A method for separating and enriching glucosinolates in broccoli seeds

Abstract

The application provides a method for separating and enriching glucosinolates in broccoli seeds, which comprises the following steps: passivating endogenous enzymes of the broccoli seeds and crushing the seeds; adding the seed powder into cellulase for enzymolysis to obtain an extract; adding barium acetate into the extract, and concentrating the extract by rotary evaporation at 50-60 DEG C to 0.1-0.2 times of the total volume of the extract to obtain a crude extract; adding the glucosinolate crude extract into an ethanol-dipotassium hydrogen phosphate aqueous two-phase system, loading the mixed solution into a flotation column, and then introducing air from the bottom, after which the ethanol-dipotassium hydrogen phosphate mixed solution is allowed to stand and separate into two layers, the two phases are separated after extraction, the upper phase material is reserved, and the concentration of glucosinolates is determined. The application adopts the aqueous two-phase system combined with the flotation technology, takes ethanol / dipotassium hydrogen phosphate as the aqueous two-phase system, and adopts the response surface method (RSM) to construct the glucosinolate separation flotation process, so that the recovery rate of glucosinolates can be significantly improved.

Description

Technical Field

[0001] This invention relates to the field of enrichment and separation, specifically to a method for the separation and enrichment of glucosinolates from broccoli seeds. Background Technology

[0002] Glucosinolates (GLS), also known as glucosinolates, glucosinolates, and black mustard glycosides, are a large class of sulfur-containing compounds. They are major secondary metabolites of cruciferous plants and are widely found in plants of the Brassica genus within the cruciferous family, such as broccoli, mustard greens, and cabbage. The core structure of GLS consists of a D-glucose linked to a sulfonate aldehyde oxime group and a side chain derived from the amino acid R. Its structural formula is as follows:

[0003]

[0004] Based on the different R groups in their side chains, glucosinolates (GLS) are further classified into three types: aliphatic glucosinolates, aromatic glucosinolates, and indole glucosinolates. To date, approximately 180 types of GLS have been reported in the literature. The diversity of glucosinolates is due to side chain modifications, extensions, and extensive secondary modifications of the amino acid precursors before the formation of the glucosinolate core structure. The diversity of the R chains of the amino acids, the formation of the core structure, and the modifications of the side chains result in the various types of GLS. Complete GLS possess chemical stability and relatively high biological activity.

[0005] The primary ecological role of glucosinolates (GSLs) is plant defense. When plant tissues are damaged, they are hydrolyzed by myrosinase into isothiocyanates (ITCs), which are toxic to herbivores and pathogens. The glucosinolate-myrosinase system provides plants with an effective defense system. More importantly, GSL compounds and their enzymatic hydrolysis products, isothiocyanates, are closely related to human health. They possess various biological activities, and some epidemiological and pharmacological studies have shown that a diet rich in cruciferous vegetables (Brassicae) may play an important role in preventing many chronic diseases, such as cardiovascular disease, diabetes (II), dementia, immune dysfunction, obesity, and certain cancers. In recent years, they have attracted great interest due to their role in cancer prevention. Some studies have shown that GSLs and their metabolites can inhibit tumor growth by inhibiting the activation of phase I enzymes (cytochrome P450 enzymes) while increasing the activity of phase II detoxification enzymes (such as glutathione transsulfurase and quinone reductase).

[0006] Glucosinosteroids (GSLs) are water-soluble, non-volatile, and thermally stable ionic compounds. Based on this characteristic, the extraction of GSLs primarily uses boiling water or aqueous organic solvents (such as methanol, ethanol, or mixtures of alcohol and water) for solid-liquid extraction. The separation and purification of GSLs often employs column chromatography, utilizing the chemical properties of GSLs themselves and selecting appropriate stationary and mobile phases to achieve enrichment. Commonly used chromatographic stationary phase materials for GSL separation and purification include DEAE Sephade x A-25 dextran gel, D315 macroporous resin, and acidic alumina. Column separation offers advantages such as relatively clear separation effects and high purity, but the process is complex, requires large amounts of solvent, is time-consuming, and has a small batch processing capacity.

