CN108103119B - Method for continuously and stably producing high-purity sulforaphene - Google Patents
Method for continuously and stably producing high-purity sulforaphene Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/002—Nitriles (-CN)
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- C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
- C07C331/16—Isothiocyanates
- C07C331/18—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms
- C07C331/22—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The technical field of high-purity sulforaphene production, and particularly relates to a method for continuously and stably producing high-purity sulforaphene. The high-purity sulforaphene is produced by taking crude myrosinase, crosslinked chitosan microspheres and radish seed meal as raw materials through pretreatment, immobilized enzyme, reverse osmosis, centrifugal extraction, vacuum concentration and the like. The invention provides a method for continuously and stably producing high-purity sulforaphene, which has the characteristics of simple, stable and continuous production process, easiness in amplification, high product yield, low equipment investment cost, low production cost, high comprehensive utilization rate of resources and the like. The high-purity sulforaphene prepared by the method has the characteristics of high product purity, stable performance and the like, and can be widely applied to the industries of medicines, health products, foods, daily chemicals and the like.
Description
Technical Field
The invention belongs to the technical field of production of high-purity sulforaphene, and particularly relates to a method for continuously and stably producing the high-purity sulforaphene.
Background
Cruciferous vegetables contain a large amount of glucosinolates and myrosinase, and when people eat the vegetables, the myrosinase in the vegetables is fully contacted with the glucosinolates, and the glucosinolates rapidly generate compounds such as isothiocyanates, thiocyanates and nitriles. Modern researches show that the isothiocyanate compounds have the effects of inducing II-phase detoxification enzyme activity, remarkably improving the capability of human bodies to eliminate carcinogens and strongly inhibiting the growth and proliferation of cancer cells, thereby endowing cruciferous vegetables with the effects of cancer prevention and cancer resistance.
Thioglycoside is widely present in seeds, roots, stems, leaves and flower buds of cruciferous plants, and has great difference in content and variety. Wherein glucoraphanin (4-methylsulfinyl-3-butenyl thioglycoside) is mainly distributed in radish seeds, and myrosinase can catalyze glucoraphanin to generate sulforaphane (4-methylsulfinyl-3-butenyl isothiocyanate) under certain conditions. The sulforaphene has pharmacological activities of antibiosis, anti-inflammation, mutation resistance, cancer resistance and the like, has wide application prospect and huge market potential.
For example, patent publication No. CN 104876843a, "a method for preparing high-purity sulforaphene from carmine radish seeds" published on 9/2/2015 discloses a method for producing high-purity sulforaphene, which comprises: preparing a sulforaphene preparation solution by taking commercially degreased carmine radish seed powder as a raw material and performing enzymolysis, centrifugation, resin separation and high performance liquid chromatography preparation separation; and finally, quickly replacing by alcohol water, concentrating and freeze-drying to obtain a high-purity sulforaphene product. The main disadvantages of this process are: firstly, the high performance liquid chromatography is used as a preparation method of a high-purity product, so that the equipment investment and the cost of the filler required in the production are high, and the large-scale production is not facilitated. ② in the process of preparing the high-purity sulforaphene freeze-dried substance, due to the nature and the technical engineering characteristics of the sulforaphene, the pre-freezing is very difficult, so that the required pre-freezing temperature is low, the pre-freezing time is long, the energy consumption is high, and the production cost is increased. ③ the common defatted carmine radish seed powder is mostly a product after oil extraction, and the temperature is higher in the oil extraction process, which causes partial inactivation of myrosinase, resulting in incomplete hydrolysis of glucoraphanin, low yield of the sulforaphene and great waste of resources. The used equipment is expensive, the technical requirement is higher, the production capacity is limited, and the industrial production is not convenient.
Disclosure of Invention
The invention aims to provide a method for continuously and stably producing high-purity sulforaphene aiming at the defects of the existing method for producing sulforaphene, and the method has the characteristics of simple, stable and continuous production process, easiness in amplification, high product yield, low equipment investment cost, low production cost, high comprehensive utilization rate of resources and the like. The high-purity sulforaphene prepared by the method has the characteristics of high product purity, stable performance and the like.
The mechanism of the invention is as follows: the method adopts a double-aqueous phase extraction technology to purify the crude myrosinase, forms mutually incompatible two-aqueous phase systems by using polyethylene glycol and ammonium sulfate in water at proper concentrations, achieves the purpose of purifying the myrosinase according to the selective distribution of separated substances between the two-phase systems, and can effectively maintain the activity and the configuration of the myrosinase. The straight-chain diamine substance is used as a flexible chain modification carrier, and the myrosinase is combined on the flexible chain to realize the flexible fixation of the enzyme. When the enzyme is flexibly immobilized, the enzyme is contacted and collided with the flexible surface of the carrier, so that the change of enzyme conformation is not easy to cause, and the reduction of enzyme activity can be reduced. In addition, because the enzyme is fixed on the flexible chain, compared with rigid immobilization, the enzyme has good degree of freedom and mobility and small steric hindrance, and the free homogeneous catalytic activity of the enzyme can be better kept when the flexible immobilized enzyme acts with a substrate. The immobilized myrosinase reaction column and the sulforaphene purification column are combined, and the generation and the separation of the sulforaphene are realized simultaneously. The obtained low-purity sulforaphene is further purified by centrifugal extraction, light and heavy two-phase solutions respectively enter an annular space type mixing area formed between a rotary drum and a shell from two feeding pipe openings according to a certain proportion, the two phases are rapidly mixed and dispersed by a turbine disc and an impeller by means of the rotation of the rotary drum, and the two-phase solutions are fully transferred. The mixed liquid enters the rotary drum under the action of the vortex disc, the mixed liquid and the rotary drum rotate synchronously in a bulkhead area formed by the fortune plate, and heavy phase liquid with high specific gravity gradually leaves away from the center of the rotary drum and leans against the wall of the rotary drum in the upward flowing process under the action of centrifugal force; the light phase liquid with smaller specific gravity gradually gets away from the wall of the rotary drum and leans to the center, and the clarified two-phase liquid finally enters the collection chamber through the weir plates and is respectively led out of the machine through the leading pipes to complete the two-phase separation process. The process is continuous and short in period from the generation, adsorption separation and centrifugal extraction of the sulforaphene, and the sulforaphene is quickly separated from a water phase system, so that the process and product stability is improved.
