A method for increasing the conversion of allyl isothiocyanate by an enzymatic reaction

Abstract

The application discloses a method for improving conversion rate of allyl isothiocyanate through enzymatic reaction, and the method comprises the following steps: firstly, extracting black mustard enzyme from broccoli seeds through ultrasonic extraction; secondly, flocculating the black mustard enzyme, alkyl glycoside and casein phosphopeptide under the action of sodium polyacrylate to form flocculation liquid, mixing the flocculation liquid with magnetic starch milk, and post-treating to obtain immobilized black mustard enzyme; and then, adding the immobilized black mustard enzyme into black mustard glycoside aqueous solution, and performing enzymolysis reaction under the condition of high-pressure pulse electric field to obtain allyl isothiocyanate. The application improves the conversion rate of allyl isothiocyanate through enzymatic reaction, and the product has high quality and wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for improving the conversion rate of allyl isothiocyanate by enzymatic reaction, belonging to the technical field. BACKGROUND

[0002] Allyl isothiocyanate is mainly used for preparing spices and mustard type flavors for pickles, cans, sauces, seasonings, etc. Allyl isothiocyanate has good antibacterial effect and is widely used in fresh meat and vegetable preservation. Currently, most of the allyl isothiocyanate on the market is prepared by chemical synthesis, and a small amount is obtained by extraction and purification from cruciferous plants. However, the allyl isothiocyanate synthesized by chemical synthesis has complex synthesis process and high reagent toxicity, and has great application risk in the food industry. Plant extraction has the problems of uncontrollability, low yield, high cost, and the purity of the product cannot be guaranteed.

[0003] Patent application CN104673850A discloses a method for preparing allyl isothiocyanate by immobilized enzyme conversion, which includes four steps of extraction of myrosinase, activation of magnetic beads, cross-linking fixation of myrosinase, and conversion of immobilized myrosinase to prepare allyl isothiocyanate. The patent uses enzymatic method to prepare allyl isothiocyanate, which has obvious advantages compared with chemical synthesis method and plant extraction method.

[0004] Myrosinase contains a large number of disulfide bonds and is a kind of enzyme that is easy to inactivate, so it is difficult to immobilize. The immobilization effect of the foregoing patent technology on myrosinase is general, which causes waste of myrosinase and affects the quality of the final product.

[0005] In addition, the adjustment of the enzymatic reaction conditions has a great influence on the product quality, and there is currently a lack of related research on improving the conversion rate of allyl isothiocyanate by enzymatic reaction. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for improving the conversion rate of allyl isothiocyanate by enzymatic reaction.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A method for improving the conversion rate of allyl isothiocyanate by enzymatic reaction, the specific steps are as follows:

[0009] (1) Extraction of myrosinase: myrosinase is obtained by ultrasonic extraction from broccoli seeds;

[0010] (2) Fixation of myrosinase: myrosinase, alkyl glycoside and casein phosphopeptide are flocculated under the action of sodium polyacrylate to form flocculation liquid, the flocculation liquid is mixed with magnetic starch milk, and then treated to obtain immobilized myrosinase;

[0011] (3) adding immobilized myrosinase into the aqueous solution of sinigrin, and performing enzymatic hydrolysis under the condition of high-voltage pulsed electric field to obtain allyl isothiocyanate.

[0012] Preferably, the specific method of step (1) is as follows: grinding broccoli seeds into powder with 80-100 mesh, then adding the powder into phosphate buffer solution, ultrasonic extraction at 35-37℃, centrifugation to obtain supernatant, concentration, and drying to obtain myrosinase.

[0013] Further preferably, the mass ratio of the powder to the phosphate buffer solution is 1:10-12, and the concentration of the phosphate buffer solution is 0.2 mol / L, and pH=7.5.

[0014] Further preferably, the process condition of ultrasonic extraction is as follows: ultrasonic extraction at 300-500 W for 60-70 minutes.

[0015] Preferably, in step (2), the flocculation process is as follows: adding myrosinase, alkyl polyglycoside, and casein phosphopeptide into phosphate buffer solution, stirring to mix, then adding sodium polyacrylate aqueous solution, vortex flocculation to obtain flocculation liquid.

[0016] Further preferably, the mass ratio of myrosinase, alkyl polyglycoside, casein phosphopeptide, phosphate buffer solution, and sodium polyacrylate aqueous solution is 1:0.3-0.4:0.1-0.2:10-12:8-10, and the mass concentration of the sodium polyacrylate aqueous solution is 3-4%.

[0017] Further preferably, the vortex rotation speed is 2000-3000 r / min, and the vortex time is 15-20 s.

[0018] Preferably, in step (2), the magnetic starch milk is prepared by the following method:

[0019] (A) adding starch into deionized water while stirring to mix, to obtain starch milk with a mass concentration of 30-40%;

[0020] (B) dissolving ferric chloride and ferrous chloride in deionized water to obtain a ferric salt solution;

[0021] (C) then mixing the starch milk with the ferric salt solution, adjusting pH to 10-12, heating and ultrasonic treatment, naturally cooling to room temperature, adjusting pH to 7, washing, and separating by a magnet to obtain magnetic starch microspheres;

[0022] (D) finally ultrasonic dispersing the magnetic starch microspheres in deionized water to obtain the magnetic starch milk.

[0023] Further preferably, in step (B), the mass ratio of ferric chloride, ferrous chloride, and deionized water is 1:1-2:10-12.

[0024] Further preferably, in step (C), the volume ratio of starch milk to iron salt solution is 1:8-10.