[0007] Aqueous two-phase systems (ATPS) are powerful platforms for the separation and purification of high-value biomolecules. This liquid-liquid extraction technology has become a powerful biological separation tool and has been widely used for the separation and purification of various biological materials, such as proteins, nucleic acids, enzymes, plant bioactive substances, and pigments. In ATPS, two miscible solutes exceeding critical concentrations can form two immiscible phases. The solubility of the separated substances differs in the two phases, thus achieving the purpose of separation and purification. Aqueous two-phase flotation, based on aqueous two-phase extraction, uses bubble mass transfer to enhance the distribution of the target analyte between the two phases. This method organically combines aqueous two-phase extraction and solvent flotation. It leverages the advantages of ATPS—simple operation, mild conditions, low energy consumption, and ease of industrial scale-up—while combining the high efficiency and rapid separation characteristics of flotation technology, making it a novel separation and enrichment technology with great development potential.

[0008] This invention aims to explore an economical and efficient process for the separation and enrichment of glucosinolates to improve the yield of glucosinolates and provide a foundation for in-depth research and subsequent development and utilization of broccoli glucosinolates. Summary of the Invention

[0009] To overcome the problems of the existing technology, the present invention provides a simple, environmentally friendly, highly efficient, and continuously operable method for the separation and enrichment of glucosinolates.

[0010] This invention provides the following technical solutions:

[0011] A method for isolating and enriching glucosinolates from broccoli seeds, comprising the following steps:

[0012] 1) Dry the broccoli seeds at 90-110℃ to deactivate endogenous enzymes, then crush them;

[0013] 2) Dissolve the seed powder in phosphate buffer, add cellulase for enzymatic hydrolysis, heat the solution to 90-100℃ for extraction after hydrolysis, collect the supernatant after centrifugation to obtain the extract;

[0014] 3) Add barium acetate to the extract, let it stand to precipitate, centrifuge and collect the supernatant, then concentrate it by rotary evaporation at 50-60℃ to 0.1-0.2 times the total volume of the extract to obtain crude liquid of glucosinolate;

[0015] 4) Add crude extract of glucosinolate to the aqueous two-phase system of ethanol-dipotassium hydrogen phosphate, pack the mixture into a flotation column and introduce air from the bottom. Then allow the ethanol-dipotassium hydrogen phosphate mixture to stand and separate into layers. After extraction, separate the two phases, retain the upper phase, and determine the concentration of glucosinolate.

[0016] Furthermore, in step 2), the seed powder is dissolved in phosphate buffer at a mass-to-volume ratio of 1g:10-20mL, with a pH value of 4.0-5.5.

[0017] Furthermore, in step 2), the mass ratio of cellulase to seed powder is 1:90-1:120, the enzymatic hydrolysis conditions are 45-60℃, the enzymatic hydrolysis time is 40-60 min, the extraction is performed twice, each time for 1-2 h, and the centrifugation conditions are: centrifugation at 6000-8000 r / min for 10-15 min.

[0018] Further, in step 3), add 0.5M barium acetate at 1 / 8-1 / 12 of the volume of the extract, let stand for 30-60 minutes, and centrifuge at 6000-8000 r / min for 10-15 minutes.

[0019] Furthermore, in step 4), the mass concentration of ethanol in the ethanol-dipotassium hydrogen phosphate mixture is 10-25%, the mass concentration of dipotassium hydrogen phosphate is 35-50%, the remainder is distilled water, the concentration of the crude glucosinolate extract is 15%-25%, the pH value is 8-9, the gas flow rate of the air is 40-50 mL / min, and the flotation time is 20-30 min.

[0020] Furthermore, in step 2), the seed powder is dissolved in phosphate buffer at a mass-to-volume ratio of 1g:10mL, with a pH of 5.