The purpose of the invention is realized as follows: a method for continuously and stably producing sulforaphene uses crude myrosinase, cross-linked chitosan microspheres and radish seed meal as raw materials, and high-purity sulforaphene is obtained by pretreatment, immobilized enzyme, reverse osmosis, centrifugal extraction, vacuum concentration and the like. The method comprises the following specific steps:
(1) preparation of myrosinase pretreatment solution
Taking commercially available crude myrosinase as a raw material, and according to the mass (g) of the crude myrosinase: volume (mL) ratio of buffer solution 1: 60-150, adding the crude myrosinase into the buffer solution, slowly stirring and uniformly mixing, and then putting into an ultrasonic processor for ultrasonic treatment for 10-15 min for activating and dispersing the crude myrosinase. And after the ultrasonic treatment is finished, pumping into a vacuum filter again, carrying out vacuum filtration, and respectively collecting filtrate and filter residue. Drying and crushing the collected filter residue to be used as a feed additive; the collected filtrate, namely the myrosinase pretreatment solution, is used for the next treatment. Wherein the buffer solution is a trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH 6.5, a sodium dihydrogen phosphate-citric acid buffer solution with pH 6.5, or a sodium citrate-citric acid buffer solution with pH 6.5.
(2) Preparation of myrosinase lyophilized powder
According to the myrosinase pretreatment solution: 20-30% of polyethylene glycol solution with the molecular weight of 600-1000: the volume ratio of the ammonium sulfate solution with the mass fraction of 15-30% is 1: 2-4: 2-4, uniformly mixing the myrosinase pretreatment solution, the polyethylene glycol solution and the ammonium sulfate solution, pumping into a separation type tubular centrifuge, centrifuging at the rotating speed of 6000-8000 r/min, and respectively collecting a centrifugal light liquid and a centrifugal heavy liquid. The collected centrifugal light liquid is rich in polyethylene glycol and can be continuously used in the step (2) after being prepared; pumping the collected heavy centrifugal liquid into a nano filter with the molecular weight cutoff of 300-600 Da, performing primary nanofiltration separation under 0.25-0.35 MPa until the volume of the nanofiltration trapped liquid is 10-20% of the original volume, and respectively collecting primary nanofiltration filtered liquid and primary nanofiltration trapped liquid. And supplementing deionized water to the original volume of the collected first nanofiltration trapped fluid, performing second nanofiltration separation under the same condition until the volume of the second nanofiltration trapped fluid is reduced to 5-10% of the original volume, and respectively collecting second nanofiltration filtered fluid and second nanofiltration trapped fluid. The collected first and second nanofiltration filtrates are rich in ammonium sulfate and can be used in the step (2) after being prepared. And pre-freezing the collected second nanofiltration trapped fluid for 2-4 h at-16 to-20 ℃, and then placing the second nanofiltration trapped fluid in a freeze dryer for freeze drying for 24-36 h at 30-60 Pa and-50 to-60 ℃ to prepare the myrosinase freeze-dried powder for next treatment.
(3) Preparation of immobilized myrosinase
Taking commercially available crosslinked chitosan microspheres as a raw material, and according to the mass (g) of the crosslinked chitosan microspheres: volume of diamine solution (mL): the volume (mL) ratio of 1.0-2.5% glutaraldehyde solution is 1: 12-30: 1.5-3.0, dispersing the crosslinked chitosan microspheres in a diamine solution, uniformly stirring, adding a glutaraldehyde solution, and stirring and reacting at 60 ℃ for 1-1.5 hours; and (4) filtering, collecting a filter cake, and according to the mass (g) of the filter cake: the volume (mL) ratio of 40-60% ethanol solution in volume fraction is 1: 3-5, washing the filter cake with an ethanol solution to obtain the grafted diamine crosslinked chitosan microsphere. According to the mass (g) of the myrosinase freeze-dried powder: tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution volume (mL) ratio of pH 6.5 of 1: adding the myrosinase freeze-dried powder into a buffer solution according to the proportion of 40-60 to obtain a myrosinase solution, and then, according to the mass (g) of the grafted diamine crosslinked chitosan microspheres: volume of myrosinase solution (mL): the volume (mL) ratio of a malonaldehyde solution with a malonaldehyde mass concentration of 15% is 1: 50-80: 4-8, adding the grafted diamine crosslinked chitosan microspheres into a myrosinase solution, soaking for 1-3 hours, adding a malondialdehyde solution, carrying out immobilized enzyme reaction at the temperature of 30-40 ℃ for 2-4 hours, carrying out suction filtration after the reaction is finished, and respectively collecting the suction filtration residues and the suction filtration liquid. And (3) washing the collected suction filtration residues with deionized water with the volume 2 times that of the suction filtration residues, removing the included free myrosinase and malonaldehyde, and respectively collecting washing residues and washing liquid. The collected washing slag, namely the prepared immobilized myrosinase, is stored for later use at the temperature of 4 ℃; and combining the collected washing liquid with the collected suction filtration liquid, concentrating, blending, and then using the washing liquid for immobilizing the myrosinase again. Wherein the diamine solution is 7.5 percent of 1, 2-ethylenediamine or 10 percent of 1, 6-hexamethylenediamine or 14.5 percent of 1, 10-decamethylenediamine in percentage by mass.
(4) Preparation of crude glucoraphanin extract
Radish seed meal is used as a raw material, and the weight (kg) of the radish seed meal is as follows: the volume (L) ratio of 50-80% ethanol solution is 1: and 6-9, continuously stirring and leaching for 4-7 hours at room temperature, adjusting the pH to 2-3 by using a citric acid solution with the mass fraction of 2-6%, pumping into a clarified tubular centrifuge, centrifuging at the rotation speed of 15000-20000 r/min, and collecting the centrifuged clear liquid and the sediments respectively. The collected sediments are rich in protein, and can be used as animal feed additives after drying treatment. And pumping the collected centrifugal clear liquid into a reverse osmosis concentrator, performing first reverse osmosis concentration under the pressure of 0.15-0.25 MPa until the volume of the first reverse osmosis trapped liquid is reduced to 10-15% of the original volume, and respectively collecting the first reverse osmosis permeate and the first reverse osmosis trapped liquid. And adding deionized water into the collected first reverse osmosis trapped fluid, supplementing the volume to the original volume, performing second reverse osmosis concentration under the same condition until the volume of the second reverse osmosis trapped fluid is reduced to 5-10% of the original volume, and collecting second reverse osmosis permeate and second reverse osmosis trapped fluid respectively. Pre-freezing the collected second reverse osmosis trapped fluid at-16 to-20 ℃ for 2-4 h, then placing the pre-frozen trapped fluid in a freeze dryer, and carrying out freeze drying for 24-36 h at 30-60 Pa and-50 to-60 ℃ to prepare a glucoraphanin crude extract for next treatment; and combining the collected second reverse osmosis permeate with the collected first reverse osmosis permeate, recovering ethanol by using a climbing film evaporator, and blending to obtain an ethanol solution with the volume fraction of 50-80%, wherein the ethanol solution can be continuously used.