[0025] Further preferably, in step (C), the process conditions for heating and ultrasonic treatment are as follows: temperature 70-75 DEG C, ultrasonic power 500-600 W, and treatment time 30-40 minutes.

[0026] Further preferably, in step (C), the pH is adjusted to 10-12 by using 0.5 mol / L sodium hydroxide solution and adjusted to 7 by using glacial acetic acid.

[0027] Further preferably, in step (C), the starch is washed 2-3 times with 90-95% (volume concentration) aqueous ethanol solution.

[0028] Further preferably, in step (D), the mass ratio of magnetic starch microspheres to deionized water is 1:10-12.

[0029] Preferably, in step (2), the volume ratio of flocculation liquid to magnetic starch milk is 1:3-4, and the mixture is stirred at 300-400 r / min for 25-35 minutes.

[0030] Preferably, in step (2), the post-treatment includes: magnetic separation and vacuum freeze-drying.

[0031] Preferably, in step (3), the amount ratio of the aqueous sinigrin solution to the immobilized sinigrin enzyme is 100 mL:20-25 mg, and the aqueous sinigrin solution is obtained by dissolving sinigrin in 10-12 times its weight of deionized water.

[0032] Preferably, in step (3), the high-voltage pulsed electric field conditions are as follows: pulse frequency 300-400 Hz, pulse width 4-5 mu s, and electric field strength 2-3 kV / cm.

[0033] Preferably, in step (3), the enzymatic reaction conditions are as follows: pH=7.5, temperature 28-30 DEG C, and time 30-40 minutes.

[0034] Further preferably, after the enzymatic reaction, dichloromethane is used for extraction for 30 minutes, the organic phase is taken, rotary evaporation is performed, and drying is carried out, to obtain allyl isothiocyanate; the amount of dichloromethane is half the volume of the enzymatic reaction product.

[0035] The present application has the following beneficial effects:

[0036] This invention employs an enzymatic method to prepare allyl isothiocyanate. First, myrosinase is extracted from broccoli seeds using ultrasound. Then, myrosinase, alkyl glycosides, and casein phosphopeptides are flocculated under the action of sodium polyacrylate to form a flocculent solution. This flocculent solution is mixed with magnetic starch milk and post-treated to obtain immobilized myrosinase. Finally, the immobilized myrosinase is added to a myrosinase aqueous solution, and the enzymatic reaction is carried out under a high-voltage pulsed electric field to obtain allyl isothiocyanate. This invention improves the conversion rate of allyl isothiocyanate through enzymatic reaction, resulting in a high-quality product with broad application prospects.

[0037] Normally, myrosinase converts glucosinolates into allyl isothiocyanate, allyl thiocyanate, allyl nitrile, 1,3-cyclothiobutyronitrile, oxazolidinyl thione, etc. This invention improves the conversion rate of myrosinase to allyl isothiocyanate by immobilizing the myrosinase and controlling the enzymatic hydrolysis conditions.

[0038] The method of this invention avoids the use of toxic components as much as possible, making it safer. For example, sodium polyacrylate is a commonly used food additive, and alkyl glycosides and casein phosphopeptides are also commonly used in the food industry.

[0039] Alkyl glycosides and casein phosphopeptides have an auxiliary promoting effect on the enzymatic hydrolysis of black myrosinase, which greatly shortens the hydrolysis time and is also conducive to improving product purity. Detailed Implementation

[0040] The present invention will be further described below with reference to embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0041] Example 1:

[0042] A method for improving the conversion rate of allyl isothiocyanate through an enzymatic reaction, the specific steps of which are as follows:

[0043] (1) Extraction of black myrosinase: Black myrosinase was obtained by ultrasonic extraction from broccoli seeds;

[0044] Specifically, broccoli seeds are crushed to 80 mesh to obtain powder. The powder is then added to phosphate buffer, extracted by ultrasonication at 35°C, the supernatant is collected by centrifugation, concentrated, and dried to obtain black mustard enzyme.

[0045] The mass ratio of powder to phosphate buffer is 1:10, the concentration of phosphate buffer is 0.2 mol / L, and the pH is 7.5.

[0046] The ultrasonic extraction process conditions are: 300W ultrasonic extraction for 60 minutes.

[0047] (2) immobilization of myrosinase: myrosinase, alkyl glycoside, casein phosphopeptide are flocculated by sodium polyacrylate (purchased from Jiangsu Baoshijia Biotechnology Co., Ltd.) to form a flocculation liquid, the flocculation liquid is mixed with magnetic starch milk, separated by magnet, vacuum freeze-dried to obtain immobilized myrosinase;

[0048] The flocculation process is as follows: myrosinase, alkyl glycoside, casein phosphopeptide are added to phosphate buffer solution, stirred and mixed, then sodium polyacrylate aqueous solution is added, vortexed to obtain a flocculation liquid;

[0049] The mass ratio of myrosinase, alkyl glycoside, casein phosphopeptide, phosphate buffer solution, and sodium polyacrylate aqueous solution is 1:0.3:0.1:10:8, and the mass concentration of sodium polyacrylate aqueous solution is 3%.

[0050] The vortex rotation speed is 2000 r / min, and the vortex time is 15 s.

