vivo, and the half-life and bioavailability are significantly improved. At the same time, the proportion of sulforaphane reaches more than 62wt%, which proves that the present application compounds the nanoparticles with good stability with Bifidobacterium longum and disperses them in physiological saline to obtain a dietary preparation that can promote the conversion of glucoraphanin existing in the human body into sulforaphane. After taking, the level of sulforaphane in the intestine can be effectively regulated, thereby maintaining intestinal health. The half-life and bioavailability of sulforaphane in Application Example 3 are improved, proving that the present application improves the stability and sustained release of nanoparticles to the highest level by strictly controlling the mass ratio of anthocyanins, broccoli seed extract, calcium chloride and binary solution; Among Application Examples 4-6, the proportion of sulforaphane in Application Example 6 is significantly higher, which proves that the present application strictly controls the concentration of nanoparticles in the dietary preparation to 0.95 g / kg and the number of viable bacteria of Bifidobacterium longum to 5×10 9 CFU / mL can further enhance the effect of effectively regulating the level of sulforaphane in the intestine and maintain intestinal health.
[0053] The embodiments of this specific implementation method are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.
Claims
1. A method for extracting broccoli seed extract, comprising the following steps: The broccoli seeds are washed, dried, crushed, sieved, then immersed in a water extract, mixed, allowed to stand, ultrasonically treated, then cooled to room temperature, centrifuged to obtain a supernatant, extracted with a resin, eluted to obtain an eluate and freeze-dried to obtain a broccoli seed extract, characterized in that the water extract comprises a main extract and water in a weight ratio of (1.25-1.85):1, the main extract comprises an organic acid and choline chloride in a weight ratio of (3-3.5):1, and the organic acid comprises one or more of citric acid, lactic acid and acetic acid.
2. The method for extracting broccoli seed extract according to claim 1, characterized in that: The water extract comprises the main extract and water in a weight ratio of 1.45:
1.
3. The method for extracting broccoli seed extract according to claim 1, characterized in that: The main extract includes organic acid and choline chloride in a weight ratio of 3.2:
1.
4. The method for extracting broccoli seed extract according to claim 3, characterized in that: The organic acid comprises acetic acid and lactic acid in a weight ratio of 1:(4-5).
5. The method for extracting broccoli seed extract according to claim 4, characterized in that: The weight ratio of the acetic acid to the lactic acid is 1:4.
8.
6. A broccoli seed extract obtained by the method for extracting broccoli seed extract according to any one of claims 1 to 5, characterized in that The extraction rate of sulforaphane is not less than 1.28wt%.
7. A dietary preparation, characterized in that The nanoparticles prepared by the broccoli seed extract of claim 6 and Bifidobacterium longum are dispersed in physiological saline to obtain a nanoparticle concentration of 0.85 g / kg-1 g / kg and a viable count of Bifidobacterium longum of 3×10 9 CFU / mL-8×10 9 CFU / mL of dietary preparation.
8. A dietary preparation according to claim 7, characterized in that: The concentration of the nanoparticles was 0.95 g / kg, and the number of viable bacteria of Bifidobacterium longum was 5×10 9 CFU / mL.
9. A dietary preparation according to claim 7, characterized in that: The method for preparing the nanoparticles comprises the following steps: Zein, propylene glycol alginate and water are mixed to obtain a binary solution, anthocyanin and broccoli seed extract are then added to the binary solution, mixed evenly and ethanol is removed by rotary evaporation, calcium chloride is then added to the system, and vacuum freeze-dried to obtain nanoparticles, wherein the mass ratio of anthocyanin, broccoli seed extract, calcium chloride to the binary solution is (1-3):50:(1-3):
950.
10. A dietary preparation according to claim 9, characterized in that: The mass ratio of anthocyanidins, broccoli seed extract, calcium chloride and binary solution is 3:50:3:950.