(5) Reactor for preparing reaction separation coupling immobilized enzyme
And filling the prepared immobilized myrosinase into a chromatographic column, and backflushing with deionized water with the same volume as that of the immobilized myrosinase to obtain an immobilized myrosinase reaction column for glucoraphanin enzymolysis. Dispersing commercially activated adsorbent resin (SP 207 resin or D101 resin or SP750 resin) in deionized water, assembling into adsorbent resin chromatographic column, back flushing with deionized water with the same volume as the adsorbent resin chromatographic column, and discharging bubbles from the adsorbent resin chromatographic column to obtain sulforaphene purification column for separating generated sulforaphene. And connecting the immobilized myrosinase reaction column with the sulforaphane purification column to assemble a reaction separation coupling immobilized enzyme reactor, and collecting for later use. Pumping the collected backwash liquid into a biochemical treatment tank for biochemical treatment, and discharging after reaching the standard.
(6) Preparation of low purity sulforaphene
According to the mass (g) of the glucoraphanin crude extract: volume (mL) of tris-hcl buffer solution at pH 6.5 was 1: 15-20, dispersing the glucoraphanin crude extract in a buffer solution to obtain a glucoraphanin crude extract solution, and then carrying out reaction on the glucoraphanin crude extract solution according to an immobilized myrosinase reaction column: the volume ratio of the glucoraphanin crude extract solution is 1: 5-8, pumping the glucoraphanin solution into the immobilized myrosinase reaction column at the speed of 6-10 times/h of the volume of the immobilized myrosinase reaction column at the flow speed, and carrying out continuous reaction and adsorption. After the reaction and the adsorption are finished, the outflow end of the immobilized myrosinase reaction column is disconnected with the sulforaphene purification column adsorbing sulforaphene. Then purifying the obtained product by a sulforaphene purification column: the volume ratio of the ethanol solution with the volume fraction of 40-60% is 1: 1-3, taking the ethanol solution as an eluent, and pumping the eluent into the sulforaphene purification column adsorbing the sulforaphene at a flow rate of 2-4 times/hour of the volume of the sulforaphene purification column to carry out sulforaphene elution separation. Pumping the collected eluent into a vacuum film evaporator, and performing vacuum film evaporation under the conditions that the vacuum degree is 0.09-0.095 MPa and the temperature is 20-30 ℃ to obtain the low-purity sulforaphene, wherein the purity of the sulforaphene is 88.2-90.5%. Wherein, the immobilized myrosinase reaction column and the sulforaphene purification column in the reaction separation coupling immobilized enzyme reactor can be repeatedly used after being activated.
(7) Preparation of high-purity sulforaphene
According to the mass (g) of the low-purity sulforaphene: volume of deionized water (mL) ratio of 1: and 10-20, dissolving the low-purity sulforaphene in deionized water to obtain a low-purity sulforaphene solution. According to the low-purity sulforaphene solution: the volume ratio of the dichloromethane is 1: 1-2, pumping the low-purity sulforaphene solution and dichloromethane into a centrifugal extractor at a flow rate of 80-100 mL/min respectively, and collecting a water phase and an oil phase respectively. Pumping the oil phase into a vacuum concentrator, and carrying out vacuum concentration under the conditions that the vacuum degree is 0.09-0.095 MPa and the temperature is 20-30 ℃ to obtain the high-purity sulforaphene. The purity of the sulforaphene is 98.6-99.7%, and the total yield is 90.7-91.5%. The recovered dichloromethane can be continuously used for centrifugal extraction, and the water phase can be used for preparing the low-purity sulforaphene solution again.
After the technical scheme is adopted, the invention mainly has the following effects:
1. the method for purifying the myrosinase by adopting the double aqueous phase extraction technology has the advantages of high mass transfer speed, short phase separation time, lower energy consumption, less separation steps, high efficiency, large processing capacity, mild conditions, easy industrial amplification, no inactivation or denaturation of the myrosinase and the like.
2. Based on the linear diamine substance as a flexible chain, the flexible fixation of myrosinase is realized, the enzyme activity recovery rate reaches 89.4%, the repeated use half-life period exceeds 50 times, and the problems of low activity, poor stability and the like of an immobilized enzyme system are obviously improved.
3. Exogenous immobilized myrosinase is used as a catalyst, so that the problem of low yield of the sulforaphene caused by low enzyme activity in the radish seed meal is solved.
4. The technological process includes the steps of sulforaphene generation, adsorption separation and centrifugal extraction to obtain a high-purity sulforaphene product, the process is continuous, the period is short, and sulforaphene can be quickly separated from a water phase system, so that the stability of the process and the product is improved.
5. The centrifugal extraction equipment is adopted to replace the traditional extraction equipment, and the centrifugal extraction equipment has the characteristics of large treatment capacity, high mass transfer efficiency, good energy-saving effect, small equipment floor area, low comprehensive investment cost and the like.
6. The technical process can obtain two kinds of sulforaphene with different purities, meet different market demands and realize maximum utilization.
7. The invention develops a set of production method which has low equipment investment cost, low production cost and high comprehensive utilization rate of resources and is suitable for industrialization, and has important application value.
8. The obtained sulforaphene product has good stability and can be stored for a long time at the temperature of minus 4 ℃.
The product prepared by the method can be widely applied to industries such as medicine, health care products, food, daily chemicals and the like.
Fourth, detailed description of the invention
The present invention will be further described with reference to the following specific embodiments.
Example 1
A method for continuously and stably producing sulforaphene comprises the following specific steps:
(1) preparation of myrosinase pretreatment solution
The method takes commercially available crude myrosinase as a raw material, and comprises the following steps of: the volume ratio of the buffer solution is 1 g: 60 mL, adding the crude myrosinase into the buffer solution, slowly stirring and uniformly mixing, and then putting into an ultrasonic processor for ultrasonic treatment for 10 min; pumping into a vacuum filter after the ultrasonic treatment is finished, carrying out vacuum filtration, and respectively collecting filtrate and filter residue; wherein the buffer solution is trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH of 6.5.
