Method for improving conversion rate of allyl isothiocyanate through enzymatic reaction

Abstract

The invention discloses a method for improving the conversion rate of allyl isothiocyanate through enzymatic reaction. The method comprises the following steps: firstly, ultrasonically extracting myrosinase from broccoli seeds; the myrosinase, alkyl glycoside and casein phosphopeptides are flocculated under the action of sodium polyacrylate to form a flocculating solution, the flocculating solution and the magnetic starch milk are mixed, post-treatment is performed, and the immobilized myrosinase is obtained; and then adding the immobilized myrosinase into an aqueous solution of sinigrin, and carrying out enzymolysis reaction under the condition of a high-voltage pulsed electric field to obtain allyl isothiocyanate. The conversion rate of the allyl isothiocyanate is improved through the enzymatic reaction, the product quality is high, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to a method for improving the conversion rate of allyl isothiocyanate by enzymatic reaction. It belongs to the technical field. Background Art

[0002] Allyl isothiocyanate is mainly used for formulating spice and mustard-type flavors, and is used in pickles, canned foods, sauces, seasonings, etc. Allyl isothiocyanate has good anti-corrosion and antibacterial effects and is widely used in the preservation of raw meat, fruits and vegetables. At present, most of the allyl isothiocyanate on the market is prepared by chemical synthesis, and a small number is obtained by extraction and purification from cruciferous plants. However, allyl isothiocyanate prepared by chemical synthesis has great application risks in the food industry due to the complex synthesis process and high toxicity of reagents. Plant extraction has problems such as uncontrollability, low yield, high cost, and the product purity cannot be guaranteed.

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

[0004] Myrosinase contains a large number of disulfide bonds and is an enzyme that is easily inactivated. The immobilization is difficult. The immobilization effect of myrosinase in the aforementioned patent technology is general, resulting in waste of myrosinase and affecting the final product quality.

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

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for improving the conversion rate of allyl isothiocyanate by enzymatic reaction.

[0007] To achieve the above purpose, the present invention 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) Immobilization of myrosinase: Myrosinase, alkyl polyglycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate to form a flocculated liquid. The flocculated liquid is mixed with magnetic starch milk and post-treated to obtain immobilized myrosinase;

[0011] (3) Add immobilized myrosinase to the aqueous solution of sinigrin, and carry out an enzymatic hydrolysis reaction under the condition of high-voltage pulsed electric field to obtain allyl isothiocyanate.

[0012] Preferably, the specific method of step (1) is: crush the broccoli seeds to 80-100 mesh to obtain a powder, then add the powder to a phosphate buffer solution, carry out ultrasonic extraction at 35-37 °C, centrifuge to take the supernatant, concentrate, and dry to obtain myrosinase.

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

[0014] More preferably, the process conditions for ultrasonic extraction are: ultrasonic extraction at 300-500 W for 60-70 minutes.

[0015] Preferably, in step (2), the flocculation process is as follows: add myrosinase, alkyl polyglycoside, and casein phosphopeptide to a phosphate buffer solution, stir and mix evenly, then add an aqueous solution of sodium polyacrylate, and carry out vortex flocculation to obtain a flocculated solution;

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

[0017] More 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) While stirring, add starch to deionized water, stir and mix evenly to obtain a starch milk with a mass concentration of 30-40%;

[0020] (B) Dissolve ferric chloride and ferrous chloride in deionized water to obtain an iron salt solution;

[0021] (C) Then mix the starch milk with the iron salt solution, adjust the pH to 10-12, carry out heating and ultrasonic treatment, naturally cool to room temperature, adjust the pH to 7, wash, and separate with a magnet to obtain magnetic starch microspheres;

[0022] (D) Finally, ultrasonically disperse the magnetic starch microspheres in deionized water to obtain the magnetic starch milk.

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

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

[0025] More preferably, in step (C), the process conditions of the heat ultrasonic treatment are as follows: temperature 70 - 75°C, ultrasonic power 500 - 600 W, treatment time 30 - 40 minutes.