[0021] Furthermore, in step 2), the mass ratio of cellulase to seed powder is 1:100, the enzymatic hydrolysis conditions are 50℃, 50 min, ultrasonic hot extraction at 90℃ twice, 1 h each time, and centrifugation conditions are 6000 r / min for 10 min.

[0022] Further, in step 3), add 0.5M barium acetate at 1 / 10 of the volume of the extract, let stand for 30 minutes, and centrifuge at 8000 r / min for 10 minutes.

[0023] Furthermore, in step 4), the mass concentration of ethanol in the ethanol-dipotassium hydrogen phosphate mixture is 16.3%, the mass concentration of dipotassium hydrogen phosphate is 43.5%, the amount of crude thioglucosinolate extract added is 25%, the remainder is distilled water, the pH is 8.54, the gas flow rate of air is 40 mL / min, and the flotation time is 20 min.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects:

[0025] 1. This invention uses broccoli seeds (a cruciferous plant) as raw material and employs an enzyme-based, ultrasound-assisted extraction method to promote the release of glucosinolates from cells, which can greatly improve the extraction yield.

[0026] 2. A two-phase flotation technology combining water and ethanol is adopted. The two-phase system is ethanol / dipotassium hydrogen phosphate. The flotation process for the separation of glucosinolates is constructed using response surface methodology (RSM). The entire process can significantly improve the recovery rate of glucosinolates. Attached Figure Description

[0027] Figure 1 Aqueous two-phase diagram of ethanol-dipotassium hydrogen phosphate;

[0028] Figure 2 Figure showing the experimental results of the effect of pH on the flotation rate of glucosinolates.

[0029] Figure 3 Figure showing the experimental results of the effect of ethanol dosage on the flotation rate of glucosinolates.

[0030] Figure 4 Figure showing the experimental results of the effect of inorganic salt dosage on the flotation rate of glucosinolates.

[0031] Figure 5 3D plot of the effect of pH and anhydrous ethanol dosage on the flotation rate of glucosinolates.

[0032] Figure 6 3D diagram showing the effect of pH and inorganic salt dosage on the flotation rate of glucosinolates.

[0033] Figure 7 3D diagram showing the effect of anhydrous ethanol and inorganic salt dosage on the flotation rate of glucosinolates.

[0034] Figure 8 HPLC chromatograms of glucosinolate extract and flotation phases. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0036] Example 1

[0037] The type of phase-separating salt has a significant impact on the aqueous two-phase extraction (APE) efficiency. The phase-separating salt used in APE requires high solubility and dissociation in water, low cost, good phase formation with organic solvents, and high partitioning effect on the separated precipitates. This invention uses inexpensive and non-toxic ethanol as the organic solvent and investigated three commonly used inorganic salts (ammonium sulfate, sodium dihydrogen phosphate, and dipotassium hydrogen phosphate). All three inorganic salts formed phases with ethanol. Equal amounts of the three salts and ethanol were added to 50 mL test tubes respectively, and the phase separation was compared. The content of the crude thioglucosinolate extract in the two phases was further determined. The results showed that the ethanol / dipotassium hydrogen phosphate APE system had a higher extraction rate of thioglucosinolates; therefore, dipotassium hydrogen phosphate was chosen as the inorganic salt for the APE system.

[0038] To determine the mass fractions of inorganic salt (dipotassium hydrogen phosphate) and anhydrous ethanol in the aqueous two-phase system, this invention plotted an aqueous two-phase phase diagram using the ethanol / dipotassium hydrogen phosphate mass fraction as the abscissa and the ethanol mass fraction as the ordinate, employing the turbidity point method. The basis for establishing the ethanol-dipotassium hydrogen phosphate aqueous two-phase system is as follows: Figure 1 As shown, the mass concentration of ethanol in the ethanol-potassium dihydrogen phosphate mixed solution is 12.5%-32%, and the mass concentration of potassium dihydrogen phosphate is 18.5%-48.5%.

