CN107988280B - Method for extracting high-purity isothiocyanate from cruciferous vegetable seeds - Google Patents
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Abstract
A process for extracting high-purity isothiocyanic ester from the seeds of cruciferous vegetables includes such steps as providing fresh seeds of cruciferous vegetables, adding water, grinding to obtain pulp, fermenting, then advanced extraction technologies and processes such as ethyl acetate extraction, nano-membrane primary purification, polyamide resin purification, HP2MGL resin secondary purification, crystallization and the like are sequentially adopted, the technical breakthrough of factory large-scale production of high-purity isothiocyanate products is obtained, the high-purity isothiocyanate extract is obtained, high-quality raw materials are provided for research of pharmacological efficacy and functional terminal product development, and the safe, simple, convenient, reasonable and economic high-value utilization of the cruciferous vegetable seed resources is really realized, and the concept of green chemical production and the strategy of circular economy are fully embodied.
Description
Technical Field
The invention relates to a method for extracting high-purity isothiocyanate products from cruciferous vegetable seeds, in particular to a method for preparing the high-purity isothiocyanate products by adding water into raw materials, grinding the raw materials into slurry, fermenting the slurry, and then sequentially performing extraction processes such as ethyl acetate extraction, nano-film primary purification, polyamide resin purification, HP2MGL resin secondary purification, crystallization and the like.
Background
Glucosinolates, an important class of secondary metabolites in plants. Isothiocyanate is the enzymolysis product of glucosinolate widely and abundantly existing in cruciferous vegetable plants under the action of myrosinase. Research shows that the content of glucosinolate and myrosinase in mature vegetable seeds such as mustard and broccoli is high, however, the glucosinolate and the myrosinase exist independently in normal vegetable tissue, and enzymatic degradation reaction can be carried out to generate isothiocyanate only when vegetable tissue cells are damaged and the glucosinolate and the myrosinase are contacted with each other. The isothiocyanate has pungent and pungent taste, is a common seasoning for Korean cuisine, Japanese cuisine, uncooked seafood, cold vegetable dish, horse meat and the like, is also an effective component of horseradish, mustard and products thereof, and the content of the isothiocyanate is one of main quality indexes of the products. With the increasing attention of people to health care, health preservation and the like, the focus of attention in the food field is on how to utilize natural bioactive ingredients to prevent sub-health under modern dietary structure and environmental conditions. Research shows that the isothiocyanate has various biological activities of bacteriostasis, sterilization, platelet aggregation inhibition, tumor prevention and treatment and the like, so that the isothiocyanate extract and functional products thereof become healthy new pets of numerous consumers at home and abroad in recent years, and have huge market potential.
At present, the extraction method of isothiocyanate mainly comprises a squeezing method, a steam distillation method, an ultrasonic or microwave assisted extraction method, an enzymatic assisted extraction method and the like, and the separation and purification method mainly comprises a solvent extraction method, a supercritical fluid extraction method, a macroporous resin adsorption method, a low-pressure silica gel column chromatography method, a high-speed counter-current chromatography method and the like, but the methods are mostly limited to experimental research on the effect of extracting or separating and purifying isothiocyanate by a single technology and process, and meanwhile, because the content of glucosinolate in plant tissues and organs (including seeds) of cruciferous vegetables is generally less than 1% compared with the substance components of the glucosides, and the differences exist due to different vegetable varieties, growth environments, planting technologies and picking parts, the technical bottlenecks of low yield, low purity, large consumption of processing raw materials and the like exist in the process of extracting and preparing isothiocyanate, for example, in the chinese published patent application CN1405316A, the crude extract of isothiocyanate extracted and prepared cannot meet the quality requirement of development of functional end products, and the market demand of the public on isothiocyanate functional end products, it is a urgent task in this field to establish a method suitable for industrial scale production of high purity isothiocyanate.
