Preparation method of isothiocyanate in papaya seeds

Abstract

The invention discloses a preparation method of isothiocyanate in papaya seeds, which comprises the following steps: weighing choline, glycerol, sugar and water, heating and stirring uniformly, and taking out when the solid disappears and the solution is colorless or yellowish viscous liquid, so as to obtain a deep eutectic solvent DES; the choline is choline chloride or acetylcholine; weighing pawpaw seeds, crushing the pawpaw seeds, screening out powder through an 80-mesh standard sieve, collecting the powder, weighing the pawpaw seed powder, putting the powder into a reactor, adding the synthesized deep eutectic solvent DES, putting the reactor into a constant-temperature mixing instrument, and carrying out enzymatic reaction, so as to obtain a pawpaw seed enzymolysis product isothiocyanate mixed solution after the reaction is finished. The method is simple in reaction steps, suitable for industrial production and high in reaction yield.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of isothiocyanate, and in particular to a method for preparing isothiocyanate in papaya seeds. Background Art

[0002] Isothiocyanates (ITCs) are a class of organic compounds. They usually have the general structural formula of RN=C=S. ITCs exist in many plants of the Cruciferae and Carica families in the form of glucosinolates. Usually, glucosinolates in plants undergo enzymatic reactions to produce a variety of ITCs. More than 120 types of isothiocyanates have been discovered, mainly in three categories: aliphatic, indole, and aromatic. Most isothiocyanates do not exist alone in plants, but exist in several forms simultaneously.

[0003] Studies have shown that the isothiocyanate content of the papaya family is the highest among all known plants, and the content of seeds is much greater than that of the pulp. Papaya seeds contain a large amount of benzyl glucosinolate (BGS), which can react to form benzyl isothiocyanate (BITC) under the hydrolysis of endogenous myrosinase in plants. Since BGS and myrosinase are in different regions of papaya seeds, myrosinase is mainly located in the gel-like part of the outer protective coating, while glucosinolate is inside the papaya seeds, the physical separation prevents the two from reacting. However, when the tissue is broken and the cells are damaged, myrosinase dissociates from the protein body and becomes free, and then contacts with glucosinolate in the cell fluid to undergo a hydrolysis reaction. The final enzymatic hydrolysis product is mainly benzyl isothiocyanate, in addition to trace amounts of other components, such as benzyl isocyanate, benzyl nitrile and benzyl thiocyanate.

[0004] Sang Luyan et al. used N235 (trioctyl tertiary amine) as an extractant when studying the extraction of isothiocyanates, and studied allyl isothiocyanate (AITC) as a typical representative. The specific steps for preparing the extractant are: adding sulfonated kerosene as a diluent to the purified N235 to prepare an extractant with a concentration of 5%. The best extraction conditions were: the concentration of the extraction solution was 0.5%, the extraction time was 10 minutes, the pH value was 9, and the phase ratio (O / W) was 3. Under this optimal extraction condition, the single extraction rate of AITC was 90.86%.

[0005] In the preparation process of isothiocyanates, some raw material solvents are highly toxic, which not only threatens the health of operators, but also poses potential risks to the environment. At the same time, the existing preparation methods cannot achieve low-temperature and high-efficiency conversion, resulting in a low yield of the target product. Summary of the invention

[0006] The invention aims to provide a method for preparing isothiocyanate in papaya seeds, which has simple reaction steps, is suitable for industrial production and has high reaction yield.

[0007] The technical solution of the present invention:

[0008] A method for preparing isothiocyanate in papaya seeds comprises the following steps:

[0009] (1) Weighing choline, glycerol, sugar and water, heating and stirring evenly, taking out the solution while hot when the solid disappears and the solution becomes a colorless or slightly yellow viscous liquid, to obtain a low eutectic solvent DES; the choline is choline chloride or acetylcholine;

[0010] (2) Weigh papaya seeds and crush them, sieve out the powder through an 80-mesh standard sieve and collect it, weigh the papaya seed powder and place it in a reactor, add the synthesized low eutectic solvent DES, keep the reactor at a constant temperature, place it in a constant temperature mixer, and perform an enzymatic reaction. After the reaction is completed, a papaya seed enzymatic hydrolysis product isothiocyanate mixed liquid is obtained.

[0011] Preferably, the ratio of papaya seed powder to the deep eutectic solvent DES is 0.1 g:5 mL.