[0051] The magnetic starch milk is prepared by the following method:

[0052] (A) While stirring, starch is added to deionized water, stirred and mixed to obtain a starch milk with a mass concentration of 30%;

[0053] (B) Iron chloride and ferrous chloride are dissolved in deionized water to obtain an iron salt solution; the mass ratio of iron chloride, ferrous chloride, and deionized water is 1:1:10;

[0054] (C) Then the starch milk and the iron salt solution are mixed in a volume ratio of 1:8, the pH is adjusted to 10 using 0.5 mol / L sodium hydroxide solution, heated and ultrasonically treated, naturally cooled to room temperature, the pH is adjusted to 7 using glacial acetic acid, washed twice with 90% volume concentration ethanol aqueous solution, and the magnetic starch microspheres are obtained by magnet separation; the process conditions for heating and ultrasonic treatment are: temperature 70℃, ultrasonic power 500W, and treatment time 30 minutes;

[0055] (D) Finally, the magnetic starch microspheres are ultrasonically dispersed in 10 times their weight of deionized water to obtain the magnetic starch milk;

[0056] The volume ratio of the flocculation liquid to the magnetic starch milk is 1:3, and the mixture is stirred at 300 r / min for 25 minutes.

[0057] (3) 200 mg of immobilized myrosinase is added to 1 L of myrosin aqueous solution, and the enzymatic reaction is carried out under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

[0058] The high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4μs, and electric field strength 2kV / cm; the enzymatic reaction conditions are: pH=7.5, temperature 28℃, and time 30 minutes.

[0059] The aqueous solution of sinigrin is obtained by dissolving sinigrin in 10 times of deionized water by weight; the sinigrin is prepared by the following method:

[0060] (a) reflux extraction of mustard seeds with 70% volume concentration of aqueous ethanol solution, 2 times of extraction, mass ratio of material to liquid is 1:5 each time, and extraction time is 2 hours each time; after extraction, centrifugal separation is performed and the supernatant is combined;

[0061] (b) under high-voltage pulse electric field conditions, the supernatant is extracted with a solvent, the organic phase is collected, and the solvent is recovered under reduced pressure to obtain a concentrated solution; wherein the volume ratio of the supernatant to the solvent is 1:5, and the solvent is obtained by mixing ethyl acetate and acetone in a volume ratio of 3:1; the high-voltage pulse electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, and electric field strength 2 kV / cm;

[0062] (c) column chromatography of the concentrated solution on a 700B anion exchange resin column, first water washing of the resin column until the effluent is clear, then elution with 1% mass concentration of aqueous sodium hydroxide solution, collection of the eluate, concentration, drying, and obtaining of sinigrin.

[0063] After the enzymatic reaction is completed, dichloromethane is used for extraction for 30 minutes, the organic phase is taken, rotary evaporation is performed, and drying is performed to obtain allyl isothiocyanate; the amount of dichloromethane is half of the volume of the enzymatic product.

[0064] Example 2:

[0065] A method for improving the conversion rate of allyl isothiocyanate in an enzymatic reaction, the specific steps are as follows:

[0066] (1) extraction of myrosinase: ultrasonic extraction of myrosinase from broccoli seeds;

[0067] Specifically, the broccoli seeds are crushed to 100 mesh to obtain a powder, and then the powder is added to a phosphate buffer solution for ultrasonic extraction at 37°C, the supernatant is taken after centrifugal separation, concentrated, and dried to obtain myrosinase;

[0068] The mass ratio of the powder to the phosphate buffer solution is 1:12, the concentration of the phosphate buffer solution is 0.2 mol / L, and the pH is 7.5;

[0069] The process conditions for ultrasonic extraction are: ultrasonic extraction at 500 W for 70 minutes.

[0070] (2) immobilization of myrosinase: myrosinase, alkyl glycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate (purchased from Jiangsu Baoshijia Biotechnology Co., Ltd.) to form a flocculation liquid, the flocculation liquid is mixed with magnetic starch milk, separated by a magnet, and vacuum freeze-dried to obtain immobilized myrosinase;

[0071] The flocculation process is as follows: the myrosinase, alkyl glycoside, casein phosphopeptide are added into the phosphate buffer solution, stirred and mixed, then the sodium polyacrylate aqueous solution is added, vortexed and flocculated to obtain a flocculation liquid;

[0072] The mass ratio of the myrosinase, alkyl glycoside, casein phosphopeptide, phosphate buffer solution, and sodium polyacrylate aqueous solution is 1:0.4:0.2:12:10, and the mass concentration of the sodium polyacrylate aqueous solution is 4%.

[0073] The vortex rotation speed is 3000 r / min, and the vortex time is 20 s.

[0074] The magnetic starch milk is prepared by the following method:

[0075] (A) The starch is added into the deionized water while stirring, and stirred and mixed to obtain a starch milk with a mass concentration of 40%;

[0076] (B) The ferric chloride and ferrous chloride are dissolved in the deionized water to obtain a ferric salt solution; the mass ratio of the ferric chloride, ferrous chloride, and deionized water is 1:2:12;

[0077] (C) Then the starch milk and the ferric salt solution are mixed according to a volume ratio of 1:10, the pH is adjusted to 12 by using a 0.5 mol / L sodium hydroxide solution, heated and ultrasonically treated, naturally cooled to room temperature, the pH is adjusted to 7 by using glacial acetic acid, washed 3 times by using a 95% volume concentration ethanol aqueous solution, and the magnetic starch microspheres are obtained by using a magnet for separation; the process conditions for the heated ultrasonic treatment are as follows: a temperature of 75℃, an ultrasonic power of 600 W, and a treatment time of 40 minutes;

[0078] (D) Finally, the magnetic starch microspheres are ultrasonically dispersed in 12 times the weight of deionized water, and the magnetic starch milk is obtained.