[0039] Influence of various factors on aqueous two-phase extraction

[0040] ① Effect of extraction pH on the flotation rate of glucosinolates

[0041] pH affects the degree of dissociation of phosphate, a buffer substance in the system, thus influencing the partition coefficient. A pH between 7 and 10 is chosen for the aqueous two-phase system. Figure 2 The results show that pH initially increases the flotation efficiency of glucosinolates, then decreases. The flotation efficiency of glucosinolates reaches its maximum at pH = 8.

[0042] ② Effect of ethanol dosage on the extraction rate of glucosinolates

[0043] The results were as follows: Ethanol mass fractions of 10%-25% were selected. Figure 3 As shown, the extraction rate of glucosinolates first increases and then decreases with the increase of anhydrous ethanol in the system, and the maximum flotation rate is achieved when the amount of anhydrous ethanol reaches 15%.

[0044] ③ Effect of inorganic salt dosage on the extraction rate of glucosinolates

[0045] The results were investigated when the inorganic salt content was 35%-50%. Figure 4 As shown, the extraction rate of glucosinolates first increases and then decreases with the increase of dipotassium hydrogen phosphate dosage. The flotation rate of glucosinolates is the highest when the dipotassium hydrogen phosphate dosage is 45%.

[0046] Experimental Results and Analysis of Variance

[0047] Based on the single-factor experiments, the mass fraction of anhydrous ethanol (10%–20%), the mass fraction of dipotassium hydrogen phosphate (40%–50%), and pH (7–9) were used as factors and levels, as shown in Table 1.

[0048]

[0049] Table 1 Factor levels in response surface BBD design experiments

[0050] Using the Box-Behnken combinatorial design (BBD) principle, 17 sets of response surface experiments with three factors and three levels were designed, as shown in Table 2.

[0051]

[0052]

[0053] Table 2. Response Surface Experiment Design and Results

[0054] The experimental results were fitted with the flotation rate of glucosinolates as the dependent variable and the mass fraction of inorganic salts, the mass fraction of anhydrous ethanol, and pH as independent variables. The fitting results are shown in Table 3.

[0055]

[0056] (***Highly significant, **significant)

[0057] Table 3. Analysis of Variance for the Regression Model of Two-Phase Aqueous Flotation

[0058] Y=+96.26-1.05*A+2.59*B+1.91*C-1.98*A*B+0.6250*A*C+0.70*B*C-2.11*A^2-6.83*B^2-1.78*C^2

[0059] Analysis of variance (ANOVA) showed that the model's F = 23.17 and P = 0.0002 < 0.01, indicating that the quadratic model used in the experiment was highly significant and statistically meaningful. ANOVA also revealed that the amount of anhydrous ethanol (B) and the extraction pH value had highly significant effects on the extraction rate of glucosinolates. The P-value for the model's lack of fit was 0.0738 > 0.05, indicating that the lack of fit was not significant and the residuals were caused by random error. Based on the F-value, the order of the three factors affecting the extraction rate of glucosinolates was: B > C > A, i.e., ethanol amount > pH > dipotassium hydrogen phosphate. The interaction terms AC and BC were not significant, while the interaction between AB and AC was significant.

[0060] Depend on Figure 5 , 6 The response surface plots (SPTs) at levels 7 and 8 visually illustrate the interaction between the amounts of dipotassium hydrogen phosphate, anhydrous ethanol, and pH. A more pronounced parabolic surface indicates a stronger interaction, while a less pronounced parabolic surface indicates a weaker interaction. Analysis of the response surface plots combined with p-values ​​shows that the parabolic surface of response curve AB is more pronounced than that of BC and AC, indicating a stronger interaction between AB and suggesting that the amount of dipotassium hydrogen phosphate (A) and anhydrous ethanol (B) has the most significant impact on the flotation rate of glucosinolates.