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

[0027] More preferably, in step (C), it is washed 2 - 3 times with an ethanol aqueous solution with a volume concentration of 90 - 95%.

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

[0029] Preferably, in step (2), the volume ratio of the flocculant to the magnetic starch milk is 1:3 to 4, and they are stirred and mixed 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 dosage ratio of the sinigrin aqueous solution to the immobilized myrosinase is 100 mL: 20 - 25 mg, and the sinigrin aqueous 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 μs, electric field strength 2 - 3 kV / cm.

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

[0034] More preferably, after the enzymatic hydrolysis reaction, it is extracted with dichloromethane for 30 minutes, the organic phase is taken, rotary evaporated, and dried to obtain allyl isothiocyanate; the dosage of dichloromethane is half of the volume of the enzymatic hydrolysis product.

[0035] The beneficial effects of the present invention:

[0036] The present invention prepares allyl isothiocyanate by an enzymatic hydrolysis method. First, myrosinase is ultrasonically extracted from broccoli seeds; then, myrosinase, alkyl polyglycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate to form a flocculated solution. The flocculated solution is mixed with magnetic starch milk and then post-treated to obtain immobilized myrosinase; then, immobilized myrosinase is added to an aqueous solution of sinigrin, and an enzymatic hydrolysis reaction is carried out under the condition of high-voltage pulsed electric field to obtain allyl isothiocyanate. The present invention improves the conversion rate of allyl isothiocyanate through enzymatic reaction, has high product quality, and broad application prospects.

[0037] Under normal circumstances, sinigrin is converted by myrosinase to produce allyl isothiocyanate, allyl thiocyanate, allyl cyanide, 1,3 - thiacyclobutane nitrile, oxazolidinethione, etc. The present invention improves the conversion rate of sinigrin to allyl isothiocyanate by immobilizing myrosinase and controlling the enzymatic hydrolysis conditions.

[0038] The method of the present invention tries to avoid the use of toxic components and has higher safety. For example, sodium polyacrylate is a commonly used food additive, and alkyl polyglycoside and casein phosphopeptide are also commonly used in the food field.

[0039] Alkyl polyglycoside and casein phosphopeptide have an auxiliary promoting effect on the enzymatic hydrolysis of myrosinase, greatly shortening the enzymatic hydrolysis time and also being beneficial to the improvement of product purity. Specific Embodiments

[0040] The present invention will be further elaborated below in conjunction with 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 by enzymatic reaction, the specific steps are as follows:

[0043] (1) Extraction of myrosinase: Myrosinase is ultrasonically extracted from broccoli seeds;

[0044] Specifically, broccoli seeds are pulverized to 80 mesh to obtain powder. Then, the powder is added to a phosphate buffer solution, ultrasonically extracted at 35°C, centrifuged to take the supernatant, concentrated, and dried to obtain myrosinase;

[0045] Among them, 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 pH = 7.5;

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

[0047] (2) Immobilization of myrosinase: Myrosinase, alkyl polyglycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate (purchased from Jiangsu Baoshi Jia Biotechnology Co., Ltd.) to form a flocculated solution. The flocculated solution is mixed with magnetic starch milk, separated by a magnet, and vacuum freeze-dried to obtain immobilized myrosinase;

[0048] Among them, the flocculation process is as follows: Myrosinase, alkyl polyglycoside, and casein phosphopeptide are added to a phosphate buffer solution, stirred and mixed evenly, and then an aqueous solution of sodium polyacrylate is added, and vortex flocculation is carried out to obtain a flocculated solution;

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

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

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

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

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

[0054] (C) Then the starch milk and the iron salt solution are mixed at a volume ratio of 1:8, the pH is adjusted to 10 with 0.5 mol / L sodium hydroxide solution, heated and ultrasonically treated, naturally cooled to room temperature, the pH is adjusted to 7 with glacial acetic acid, and washed 2 times with an ethanol aqueous solution with a volume concentration of 90%, and magnetic starch microspheres are obtained by magnetic separation; the process conditions for heating and ultrasonic treatment are: temperature 70 °C, ultrasonic power 500 W, 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 flocculated solution to the magnetic starch milk is 1:3, and they are stirred and mixed at 300 r / min for 25 minutes.