[0079] The volume ratio of the flocculation liquid and the magnetic starch milk is 1:4, and the stirring and mixing are performed at 400 r / min for 35 minutes.

[0080] (3) 250 mg of immobilized myrosinase is added into 1 L of an aqueous solution of sinigrin, and an enzymatic reaction is performed under high-pressure pulse electric field conditions to obtain allyl isothiocyanate.

[0081] The high-pressure pulse electric field conditions are as follows: a pulse frequency of 400 Hz, a pulse width of 5 μs, and an electric field strength of 3 kV / cm; and the enzymatic reaction conditions are as follows: a pH of 7.5, a temperature of 30℃, and a time of 40 minutes.

[0082] The aqueous solution of sinigrin is obtained by dissolving sinigrin in 12 times the weight of deionized water; and the sinigrin is prepared by the following method:

[0083] (a) reflux extraction of mustard seed with 80% volume concentration of ethanol aqueous solution, 3 times, mass ratio of material to liquid 1:7, each time for 3 hours; after extraction, centrifugal separation and collection of supernatant;

[0084] (b) under high-voltage pulsed electric field, the supernatant is extracted with a solvent, the organic phase is collected, and the solvent is recovered under reduced pressure to obtain a concentrated solution; wherein the volume ratio of the supernatant to the solvent is 1:6, and the solvent is obtained by mixing ethyl acetate and acetone in a volume ratio of 4:1; the high-voltage pulsed electric field conditions are: pulse frequency 400 Hz, pulse width 5 μs, electric field strength 3 kV / cm;

[0085] (c) column chromatography of the concentrated solution on a 700B anion exchange resin column, first water washing of the resin column until the effluent is clear, then elution with 2% mass concentration of sodium hydroxide aqueous solution, collection of the eluate, concentration, drying, and obtaining of sinigrin.

[0086] After the enzymatic reaction is completed, dichloromethane is used for extraction for 30 minutes, the organic phase is taken, rotary evaporation is performed, and drying is performed to obtain allyl isothiocyanate; the amount of dichloromethane is half of the volume of the enzymatic product.

[0087] Example 3:

[0088] A method for improving the conversion rate of allyl isothiocyanate in an enzymatic reaction, the specific steps being as follows:

[0089] (1) extraction of myrosinase: ultrasonic extraction of myrosinase from broccoli seeds;

[0090] Specifically, the broccoli seeds are crushed to 90 mesh to obtain a powder, and then the powder is added to a phosphate buffer solution for ultrasonic extraction at 36°C, and the supernatant is obtained by centrifugal separation, concentrated, and dried to obtain myrosinase;

[0091] The mass ratio of the powder to the phosphate buffer solution is 1:11, the concentration of the phosphate buffer solution is 0.2 mol / L, and the pH is 7.5;

[0092] The process conditions for ultrasonic extraction are: 400 W ultrasonic extraction for 65 minutes.

[0093] (2) immobilization of myrosinase: myrosinase, alkyl glycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate (purchased from Jiangsu Baoshijia Biotechnology Co., Ltd.) to form a flocculation liquid, the flocculation liquid is mixed with magnetic starch milk, separated by a magnet, and vacuum freeze-dried to obtain immobilized myrosinase;

[0094] The flocculation process is as follows: myrosinase, alkyl glycoside, and casein phosphopeptide are added to a phosphate buffer solution, stirred and mixed, then sodium polyacrylate aqueous solution is added, vortexed to flocculate, and a flocculation liquid is obtained;

[0095] The mass ratio of black mustard enzyme, alkyl glycoside, casein phosphopeptide, phosphate buffer, and polyacrylic acid sodium aqueous solution is 1:0.35:0.15:11:9, and the mass concentration of the polyacrylic acid sodium aqueous solution is 3.5%.

[0096] The vortex rotation speed is 2000 r / min, and the vortex time is 18 s.

[0097] The magnetic starch milk is prepared by the following method:

[0098] (A) The starch is added to the deionized water while stirring, and stirred and mixed uniformly to obtain a starch milk with a mass concentration of 35%;

[0099] (B) The ferric chloride and ferrous chloride are dissolved in the deionized water to obtain a ferric salt solution; the mass ratio of the ferric chloride, ferrous chloride, and deionized water is 1:1.5:11;

[0100] (C) Then the starch milk and the ferric salt solution are mixed in a volume ratio of 1:9, the pH is adjusted to 11 by using a 0.5 mol / L sodium hydroxide solution, heated and ultrasonically treated, naturally cooled to room temperature, the pH is adjusted to 7 by using glacial acetic acid, washed twice by using a 92% volume concentration ethanol aqueous solution, and the magnetic starch microspheres are obtained by magnetic separation; the process conditions for the heated ultrasonic treatment are: a temperature of 72°C, an ultrasonic power of 600 W, and a treatment time of 35 minutes;

[0101] (D) Finally, the magnetic starch microspheres are ultrasonically dispersed in 11 times the weight of deionized water, and the magnetic starch milk is obtained.

[0102] The volume ratio of the flocculation liquid to the magnetic starch milk is 1:3.5, and the mixture is stirred at 400 r / min for 30 minutes.

[0103] (3) 220 mg of immobilized black mustard enzyme is added to 1 L of the black mustard glycoside aqueous solution, and the enzymatic reaction is carried out under the condition of a high-pressure pulsed electric field to obtain allyl isothiocyanate.

[0104] The high-pressure pulsed electric field condition is: a pulse frequency of 400 Hz, a pulse width of 4 μs, and an electric field intensity of 2 kV / cm; and the enzymatic reaction condition is: a pH of 7.5, a temperature of 29°C, and a time of 35 minutes.