(2) Preparation of myrosinase lyophilized powder
According to the myrosinase pretreatment solution: 20 percent of polyethylene glycol solution with the molecular weight of 600-1000: the volume ratio of the ammonium sulfate solution with the mass fraction of 15-30% is 1: 2: 2, uniformly mixing the myrosinase pretreatment solution, the polyethylene glycol solution and the ammonium sulfate solution, pumping into a separation type tubular centrifuge, centrifuging at the rotating speed of 6000r/min, and respectively collecting a centrifugal light liquid and a centrifugal heavy liquid; pumping the collected centrifugal heavy liquid into a nanofiltration device with the interception molecular weight of 300Da, carrying out primary nanofiltration separation under 0.25MPa until the volume of the nanofiltration intercepted liquid is 10 percent of the original volume, and respectively collecting primary nanofiltration filtered liquid and primary nanofiltration intercepted liquid; supplementing deionized water to the collected first nanofiltration trapped fluid to the original volume, carrying out second nanofiltration separation under the same condition until the volume of the second nanofiltration trapped fluid is reduced to 5% of the original volume, and respectively collecting second nanofiltration filtered fluid and second nanofiltration trapped fluid; and pre-freezing the collected second nanofiltration trapped fluid at-16 ℃ for 2h, and then placing the second nanofiltration trapped fluid in a freeze dryer for freeze drying at 30Pa and-50 ℃ for 24h to prepare the myrosinase freeze-dried powder for the next treatment.
(3) Preparation of immobilized myrosinase
The method comprises the following steps of taking commercially available crosslinked chitosan microspheres as raw materials, and according to the mass of the crosslinked chitosan microspheres: volume of diamine solution: the volume ratio of the glutaraldehyde solution with the mass fraction of 1.0% is 1 g: 12 mL of: 1.5 mL, firstly dispersing the crosslinked chitosan microspheres in a diamine solution, uniformly stirring, then adding a glutaraldehyde solution, and stirring and reacting for 1h at 60 ℃; and (3) filtering, collecting a filter cake, and according to the mass of the filter cake: the volume ratio of the ethanol solution with the volume fraction of 40% is 1 g: 3 mL, washing the filter cake with an ethanol solution to obtain the grafted diamine crosslinked chitosan microspheres; according to the mass of the myrosinase freeze-dried powder: the volume ratio of the trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH of 6.5 is 1 g: adding the myrosinase freeze-dried powder into a buffer solution according to the proportion of 40mL to obtain a myrosinase solution, and then according to the mass of the grafted diamine crosslinked chitosan microspheres: volume of myrosinase solution: the volume ratio of the malonaldehyde solution with the malonaldehyde mass concentration of 15% is 1 g: 50 mL of: adding grafted diamine crosslinked chitosan microspheres into a myrosinase solution according to the proportion of 4mL, soaking for 1h, adding a malonaldehyde solution, carrying out immobilized enzyme reaction for 2h at the temperature of 30 ℃, carrying out suction filtration after the reaction is finished, respectively collecting suction filtration residues and suction filtration liquid, washing the suction filtration residues with deionized water 2 times the volume of the suction filtration residues, and respectively collecting washing residues and washing liquid; the collected washing slag, namely the prepared immobilized myrosinase, is stored for later use at the temperature of 4 ℃; wherein the diamine solution is 1, 2-ethylenediamine with the mass fraction of 7.5%.
(4) Preparation of crude glucoraphanin extract
Radish seed meal is used as a raw material, and the weight of the radish seed meal is as follows: the volume fraction of 50% ethanol solution is 1 kg: continuously stirring and leaching for 4h at room temperature according to the proportion of 6L, adjusting the pH to 2 by using a citric acid solution with the mass fraction of 2%, then pumping into a clarification type tubular centrifuge, centrifuging at the rotation speed of 15000r/min, and respectively collecting a centrifugal clear liquid and sediments; pumping the collected centrifugal clear liquid into a reverse osmosis concentrator, performing first reverse osmosis concentration under 0.15MPa until the volume of first reverse osmosis trapped liquid is reduced to 10% of the original volume, and respectively collecting first reverse osmosis permeate and first reverse osmosis trapped liquid; adding deionized water into the collected first reverse osmosis trapped fluid, supplementing to the original volume, performing second reverse osmosis concentration under the same condition until the volume of the second reverse osmosis trapped fluid is reduced to 5% of the original volume, and respectively collecting second reverse osmosis permeate and second reverse osmosis trapped fluid; and collecting the second reverse osmosis trapped fluid, pre-freezing for 2 hours at-16 ℃, and then placing in a freeze drier for freeze drying for 24 hours at 30Pa and-50 ℃ to prepare the glucoraphanin crude extract for the next treatment.
(5) Preparation reaction separation coupling immobilized enzyme reactor
Loading the prepared immobilized myrosinase into a chromatographic column, and backflushing with deionized water with the same volume as that of the immobilized myrosinase to obtain an immobilized myrosinase reaction column; dispersing activated adsorption resin sold in the market, namely SP207 resin in deionized water, assembling into an adsorption resin chromatographic column, and performing back flushing by using the deionized water with the same volume as the adsorption resin chromatographic column to obtain a sulforaphene purification column; and connecting the immobilized myrosinase reaction column with the sulforaphene purification column to assemble a reaction separation coupling immobilized enzyme reactor, and collecting for later use.
(6) Preparation of low-purity sulforaphene
According to the mass of the glucoraphanin crude extract: the volume of the tris-hcl buffer solution at pH 6.5 was 1 g: dispersing the glucoraphanin crude extract into a buffer solution according to the proportion of 15mL to obtain a glucoraphanin crude extract solution, and then carrying out reaction on the glucoraphanin crude extract solution according to an immobilized myrosinase reaction column: the volume ratio of the glucoraphanin crude extract solution is 1: 5, pumping the glucoraphanin solution into the immobilized myrosinase reaction column at the speed of 6 times of the volume of the immobilized myrosinase reaction column per hour of the flow speed, and carrying out continuous reaction and adsorption; after the reaction and the adsorption are finished, disconnecting the outflow end of the immobilized myrosinase reaction column from the sulforaphene purification column adsorbing sulforaphene; then purifying with a sulforaphene purification column: the volume ratio of the ethanol solution with the volume fraction of 40 percent is 1: 1, taking the ethanol solution as an eluent, pumping the eluent into a sulforaphene purification column adsorbing the sulforaphene at a flow rate of 2 times/hour of the volume of the sulforaphene purification column, and eluting and separating the sulforaphene; pumping the collected eluent into a vacuum film evaporator, and performing vacuum film evaporation under the conditions that the vacuum degree is 0.09MPa and the temperature is 20 ℃ to obtain the low-purity sulforaphene with the purity of 88.2 percent.