[0057] (3) Add 200 mg of immobilized myrosinase to 1 L of sinigrin aqueous solution, and carry out an enzymatic hydrolysis reaction under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

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

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

[0060] (a) Reflux extraction of mustard seeds with an aqueous ethanol solution with a volume concentration of 70% is carried out 2 times. Each time, the mass ratio of the material to the liquid is 1:5, and the extraction time is 2 hours each time; after the extraction is completed, centrifuge and combine the supernatant;

[0061] (b) Under the condition of high-voltage pulsed electric field, extract the supernatant with a solvent, collect the organic phase, recover the solvent under reduced pressure to obtain a concentrated solution; among them, 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 a volume ratio of 3:1; the conditions of the high-voltage pulsed electric field are: pulse frequency 300 Hz, pulse width 4 μs, electric field strength 2 kV / cm;

[0062] (c) Take the concentrated solution and perform column chromatography on a 700B anion exchange resin column. First, wash the resin column with water until the effluent is clear, then elute with a 1% sodium hydroxide aqueous solution by mass, collect the eluate, concentrate, and dry to obtain sinigrin.

[0063] After the enzymatic reaction is completed, extract with dichloromethane for 30 minutes, take the organic phase, rotary evaporate, and dry to obtain allyl isothiocyanate; the amount of dichloromethane used is half of the volume of the enzymatic reaction product.

[0064] Example 2:

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

[0066] (1) Extraction of myrosinase: Myrosinase is obtained by ultrasonic extraction from broccoli seeds;

[0067] Specifically, crush broccoli seeds to 100 meshes to obtain a powder, then add the powder to a phosphate buffer solution, perform ultrasonic extraction at 37 °C, centrifuge and take the supernatant, concentrate, and dry to obtain myrosinase;

[0068] Among them, 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 pH = 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 Baoshi Jia Biotechnology Co., Ltd.) to form a flocculated solution. Mix the flocculated solution with magnetic starch milk, separate with a magnet, and vacuum freeze-dry to obtain immobilized myrosinase;

[0071] Among them, the flocculation process is as follows: Add myrosinase, alkyl polyglycoside, and casein phosphopeptide into phosphate buffer solution, stir and mix evenly, then add an aqueous solution of sodium polyacrylate, and vortex for flocculation to obtain a flocculated solution;

[0072] The mass ratio of myrosinase, alkyl polyglycoside, casein phosphopeptide, phosphate buffer solution, and aqueous solution of sodium polyacrylate is 1:0.4:0.2:12:10, and the mass concentration of the aqueous solution of sodium polyacrylate 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) While stirring, add starch into deionized water, stir and mix evenly to obtain a starch milk with a mass concentration of 40%;

[0076] (B) Dissolve 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 1:2:12;

[0077] (C) Then mix the starch milk and the iron salt solution according to a volume ratio of 1:10, adjust the pH = 12 with 0.5 mol / L sodium hydroxide solution, perform heat ultrasonic treatment, naturally cool to room temperature, adjust the pH = 7 with glacial acetic acid, wash 3 times with an aqueous ethanol solution with a volume concentration of 95%, and separate with a magnet to obtain magnetic starch microspheres; the process conditions of the heat ultrasonic treatment are: temperature 75 °C, ultrasonic power 600 W, and treatment time 40 minutes;

[0078] (D) Finally, ultrasonically disperse the magnetic starch microspheres in 12 times their weight of deionized water to obtain the described magnetic starch milk;

[0079] The volume ratio of the flocculated solution to the magnetic starch milk is 1:4, and stir and mix at 400 r / min for 35 minutes.

[0080] (3) Add 250 mg of immobilized myrosinase to 1 L of sinigrin aqueous solution, and carry out an enzymatic hydrolysis reaction under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

[0081] Among them, the high-voltage pulsed electric field conditions are: pulse frequency 400 Hz, pulse width 5 μs, and electric field strength 3 kV / cm; the enzymatic hydrolysis reaction conditions are: pH = 7.5, temperature 30 °C, and time 40 minutes.