[0061] The response surface methodology was designed using Design Expert 13 software to further optimize the aqueous two-phase flotation process. The optimal aqueous two-phase flotation process for glucosinolates was determined to be: flotation for 20 min under the following conditions: 43.54% dipotassium hydrogen phosphate, 16.3% anhydrous ethanol, and pH 8.54.

[0062] Example 2

[0063] Method for determining the yield of glucosinolates: Add 1 mL of the extract from both the upper and lower phases (upper phase diluted 10 times, lower phase undiluted) to a test tube containing 2 mL of 0.1% sodium carboxymethyl cellulose solution and 1 mL of palladium chloride colorimetric solution. After thorough mixing, let stand at room temperature for 1.5 h, and measure the absorbance at 540 nm using a spectrophotometer. Calculate the extraction rate of glucosinolates in the aqueous two-phase system using the following formula:

[0064]

[0065]

[0066]

[0067] In the formula, E is the flotation efficiency, C0 is the mass concentration (w / w) of glucosinolates in the lower phase before flotation, and C is the mass concentration (w / w) of glucosinolates in the lower phase after flotation; α is the enrichment factor, and C... tR represents the mass concentration (w / w) of glucosinolates in the upper phase after flotation; where R is the phase ratio and V is the glucosinolate concentration. t V is the volume of the upper phase after flotation, and V is the volume of the lower phase after flotation.

[0068] The content of glucosinolates in the upper and lower phases was determined by palladium chloride colorimetric method. Under the process conditions of Example 1, glucosinolates were almost completely transferred to the upper phase. The optimal flotation rate of glucosinolates was 96.9%, which was basically consistent with the model prediction of 97.26%, indicating that the selected model was reasonable. Compared with the optimal extraction rate of 64.2% constructed in the laboratory before, the extraction rate of glucosinolates was increased by 52%.

[0069] Example 3

[0070] This invention provides a method for the isolation and enrichment of glucosinolates from broccoli seeds.

[0071] 1) A method for separating and purifying glucosinolates using aqueous two-phase flotation: Broccoli seeds are dried in an oven at 100℃ for 1 hour to deactivate endogenous enzymes. The broccoli seeds are then pulverized using a grinder and defatted with petroleum ether. After defatting, the seeds are placed in a fume hood to air dry naturally, passed through a 40-60 mesh sieve, and sealed for later use.

[0072] 2) Take 15g of pretreated defatted broccoli powder, add 150mL of cellulase solution (the mass ratio of enzyme to powder is 1:100), and enzymatically hydrolyze at 50℃ for 50min. After enzymatic hydrolysis, ultrasonically extract at 90℃ for 1h, centrifuge and collect the extract under the following conditions: 6000r / min for 10min. Repeat the extraction once using the same method, and combine the two extracts.

[0073] 3) Add 0.5M barium acetate at 1 / 10 of the volume of the extract, let stand for 30 min to remove protein from the extract, centrifuge at 8000 rpm for 10 min to collect the supernatant, concentrate by rotary evaporation to obtain a deep yellow crude extract of glucosinolates with a yield of up to 69.5 mg / g, and store at 4℃ for later use.

[0074] Under the same conditions, the yield of glucosinolates extracted without enzymatic ultrasonic extraction was 38.15 mg / g, while the yield of glucosinolates extracted by enzyme-assisted ultrasonic extraction reached 71.9 mg / g. This indicates that the improved method can promote the release of glucosinolates from cells and greatly improve the yield of the target substance.

[0075] 4) The mass of the ethanol-dipotassium hydrogen phosphate aqueous two-phase system was fixed at 40.0 g. 17.4 g of dipotassium hydrogen phosphate and 6.5 g of ethanol solution were added to separate 50 mL centrifuge tubes, along with 10 g of crude glucosinolate extract (0.5 mg / mL). The remainder was distilled water, pH 8.54. The mixture was vortexed and then packed into a flotation column. Air was introduced at a flow rate of 40 mL / min, and flotation was performed for 20 min. After flotation, the mixture was allowed to stand until the volumes of the upper and lower phases no longer changed. After flotation, the two phases were separated, and the upper phase was retained. The extraction rate of glucosinolate was finally determined to be 97.3% using the palladium chloride method.