Therefore, the invention takes fresh mature mustard seeds or broccoli seeds with rich resources and higher glucosinolate content as raw materials, firstly adds water into the raw materials, grinds the raw materials into pulp, performs fermentation treatment, then innovatively integrates advanced technologies and processes such as ethyl acetate extraction, nano-membrane primary purification, polyamide resin purification, HP2MGL resin secondary purification, crystallization and the like, obtains the technical breakthrough of factory large-scale production of high-purity isothiocyanate products, creates a method for obtaining the high-purity isothiocyanate extract, provides high-quality raw materials for research of pharmacological efficacy and development of functional terminal products, truly realizes high-value utilization of safe, simple, convenient, reasonable and economic cruciferous vegetable seed resources, and fully embodies the concept of green chemical production and the strategy of circular economy.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the technology, the method for extracting the high-purity isothiocyanate from the cruciferous vegetable seeds is green, efficient and suitable for industrial production, so that the isothiocyanate in the vegetable seeds can be extracted to the maximum extent, and the yield and the purity of the isothiocyanate can be obviously improved.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for extracting high-purity isothiocyanate from cruciferous vegetable seeds comprises the following steps:
a. raw material treatment: taking fresh mature cruciferous vegetable seeds, adding deionized water, grinding into slurry, and then grinding the slurry by using a weight ratio of 2: adjusting the pH value of the mixture of the citric acid and the Vc of 1 to 5.5-6.5, and carrying out sealed fermentation at the temperature of 50-60 ℃ for 16-20 h to obtain fermentation slurry;
the mixture of citric acid and Vc is added in the raw material treatment, on one hand, the pH value of fermentation liquor is adjusted by citric acid, and on the other hand, the efficiency of producing isothiocyanate by carrying out enzymolysis on glucosinolate is improved by Vc; wherein, the formula (0.5: 1, 1:1, 1.5:1, 2.0:1, 2.5: 1) of the citric acid and the Vc and the optimized technological parameters of the pH value (5.0, 5.5, 6.0, 6.5, 7.0), the temperature (30, 40, 50, 60, 70 ℃) and the time (8, 12, 16, 20, 24 h) during the fermentation are obtained by the experimental optimization screening of a response surface method.
b. And (3) ethyl acetate extraction: adding ethyl acetate with the volume of 1-1.4 times of that of the fermentation slurry, shaking for 25-35 min, standing for extraction at the temperature of 20-30 ℃ for 2 times under the time of 50-70 min, combining ethyl acetate extract, carrying out reduced pressure concentration, recovering ethyl acetate, and carrying out vacuum freeze drying to obtain an isothiocyanate crude extract;
c. primary purification of the nano film: preparing the obtained crude isothiocyanate extract into a solution with the mass concentration of 1.5-2.5% by using deionized water, performing nanofiltration separation by using a nano-membrane with the molecular weight cutoff of 2000Da, adding deionized water with the weight of 10-20% of the weight of the pre-filtration solution for dialysis once when the weight of the filtrate is 80-90% of the weight of the pre-filtration solution, collecting the filtrate and the dialysate, combining the filtrate and the dialysate to obtain a nanofiltration separation solution, performing nanofiltration concentration by using a nano-membrane with the molecular weight cutoff of 300Da, and collecting a nanofiltration concentrated solution;
d. and (3) purifying the polyamide resin: adding deionized water into the nanofiltration concentrated solution to prepare a solution with the mass concentration of 1.5-2.5%, separating and purifying isothiocyanate through a polyamide resin chromatographic column at room temperature to obtain an eluent, concentrating the eluent under reduced pressure until the content of solid matters is 25-35%, recovering edible ethanol, and collecting the reduced-pressure concentrated solution;
e. HP2MGL resin repurification: and adding deionized water into the obtained reduced-pressure concentrated solution to prepare a solution with the mass concentration of 1.5-2.5%, separating and purifying isothiocyanate through an HP2MGL resin chromatographic column at room temperature, collecting eluent, carrying out reduced-pressure concentration, recovering edible ethanol, filtering the concentrated solution while the concentrated solution is hot, crystallizing the filtrate, standing overnight, and finally carrying out vacuum freeze drying to obtain the high-purity isothiocyanate extract.