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

[0083] (a) The mustard seeds are reflux-extracted with an 80% ethanol aqueous solution by volume for 3 times. The mass ratio of material to liquid is 1:7 each time, and the extraction time is 3 hours each time. After extraction, centrifuge and combine the supernatant;

[0084] (b) Under the condition of 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. Among them, the volume ratio of the supernatant to the solvent is 1:6, and the solvent is a mixture of ethyl acetate and acetone in a volume ratio of 4:1. The conditions of the high-voltage pulsed electric field are: pulse frequency 400 Hz, pulse width 5 μs, and electric field strength 3 kV / cm;

[0085] (c) The concentrated solution is subjected to column chromatography on a 700B anion exchange resin column. First, wash the resin column with water until the effluent is clear, then elute with a 2% sodium hydroxide aqueous solution by mass, collect the eluate, concentrate, and dry to obtain sinigrin.

[0086] After the enzymatic reaction is completed, extract with dichloromethane for 30 minutes, take the organic phase, rotary evaporate, and dry to obtain allyl isothiocyanate. The amount of dichloromethane used is half of the volume of the enzymatic reaction product.

[0087] Example 3:

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

[0089] (1) Extraction of myrosinase: Myrosinase is obtained by ultrasonic extraction from broccoli seeds;

[0090] Specifically, the broccoli seeds are crushed to 90 meshes to obtain a powder. Then, the powder is added to a phosphate buffer solution, and ultrasonic extraction is carried out at 36 °C. Centrifuge and take the supernatant, concentrate, and dry to obtain myrosinase;

[0091] Among them, 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 pH = 7.5;

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

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

[0094] Among them, the flocculation process is as follows: Myrosinase, alkyl polyglycoside, and casein phosphopeptide are added to a phosphate buffer solution, stirred and mixed evenly, and then an aqueous solution of sodium polyacrylate is added, and vortex flocculation is carried out to obtain a flocculated solution;

[0095] The mass ratio of myrosinase, alkyl glycoside, casein phosphopeptide, phosphate buffer solution, and aqueous sodium polyacrylate solution is 1:0.35:0.15:11:9, and the mass concentration of the aqueous sodium polyacrylate 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) While stirring, add starch to deionized water and stir evenly to obtain a starch milk with a mass concentration of 35%.

[0099] (B) Dissolve 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 1:1.5:11.

[0100] (C) Then mix the starch milk and the iron salt solution in a volume ratio of 1:9, adjust the pH = 11 with 0.5 mol / L sodium hydroxide solution, perform heat ultrasonic treatment, naturally cool to room temperature, adjust the pH = 7 with glacial acetic acid, wash 2 times with an aqueous ethanol solution with a volume concentration of 92%, and obtain magnetic starch microspheres by magnetic separation; the process conditions for heat ultrasonic treatment are: temperature 72 °C, ultrasonic power 600 W, treatment time 35 minutes.

[0101] (D) Finally, ultrasonically disperse the magnetic starch microspheres in 11 times their weight of deionized water to obtain the described magnetic starch milk.

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

[0103] (3) Add 220 mg of immobilized myrosinase to 1 L of sinigrin aqueous solution, and carry out an enzymatic hydrolysis reaction under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

[0104] Among them, the high-voltage pulsed electric field conditions are: pulse frequency 400 Hz, pulse width 4 μs, electric field strength 2 kV / cm; the enzymatic hydrolysis reaction conditions are: pH = 7.5, temperature 29 °C, time 35 minutes.

[0105] The sinigrin aqueous solution is obtained by dissolving sinigrin in 11 times its weight of deionized water; sinigrin is prepared by the following method:

[0106] (a) Use an aqueous ethanol solution with a volume concentration of 75% to reflux and extract mustard seeds, extract 2 times, with a material-liquid mass ratio of 1:6 each time and an extraction time of 2 hours each time; after extraction, centrifuge and combine the supernatant.