[0012] Preferably, the temperature of the heating and stirring in step (1) is 90°C.

[0013] Preferably, in step (1), the molar ratio of choline, glycerol and sugar is 1-4:1:4-1.

[0014] Preferably, the amount of water added in step (1) is 10-50% of the total mass of choline, glycerol and sugar, and the water is deionized water.

[0015] Preferably, the sugar in step (1) is sucrose, D-fructose, maltose or lactose.

[0016] Preferably, the preparation method of acetylcholine is as follows: anhydrous ethanol is added to choline chloride and anhydrous sodium acetate, covered with tin foil and stirred for 48 to 96 hours, and when a white solid precipitate is precipitated from the lower layer after standing and stratification, the supernatant is taken and rotary evaporated, and the ethanol is separated at a temperature of 38 to 42° C., and the water is separated at 60° C., and acetylcholine is finally obtained when it becomes a white viscous state.

[0017] Preferably, in the process of preparing acetylcholine, the molar ratio of choline chloride, anhydrous sodium acetate and anhydrous ethanol is 1:1.2:100.

[0018] Preferably, the enzymatic reaction conditions in step (2) are: temperature 40-80°C, time 20-120 min, and constant temperature stirring speed 1200 r / min.

[0019] Preferably, after the enzymatic reaction in step (2) is completed, the isothiocyanate mixed solution of the enzymatic hydrolysis product of papaya seeds is diluted with deionized water, the volume ratio of the isothiocyanate mixed solution of the enzymatic hydrolysis product of papaya seeds: water is 1:4, and the dilution is filtered through a 0.45 μm acetate fiber microporous filter membrane to obtain a dilution of the isothiocyanate mixed solution.

[0020] In DES, choline chloride or acetylcholine act as hydrogen bond acceptors, and sugar acts as a hydrogen bond donor. Glycerol is added because it is an enzymatic reaction. This enzyme already exists in papaya seeds. The addition of glycerol is to maintain the stability of the enzyme in papaya seeds. After adding glycerol, the viscosity of DES is further reduced. Glycerol can also act as a hydrogen bond donor.

[0021] The DES system is relatively viscous. Adding water can further reduce the viscosity of the DES system. The enzymatic reaction also requires water, so the water content needs to be adjusted.

[0022] Beneficial effects of the present invention: The present invention adopts a low eutectic solvent (DES) as a preparation solvent. This solvent has the advantages of good biocompatibility, non-flammability, non-volatility, etc. Compared with traditional organic solvents, it is more environmentally friendly and reduces pollution to the environment. At the same time, the preparation raw materials of the DES used are easy to obtain and have low cost. Since environmentally friendly DES is used as a preparation solvent, the use of harmful chemicals is reduced and the safety of the operation is improved. The operation process of the present invention is simple and easy to control, and provides a green, efficient and low-cost method for preparing isothiocyanates, which is of great significance for promoting its industrial production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Diagram of the extraction of isothiocyanates from different sugar DES;

[0024] Figure 2 This is a diagram showing the extraction of isothiocyanate by DES at different raw material ratios;

[0025] Figure 3 The figure shows the extraction of isothiocyanate by DES with different water contents;

[0026] Figure 4 The figure shows the extraction of isothiocyanate at different enzymatic reaction times;

[0027] Figure 5 This is a diagram showing the extraction of isothiocyanate at different enzymatic reaction temperatures. DETAILED DESCRIPTION

[0028] Isothiocyanate Detection Method

[0029] HPLC analysis

[0030] The detailed analysis conditions are as follows: column model Diamonsil C18 (2) (5u 200×4.6mm); column temperature 25°C; mobile phase ratio 50% water 50% acetonitrile; isocratic elution time 30min; mobile phase flow rate 1mL / min; injection volume 10μL; detector wavelength 191nm.

[0031] Drawing of the standard curve of isothiocyanate

[0032] Prepare about 30mL of acetonitrile, add a drop of isothiocyanate standard and dilute 10 times to obtain a mother solution of a certain concentration. Take five centrifuge tubes, take the mother solution and dilute it with acetonitrile 0 times, 1 times, 2 times, 3 times, and 4 times to obtain 5 isothiocyanate solutions of different concentrations, take 0.5mL in a liquid phase vial, detect the peak area value of the target by high performance liquid chromatography (HPLC), and draw a concentration-peak area standard curve. After that, the enzymatic reaction solution obtained from each reaction is detected by HPLC to obtain the peak area, and the corresponding concentration is found in the standard curve. Calculate the yield according to the formula.