In the step a, the cruciferous vegetable seeds are selected from mustard seeds or broccoli seeds, and the addition amount of the deionized water is 0.8-1.2 times of the weight of the cruciferous vegetable seeds.
And c, performing nanofiltration separation at the temperature of 35-40 ℃ and under the pressure of 2.4-2.8 MPa in the step c, and performing nanofiltration concentration at the temperature of 35-40 ℃ and under the pressure of 2.8-3.2 MPa.
And d, separating and purifying the isothiocyanate through the polyamide resin chromatographic column in the step d, namely enabling a solution with the mass concentration of 1.5-2.5% and the volume of 7-9 BV to pass through the polyamide resin chromatographic column at the flow rate of 2.0-3.0 BV/h, adsorbing the resin to be saturated, standing for 30-50 min, eluting with edible ethanol with the mass concentration of 3.0-4.0 BV of 15-25% at the flow rate of 1.5-2.5 BV/h to remove impurities, eluting with edible ethanol with the mass concentration of 4-6 BV of 60-80% at the flow rate of 1.0-2.0 BV/h to elute the isothiocyanate, and collecting eluent.
And e, separating and purifying the isothiocyanate through the HP2MGL resin chromatographic column in the step e, namely enabling 5-7 BV of a solution with the mass concentration of 1.5-2.5% to pass through the HP2MGL resin chromatographic column at the flow rate of 1.5-2.5 BV/h, enabling the resin to be adsorbed to be saturated, standing for 30-50 min, then eluting with 2.5-3.5 BV of edible ethanol with the mass concentration of 25-35% at the flow rate of 1.0-2.0 BV/h to remove impurities, eluting with 3-5 BV of edible ethanol with the volume concentration of 65-75% at the flow rate of 0.8-1.2 BV/h to elute the isothiocyanate, and collecting eluent.
The vacuum degree of the decompression concentration in the steps b, d and e is-0.05 to-0.09 MPa, and the temperature is 50 to 60 ℃. The vacuum degree of vacuum freeze drying in the step b and the step e is-0.05 to-0.09 MPa, and the temperature is-38 to-42 ℃.
And e, crystallizing the filtrate in the step e at the temperature of 0-4 ℃.
The technological parameters of the ethyl acetate extraction, the nano-film primary purification, the polyamide resin purification and the HP2MGL resin secondary purification are optimized and screened by a single-factor experiment, an orthogonal experiment and an optimal combination verification experiment. Wherein:
1) the ethyl acetate extraction is based on the principle that isothiocyanate and ethyl acetate are similar in molecular polarity and easy to dissolve in ethyl acetate, and the isothiocyanate substances are conveniently, simply and maximally extracted from the raw material fermentation slurry by utilizing the ethyl acetate, and other substances which are insoluble in the ethyl acetate are separated out; the optimization process technical parameter takes the yield of the isothiocyanate as a research index, and the optimization screening is realized through orthogonal test and optimal combined verification experiment on the basis of researching and analyzing the usage amount (1: 1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1: 2.0) of ethyl acetate of raw material slurry in unit volume, shaking time (10, 20, 30, 40, 50, 60 min), standing extraction time (15, 30, 45, 60, 75, 90 min), extraction temperature (4, 20, 30, 40, 50, 60 ℃), extraction frequency (1, 2, 3 times) and other single factors on the extraction effect of the isothiocyanate.