[0107] (b) Under the condition of 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 3:1; the conditions of the high-voltage pulsed electric field are: pulse frequency 400 Hz, pulse width 5 μs, and electric field strength 2 kV / cm;

[0108] (c) The concentrated solution is subjected to column chromatography on a 700B anion exchange resin column. First, the resin column is washed with water until the effluent is clear, and then eluted with a 1.5% sodium hydroxide aqueous solution by mass concentration. The eluate is collected, concentrated, and dried to obtain sinigrin.

[0109] After the enzymatic hydrolysis reaction is completed, it is extracted with dichloromethane for 30 minutes, the organic phase is taken, rotary evaporated, and dried to obtain allyl isothiocyanate; the amount of dichloromethane used is half of the volume of the enzymatic hydrolysis product.

[0110] Comparative Example 1

[0111] A preparation method of allyl isothiocyanate, the specific steps are 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 meshes to obtain a powder. Then, the powder is added to a phosphate buffer solution, ultrasonically extracted at 35 °C, centrifuged to take the supernatant, concentrated, and dried to obtain myrosinase;

[0114] Among them, 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 pH = 7.5;

[0115] The process conditions for ultrasonic extraction are: 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 magnetic starch milk, separated by a magnet, and vacuum freeze-dried to obtain immobilized myrosinase;

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

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

[0119] (A) While stirring, starch is added to deionized water and stirred evenly to obtain a 30% starch milk by mass concentration;

[0120] (B) Dissolve 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 mix the starch milk and the iron salt solution at a volume ratio of 1:8, adjust the pH to 10 with 0.5 mol / L sodium hydroxide solution, perform heat ultrasonic treatment, naturally cool to room temperature, adjust the pH to 7 with glacial acetic acid, wash twice with 90% ethanol aqueous solution by volume, and separate with a magnet to obtain magnetic starch microspheres; the process conditions for heat ultrasonic treatment are: temperature 70 °C, ultrasonic power 500 W, treatment time 30 minutes;

[0122] (D) Finally, ultrasonically disperse the magnetic starch microspheres in 10 times their weight of deionized water to obtain the magnetic starch milk;

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

[0124] (3) Add 200 mg of immobilized myrosinase to 1 L of sinigrin aqueous solution, and carry out an enzymatic hydrolysis reaction under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

[0125] Among them, the high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, electric field strength 2 kV / cm; the enzymatic hydrolysis reaction conditions are: pH = 7.5, temperature 28 °C, time 30 minutes.

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

[0127] (a) Reflux extract mustard seeds with 70% ethanol aqueous solution by volume, extract twice, with a material-liquid mass ratio of 1:5 each time and an extraction time of 2 hours each time; after extraction, centrifuge and combine the supernatant;

[0128] (b) Under high-voltage pulsed electric field conditions, extract the supernatant with a solvent, collect the organic phase, and recover the solvent under reduced pressure to obtain a concentrated solution; among them, the volume ratio of the supernatant to the solvent is 1:5, and the solvent is a mixture of ethyl acetate and acetone in a volume ratio of 3:1; the high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, electric field strength 2 kV / cm;

[0129] (c) Take the concentrated solution and perform column chromatography on a 700B anion exchange resin column. First, wash the resin column with water until the effluent is clear, then elute with 1% sodium hydroxide aqueous solution by mass concentration, collect the eluate, concentrate, and dry to obtain sinigrin.

[0130] After the enzymatic reaction is completed, extract with dichloromethane for 30 minutes, take the organic phase, rotary evaporate, and dry to obtain allyl isothiocyanate; the amount of dichloromethane used is half of the volume of the enzymatic reaction product.