[0076] Further analysis and identification of glucosinolates in the extract and flotation phases were performed using high-performance liquid chromatography (HPLC). The specific determination method is as follows: A standard solution was prepared using potassium myrosinase as a standard. Simultaneously, 1 mL of the upper phase solution was accurately transferred and diluted to a 10 mL volumetric flask with deionized water, filtered through a 0.45 μm aqueous filter membrane, and analyzed by HPLC. The lower phase solution did not require dilution and was filtered through a 0.45 μm aqueous filter membrane before HPLC analysis. HPLC detection conditions: mobile phase: aqueous phase (A) containing 20 mM ammonium acetate and 0.05% trifluoroacetic acid, and organic phase methanol (B); flow rate: 1 mL / min; WVL: 227 nm; gradient program: 5% B for the first 8 min; increasing to 60% from 8-15 min; 60%-5% B from 15-16 min; 5% B from 16-20 min; injection volume: 20 μL. The HPLC identification results are as follows: Figure 8 As shown, A is the HPLC chromatogram of potassium glucosinolate standard, B is the HPLC chromatogram of crude glucosinolate extract, C is the HPLC chromatogram of the upper flotation phase, and D is the HPLC chromatogram of the lower flotation phase. Figure 8 It can be seen that by comparing the retention times of potassium glucosinolate standard, the determination can be made. Figure 8 In C, the chromatographic peak of the upper flotation phase at 4 min is glucosinolate, while that of the lower flotation phase is... Figure 8 The absence of a chromatographic peak at position D further demonstrates that this method can transfer glucosinolates from the extract to the upper phase, thus achieving enrichment and purification.

[0077] Example 4

[0078] This invention provides a method for the isolation and enrichment of glucosinolates from broccoli seeds.

[0079] (1) A method for separating and purifying glucosinolates by aqueous two-phase flotation: Broccoli seeds are dried in an oven at 100℃ for 1 hour to deactivate endogenous enzymes. The broccoli seeds are then crushed using a pulverizer and defatted with petroleum ether. After defatting, the seeds are placed in a fume hood to air dry naturally, passed through a 40-60 mesh sieve, and sealed for later use.

[0080] (2) Take 15g of pretreated defatted broccoli powder, add 150mL of cellulase solution (the mass ratio of enzyme to powder is 1:90), and enzymatically hydrolyze at 50℃ for 50min. After enzymatic hydrolysis, ultrasonically extract at 90℃ for 1h, centrifuge and collect the extract. Repeat the extraction once using the same method, combine the two extracts, add 0.5M barium acetate at 1 / 10 of the extract volume, let stand for 30min to remove protein from the extract, centrifuge at 8000rpm for 10min and collect the supernatant, concentrate by rotary evaporation to obtain a deep yellow crude extract of glucosinolates with a yield of up to 69.5mg / g, and store at 4℃ for later use.

[0081] (3) The mass of the ethanol-dipotassium hydrogen phosphate aqueous two-phase system was fixed at 40.0 g. 17.4 g of dipotassium hydrogen phosphate and 6.5 g of ethanol solution were added to a 50 mL centrifuge tube, followed by 10 g of 0.5 mg / mL glucosinolate extract. The remainder was distilled water with a pH of 8.5. The mixture was then placed on a vortex mixer and mixed thoroughly. The mixture was then loaded into a flotation column and air was introduced at a flow rate of 40 mL / min. The flotation was carried out for 20 min, and then allowed to stand until the volumes of the upper and lower phases no longer changed. After the flotation was completed, the two phases were separated, and the upper phase was retained. The extraction rate of glucosinolate was found to be 95.5%.