2) The primary purification of the nano-film is based on the molecular weight of isothiocyanate substances, the nano-film with proper film aperture and the technical parameters of nanofiltration are selected by utilizing the molecular sieve effect of the nano-film, other substances with molecular weight larger or smaller than that of the isothiocyanate substances are separated out to the maximum extent by nanofiltration separation or nanofiltration concentration respectively, and meanwhile, the yield and the purity of the isothiocyanate are improved to the maximum extent, and excellent raw material substrates are provided for the subsequent separation and purification of high-purity isothiocyanate products by resin; the optimization process technical parameters take yield and purity of isothiocyanate as investigation indexes, and optimization screening is carried out through orthogonal test and optimal combined verification experiment on the basis of researching and analyzing single-factor extraction effect of feed liquid mass concentration (0.5, 1.5, 2.5 and 3.5%), molecular weight cut-off of nano separation membrane (1000, 2000, 2500 and 3500 Da), nano filtration separation temperature (30, 35, 40 and 45 ℃), nano filtration separation pressure (2.0, 2.4, 2.8 and 3.2 MPa), dialysis amount (10, 20, 30 and 40%) and dialysis frequency (1 and 2) and molecular weight cut-off of nano concentration membrane (reverse osmosis membrane, 200 and 300 Da), nano filtration concentration temperature (30, 35, 40 and 45 ℃), nano filtration separation pressure (2.4, 2.8, 3.2 and 3.6 MPa), dialysis amount (10, 20, 30 and 40%) and dialysis frequency (1 and the like.
3) The optimized technological parameters of polyamide resin purification are that on the basis of comparing and researching the static adsorption and desorption rates of 10 resins such as D101, ADS-7, AB-8, NKA-9, HPD-400, HZ16, HZ816, HZ906, HZ806, polyamide and the like on isothiocyanate, the polyamide resin with highest isothiocyanate yield is preferably selected, then the yield and purity of isothiocyanate are used as research indexes, in the research and analysis of the mass concentration, the sample volume and the sample flow rate of the sample loading material liquid, the standing time for leaching and removing impurities, the mass concentration of ethanol, the leaching volume and the leaching flow rate, and on the basis of the separation and purification effects of isothiocyanate on single factors such as mass concentration of isothiocyanate elution ethanol, elution volume, elution flow rate and the like, optimizing and screening technological parameters of dynamic separation and purification of isothiocyanate of optimal resin polyamide through an orthogonal test and an optimal combination verification test;
4) the reason why the HP2MGL resin is re-purified is a process technology which is necessary to re-purify the isothiocyanate obtained after the polyamide resin is purified in order to obtain the isothiocyanate with the purity of more than 95 percent, is that although the molecular polarities of the HP2MGL resin and the isothiocyanate are more similar to those of the polyamide resin, researches show that the nanofiltration concentrated solution contains more other impurities, the HP2MGL resin is only used for once purification, and the isothiocyanate with the purity of more than 95 percent cannot be obtained, and the HP2MGL resin is used for re-purifying the isothiocyanate obtained after the polyamide resin is purified, so that the isothiocyanate with the purity of more than 95 percent can be obtained; the optimized technological parameters are that on the basis of comparing and researching the static adsorption and desorption rates of 8 resins such as LS300, LS300B, LS303, LS306, DM11, HZ 832, HP-20, HP2MGL and the like on isothiocyanate, the HP2MGL resin with the highest isothiocyanate yield is preferred; and then, taking the yield and the purity of the isothiocyanate as investigation indexes, and optimizing and screening the technological parameters of the dynamic separation and purification of the isothiocyanate by using the optimal resin HP2MGL through an orthogonal test and an optimal combined verification test on the basis of researching and analyzing the standing time, the ethanol mass concentration, the elution volume and the elution flow rate of the sample liquid for removing impurities, and the separation and purification effects of the isothiocyanate by using single factors such as the mass concentration of the sample liquid for eluting, the ethanol mass concentration for eluting, the elution volume and the elution flow rate of the isothiocyanate for eluting and removing the impurities.
Compared with the prior art, the invention has the advantages that:
1. according to the invention, mustard seeds or green Chinese flowering rape seeds which have large resource quantity, high glucosinolate and endogenous myrosinase content but are usually discarded as a byproduct of vegetable production and consumption are taken as raw materials, according to a biochemical mechanism of isothiocyanate generated by glucosinolate enzymolysis, the effect of endogenous myrosinase is fully exerted by adding a citric acid and Vc formula mixture and optimizing fermentation process technical parameters in raw material slurry, enzymatic reaction is stimulated to the maximum extent, the efficiency of isothiocyanate generated by enzymolysis is obviously improved, and the problems of increased processing cost, safe exogenous material addition, lower enzymolysis efficiency and the like in fermentation by utilizing exogenous myrosinase are avoided.