[0131] Comparative Example 2

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

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

[0134] Specifically, crush the broccoli seeds to 80 mesh to obtain a powder, then add the powder to a phosphate buffer solution, extract by ultrasonic wave at 35 °C, centrifuge to take the supernatant, concentrate, and dry to obtain myrosinase;

[0135] Among them, 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 pH = 7.5;

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

[0137] (2) Immobilization of myrosinase: Myrosinase and alkyl polyglycoside are flocculated under the action of sodium polyacrylate (purchased from Jiangsu Baoshi Jia Biotechnology Co., Ltd.) to form a flocculated liquid. Mix the flocculated liquid with magnetic starch milk, separate with a magnet, and vacuum freeze-dry to obtain immobilized myrosinase;

[0138] Among them, the flocculation process is as follows: Add myrosinase and alkyl polyglycoside to a phosphate buffer solution, stir and mix evenly, then add an aqueous solution of sodium polyacrylate, and vortex to flocculate to obtain a flocculated liquid;

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

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

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

[0142] (A) While stirring, add starch to deionized water, stir and mix evenly to obtain a starch milk with a mass concentration of 30%;

[0143] (B) Dissolve 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;

[0144] (C) Then mix the starch milk with the iron salt solution at a volume ratio of 1:8, adjust the pH to 10 using 0.5 mol / L sodium hydroxide solution, perform heat ultrasonic treatment, naturally cool to room temperature, adjust the pH to 7 using glacial acetic acid, wash twice with 90% ethanol aqueous solution by volume, and obtain magnetic starch microspheres by magnetic separation; the process conditions for heat ultrasonic treatment are: temperature 70 °C, ultrasonic power 500 W, treatment time 30 minutes;

[0145] (D) Finally, ultrasonically disperse the magnetic starch microspheres in deionized water with a weight 10 times that of the microspheres to obtain the magnetic starch milk described above;

[0146] The volume ratio of the flocculant solution to the magnetic starch milk is 1:3, and they are stirred and mixed at 300 r / min for 25 minutes.

[0147] (3) Add 200 mg of immobilized myrosinase to 1 L of sinigrin aqueous solution, and perform enzymatic hydrolysis reaction under high-voltage pulsed electric field conditions to obtain allyl isothiocyanate.

[0148] Among them, the high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, electric field strength 2 kV / cm; the enzymatic hydrolysis reaction conditions are: pH = 7.5, temperature 28 °C, time 30 minutes.

[0149] The sinigrin aqueous solution is obtained by dissolving sinigrin in deionized water with a weight 10 times that of sinigrin; sinigrin is prepared by the following method:

[0150] (a) Reflux extract mustard seeds with 70% ethanol aqueous solution by volume, extract twice, with a material-liquid mass ratio of 1:5 each time and an extraction time of 2 hours each time; after extraction, centrifuge and combine the supernatant;

[0151] (b) Under high-voltage pulsed electric field conditions, extract the supernatant with a solvent, collect the organic phase, and recover the solvent under reduced pressure to obtain a concentrated solution; among them, the volume ratio of the supernatant to the solvent is 1:5, and the solvent is a mixture of ethyl acetate and acetone in a volume ratio of 3:1; the high-voltage pulsed electric field conditions are: pulse frequency 300 Hz, pulse width 4 μs, electric field strength 2 kV / cm;

[0152] (c) Take the concentrated solution and perform column chromatography on a 700B anion exchange resin column. First, wash the resin column with water until the effluent is clear, then elute with 1% sodium hydroxide aqueous solution by mass concentration, collect the eluate, concentrate, and dry to obtain sinigrin.

[0153] After the enzymatic hydrolysis reaction, extract with dichloromethane for 30 minutes, take the organic phase, perform rotary evaporation, and dry to obtain allyl isothiocyanate; the amount of dichloromethane used is half of the volume of the enzymatic hydrolysis product.

[0154] Comparative Example 3

[0155] A preparation method of allyl isothiocyanate is as follows:

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

[0157] Specifically, the broccoli seeds are ground to 80 mesh to obtain a powder. Then the powder is added to a phosphate buffer solution, and ultrasonic extraction is carried out at 35°C. After centrifugation, the supernatant is taken, concentrated, and dried to obtain myrosinase.

[0158] Among them, 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 pH = 7.5.