(7) Preparation of high-purity sulforaphene
According to the mass of the low-purity sulforaphene: the proportion of the volume of the deionized water is 1 g: dissolving the low-purity sulforaphene into deionized water according to the proportion of 10mL to obtain a low-purity sulforaphene solution; according to the low-purity sulforaphene solution: the volume ratio of the dichloromethane is 1: 1, pumping the low-purity sulforaphene solution and dichloromethane into a centrifugal extractor at a flow rate of 80mL/min respectively, and collecting a water phase and an oil phase respectively; pumping the oil phase into a vacuum concentrator, and carrying out vacuum concentration under the conditions that the vacuum degree is 0.09MPa and the temperature is 20 ℃ to obtain high-purity sulforaphene; the purity of the sulforaphene reaches 98.6 percent, and the total yield reaches 90.7 percent.
Example 2
A method for continuously and stably producing sulforaphene comprises the following specific steps:
(1) preparation of myrosinase pretreatment solution
Taking commercially available crude myrosinase as a raw material, and according to the mass of the crude myrosinase: the volume ratio of the buffer solution is 1 g: 100mL, adding the crude myrosinase into the buffer solution, slowly stirring and uniformly mixing, and then putting into an ultrasonic processor for ultrasonic treatment for 12 min; pumping into a vacuum filter after the ultrasonic treatment is finished, carrying out vacuum filtration, and respectively collecting filtrate and filter residue; wherein the buffer solution is a sodium dihydrogen phosphate-citric acid buffer solution with pH of 6.5.
(2) Preparation of myrosinase lyophilized powder
According to the myrosinase pretreatment solution: 25% by mass of polyethylene glycol solution with molecular weight of 600-1000: the volume ratio of the ammonium sulfate solution with the mass fraction of 20 percent is 1: 3: 3, uniformly mixing the myrosinase pretreatment solution, the polyethylene glycol solution and the ammonium sulfate solution, pumping into a separation type tubular centrifuge, centrifuging at the rotating speed of 7000r/min, and respectively collecting a centrifugal light solution and a centrifugal heavy solution; pumping the collected heavy centrifugal liquid into a nano filter with the molecular weight cutoff of 400Da, performing primary nanofiltration separation under 0.30MPa until the volume of the nanofiltration trapped liquid is 15% of the original volume, and respectively collecting primary nanofiltration filtered liquid and primary nanofiltration trapped liquid; supplementing deionized water to the collected first nanofiltration trapped fluid to the original volume, carrying out second nanofiltration separation under the same condition until the volume of the second nanofiltration trapped fluid is reduced to 8% of the original volume, and respectively collecting second nanofiltration filtered fluid and second nanofiltration trapped fluid; and pre-freezing the collected second nanofiltration trapped fluid for 3h at-18 ℃, and then placing the second nanofiltration trapped fluid in a freeze dryer for freeze drying for 30h at 45Pa and-55 ℃, thus preparing the myrosinase freeze-dried powder for the next treatment.
(3) Preparation of immobilized myrosinase
The method is characterized in that cross-linked chitosan microspheres sold in the market are used as raw materials, and the mass of the cross-linked chitosan microspheres is as follows: volume of diamine solution: the volume ratio of the glutaraldehyde solution with the mass fraction of 1.8% is 1 g: 16 mL of: 2.2 mL, dispersing the crosslinked chitosan microspheres in a diamine solution, uniformly stirring, adding a glutaraldehyde solution, and stirring and reacting at 60 ℃ for 1.2 hours; and (3) filtering, collecting a filter cake, and according to the mass of the filter cake: the volume ratio of 50% ethanol solution is 1 g: washing the filter cake with ethanol solution at a ratio of 4mL to obtain the grafted diamine crosslinked chitosan microspheres; according to the mass of the myrosinase freeze-dried powder: the volume ratio of the trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH 6.5 is 1 g: adding 50 mL of myrosinase freeze-dried powder into a buffer solution to obtain a myrosinase solution, and then according to the mass of the grafted diamine crosslinked chitosan microspheres: volume of myrosinase solution: the volume ratio of the malonaldehyde solution with the malonaldehyde mass concentration of 15% is 1 g: 65 mL of: adding grafted diamine crosslinked chitosan microspheres into a myrosinase solution according to the proportion of 6 mL, soaking for 2h, adding a malonaldehyde solution, carrying out immobilized enzyme reaction for 3h at the temperature of 35 ℃, carrying out suction filtration after the reaction is finished, respectively collecting suction filtration residues and suction filtration liquid, washing the suction filtration residues with deionized water of which the volume is 2 times that of the suction filtration residues, and respectively collecting washing residues and washing liquid; the collected washing slag, namely the prepared immobilized myrosinase, is stored for later use at the temperature of 4 ℃; wherein the diamine solution is 1, 6-hexamethylene diamine with the mass fraction of 10%.
(4) Preparation of crude glucoraphanin extract
Radish seed meal is used as a raw material, and the mass of the radish seed meal is as follows: the volume ratio of the 65% ethanol solution is 1 kg: 7L, continuously stirring and leaching for 5 hours at room temperature, adjusting the pH to 2.5 by using a citric acid solution with the mass fraction of 4%, pumping into a clarification type tubular centrifuge, centrifuging at the rotating speed of 18000r/min, and respectively collecting a centrifuged clear liquid and a sediment; pumping the collected centrifugal clear liquid into a reverse osmosis concentrator, performing first reverse osmosis concentration under 0.20MPa until the volume of the first reverse osmosis trapped liquid is reduced to 12% of the original volume, and respectively collecting first reverse osmosis permeate and first reverse osmosis trapped liquid; adding deionized water into the collected first reverse osmosis trapped fluid, supplementing to the original volume, performing second reverse osmosis concentration under the same condition until the volume of the second reverse osmosis trapped fluid is reduced to 8% of the original volume, and respectively collecting second reverse osmosis permeate and second reverse osmosis trapped fluid; collecting the second reverse osmosis trapped fluid, pre-freezing at-18 deg.C for 3 hr, and freeze-drying in freeze-drying machine at 45Pa and-55 deg.C for 30 hr to obtain glucoraphanin crude extract for further treatment.
(5) Reactor for preparing reaction separation coupling immobilized enzyme
Loading the prepared immobilized myrosinase into a chromatographic column, and backflushing with deionized water with the same volume as that of the immobilized myrosinase to obtain an immobilized myrosinase reaction column; dispersing activated adsorption resin sold in the market, namely D101 resin, in deionized water, assembling into an adsorption resin chromatographic column, and performing back flushing by using the deionized water with the same volume as the adsorption resin chromatographic column to obtain a sulforaphene purification column; and connecting the immobilized myrosinase reaction column with the sulforaphene purification column to assemble a reaction separation coupling immobilized enzyme reactor, and collecting for later use.