2. According to the invention, advanced technologies and processes such as ethyl acetate extraction, nano-membrane primary purification, polyamide resin purification, HP2MGL resin secondary purification, crystallization and the like are innovatively integrated, so that the purity of isothiocyanate is gradually improved while the yield of isothiocyanate in the separation and purification process is ensured to the maximum extent, and a high-purity isothiocyanate product with purity of more than 95% is finally obtained; meanwhile, the single process technology used by the invention is mature and common technology suitable for factory large-scale production, and if the technology is applied according to the complete process flow and technical parameters of the invention, the breakthrough of factory large-scale production of isothiocyanate high-purity products with purity of more than 95% can be realized, and the problems of low yield, low purity, large consumption of processing raw materials and the like of the traditional production mode are solved.
3. The invention is popularized and applied, can greatly improve the high-valued utilization efficiency of vegetable resources such as mustard seeds or broccoli seeds and the like, meets the quality requirement of development of the functional terminal products of the isothiocyanate and meets the market demand of the general public on the functional terminal products of the isothiocyanate.
Detailed Description
Example 1
Taking fresh mature mustard seeds, adding deionized water with the weight of 0.8 time, grinding into slurry, then adjusting the pH to 5.5 by using a mixture of citric acid and Vc with the mass ratio of 2:1, sealing by using a plastic film, fermenting for 20 hours at 50 ℃ to obtain fermented slurry, adding ethyl acetate with the volume of 1 time into the fermented slurry, extracting for 2 times under the conditions of shaking for 25 min, standing for 20 ℃ and standing for extraction for 70min, combining extract liquor, carrying out reduced pressure concentration at-0.09 MPa and 50 ℃, recovering ethyl acetate, and carrying out vacuum freeze drying on concentrated solution at-0.05 MPa and-42 ℃ to obtain an isothiocyanate crude extract; preparing the crude extract into a solution with the mass concentration of 1.5% by using deionized water, performing nanofiltration separation on a nano-film with the molecular weight cutoff of 2000Da at the temperature of 35 ℃ and under the pressure of 2.8MPa, adding deionized water with the weight of 10% of the weight of the solution before filtration for dialysis for 1 time when the weight of the filtrate is 90% of the weight of the solution before filtration, combining the filtrate and dialysate, performing nanofiltration concentration on the filtrate by using a nano-film with the molecular weight cutoff of 300Da at the temperature of 35 ℃ and under the pressure of 3.2MPa, and collecting a nanofiltration concentrated solution; adding deionized water into the nanofiltration concentrated solution to prepare a solution with the mass concentration of 1.5%, passing 7BV of the solution through a polyamide resin chromatographic column at the flow rate of 3.0BV/h at room temperature to adsorb the resin to saturation, standing for 50min, leaching with 3.0BV of edible ethanol with the mass concentration of 15% at the flow rate of 2.5BV/h to remove impurities, eluting isothiocyanate with 4BV of edible ethanol with the mass concentration of 60% at the flow rate of 2.0BV/h, collecting eluent, concentrating under reduced pressure at-0.05 MPa and 60 ℃ until the solid content is 25.0%, recovering the edible ethanol, and collecting the reduced-pressure concentrated solution; and adding deionized water into the reduced pressure concentrated solution to prepare a solution with the mass concentration of 1.5%, passing 5BV of the solution through an HP2MGL resin chromatographic column at the flow rate of 1.5BV/h at room temperature to adsorb the resin to saturation, standing for 30min, leaching with 2.5BV of edible ethanol with the mass concentration of 25% at the flow rate of 2.0BV/h to remove impurities, eluting the isothiocyanate with 3BV of 65% at the flow rate of 1.2BV/h, collecting the eluent, carrying out reduced pressure concentration at-0.05 MPa and 60 ℃, recovering the edible ethanol, filtering the concentrated solution while hot, crystallizing the filtrate, standing overnight, and finally carrying out vacuum freeze drying at-0.05 MPa and-42 ℃ to obtain the high-purity isothiocyanate extract.