[0033] The isothiocyanate yield is calculated according to formula (1):

[0034]

[0035] c, concentration of enzyme reaction solution, g / L;

[0036] V1, total volume of enzymatic reaction solution, mL;

[0037] m, mass of papaya seed powder in enzymatic reaction solution, mg.

[0038] Example

[0039] Example 1

[0040] 1.1 Preparation of acetylcholine

[0041] Weigh a certain molar ratio of choline chloride and anhydrous sodium acetate (1:1.2) into a 1L beaker, add about 800mL of anhydrous ethanol, cover with tin foil to block light and prevent ethanol from absorbing water vapor in the air. Stir the mixture under magnetic stirring for three days until a large amount of white solid precipitates on the lower layer after stratification. Take the supernatant and evaporate it by rotary evaporation. Separate ethanol at a temperature of about 40°C and separate water at 60°C. Finally, obtain homemade acetylcholine when it becomes white and viscous.

[0042] 1.2DES Synthesis Method

[0043] Weigh choline chloride / acetylcholine, glycerol, and sugar in a molar ratio of 1:1:1, add 35% of the total mass of water, and place in a 50mL round-bottom flask. Mix evenly at a certain temperature using a magnetic heating stirrer. When the solid disappears and the solution becomes a colorless / slightly yellow viscous liquid, take it out while it is hot.

[0044] 1.3 Preparation of isothiocyanates

[0045] Weigh 500g of papaya seeds and crush them with a multifunctional food processor, sieve out fine powder through an 80-mesh standard sieve and collect it, accurately weigh 0.1g of papaya seed powder using an analytical balance and place it in a 10mL centrifuge tube, add 5mL of the synthesized hydrophilic DES solution prepared in the previous step. Place the centrifuge tube in a constant temperature mixer, and its initial enzymatic reaction conditions are: temperature 40°C, speed 1200r / min, time 2h. After the reaction is completed, 2mL is diluted to 10mL with deionized water, and 0.5mL of the sample is taken with a disposable syringe, filtered through a 0.45μm acetate fiber microporous filter membrane and stored in a liquid phase vial.

[0046] Examples 2 to 6 are all optimized based on this example.

[0047] Example 2 Effect of different choline / sugar DES on isothiocyanate yield

[0048] Choline chloride / acetylcholine, glycerol and different sugars in a molar ratio of 1:1:1 were weighed into a 50 mL centrifuge tube, 35% of the total mass of water was added, and the centrifuge tube was placed in a constant temperature mixer to synthesize eight kinds of DES respectively. The papaya seed enzymatic hydrolysis product isothiocyanate was prepared according to the method of Example 1.3, the content of isothiocyanate was detected respectively, and the yield was calculated, and the effects of different sugars in choline in DES on the isothiocyanate yield were compared.

[0049] The specific experimental data are shown in Table 1 and Table 2:

[0050] Table 1 Extraction of isothiocyanates by DES (choline chloride) with different sugars

[0051]

[0052] Table 2 Extraction of isothiocyanate by different sugars DES (acetylcholine)

[0053]

[0054] According to Table 1, Table 2 and Figure 1 The results show that the yield of isothiocyanate using different sugar DES systems is ranked as follows: fructose > lactose > sucrose > maltose. The yield of isothiocyanate prepared by fructose DES system is significantly better than that of the other three sugar DES systems, so the fructose DES system is used to optimize other conditions.

[0055] Example 3 Effect of different ratios of choline and sugar in DES on the yield of isothiocyanate

[0056] Choline chloride / acetylcholine and sugars with different ratios were weighed, and the ratio of glycerol was unchanged compared with Example 1, and put into a 50 mL centrifuge tube, 35% of the total mass of water was added, and the centrifuge tube was placed in a constant temperature mixer to synthesize six DES solvents with ratios of 1:4, 1:3, 1:2, 2:1, 3:1, and 4:1, respectively. The papaya seed enzymatic hydrolysis product isothiocyanate was prepared according to the method described in Example 1.3, the content of isothiocyanate was detected and the yield was calculated, and the effects of different ratios of choline and fructose in DES on the isothiocyanate yield were compared.

