A method for extracting papain using low-temperature plasma-assisted deep eutectic solvent

Abstract

The present invention discloses a method for extracting papain with a low-temperature plasma-assisted low-eutectic solvent. The extraction method comprises the following steps: using papaya peel as a processing raw material, putting it into a low-eutectic solvent, using low-temperature plasma to perform a mild and strong wall-breaking treatment on it, and the low-eutectic solvent dissolves the protease to obtain an extract; adding a protective agent to the extract, and then separating and freeze-drying through a multi-stage ceramic membrane to obtain papain. Compared with the prior art, the method of the present invention uses a low-toxic and environmentally friendly low-eutectic solvent in combination with low-temperature plasma to extract papain. During the treatment process, the low-temperature plasma causes the cell wall to rupture in a relatively short time, increases the extraction rate, effectively shortens the extraction time, and improves the enzyme activity. It has the characteristics of low temperature, fast and no residue, and simple operation. The low-eutectic solvent replaces the organic solvent, which is not only environmentally friendly and efficient, low cost, simple to operate, but also can be effectively recycled and suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to the field of natural product extraction, and in particular to a method for extracting papain with the assistance of a low-temperature plasma and a deep eutectic solvent. Background Art

[0002] Papaya, also known as papaya, is a tropical and subtropical fruit. It is widely planted in southern provinces of my country, with an annual output of about 800,000 tons. Papain, also known as papain, is a type of thiol protease. It is widely found in the roots, stems, leaves and fruits of papaya. Its active center contains cysteine, and it has the characteristics of high enzyme activity, good thermal stability, natural hygiene and safety. It can hydrolyze the carboxyl end of arginine and lysine in proteins and polypeptides, and can preferentially hydrolyze the peptide bond of amino acids with two carboxyl groups or aromatic L-amino acids at the N-terminus of the peptide bond. Therefore, it is widely used in food, medicine, feed, daily chemical and other industries.

[0003] Papain is widely present in papaya stems, leaves and fruits, with the highest content in the latex of immature fruits. Papaya peels are generally treated as waste, but studies have shown that the activity of papain extracted from different parts of papaya is different. The activity and content of papain in papaya peel are the highest, followed by papaya pulp protease and papaya pulp protease. Extracting papain from papaya peel can not only improve the comprehensive utilization value of papaya peel, but also improve the economic benefits of protease at multiple levels.

[0004] At present, the extraction process of papain mainly includes affinity chromatography separation, ultrafiltration membrane separation, precipitation separation, extraction separation and flocculation. These traditional separation methods are relatively complicated, and have the disadvantages of high cost, serious pollution, large amount of reagents used, etc. In recent years, the emerging two-phase aqueous systems such as polymer / salt and polymer / polymer separation and purification of papain have certain limitations, such as high viscosity and non-degradability, which cause certain degree of harm to people and the environment. These extraction methods have some limitations and are difficult to carry out industrial production.

[0005] At the same time, under the green chemistry initiative, avoiding the use of harmful traditional organic solvents and developing green and environmentally friendly chemical reaction processes have become inevitable choices. Therefore, it is of great significance to find a simple, low-cost, mild, and green method to extract high-purity and high-quality papain. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for extracting papain using a low-melting solvent assisted by low-temperature plasma. Compared with traditional extraction techniques, the extraction of papain using a low-melting solvent technique is green and environmentally friendly, no waste acid or waste alkali is produced, it is recyclable and degradable, and it is low-cost and easy to operate. Low-temperature plasma is a new type of non-thermal extraction technology, which contains a variety of active ingredients, such as reactive oxygen (O, O3, H2O2, -OH), reactive nitrogen (NO, NO2- and NO3), charged particles, electrons and photons, etc., and has strong biological activity and oxidizing properties, so that high-energy particles bombard biological cells to cause etching on their surfaces. This technology has the characteristics of being green, safe, fast and convenient. At the same time, the use of low-temperature plasma to assist in the extraction of papain can improve the enzyme yield and enzyme activity, the process steps are simple, suitable for large-scale industrial production, and has great application prospects. The method comprises the following steps:

[0007] 1) mixing papaya peel and a deep eutectic solvent aqueous solution, and performing low-temperature plasma extraction to obtain an extract;

[0008] 2) Add a protective agent to the extract, and then separate it through multi-stage membranes and freeze-dry it to obtain papain.

[0009] The invention uses a low eutectic solvent mixed with papaya pulp. The low eutectic solvent has good solubility for protein, avoids the use of a large amount of organic solvents in the traditional method, and achieves a green and environmentally friendly effect while reducing production costs.