The detection shows that the yield of the extract is 73.46 percent of the total amount of the isothiocyanate in the fermentation slurry, and the purity reaches 98.12 percent.
Example 2
Adding 1.2 times of deionized water into fresh mature mustard seeds, grinding the mixture into slurry, adjusting the pH value to 6.5 by using a mixture of citric acid and Vc with the mass ratio of 2:1, sealing the mixture by using a plastic film, fermenting the mixture for 16 hours at 60 ℃ to obtain fermented slurry, adding 1.4 times of ethyl acetate into the fermented slurry, extracting the mixture for 2 times at the temperature of 30 ℃ and the shaking time for 35min, standing and extracting the mixture for 50min, combining extract liquor, carrying out reduced pressure concentration at the temperature of-0.05 MPa and 60 ℃, recovering the ethyl acetate, and carrying out vacuum freeze drying on the concentrated solution at the temperature of-0.09 MPa and-38 ℃ to obtain an isothiocyanate crude extract; preparing the crude extract into a solution with the mass concentration of 2.5% by using deionized water, performing nanofiltration separation by using a nano-membrane with the molecular weight cutoff of 2000Da at the conditions of 40 ℃ and 2.4MPa, adding deionized water with the weight of 20% of the weight of the solution before filtration for dialysis for 1 time when the weight of the filtrate is 80% of the weight of the solution before filtration, combining the filtrate and dialysate, performing nanofiltration concentration by using a nano-membrane with the molecular weight cutoff of 300Da at the conditions of 40 ℃ and 2.8MPa, and collecting the nanofiltration concentrated solution; preparing a nanofiltration concentrated solution into a solution with the mass concentration of 2.5%, passing 9BV of the solution through a polyamide resin chromatographic column at the flow rate of 2.0BV/h at room temperature to adsorb the resin to saturation, standing for 30min, leaching with 4.0BV of edible ethanol with the mass concentration of 20% at the flow rate of 1.5BV/h to remove impurities, eluting isothiocyanate with 6BV of edible ethanol with the mass concentration of 80% at the flow rate of 1.0BV/h, collecting eluent, concentrating under reduced pressure at-0.09 MPa and 50 ℃ until the solid content is 35.0%, recovering the edible ethanol, and collecting a reduced-pressure concentrated solution; and adding deionized water into the reduced pressure concentrated solution to prepare a solution with the mass concentration of 2.5%, passing 7BV of the solution through an HP2MGL resin chromatographic column at the flow rate of 2.5BV/h at room temperature, adsorbing the resin to saturation, standing for 50min, leaching with 3.5BV of edible ethanol with the mass concentration of 35% at the flow rate of 1.0BV/h to remove impurities, eluting the isothiocyanate with 5BV of edible ethanol with the mass concentration of 75% at the flow rate of 0.8BV/h, collecting the eluent, carrying out reduced pressure concentration at-0.09 MPa and 50 ℃, recovering the edible ethanol, filtering the concentrated solution while hot, crystallizing the filtrate, standing overnight, and finally carrying out vacuum freeze drying at-0.09 MPa and-38 ℃ to obtain the high-purity isothiocyanate extract.
The detection shows that the yield of the extract is 77.65% of the total amount of isothiocyanate in the fermentation slurry, and the purity reaches 95.76%.