[0057] The specific experimental data are shown in Table 3 and Table 4 below:

[0058] Table 3 Extraction of isothiocyanates by DES (choline chloride) with different raw material ratios

[0059]

[0060] Table 4 Extraction of isothiocyanate by DES (acetylcholine) with different raw material ratios

[0061]

[0062] According to Table 3, Table 4 and Figure 2 The results showed that the yield of DES systems with different choline and fructose ratios was ranked as follows: 3:1>4:1>2:1>1:2>1:3>1:4. The yield of acetylcholine-fructose DES with acetylcholine:fructose:glycerol ratio of 3:1:1 was significantly better than that of other groups. Therefore, the DES system with acetylcholine:fructose:glycerol ratio of 3:1:1 was applied to the optimization of other conditions.

[0063] Example 4 Effect of water content in DES on isothiocyanate yield

[0064] Acetylcholine, fructose and glycerol in a molar ratio of 3:1:1 were weighed into a 50 mL centrifuge tube, and different amounts of water were added. The centrifuge tube was placed in a constant temperature mixer, and five DES solvents prepared by adding 10%, 20%, 30%, 40% and 50% of water to acetylcholine, fructose and glycerol, respectively. The papaya seed enzymatic hydrolysate isothiocyanate was prepared according to the method described in Example 1.3, and the content of isothiocyanate was detected and the yield was calculated, and the effect of water content in hydrophilic DES on the isothiocyanate yield was compared.

[0065] The specific experimental data are shown in Table 5:

[0066] Table 5 Extraction of isothiocyanates by DES with different water contents

[0067]

[0068] According to Table 5 and Figure 3 The results show that the yield of DES systems with different water contents is ranked as follows: 40%>30%>50%>20%>10%. Under the same conditions, the yield of DES system with 40% water content is significantly better than that of DES systems with other water contents. Therefore, the fructose DES system with 40% water content is used to optimize other conditions.

[0069] Example 5 Effect of different enzymatic reaction times on isothiocyanate yields

[0070] Acetylcholine, fructose and glycerol in a molar ratio of 3:1:1 were weighed into a 50 mL centrifuge tube, 40% of the total mass of water was added, and the centrifuge tube was placed in a constant temperature mixer to synthesize DES. The enzymatic reaction was carried out for 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min according to the method described in Example 1.3 to prepare papaya seed enzymatic hydrolysis product isothiocyanate, the isothiocyanate content was detected and the yield was calculated, and the effect of the enzymatic reaction time on the isothiocyanate yield was compared.

[0071] The specific experimental data are shown in Table 6:

[0072] Table 6 Extraction of isothiocyanates at different enzymatic reaction times

[0073]

[0074]

[0075] According to Table 6 and Figure 4 The results showed that the yields of different enzymatic reaction times were ranked as follows: 40min>80min>60min>100min>120min>20min. Under the same conditions, the yield of enzymatic reaction for 40min was significantly better than that of isothiocyanate at other enzymatic reaction times, so the enzymatic reaction for 40min was used to optimize other conditions.

[0076] Example 6 Effect of different enzymatic reaction temperatures on isothiocyanate yields

[0077] Weigh acetylcholine, fructose, and glycerol in a molar ratio of 3:1:1 into a 50 mL centrifuge tube, and add 40% of the total mass of water. Place the centrifuge tube in a constant temperature mixer to synthesize DES. According to the method described in Example 1.3, enzymatic reactions were performed at temperatures of 25°C, 40°C-80°C (every 10°C was a temperature interval) to prepare papaya seed enzymatic hydrolysate isothiocyanate, and the content of isothiocyanate was detected and the yield was calculated, and the effect of the temperature of the enzymatic reaction on the isothiocyanate yield was compared.

[0078] The specific experimental data are shown in Table 7:

[0079] Table 7 Extraction of isothiocyanates at different enzymatic reaction temperatures

[0080]

[0081] According to Table 7 and Figure 5 The results showed that the yields of different enzymatic reaction temperatures were ranked as follows: 40℃>25℃>60℃>50℃>70℃. No target substance was detected at an enzymatic reaction temperature of 80℃, which may be due to enzyme inactivation caused by excessive temperature. Under the same conditions, the yield of enzymatic reaction at 40℃ was significantly better than that of other enzymatic reaction temperatures.

[0082] The optimal embodiment 7 is obtained based on embodiments 1-6.