(6) Preparation of low purity sulforaphene
According to the mass of the crude glucoraphanin extract: the volume of the tris-hcl buffer solution at pH 6.5 was 1 g: dispersing the glucoraphanin crude extract into a buffer solution according to the proportion of 18mL to obtain a glucoraphanin crude extract solution, and then carrying out reaction on the glucoraphanin crude extract solution according to the following steps of: the volume ratio of the glucoraphanin crude extract solution is 1: 6, pumping the glucoraphanin solution into the immobilized myrosinase reaction column at the speed of 8 times of the volume of the immobilized myrosinase reaction column at the flow speed for continuous reaction and adsorption; after the reaction and the adsorption are finished, disconnecting the outflow end of the immobilized myrosinase reaction column from the sulforaphene purification column adsorbing sulforaphene; then purifying with a sulforaphene purification column: the volume ratio of the ethanol solution with the volume fraction of 50 percent is 1: 2, taking the ethanol solution as an eluent, pumping the eluent into the sulforaphene purification column adsorbing the sulforaphene at a flow rate which is 3 times/h of the volume of the sulforaphene purification column, and eluting and separating the sulforaphene; pumping the collected eluent into a vacuum film evaporator, and performing vacuum film evaporation at the vacuum degree of 0.092MPa and the temperature of 25 ℃ to obtain the low-purity sulforaphene with the purity of 90.5%.
(7) Preparation of high purity sulforaphene
According to the mass of the low-purity sulforaphene: the volume ratio of deionized water is 1 g: dissolving low-purity sulforaphene into deionized water according to the proportion of 15mL to obtain a low-purity sulforaphene solution; according to the low-purity sulforaphene solution: the volume ratio of the dichloromethane is 1: 1.5, pumping the low-purity sulforaphene solution and dichloromethane into a centrifugal extractor at the flow rate of 90mL/min respectively, and collecting a water phase and an oil phase respectively; pumping the oil phase into a vacuum concentrator, and concentrating under vacuum at 25 deg.C and 0.092MPa to obtain high-purity sulforaphene; the purity of the sulforaphene reaches 99.7 percent, and the total yield reaches 91.5 percent.
Example 3
A method for continuously and stably producing sulforaphene comprises the following specific steps:
(1) preparation of myrosinase pretreatment solution
Taking commercially available crude myrosinase as a raw material, and according to the mass of the crude myrosinase: the volume ratio of the buffer solution is 1 g: 150 mL, adding the crude myrosinase into the buffer solution, slowly stirring and uniformly mixing, and then putting the mixture into an ultrasonic processor for ultrasonic treatment for 10 min; pumping into a vacuum filter after the ultrasonic treatment is finished, carrying out vacuum filtration, and respectively collecting filtrate and filter residue; wherein the buffer solution is a sodium citrate-citric acid buffer solution with pH of 6.5.
(2) Preparation of myrosinase lyophilized powder
According to the myrosinase pretreatment solution: 30 percent of polyethylene glycol solution with molecular weight of 600-1000: the volume ratio of the ammonium sulfate solution with the mass fraction of 30 percent is 1: 4: 4, uniformly mixing the myrosinase pretreatment solution, the polyethylene glycol solution and the ammonium sulfate solution, pumping into a separation type tubular centrifuge, centrifuging at the rotating speed of 8000r/min, and respectively collecting a centrifugal light liquid and a centrifugal heavy liquid; pumping the collected heavy centrifugal liquid into a nano filter with the molecular weight cutoff of 600Da, performing primary nanofiltration separation under 0.35MPa until the volume of the nanofiltration trapped liquid is 20% of the original volume, and respectively collecting primary nanofiltration filtered liquid and primary nanofiltration trapped liquid; supplementing deionized water to the collected first nanofiltration trapped fluid to the original volume, carrying out second nanofiltration separation under the same condition until the volume of the second nanofiltration trapped fluid is reduced to 10% of the original volume, and respectively collecting second nanofiltration filtered fluid and second nanofiltration trapped fluid; and pre-freezing the collected second nanofiltration trapped fluid for 4h at-20 ℃, and then placing the second nanofiltration trapped fluid in a freeze dryer for freeze drying for 36h at 60Pa and-60 ℃ to prepare the myrosinase freeze-dried powder for next treatment.
(3) Preparation of immobilized myrosinase
The method comprises the following steps of taking commercially available crosslinked chitosan microspheres as raw materials, and according to the mass of the crosslinked chitosan microspheres: volume of diamine solution: the volume ratio of the glutaraldehyde solution with the mass fraction of 2.5% is 1 g: 30 mL of: 3.0 mL, firstly dispersing the crosslinked chitosan microspheres in a diamine solution, uniformly stirring, then adding a glutaraldehyde solution, and stirring and reacting for 1.5 hours at 60 ℃; and (3) filtering, collecting a filter cake, and according to the mass of the filter cake: the volume ratio of the ethanol solution with the volume fraction of 60 percent is 1 g: washing the filter cake with an ethanol solution at a ratio of 5mL to obtain the grafted diamine crosslinked chitosan microspheres; according to the mass of the myrosinase freeze-dried powder: the volume ratio of the trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH of 6.5 is 1 g: 60 mL, firstly adding the myrosinase freeze-dried powder into a buffer solution to obtain a myrosinase solution, and then according to the mass of the grafted diamine crosslinked chitosan microspheres: volume of myrosinase solution: the volume ratio of the malonaldehyde solution with the malonaldehyde mass concentration of 15% is 1 g: 80mL of: adding grafted diamine crosslinked chitosan microspheres into a myrosinase solution according to the proportion of 8mL, soaking for 3h, adding a malonaldehyde solution, carrying out immobilized enzyme reaction for 4h at the temperature of 40 ℃, carrying out suction filtration after the reaction is finished, respectively collecting suction filtration residues and suction filtration liquid, washing the suction filtration residues with deionized water 2 times the volume of the suction filtration residues, and respectively collecting washing residues and washing liquid; the collected washing slag, namely the prepared immobilized myrosinase, is stored for later use at the temperature of 4 ℃; wherein, 14.5 percent of 1, 10-decamethylene diamine.