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

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

[0161] Among them, the flocculation process is as follows: Myrosinase, alkyl polyglycoside, and casein phosphopeptide are added to a phosphate buffer solution, stirred and mixed evenly, and then an aqueous solution of sodium polyacrylate is added, and vortex flocculation is carried out to obtain a flocculated solution.

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

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

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

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

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

[0167] (C) Then mix the starch milk and the iron salt solution at a volume ratio of 1:8, adjust the pH to 10 using 0.5 mol / L sodium hydroxide solution, perform heat ultrasonic treatment, naturally cool to room temperature, adjust the pH to 7 using glacial acetic acid, wash twice with 90% ethanol aqueous solution by volume, and obtain magnetic starch microspheres by magnetic separation; the process conditions for heat ultrasonic treatment are: temperature 70 °C, ultrasonic power 500 W, treatment time 30 minutes;

[0168] (D) Finally, ultrasonically disperse the magnetic starch microspheres in deionized water with a weight 10 times that of the microspheres to obtain the magnetic starch milk described above;

[0169] The volume ratio of the flocculant solution to the magnetic starch milk is 1:3, and they are stirred and mixed at 300 r / min for 25 minutes.

[0170] (3) Add 200 mg of immobilized myrosinase to 1 L of sinigrin aqueous solution for enzymatic hydrolysis reaction to obtain allyl isothiocyanate.

[0171] Among them, the conditions for the enzymatic hydrolysis reaction are: pH = 7.5, temperature 28 °C, time 30 minutes.

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

[0173] (a) Reflux extract mustard seeds with 70% ethanol aqueous solution by volume for 2 times, with a material-liquid mass ratio of 1:5 each time and an extraction time of 2 hours each time; after extraction, centrifuge and combine the supernatant;

[0174] (b) Under the conditions of high-voltage pulsed electric field, extract the supernatant with a solvent, collect the organic phase, and recover the solvent under reduced pressure to obtain a concentrated solution; among them, the volume ratio of the supernatant to the solvent is 1:5, and the solvent is a mixture of ethyl acetate and acetone in a volume ratio of 3:1; the conditions of high-voltage pulsed electric field are: pulse frequency 300 Hz, pulse width 4 μs, electric field strength 2 kV / cm;

[0175] (c) Take the concentrated solution and perform column chromatography on a 700B anion exchange resin column. First, wash the resin column with water until the effluent is clear, then elute with 1% sodium hydroxide aqueous solution by mass concentration, collect the eluate, concentrate, and dry to obtain sinigrin.

[0176] After the enzymatic hydrolysis reaction, extract with dichloromethane for 30 minutes, take the organic phase, rotary evaporate, and dry to obtain allyl isothiocyanate; the amount of dichloromethane used is half of the volume of the enzymatic hydrolysis product.

[0177] Calculate the conversion rate of allyl isothiocyanate (calculated based on the molar amount of sinigrin) for Examples 1 to 3 and Comparative Examples 1 to 3 respectively, and test the product purity.

[0178] The purity test method is as follows:

[0179] Accurately weigh about 3 g of the product, add it to 30 mL of water, stir to disperse evenly, accurately add 25.0 mL of piperidine standard solution, stopper, shake for 3 minutes, let stand for 40 minutes, filter to obtain the filtrate. Using bromocresol green-methyl red as the indicator (obtained by dissolving 0.1000 g of bromocresol green and 0.0200 g of methyl red in 100 mL of absolute ethanol), titrate the filtrate with hydrochloric acid standard solution until the color changes to wine red as the end point. Calculate the isothiocyanate content (calculated as allyl isothiocyanate) according to the formula: Isothiocyanate content (%) = 9.915×(c1V1 - c2V2) / W, where c1 is the concentration of the piperidine standard solution (mol / L), V1 is the added volume of the piperidine 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 hydrochloric acid standard solution: Measure 4.5 mL of concentrated hydrochloric acid (mass concentration 37%), dissolve it in 1000 mL of distilled water to obtain hydrochloric acid standard solution. Accurately weigh 0.1000 g of anhydrous sodium carbonate with constant weight of 280, dissolve it with 50 mL of distilled water, add 5 drops of bromocresol green-methyl red indicator, titrate with hydrochloric acid solution until the color changes from green to wine red, boil for 2 minutes, cool and continue titrating until it turns wine red, 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 piperidine standard solution: Measure 2.5 mL of freshly distilled piperidine, add it to a 500 mL volumetric flask, add absolute ethanol to make up the volume, shake well, and use bromocresol green-methyl red as the indicator, titrate with hydrochloric acid standard solution to determine the accurate concentration.