[0105] The black mustard glycoside aqueous solution is obtained by dissolving the black mustard glycoside in 11 times the weight of deionized water; and the black mustard glycoside is prepared by the following method:

[0106] (a) The mustard seed is extracted by using a 75% volume concentration ethanol aqueous solution by reflux extraction, the extraction is carried out twice, the material liquid mass ratio is 1:6 for each extraction, and the extraction time is 2 hours for each extraction; and after the extraction is completed, the supernatant is centrifuged and combined;

[0107] (b) extracting the supernatant with a solvent under high-voltage pulsed electric field, collecting the organic phase, and recovering the solvent under reduced pressure to obtain a concentrated solution; wherein the volume ratio of the supernatant to the solvent is 1:6, the solvent is obtained by mixing ethyl acetate and acetone at a volume ratio of 3:1, and the high-voltage pulsed electric field is at a pulse frequency of 400 Hz, a pulse width of 5 μs, and an electric field strength of 2 kV / cm;

[0108] (c) subjecting the concentrated solution to column chromatography on a 700B anion exchange resin, first washing the column with water until the effluent is clear, then eluting with a 1.5% sodium hydroxide aqueous solution, collecting the eluate, concentrating, and drying to obtain sinigrin.

[0109] After the enzymatic reaction is completed, dichloromethane is used for extraction for 30 minutes, the organic phase is taken, rotary evaporation is performed, and drying is performed to obtain allyl isothiocyanate; the amount of dichloromethane is half the volume of the enzymatic product.

[0110] Comparative Example 1

[0111] A method for preparing allyl isothiocyanate, the specific steps being as follows:

[0112] (1) Extraction of myrosinase: myrosinase is obtained by ultrasonic extraction from broccoli seeds;

[0113] Specifically, the broccoli seeds are ground to 80 mesh to obtain a powder, the powder is then added to a phosphate buffer solution, ultrasonic extraction is performed at 35°C, the supernatant is taken after centrifugation, concentration and drying are performed to obtain myrosinase;

[0114] The mass ratio of the powder to the phosphate buffer solution is 1:10, the concentration of the phosphate buffer solution is 0.2 mol / L, and the pH is 7.5.

[0115] The process conditions for ultrasonic extraction are as follows: ultrasonic extraction at 300 W for 60 minutes.

[0116] (2) Immobilization of myrosinase: myrosinase, alkyl glycoside, and casein phosphopeptide are added to a phosphate buffer solution to obtain a myrosinase solution, the myrosinase solution is mixed with a magnetic starch milk, magnetic separation is performed, and vacuum freeze-drying is performed to obtain immobilized myrosinase.

[0117] The mass ratio of myrosinase, alkyl glycoside, casein phosphopeptide, and phosphate buffer solution is 1:0.3:0.1:10.

[0118] The magnetic starch milk is prepared by the following method:

[0119] (A) While stirring, starch is added to deionized water, and stirring is performed until uniform to obtain a starch milk with a mass concentration of 30%;

[0120] (B) dissolving ferric chloride and ferrous chloride in deionized water to obtain an iron salt solution; the mass ratio of ferric chloride, ferrous chloride and deionized water is 1:1:10;

[0121] (C) then mixing the starch milk with the iron salt solution according to a volume ratio of 1:8, adjusting pH to 10 by using 0.5 mol / L sodium hydroxide solution, heating and ultrasonic treating, naturally cooling to room temperature, adjusting pH to 7 by using glacial acetic acid, washing twice by using 90% volume concentration ethanol aqueous solution, and obtaining the magnetic starch microspheres by magnetic separation; the process conditions of heating and ultrasonic treating are: temperature 70°C, ultrasonic power 500 W, and treating time 30 minutes;

[0122] (D) finally ultrasonic dispersing the magnetic starch microspheres in 10 times weight of deionized water to obtain the magnetic starch milk;

[0123] The volume ratio of the myrosinase solution and the magnetic starch milk is 1:3, and the mixture is stirred at 300 r / min for 25 minutes.

[0124] (3) adding 200 mg of immobilized myrosinase into 1 L of the aqueous myrosin solution, and carrying out enzymolysis reaction under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

[0125] The high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, and electric field intensity 2 kV / cm; and the enzymolysis reaction conditions are: pH 7.5, temperature 28°C, and time 30 minutes.

[0126] The aqueous myrosin solution is obtained by dissolving myrosin in 10 times weight of deionized water; and the myrosin is prepared by the following method:

[0127] (a) reflux extracting mustard seeds by using 70% volume concentration ethanol aqueous solution, extracting twice, and the material-liquid mass ratio is 1:5 each time, and the extraction time is 2 hours each time; and after the extraction, centrifuging and combining the supernatants;

[0128] (b) under high-voltage pulsed electric field conditions, extracting the supernatant by using a solvent, collecting the organic phase, and recovering the solvent under reduced pressure to obtain a concentrated solution; wherein the volume ratio of the supernatant and the solvent is 1:5, and the solvent is obtained by mixing ethyl acetate and acetone according to a volume ratio of 3:1; and the high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, and electric field intensity 2 kV / cm;

[0129] (c) taking the concentrated solution to column chromatography on a 700B anion exchange resin column, first washing the column with water until the effluent is clear, then eluting with 1% mass concentration sodium hydroxide aqueous solution, collecting the eluate, concentrating, and drying to obtain myrosin.