Example 3
Taking fresh mature broccoli seeds, adding 1.0 time of deionized water by weight, grinding into slurry, then adjusting the pH value to 6.0 by using a mixture of citric acid and Vc in a mass ratio of 2:1, sealing by using a plastic film, and fermenting at 55 ℃ for 18 hours to obtain fermentation slurry; adding ethyl acetate 1.2 times the volume of the fermented slurry into the fermented slurry, shaking for 30min, standing at 25 deg.C for 2 times, standing for 60min, extracting, mixing extractive solutions, concentrating under reduced pressure at-0.07 MPa and 55 deg.C, recovering ethyl acetate, and vacuum freeze drying the concentrated solution at-0.07 MPa and-40 deg.C to obtain crude isothiocyanate extract; preparing the crude extract into a solution with the mass concentration of 2.0% by using deionized water, performing nanofiltration separation by using a nano-membrane with the molecular weight cutoff of 2000Da at 37.5 ℃ under the condition of 2.6MPa, adding deionized water with the weight of 15% of the weight of the solution before filtration for dialysis for 1 time when the weight of the filtrate is 85% of that of the solution before filtration, combining the filtrate and the dialysate, performing nanofiltration concentration by using a nano-membrane with the molecular weight cutoff of 300Da at 37.5 ℃ under the condition of 3.0MPa, and collecting nanofiltration concentrated solution; preparing a nanofiltration concentrated solution into a solution with the mass concentration of 2.0% by using deionized water, passing 8BV of the solution through a polyamide resin chromatographic column at the flow rate of 2.5BV/h at room temperature to adsorb the resin to saturation, standing for 40min, leaching with 3.5BV of edible ethanol with the mass concentration of 20% at the flow rate of 2.0BV/h to remove impurities, eluting isothiocyanate with 5BV of edible ethanol with the mass concentration of 70% at the flow rate of 1.5BV/h, collecting eluent, concentrating under reduced pressure at-0.07 MPa and 55 ℃ until the solid content is 30%, recovering the edible ethanol, and collecting a reduced-pressure concentrated solution; preparing the reduced pressure concentrated solution into a solution with the mass concentration of 2.0% by using deionized water, passing 6BV of the solution through an HP2MGL resin chromatographic column at the flow rate of 2.0BV/h at room temperature, adsorbing the resin to saturation, standing for 40min, leaching with 3.0BV of edible ethanol with the mass concentration of 30% at the flow rate of 1.5BV/h to remove impurities, eluting isothiocyanate with 4BV of edible ethanol with the mass concentration of 70% at the flow rate of 1.0BV/h, collecting eluent, carrying out reduced pressure concentration at-0.07 MPa and 55 ℃, recovering the edible ethanol, filtering the hot concentrated solution, crystallizing filtrate, standing overnight, and finally carrying out vacuum freeze drying at-0.07 MPa and-40 ℃ to obtain the high-purity isothiocyanate extract.
The detection shows that the yield of the extract is 75.67 percent of the total amount of the isothiocyanate in the fermentation slurry, and the purity reaches 97.84 percent.
Claims (10)
1. A method for extracting high-purity isothiocyanate from cruciferous vegetable seeds is characterized by comprising the following steps:
a. raw material treatment: taking fresh mature cruciferous vegetable seeds, adding deionized water, grinding into slurry, and then grinding the slurry by using a weight ratio of 2: adjusting the pH value of the mixture of the citric acid and the Vc of 1 to 5.5-6.5, and carrying out sealed fermentation at the temperature of 50-60 ℃ for 16-20 h to obtain fermentation slurry;
b. and (3) ethyl acetate extraction: adding ethyl acetate with the volume of 1-1.4 times of that of the fermentation slurry, shaking for 25-35 min, standing for extraction at the temperature of 20-30 ℃ for 2 times under the time of 50-70 min, combining ethyl acetate extract, carrying out reduced pressure concentration, recovering ethyl acetate, and carrying out vacuum freeze drying to obtain an isothiocyanate crude extract;
c. primary purification of the nano film: preparing the obtained crude isothiocyanate extract into a solution with the mass concentration of 1.5-2.5% by using deionized water, performing nanofiltration separation by using a nano-membrane with the molecular weight cutoff of 2000Da, adding deionized water with the weight of 10-20% of the weight of the pre-filtration solution for dialysis once when the weight of the filtrate is 80-90% of the weight of the pre-filtration solution, collecting the filtrate and the dialysate, combining the filtrate and the dialysate to obtain a nanofiltration separation solution, performing nanofiltration concentration by using a nano-membrane with the molecular weight cutoff of 300Da, and collecting a nanofiltration concentrated solution;
d. and (3) purifying the polyamide resin: preparing the nanofiltration concentrated solution into a solution with the mass concentration of 1.5-2.5%, separating and purifying isothiocyanate through a polyamide resin chromatographic column at room temperature to obtain an eluent, concentrating the eluent under reduced pressure until the content of solid matters is 25-35%, recovering edible ethanol, and collecting the reduced-pressure concentrated solution;
e. HP2MGL resin repurification: preparing the obtained reduced pressure concentrated solution into a solution with the mass concentration of 1.5-2.5%, separating and purifying isothiocyanate through an HP2MGL resin chromatographic column at room temperature, collecting eluent, carrying out reduced pressure concentration, recovering edible ethanol, filtering the concentrated solution while the concentrated solution is hot, crystallizing the filtrate, standing overnight, and finally carrying out vacuum freeze drying to obtain the high-purity isothiocyanate extract with the purity of more than 95%.