[0082] Example 5

[0083] This invention provides a method for the isolation and enrichment of glucosinolates from broccoli seeds.

[0084] (1) A method for separating and purifying glucosinolates by aqueous two-phase flotation: Broccoli seeds are dried in an oven at 100℃ for 1 hour to deactivate endogenous enzymes. The broccoli seeds are then crushed using a pulverizer and defatted with petroleum ether. After defatting, the seeds are placed in a fume hood to air dry naturally, passed through a 40-60 mesh sieve, and sealed for later use.

[0085] (2) Take 15g of pretreated defatted broccoli powder, add 150mL of cellulase solution (the mass ratio of enzyme to powder is 1:100), and enzymatically hydrolyze at 50℃ for 50min. After enzymatic hydrolysis, ultrasonically extract at 90℃ for 1h, centrifuge and collect the extract. Repeat the extraction once using the same method, combine the two extracts, add 0.5M barium acetate at 1 / 10 of the extract volume, let stand for 30min to remove protein from the extract, centrifuge at 8000rpm for 10min and collect the supernatant, concentrate by rotary evaporation to obtain a deep yellow crude extract of glucosinolates with a yield of up to 69.5mg / g, and store at 4℃ for later use.

[0086] (3) The mass of the ethanol-dipotassium hydrogen phosphate aqueous two-phase system was fixed at 40.0 g. 20 g of dipotassium hydrogen phosphate and 6 g of ethanol solution were added to a 50 mL centrifuge tube, followed by 10 g of 0.5 mg / mL glucosinolate extract. The remainder was distilled water, with a pH of 7. The mixture was vortexed and then loaded into a flotation column. Air was introduced at a flow rate of 40 mL / min, and flotation was performed for 20 min. After flotation, the mixture was allowed to stand until the volumes of the upper and lower phases no longer changed. After flotation, the two phases were separated, and the upper phase was retained. The extraction rate of glucosinolate was found to be 90.2%.

[0087] Example 6

[0088] This invention provides a method for the isolation and enrichment of glucosinolates from broccoli seeds.

[0089] (1) A method for separating and purifying glucosinolates by aqueous two-phase flotation: Broccoli seeds are dried in an oven at 100℃ for 1 hour to deactivate endogenous enzymes. The broccoli seeds are then crushed using a pulverizer and defatted with petroleum ether. After defatting, the seeds are placed in a fume hood to air dry naturally, passed through a 40-60 mesh sieve, and sealed for later use.

[0090] (2) Take 15g of pretreated defatted broccoli powder, add 150mL of cellulase solution (the mass ratio of enzyme to powder is 1:100), and enzymatically hydrolyze at 50℃ for 50min. After enzymatic hydrolysis, ultrasonically extract at 90℃ for 1h, centrifuge and collect the extract. Repeat the extraction once using the same method, combine the two extracts, add 0.5M barium acetate at 1 / 10 of the extract volume, let stand for 30min to remove protein from the extract, centrifuge at 8000rpm for 10min and collect the supernatant, concentrate by rotary evaporation to obtain a deep yellow crude extract of glucosinolates with a yield of up to 69.5mg / g, and store at 4℃ for later use.

[0091] (3) The mass of the ethanol-dipotassium hydrogen phosphate aqueous two-phase system was fixed at 40.0 g. 16 g of dipotassium hydrogen phosphate and 6 g of ethanol solution were added to a 50 mL centrifuge tube, along with 10 g of 0.5 mg / mL glucosinolate extract. The remainder was distilled water, with a pH of 9. The mixture was vortexed and then loaded into a flotation column. Air was introduced at a flow rate of 40 mL / min, and flotation was performed for 20 min. After flotation, the mixture was allowed to stand until the volumes of the upper and lower phases no longer changed. After flotation, the two phases were separated, and the upper phase was retained. The extraction rate of glucosinolate was found to be 93.3%.