[0130] After the enzymatic reaction, dichloromethane is used to extract for 30 minutes, and the organic phase is taken, rotary evaporated, and dried to obtain allyl isothiocyanate; the amount of dichloromethane is half of the volume of the enzymatic product.

[0131] Comparative Example 2

[0132] A method for preparing allyl isothiocyanate, and the specific steps are as follows:

[0133] (1) Extraction of myrosinase: myrosinase is obtained by ultrasonic extraction from broccoli seeds;

[0134] Specifically, the broccoli seeds are crushed to 80 mesh to obtain a powder, and then the powder is added to a phosphate buffer solution for ultrasonic extraction at 35°C. The supernatant is obtained by centrifugation, concentrated, and dried to obtain myrosinase;

[0135] The mass ratio of the powder to the phosphate buffer solution is 1:10, the concentration of the phosphate buffer solution is 0.2 mol / L, and the pH is 7.5;

[0136] The process conditions for ultrasonic extraction are 300W ultrasonic extraction for 60 minutes.

[0137] (2) Immobilization of myrosinase: myrosinase and alkyl glycoside are flocculated under the action of sodium polyacrylate (purchased from Jiangsu Baoshijia Biotechnology Co., Ltd.) to form a flocculation liquid. The flocculation liquid is mixed with a magnetic starch milk, separated by a magnet, and vacuum freeze-dried to obtain immobilized myrosinase;

[0138] The flocculation process is as follows: myrosinase and alkyl glycoside are added to a phosphate buffer solution, stirred and mixed, and then sodium polyacrylate aqueous solution is added, vortexed to obtain a flocculation liquid;

[0139] The mass ratio of myrosinase, alkyl glycoside, phosphate buffer solution, and sodium polyacrylate aqueous solution is 1:0.3:10:8, and the mass concentration of the sodium polyacrylate aqueous solution is 3%.

[0140] The vortex rotation speed is 2000 r / min, and the vortex time is 15 s.

[0141] The magnetic starch milk is prepared by the following method:

[0142] (A) While stirring, starch is added to deionized water, stirred and mixed to obtain a starch milk with a mass concentration of 30%;

[0143] (B) Iron chloride and ferrous chloride are dissolved in deionized water to obtain an iron salt solution; the mass ratio of iron chloride, ferrous chloride, and deionized water is 1:1:10;

[0144] (C) Then the starch milk is mixed with the iron salt solution according to the volume ratio of 1:8, the pH is adjusted to 10 by using 0.5 mol / L sodium hydroxide solution, heated and ultrasonically treated, naturally cooled to room temperature, the pH is adjusted to 7 by using glacial acetic acid, washed twice by using 90% volume concentration ethanol aqueous solution, and the magnetic starch microspheres are obtained by magnetic separation; the process conditions of the heated and ultrasonic treatment are: temperature 70°C, ultrasonic power 500 W, and treatment time 30 minutes;

[0145] (D) Finally, the magnetic starch microspheres are ultrasonically dispersed in 10 times weight of deionized water, and the magnetic starch milk is obtained.

[0146] The volume ratio of the flocculation liquid to the magnetic starch milk is 1:3, and the mixture is stirred at 300 r / min for 25 minutes.

[0147] (3) 200 mg of the immobilized myrosinase is added into 1 L of the aqueous myrosin solution, and the enzymatic reaction is carried out under the condition of high-voltage pulsed electric field to obtain allyl isothiocyanate.

[0148] The high-voltage pulsed electric field condition is: pulse frequency 300 Hz, pulse width 4 μs, and electric field intensity 2 kV / cm; and the enzymatic reaction condition is: pH 7.5, temperature 28°C, and time 30 minutes.

[0149] The aqueous myrosin solution is obtained by dissolving myrosin in 10 times weight of deionized water; and the myrosin is prepared by the following method:

[0150] (a) The mustard seed is extracted by using 70% volume concentration ethanol aqueous solution under reflux, and the extraction is carried out twice, and the mass ratio of the material to the liquid is 1:5 each time, and the extraction time is 2 hours each time; and after the extraction is completed, the supernatant is obtained by centrifugation.

[0151] (b) The supernatant is extracted by using a solvent under the condition of high-voltage pulsed electric field, the organic phase is collected, and the solvent is recovered under reduced pressure to obtain a concentrated liquid; the volume ratio of the supernatant to the solvent is 1:5, and the solvent is obtained by mixing ethyl acetate and acetone according to the volume ratio of 3:1; and the high-voltage pulsed electric field condition is: pulse frequency 300 Hz, pulse width 4 μs, and electric field intensity 2 kV / cm.

[0152] (c) The concentrated liquid is subjected to column chromatography by using a 700B anion exchange resin column, the resin column is washed with water until the effluent is clear, and then eluted with 1% mass concentration sodium hydroxide aqueous solution, and the eluate is collected, concentrated, and dried to obtain myrosin.

[0153] After the enzymatic reaction is completed, the reaction product is extracted by using dichloromethane for 30 minutes, the organic phase is taken, rotary evaporated, and dried to obtain allyl isothiocyanate; and the amount of dichloromethane is half of the volume of the enzymatic reaction product.

[0154] Comparative Example 3

[0155] A method for preparing allyl isothiocyanate, the specific steps are as follows:

[0156] (1) Extraction of myrosinase: myrosinase is obtained by ultrasonic extraction from broccoli seeds;

[0157] Specifically, the broccoli seeds are crushed to 80 mesh to obtain a powder, and then the powder is added to a phosphate buffer solution, ultrasonic extraction is performed at 35°C, the supernatant is obtained by centrifugation, concentrated, and dried to obtain myrosinase;

[0158] The mass ratio of the powder to the phosphate buffer solution is 1:10, the concentration of the phosphate buffer solution is 0.2 mol / L, and the pH is 7.5;

[0159] The process conditions for ultrasonic extraction are 300W ultrasonic extraction for 60 minutes.