2. The method of claim 1, wherein the cruciferous vegetable seed in step a is selected from the group consisting of mustard seed and broccoli seed.
3. The method for extracting high-purity isothiocyanate from cruciferous vegetable seeds according to claim 1, wherein the deionized water is added in an amount of 0.8 to 1.2 times the weight of the cruciferous vegetable seeds in the step a.
4. The method for extracting high-purity isothiocyanate from cruciferous vegetable seeds as claimed in claim 1, wherein the nanofiltration separation in step c is performed at a temperature of 35 to 40 ℃ and a pressure of 2.4 to 2.8 MPa.
5. The method for extracting high-purity isothiocyanate from cruciferous vegetable seeds as claimed in claim 1, wherein the nanofiltration concentration in the step c is performed at a temperature of 35 to 40 ℃ and a pressure of 2.8 to 3.2 MPa.
6. The method for extracting high purity isothiocyanate from cruciferous vegetable seeds as claimed in claim 1, wherein the separation and purification of isothiocyanate through polyamide resin chromatography column in step d is carried out by passing 7-9 BV of 1.5-2.5% solution through polyamide resin chromatography column at flow rate of 2.0-3.0 BV/h to make resin adsorbed to saturation, standing for 30-50 min, then removing impurities with 3.0-4.0 BV of 15-25% edible ethanol at flow rate of 1.5-2.5 BV/h, eluting isothiocyanate with 4-6 BV of 60-80% edible ethanol at flow rate of 1.0-2.0 BV/h, and collecting eluent.
7. The method for extracting high purity isothiocyanate from cruciferous vegetable seeds as claimed in claim 1, wherein the separation and purification of isothiocyanate by HP2MGL resin chromatography column in step e is carried out by passing 5-7 BV 1.5-2.5% solution through HP2MGL resin chromatography column at a flow rate of 1.5-2.5 BV/h to make resin adsorbed to saturation, standing for 30-50 min, eluting with 2.5-3.5 BV 25-35% edible ethanol at a flow rate of 1.0-2.0 BV/h to remove impurities, eluting with 3-5 BV 65-75% edible ethanol at a flow rate of 0.8-1.2 BV/h to collect eluent.
8. The method for extracting high purity isothiocyanate from cruciferous vegetable seeds according to claim 1, wherein the vacuum degree of the vacuum concentration in the step b, the step d and the step e is-0.05 to-0.09 MPa, and the temperature is 50 to 60 ℃.
9. The method for extracting high purity isothiocyanate from cruciferous vegetable seeds according to claim 1, wherein the vacuum degree of the vacuum freeze-drying in the step b and the step e is-0.05 to-0.09 MPa, and the temperature is-38 to-42 ℃.
10. The method for extracting high-purity isothiocyanate from cruciferous vegetable seeds as set forth in claim 1, wherein the temperature for crystallizing the filtrate in the step e is 0 to 4 ℃.
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