[0092] Example 7

[0093] Example 7 differs from Example 3 in that the concentration of glucosinolates is 0.3 mg / mL, while the other treatment methods are the same as in Example 3. The yield of glucosinolates in the upper phase after extraction with an ethanol-dipotassium hydrogen phosphate aqueous two-phase system was measured to be 95.8%.

[0094] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for isolating and enriching glucosinolates from broccoli seeds, characterized in that, Includes the following steps: 1) Dry the broccoli seeds at 90-110℃ to deactivate endogenous enzymes, then crush them; 2) Dissolve the seed powder in phosphate buffer, add cellulase for enzymatic hydrolysis, heat the solution to 90-100℃ and sonicate for extraction, centrifuge and collect the supernatant to obtain the extract; 3) Add barium acetate to the extract, let it stand to precipitate, centrifuge and collect the supernatant, then concentrate it by rotary evaporation at 50-60℃ to 0.1-0.2 times the total volume of the extract to obtain the crude extract of glucosinolates; 4) Add crude thioglucosinolate extract to the ethanol-dipotassium hydrogen phosphate aqueous two-phase system, load the mixture into a flotation column, and introduce air from the bottom. Then, allow the ethanol-dipotassium hydrogen phosphate mixture to stand and separate into layers. After extraction, separate the two phases, retain the upper phase, and determine the concentration of thioglucosinolate. The mass concentration of ethanol in the ethanol-dipotassium hydrogen phosphate mixture is 10-25%, the mass concentration of dipotassium hydrogen phosphate is 35-50%, and the remainder is distilled water. The concentration of crude thioglucosinolate extract is 15%-25%, the pH value is 8-9, the air flow rate is 40-50 mL / min, and the flotation time is 20-30 min.

2. The method for separating and enriching glucosinolates from broccoli seeds according to claim 1, characterized in that, In step 2), the seed powder is dissolved in phosphate buffer at a mass-to-volume ratio of 1g:10-20mL, with a pH of 4.0-5.

5.

3. The method for separating and enriching glucosinolates from broccoli seeds according to claim 1, characterized in that, In step 2), the mass ratio of cellulase to seed powder is 1:90-1:120, the enzymatic hydrolysis conditions are 45-60℃, the enzymatic hydrolysis time is 40-60 min, the extraction is performed twice, each time for 1-2 h, and the centrifugation conditions are 6000-8000 r / min for 10-15 min.

4. The method for separating and enriching glucosinolates from broccoli seeds according to claim 1, characterized in that, In step 3), add 0.5M barium acetate at 1 / 8-1 / 12 of the volume of the extract, let stand for 30-60 min, and centrifuge at 6000-8000 r / min for 10-15 min.

5. The method for separating and enriching glucosinolates from broccoli seeds according to claim 2, characterized in that, In step 2), the seed powder is dissolved in phosphate buffer at a mass-to-volume ratio of 1g:10mL, with a pH of 5.

6. The method for separating and enriching glucosinolates from broccoli seeds according to claim 3, characterized in that, In step 2), the mass ratio of cellulase to seed powder is 1:100, the enzymatic hydrolysis conditions are 50℃, 50 min, ultrasonic hot extraction at 90℃ twice, 1 h each time, and centrifugation conditions are 6000 r / min for 10 min.

7. The method for separating and enriching glucosinolates from broccoli seeds according to claim 4, characterized in that, In step 3), add 0.5M barium acetate at 1 / 10 of the extract volume, let stand for 30 min, and centrifuge at 8000 r / min for 10 min.

8. The method for separating and enriching glucosinolates from broccoli seeds according to claim 1, characterized in that, In step 4), the mass concentration of ethanol in the ethanol-dipotassium hydrogen phosphate mixture is 16.3%, the mass concentration of dipotassium hydrogen phosphate is 43.5%, the amount of crude glucosinolate extract added is 25%, the remainder is distilled water, the pH is 8.54, the gas flow rate of air is 40 mL / min, and the flotation time is 20 min.

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