(4) Preparation of crude glucoraphanin extract
Radish seed meal is used as a raw material, and the mass of the radish seed meal is as follows: the volume ratio of the 80% ethanol solution is 1 kg: continuously stirring and leaching for 7h at room temperature according to the proportion of 9L, adjusting the pH to 3 by using a citric acid solution with the mass fraction of 6%, pumping into a clarification type tubular centrifuge, centrifuging at the rotation speed of 20000r/min, and respectively collecting a centrifuged clear liquid and a sediment; pumping the collected centrifugal clear liquid into a reverse osmosis concentrator, performing first reverse osmosis concentration under 0.25MPa until the volume of the first reverse osmosis trapped liquid is reduced to 15% of the original volume, and respectively collecting first reverse osmosis permeate and first reverse osmosis trapped liquid; adding deionized water into the collected first reverse osmosis trapped fluid, supplementing the volume to the original volume, performing second reverse osmosis concentration under the same condition until the volume of the second reverse osmosis trapped fluid is reduced to 10% of the original volume, and respectively collecting a second reverse osmosis permeate and a second reverse osmosis trapped fluid; collecting the second reverse osmosis trapped fluid, pre-freezing at-20 deg.C for 4 hr, and freeze-drying in freeze drier at-60 Pa and-60 deg.C for 36 hr to obtain glucoraphanin crude extract for further treatment.
(5) Reactor for preparing reaction separation coupling immobilized enzyme
Loading the prepared immobilized myrosinase into a chromatographic column, and backflushing with deionized water with the same volume as that of the immobilized myrosinase to obtain an immobilized myrosinase reaction column; dispersing activated adsorption resin, namely SP750 resin, which is sold in the market into deionized water, assembling into an adsorption resin chromatographic column, and performing back flushing by using the deionized water with the same volume as the adsorption resin chromatographic column to obtain a sulforaphene purification column; and connecting the immobilized myrosinase reaction column with the sulforaphane purification column to assemble a reaction separation coupling immobilized enzyme reactor, and collecting for later use.
(6) Preparation of low purity sulforaphene
According to the mass of the crude glucoraphanin extract: the volume of the tris-hcl buffer solution at pH 6.5 was 1 g: dispersing the glucoraphanin crude extract into a buffer solution according to the proportion of 20 mL to obtain a glucoraphanin crude extract solution, and then carrying out reaction on the glucoraphanin crude extract solution according to an immobilized myrosinase reaction column: the volume ratio of the glucoraphanin crude extract solution is 1: 8, pumping the glucoraphanin solution into the immobilized myrosinase reaction column at the speed of 6-10 times/h of the volume of the immobilized myrosinase reaction column at the flow speed, and carrying out continuous reaction and adsorption; after the reaction and the adsorption are finished, disconnecting the outflow end of the immobilized myrosinase reaction column from the sulforaphene purification column adsorbing sulforaphene; then purifying the obtained product by a sulforaphene purification column: the volume ratio of the ethanol solution with the volume fraction of 60% is 1: 3, taking the ethanol solution as eluent, pumping the eluent into the sulforaphene purification column adsorbing the sulforaphene, and eluting and separating the sulforaphene, wherein the flow rate of the eluent is 4 times/h of the volume of the sulforaphene purification column; pumping the collected eluent into a vacuum film evaporator, and performing vacuum film evaporation at the vacuum degree of 0.095MPa and the temperature of 30 ℃ to obtain the low-purity sulforaphene with the purity of 90.5%.
(7) Preparation of high purity sulforaphene
According to the mass of the low-purity sulforaphene: the proportion of the volume of the deionized water is 1 g: dissolving the low-purity sulforaphene into deionized water according to the proportion of 20 mL to obtain a low-purity sulforaphene solution; according to the low-purity sulforaphene solution: the volume ratio of the dichloromethane is 1: 2, pumping the low-purity sulforaphene solution and dichloromethane into a centrifugal extractor at a flow rate of 100mL/min respectively, and collecting a water phase and an oil phase respectively; pumping the oil phase into a vacuum concentrator, and vacuum concentrating at 30 deg.C under vacuum degree of 0.095MPa to obtain high-purity sulforaphene; the purity of the sulforaphene reaches 99.0 percent, and the total yield reaches 91.2 percent.
Claims (1)
1. A method for continuously and stably producing high-purity sulforaphene is characterized by comprising the following specific steps:
(1) preparation of myrosinase pretreatment solution
The method takes commercially available crude myrosinase as a raw material, and comprises the following steps of: the volume ratio of the buffer solution is 1 g: adding the crude myrosinase into a buffer solution according to the proportion of 60-150 mL, slowly stirring and uniformly mixing, and then putting into an ultrasonic processor for ultrasonic treatment for 10-15 min; pumping into a vacuum filter after the ultrasonic treatment is finished, carrying out vacuum filtration, and respectively collecting filtrate and filter residue; wherein the buffer solution is a trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH 6.5, a sodium dihydrogen phosphate-citric acid buffer solution with pH 6.5, or a sodium citrate-citric acid buffer solution with pH 6.5;
(2) preparation of myrosinase lyophilized powder
According to the myrosinase pretreatment solution: 20-30% of polyethylene glycol solution with the molecular weight of 600-1000: the volume ratio of the ammonium sulfate solution with the mass fraction of 15-30% is 1: 2-4: 2-4, uniformly mixing the myrosinase pretreatment solution, the polyethylene glycol solution and the ammonium sulfate solution, pumping into a separation type tubular centrifuge, centrifuging at the rotating speed of 6000-8000 r/min, and respectively collecting a centrifugal light solution and a centrifugal heavy solution; pumping the collected heavy centrifugal liquid into a nano filter with the molecular weight cutoff of 300-600 Da, performing primary nanofiltration separation under 0.25-0.35 MPa until the volume of the nanofiltration trapped liquid is 10-20% of the original volume, and respectively collecting a primary nanofiltration filtered liquid and a primary nanofiltration trapped liquid; supplementing deionized water to the original volume of the collected first nanofiltration trapped fluid, carrying out second nanofiltration separation under the same condition until the volume of the second nanofiltration trapped fluid is reduced to 5-10% of the original volume, and respectively collecting second nanofiltration filtered fluid and second nanofiltration trapped fluid; pre-freezing the collected second nanofiltration trapped fluid for 2-4 h at-16 to-20 ℃, then placing the second nanofiltration trapped fluid in a freeze dryer, and carrying out freeze drying for 24-36 h at 30-60 Pa and-50 to-60 ℃ to prepare myrosinase freeze-dried powder for next treatment;