[0182] The results are shown in Table 1.

[0183] Table 1. Conversion rate and purity of allyl isothiocyanate

[0184] Conversion rate (%) 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, for the methods of Examples 1 - 3, the conversion rate of allyl isothiocyanate is high and the purity is high.

[0186] For Comparative Example 1, the flocculation step in the myrosinase immobilization process is omitted; for Comparative Example 2, casein phosphopeptide is omitted; for Comparative Example 3, the high-voltage pulsed electric field treatment is omitted during enzymatic hydrolysis. The product conversion rate and purity are significantly deteriorated, indicating that the immobilization method of myrosinase, auxiliary enzymatic hydrolysis means such as casein phosphopeptide, and the high-voltage pulsed electric field treatment during enzymatic hydrolysis act synergistically to promote the conversion of sinigrin to allyl isothiocyanate and improve the product purity.

[0187] Although the specific embodiments of the present invention are described above, they do not limit the protection scope of the present invention. Based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. A method for improving the conversion rate of allyl isothiocyanate by enzymatic reaction, characterized in that: The specific steps are as follows: (1) Extraction of myrosinase: Myrosinase was obtained from broccoli seeds by ultrasonic extraction; (2) Immobilization of myrosinase: Myrosinase, alkyl glycoside, and casein phosphopeptide are flocculated under the action of sodium polyacrylate to form a flocculated liquid, and the flocculated liquid is mixed with magnetic starch emulsion and post-treated to obtain immobilized myrosinase; (3) adding immobilized myrosinase to the myrosinase aqueous solution, and performing enzymatic hydrolysis under high-voltage pulse electric field conditions to obtain allyl isothiocyanate.

2. The method according to claim 1, characterized in that The specific method of step (1) is as follows: broccoli seeds are crushed into 80-100 meshes to obtain powder, the powder is then added to phosphate buffer, ultrasonically extracted at 35-37° C., centrifuged to obtain the supernatant, concentrated, and dried to obtain myrosinase.

3. The method according to claim 1, characterized in that: In step (2), the flocculation process is as follows: adding myrosinase, alkyl glycoside, and casein phosphopeptide into phosphate buffer, stirring and mixing, then adding sodium polyacrylate aqueous solution, vortexing and flocculating to obtain a flocculated liquid.

4. The method according to claim 1, characterized in that: In step (2), the magnetic starch emulsion is prepared by the following method: (A) adding starch into deionized water while stirring, stirring and mixing, to obtain starch milk with a mass concentration of 30 to 40%; (B) dissolving ferric chloride and ferrous chloride in deionized water to obtain an iron salt solution; (C) then mixing the starch milk with the iron salt solution, adjusting the pH to 10-12, heating and ultrasonic treatment, naturally cooling to room temperature, adjusting the pH to 7, washing, and separating with a magnet to obtain magnetic starch microspheres; (D) Finally, the magnetic starch microspheres are ultrasonically dispersed in deionized water to obtain the magnetic starch emulsion.

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

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

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

8. The method according to claim 1, characterized in that In step (3), the high voltage pulse electric field conditions are: pulse frequency 300-400 Hz, pulse width 4-5 μs, and electric field strength 2-3 kV / cm.

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

10. The method according to claim 9, characterized in that After the enzymatic hydrolysis reaction is completed, dichloromethane is used for extraction for 30 minutes, and the organic phase is taken, rotary evaporated, and dried to obtain allyl isothiocyanate; the amount of dichloromethane used is half the volume of the enzymatic hydrolysis product.