[0160] (2) Immobilization of myrosinase: myrosinase, alkyl glycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate (purchased from Jiangsu Baoshijia Biotechnology Co., Ltd.) to form a flocculation liquid, the flocculation liquid is mixed with magnetic starch milk, separated by a magnet, and vacuum freeze-dried to obtain immobilized myrosinase;

[0161] The flocculation process is as follows: myrosinase, alkyl glycoside, and casein phosphopeptide are added to a phosphate buffer solution, stirred and mixed, then sodium polyacrylate aqueous solution is added, vortexed to flocculate, and a flocculation liquid is obtained;

[0162] The mass ratio of myrosinase, alkyl glycoside, casein phosphopeptide, phosphate buffer solution, and sodium polyacrylate aqueous solution is 1:0.3:0.1:10:8, and the mass concentration of the sodium polyacrylate aqueous solution is 3%.

[0163] The vortex rotation speed is 2000r / min, and the vortex time is 15s.

[0164] The magnetic starch milk is prepared by the following method:

[0165] (A) While stirring, starch is added to deionized water, stirred and mixed to obtain a starch milk with a mass concentration of 30%;

[0166] (B) Iron chloride and ferrous chloride are dissolved in deionized water to obtain an iron salt solution; the mass ratio of iron chloride, ferrous chloride, and deionized water is 1:1:10;

[0167] (C) Then the starch milk is mixed with the iron salt solution according to the volume ratio of 1:8, the pH is adjusted to 10 by using 0.5 mol / L sodium hydroxide solution, the ultrasonic treatment is performed under heating, the natural cooling is performed to room temperature, the pH is adjusted to 7 by using glacial acetic acid, the washing is performed twice by using 90% volume concentration ethanol aqueous solution, the magnetic separation is performed to obtain the magnetic starch microspheres; the process conditions of the ultrasonic treatment under heating are as follows: the temperature is 70°C, the ultrasonic power is 500 W, and the treatment time is 30 minutes;

[0168] (D) Finally, the magnetic starch microspheres are dispersed in 10 times weight of deionized water by ultrasonic treatment to obtain the magnetic starch milk.

[0169] The volume ratio of the flocculation liquid to the magnetic starch milk is 1:3, and the mixing is performed under stirring at 300 r / min for 25 minutes.

[0170] (3) 200 mg of the immobilized myrosinase is added into 1 L of the aqueous myrosinase solution, and the enzymatic reaction is performed to obtain allyl isothiocyanate.

[0171] The enzymatic reaction conditions are as follows: the pH is 7.5, the temperature is 28°C, and the time is 30 minutes.

[0172] The aqueous myrosinase solution is obtained by dissolving myrosinase in 10 times weight of deionized water; the myrosinase is prepared by the following method:

[0173] (a) The mustard seed is extracted by using 70% volume concentration ethanol aqueous solution under reflux, the extraction is performed twice, the mass ratio of the material to the liquid is 1:5 during each extraction, and the extraction time is 2 hours during each extraction; after the extraction is completed, the supernatant is obtained by centrifugation;

[0174] (b) The supernatant is extracted by using a solvent under high-voltage pulsed electric field conditions, the organic phase is collected, and the solvent is recovered under reduced pressure to obtain a concentrated liquid; the volume ratio of the supernatant to the solvent is 1:5, the solvent is obtained by mixing ethyl acetate and acetone according to the volume ratio of 3:1, and the high-voltage pulsed electric field conditions are as follows: the pulse frequency is 300 Hz, the pulse width is 4 μs, and the electric field strength is 2 kV / cm;

[0175] (c) The concentrated liquid is subjected to column chromatography on a 700B anion exchange resin column, the resin column is washed with water until the effluent is clear, then eluted with 1% mass concentration sodium hydroxide aqueous solution, the eluate is collected, concentrated, and dried to obtain myrosinase.

[0176] After the enzymatic reaction is completed, the extraction is performed by using dichloromethane for 30 minutes, the organic phase is taken, rotary evaporation is performed, and drying is performed to obtain allyl isothiocyanate; the amount of dichloromethane is half of the volume of the enzymatic reaction product.

[0177] The conversion rates of the allyl isothiocyanate in Examples 1-3 and Comparative Examples 1-3 are calculated (based on the molar amount of myrosinase), and the product purity is tested.

[0178] The purity test method is as follows:

[0179] Accurately take 3g of the product, add it into 30mL of water, stir and disperse uniformly, accurately add 25.0mL of hexahydropyridine standard solution, add a stopper, shake for 3 minutes, stand for 40 minutes, filter to obtain the filtrate, use bromocresol green-methyl red as the indicator (obtained by dissolving 0.1000g of bromocresol green and 0.0200g of methyl red in 100mL of anhydrous ethanol), titrate the filtrate with the hydrochloric acid standard solution until the wine red color is the end point, and calculate the isothiocyanate content (calculated as allyl isothiocyanate) according to the formula: isothiocyanate content (%) = 9.915 x (c1V1-c2V2) / W, wherein c1 is the concentration of the hexahydropyridine standard solution (mol / L), V1 is the added volume of the hexahydropyridine standard solution (mL), c2 is the concentration of the hydrochloric acid standard solution (mol / L), V2 is the volume of the hydrochloric acid standard solution consumed during titration (mL), and W is the sample weight of the product (g).