(3) preparation of immobilized myrosinase
The method comprises the following steps of taking commercially available crosslinked chitosan microspheres as raw materials, and according to the mass of the crosslinked chitosan microspheres: volume of diamine solution: the volume ratio of 1.0-2.5% glutaraldehyde solution is 1 g: 12-30 mL: 1.5-3.0 mL, dispersing the crosslinked chitosan microspheres in a diamine solution, uniformly stirring, adding a glutaraldehyde solution, and stirring and reacting at 60 ℃ for 1-1.5 hours; and (3) filtering and collecting a filter cake, and according to the mass of the filter cake: the volume ratio of 40-60% ethanol solution is 1 g: washing the filter cake with an ethanol solution in a ratio of 3-5 mL to obtain the grafted diamine crosslinked chitosan microsphere; according to the mass of the myrosinase freeze-dried powder: the volume ratio of the trihydroxymethylaminomethane-hydrochloric acid buffer solution with pH 6.5 is 1 g: adding the myrosinase freeze-dried powder into a buffer solution according to the proportion of 40-60 mL to obtain a myrosinase solution, and then according to the mass of grafted diamine crosslinked chitosan microspheres: volume of myrosinase solution: the volume ratio of the malonaldehyde solution with the malonaldehyde mass concentration of 15% is 1 g: 50-80 mL: adding grafted diamine crosslinked chitosan microspheres into a myrosinase solution according to the proportion of 4-8 mL, soaking for 1-3 h, adding a malonaldehyde solution, carrying out immobilized enzyme reaction for 2-4 h at the temperature of 30-40 ℃, carrying out suction filtration after the reaction is finished, respectively collecting suction filtration residues and suction filtration liquid, washing the suction filtration residues with deionized water of which the volume is 2 times that of the suction filtration residues, and respectively collecting washing residues and washing liquid; the collected washing slag, namely the prepared immobilized myrosinase, is stored for later use at the temperature of 4 ℃; wherein the diamine solution is 7.5 percent of 1, 2-ethylenediamine, 10 percent of 1, 6-hexanediamine or 14.5 percent of 1, 10-decanediamine by mass fraction;
(4) preparation of crude glucoraphanin extract
Radish seed meal is used as a raw material, and the mass of the radish seed meal is as follows: the volume fraction of 50-80% ethanol solution is 1 kg: continuously stirring and leaching for 4-7 hours at room temperature according to the proportion of 6-9L, adjusting the pH to 2-3 by using a citric acid solution with the mass fraction of 2-6%, then pumping into a clarified tubular centrifuge, centrifuging at the rotation speed of 15000-20000 r/min, and respectively collecting a centrifuged clear liquid and a sediment; pumping the collected centrifugal clear liquid into a reverse osmosis concentrator, performing first reverse osmosis concentration under the pressure of 0.15-0.25 MPa until the volume of the first reverse osmosis trapped liquid is reduced to 10-15% of the original volume, and respectively collecting first reverse osmosis permeate and first reverse osmosis trapped liquid; adding deionized water into the collected first reverse osmosis trapped fluid, supplementing the volume to the original volume, performing second reverse osmosis concentration under the same condition until the volume of the second reverse osmosis trapped fluid is reduced to 5-10% of the original volume, and respectively collecting a second reverse osmosis permeate and a second reverse osmosis trapped fluid; pre-freezing the collected second reverse osmosis trapped fluid at-16 to-20 ℃ for 2-4 h, then placing the pre-frozen trapped fluid in a freeze dryer, and carrying out freeze drying for 24-36 h at 30-60 Pa and-50 to-60 ℃ to prepare a glucoraphanin crude extract for next treatment;
(5) reactor for preparing reaction separation coupling immobilized enzyme
Loading the prepared immobilized myrosinase into a chromatographic column, and backflushing with deionized water with the same volume as that of the immobilized myrosinase to obtain an immobilized myrosinase reaction column; dispersing commercially activated adsorbent resin, namely SP207 resin or D101 resin or SP750 resin, in deionized water, assembling into an adsorbent resin chromatographic column, and back-flushing with deionized water with the same volume as the adsorbent resin chromatographic column to obtain a sulforaphene purification column; connecting the immobilized myrosinase reaction column with a sulforaphene purification column to assemble a reaction separation coupling immobilized enzyme reactor, and collecting for later use;
(6) preparation of low purity sulforaphene
According to the mass of the crude glucoraphanin extract: the volume of the tris-hcl buffer solution at pH 6.5 was 1 g: dispersing the glucoraphanin crude extract into a buffer solution according to the proportion of 15-20 mL to obtain a glucoraphanin crude extract solution, and then carrying out reaction on the glucoraphanin crude extract solution according to an immobilized myrosinase reaction column: the volume ratio of the glucoraphanin crude extract solution is 1: 5-8, pumping the glucoraphanin solution into the immobilized myrosinase reaction column at the speed of 6-10 times of the volume of the immobilized myrosinase reaction column per hour of the flow speed, and carrying out continuous reaction and adsorption; after the reaction and the adsorption are finished, disconnecting the outflow end of the immobilized myrosinase reaction column from the sulforaphene purification column adsorbing sulforaphene; then purifying with a sulforaphene purification column: the volume ratio of the ethanol solution with the volume fraction of 40-60% is 1: 1-3, taking the ethanol solution as an eluent, and pumping the eluent into a sulforaphene purification column adsorbing sulforaphene at a flow rate of 2-4 times/hour of the volume of the sulforaphene purification column to perform sulforaphene elution separation; pumping the collected eluent into a vacuum film evaporator, and performing vacuum film evaporation under the conditions that the vacuum degree is 0.09-0.095 MPa and the temperature is 20-30 ℃ to obtain the low-purity sulforaphene, wherein the purity of the sulforaphene reaches 88.2-90.5%;
(7) preparation of high purity sulforaphene
According to the mass of the low-purity sulforaphene: the volume ratio of deionized water is 1 g: dissolving low-purity sulforaphene into deionized water at a ratio of 10-20 mL to obtain a low-purity sulforaphene solution; according to the low-purity sulforaphene solution: the volume ratio of the dichloromethane is 1: 1-2, pumping the low-purity sulforaphene solution and dichloromethane into a centrifugal extractor at a flow rate of 80-100 mL/min respectively, and collecting a water phase and an oil phase respectively; pumping the oil phase into a vacuum concentrator, and carrying out vacuum concentration under the conditions that the vacuum degree is 0.09-0.095 MPa and the temperature is 20-30 ℃ to obtain high-purity sulforaphene; the purity of the sulforaphene is 98.6-99.7%, and the total yield is 90.7-91.5%.
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