[0083] Best Embodiment 7

[0084] 7.1 Preparation of acetylcholine

[0085] Weigh a certain molar ratio of choline chloride and anhydrous sodium acetate (1:1.2) into a 1L beaker, add about 800mL of anhydrous ethanol, cover with tin foil to block light and prevent ethanol from absorbing water vapor in the air. Stir the mixture under magnetic stirring for three days until a large amount of white solid precipitates on the lower layer after stratification. Take the supernatant and evaporate it by rotary evaporation. Separate ethanol at a temperature of about 40°C and separate water at 60°C. Finally, obtain homemade acetylcholine when it becomes white and viscous.

[0086] 7.2DES Synthesis Method

[0087] Weigh choline chloride / acetylcholine, glycerol, and fructose in a molar ratio of 3:1:1, add 40% of the total mass of water, and place in a 50mL round-bottom flask. Mix evenly at a certain temperature using a magnetic heating stirrer. When the solid disappears and the solution becomes a colorless / slightly yellow viscous liquid, remove it while it is hot.

[0088] 7.3 Preparation of isothiocyanates

[0089] Weigh 500g of papaya seeds and crush them with a multifunctional food processor, sieve out fine powder through an 80-mesh standard sieve and collect it, accurately weigh 0.1g of papaya seed powder using an analytical balance and place it in a 10mL centrifuge tube, add 5mL of the synthesized hydrophilic DES solution. Place the centrifuge tube in a constant temperature mixer, and its initial enzymatic reaction conditions are: temperature 40°C, speed 1200r / min, time 40min. After the reaction is completed, add deionized water to dilute to 10mL, take 0.5mL of sample with a disposable syringe, filter through a 0.45μm acetate fiber microporous filter membrane and store in a liquid phase vial.

[0090] The maximum yield of isothiocyanate was 1.80%.

[0091] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical content of the present invention.

Claims

1. A method for preparing isothiocyanate in papaya seeds, characterized in that: The following steps are involved: (1) Weighing choline, glycerol, sugar and water, heating and stirring evenly, taking out the solution while hot when the solid disappears and the solution becomes a colorless or slightly yellow viscous liquid, to obtain a low eutectic solvent DES; the choline is choline chloride or acetylcholine; (2) Weigh papaya seeds and crush them, sieve out the powder through an 80-mesh standard sieve and collect it, weigh the papaya seed powder and place it in a reactor, add the synthesized low eutectic solvent DES, keep the reactor at a constant temperature, and perform an enzymatic reaction. After the reaction is completed, a papaya seed enzymatic hydrolysis product isothiocyanate mixed liquid is obtained.

2. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: The ratio of papaya seed powder to deep eutectic solvent DES is 0.1 g:5 mL.

3. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: The temperature of the heating and stirring in step (1) is 90°C.

4. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: In step (1), the molar ratio of choline, glycerol and sugar is 1-4:1:4-1.

5. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: In step (1), the amount of water added is 10-50% of the total mass of choline, glycerol and sugar, and the water is deionized water.

6. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: The sugar in step (1) is sucrose, D-fructose, maltose or lactose.

7. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: The preparation method of acetylcholine is as follows: anhydrous ethanol is added to choline chloride and anhydrous sodium acetate, and the mixture is covered with tin foil and stirred for 48 to 96 hours. When a white solid precipitates out from the lower layer after standing for stratification, the supernatant is rotary evaporated, and the ethanol is separated at a temperature of 38 to 42°C, and the water is separated at 60°C, and acetylcholine is finally obtained when the mixture becomes a white viscous state.

8. The method for preparing isothiocyanate in papaya seeds according to claim 7, characterized in that: The molar ratio of choline chloride, anhydrous sodium acetate and anhydrous ethanol is 1:1.2:

100.

9. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: The enzymatic reaction conditions in step (2) are: temperature 40-80° C., time 20-120 min, and constant temperature stirring speed 1200 r / min.

10. The method for preparing isothiocyanate in papaya seeds according to claim 1, characterized in that: After the enzymatic reaction in step (2) is completed, the isothiocyanate mixed solution of the enzymatic hydrolyzed product of papaya seeds is diluted with deionized water, the volume ratio of the isothiocyanate mixed solution of the enzymatic hydrolyzed product of papaya seeds to water is 1:4, and the dilution is filtered through a 0.45 μm acetate fiber microporous filter membrane to obtain a dilution of the isothiocyanate mixed solution.

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