[0180] Preparation of the hydrochloric acid standard solution: take 4.5mL of concentrated hydrochloric acid (mass concentration 37%) and dissolve it in 1000mL of distilled water to obtain the hydrochloric acid standard solution. Accurately take 280 constant-weight reference anhydrous sodium carbonate 0.1000g, dissolve it with 50mL of distilled water, add 5 drops of bromocresol green-methyl red indicator, titrate with the hydrochloric acid solution until the green color turns wine red, boil for 2 minutes, continue to titrate until the wine red color appears after cooling, record the volume of the hydrochloric acid standard solution consumed, and calculate the accurate concentration of the prepared hydrochloric acid standard solution.

[0181] Preparation of the hexahydropyridine standard solution: take 2.5mL of freshly distilled hexahydropyridine and add it to a 500mL volumetric flask, add anhydrous ethanol to constant volume, shake well, and titrate with the hydrochloric acid standard solution to determine the accurate concentration with bromocresol green-methyl red as the indicator.

[0182] The results are shown in Table 1.

[0183] Table 1. Allyl isothiocyanate conversion rate and purity

[0184] Conversion (%) Purity (%) Example 1 98.5 99.2 Example 2 98.9 99.3 Example 3 99.4 99.5 Comparative Example 1 90.2 95.1 Comparative Example 2 93.2 94.5 Comparative Example 3 93.8 93.2

[0185] As can be seen from Table 1, the methods of Examples 1-3 have high allyl isothiocyanate conversion rate and high purity.

[0186] The myrosinase immobilization step of Comparative Example 1 omits flocculation, Comparative Example 2 omits casein phosphopeptide, and Comparative Example 3 omits high-voltage pulsed electric field treatment during enzymolysis, and the product conversion rate and purity are all significantly worse, indicating that the myrosinase immobilization method, casein phosphopeptide and other auxiliary enzymolysis means, and high-voltage pulsed electric field treatment during enzymolysis synergistically promote the conversion of myrosin from allyl isothiocyanate and improve the product purity.

[0187] Although the specific embodiments of the present application have been described above, the above description is not intended to limit the scope of the present application. Various modifications or changes can be made to the present application by those skilled in the art without departing from the technical solutions of the present application, and such modifications or changes are still within the scope of the present application.

Claims

1. A method for improving the conversion rate of allyl isothiocyanate through an enzymatic reaction, characterized in that, The specific steps are as follows: (1) Extraction of black myrosinase: Black myrosinase was obtained by ultrasonic extraction from broccoli seeds; (2) Immobilization of black myrosinase: Black myrosinase, alkyl glycosides and casein phosphopeptides are flocculated under the action of sodium polyacrylate to form a flocculent liquid. The flocculent liquid is mixed with magnetic starch milk and then post-treated to obtain immobilized black myrosinase. (3) Immobilized black myrosinase was added to the aqueous solution of black myrosinase and enzymatically hydrolyzed under high voltage pulse electric field to obtain allyl isothiocyanate; In step (2), the magnetic starch milk is prepared by the following method: (A) While stirring, add starch to deionized water and stir until well mixed to obtain a starch milk with a mass concentration of 30-40%; (B) Dissolve ferric chloride and ferrous chloride in deionized water to obtain an iron salt solution; (C) Then the starch milk was mixed with the iron salt solution, the pH was adjusted to 10-12, heated and sonicated, cooled naturally to room temperature, the pH was adjusted to 7, washed, and separated by a magnet to obtain magnetic starch microspheres; (D) Finally, the magnetic starch microspheres are ultrasonically dispersed in deionized water to obtain the magnetic starch milk. In step (3), the high-voltage pulse electric field conditions are: pulse frequency 300-400Hz, pulse width 4-5μs, and electric field strength 2-3kV / cm.

2. The method according to claim 1, characterized in that, The specific method of step (1) is as follows: crush broccoli seeds to 80-100 mesh to obtain powder, then add the powder to phosphate buffer, extract by ultrasonication at 35-37℃, centrifuge to obtain supernatant, concentrate, and dry to obtain black mustard enzyme.

3. The method according to claim 1, characterized in that, In step (2), the flocculation process is as follows: black myrosinase, alkyl glycosides and casein phosphopeptides are added to phosphate buffer, stirred and mixed, and then sodium polyacrylate aqueous solution is added and vortexed to flocculate to obtain flocculent liquid.

4. The method according to claim 1, characterized in that, In step (2), the volume ratio of flocculant to magnetic starch milk is 1:3-4, and the mixture is stirred at 300-400 r / min for 25-35 minutes.

5. The method according to claim 1, characterized in that, In step (2), the post-processing includes: magnet separation and vacuum freeze drying.

6. The method according to claim 1, characterized in that, In step (3), the ratio of the amount of myrosin aqueous solution to the amount of immobilized myrosinase is 100 mL: 20-25 mg. The myrosin aqueous solution is obtained by dissolving myrosin in 10-12 times its weight of deionized water.

7. The method according to claim 1, characterized in that, In step (3), the enzymatic hydrolysis reaction conditions are: pH=7.5, temperature 28~30℃, and time 30~40 minutes.

8. The method according to claim 7, characterized in that, After the enzymatic hydrolysis reaction was completed, the product was extracted with dichloromethane for 30 minutes. The organic phase was collected, rotary evaporated, and dried to obtain allyl isothiocyanate. The amount of dichloromethane used was half the volume of the enzymatic hydrolysis product.