[0010] The low eutectic solvent composed of a hydrogen bond acceptor and a hydrogen bond donor used in the present invention is a new type of solvent, which is simple and fast to synthesize, green and pollution-free, biodegradable, and inexpensive. Hydrogen bond donors with a higher tendency to form hydrogen bonds are often more conducive to improving enzyme activity, so acceptors and donors with a higher tendency to form hydrogen bonds are selected: choline chloride and amino acids are selected as hydrogen bond acceptors, and glucose, glycerol, polyethylene glycol, ethylene glycol, n-propanol, propylene glycol, and butanediol are selected as hydrogen bond donors.

[0011] Furthermore, the low eutectic solvent is composed of a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond acceptor is one of choline chloride and amino acid, and the hydrogen bond donor is one or more of glucose, glycerol, polyethylene glycol, ethylene glycol, n-propanol, propylene glycol, and butanediol.

[0012] Preferably, the hydrogen bond acceptor of the deep eutectic solvent is choline chloride. Choline chloride has the following advantages:

[0013] (1) Low cost. According to the synthesis mechanism of DESs and the principles of green chemistry, cheap, readily available and environmentally friendly green materials are preferred. They are quite cheap and are often used as hydrogen bond acceptors in the synthesis of DESs.

[0014] (2) Choline chloride-based low eutectic solvents have a good protective effect on proteins.

[0015] (3) DESs based on choline chloride have a large number of hydrogen bonds, which is beneficial for protein extraction.

[0016] (4) Reusable. It can be reused for more than 5 cycles without any significant loss.

[0017] Further preferably, when the hydrogen bond acceptor of the low eutectic solvent is choline chloride, the hydrogen bond donor of the low eutectic solvent is glycerol.

[0018] Furthermore, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-4).

[0019] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:3.

[0020] Furthermore, relative to 1 g of papaya peel sample, the amount of the low eutectic solvent used is 10-60 mL.

[0021] Preferably, relative to 1 g of papaya peel sample, the amount of the low eutectic solvent used is 40 mL.

[0022] Furthermore, the preparation method of the low eutectic solvent is to mix the hydrogen bond acceptor and the hydrogen bond donor in a molar ratio of 1:(1-4), and stir at 60-100° C. for 1-2 hours until a clear homogeneous phase is obtained, thereby obtaining a low eutectic solvent.

[0023] Preferably, the preparation method of the low eutectic solvent is to mix the hydrogen bond acceptor and the hydrogen bond donor in a molar ratio of 1:(1-4), and stir at 65° C. for 1-2 hours until a clear homogeneous phase is obtained.

[0024] The present invention utilizes low-temperature plasma to assist a low eutectic solvent to extract papain. Compared with a single extraction method using a low eutectic solvent, the present invention uses low-temperature plasma treatment to allow high-energy particles to bombard biological cells, thereby etching their surfaces and causing cell wall ruptures in a relatively short period of time, thereby increasing the contact area between the protein and the low eutectic solvent, increasing the rate at which the low eutectic solvent dissolves the protein, and shortening the extraction time. The invention has the characteristics of low temperature, rapidity, no residue, simple operation, high efficiency, and environmental protection.

[0025] Furthermore, the working gas of the low-temperature plasma is selected from one of argon, helium, air, or a mixture of helium / argon and oxygen in any proportion.

[0026] Preferably, the working gas of the low-temperature plasma is air.

[0027] Furthermore, the low-temperature plasma discharge form is selected from one of glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge.

[0028] Preferably, the low-temperature plasma discharge form is dielectric barrier discharge.

[0029] Furthermore, the low-temperature plasma operating voltage is 15-45V, and the processing time is 1-4min.

[0030] Preferably, the low-temperature plasma treatment time is 2 minutes.

[0031] Furthermore, the protective agent is cysteine, and the concentration of the protective agent in the papaya pulp solution is 0.01-1.5 mol / L; or the protective agent is a mixture of DTNB nitrobenzoic acid disulfide compound and one or more of EDTA, ascorbic acid, cyclodextrin or maltodextrin, and the concentration of the protective agent in the papaya pulp solution is 0.01-1.5 mol / L; the protective agent is added to the extract to protect papain and prevent the enzyme from being inactivated during subsequent reactions.

[0032] Furthermore, the membrane of the multi-stage membrane separation technology is a ceramic membrane, and the extract with the added protective agent is microfiltered using a 0.02-micron ceramic membrane to remove impurities and insoluble components in the extract. The filtrate after filtration is ultrafiltered using a 14-nanometer ceramic membrane to remove colloids, polypeptides and other macromolecular organic matter to obtain a filtrate, and finally a 10-nanometer ceramic membrane is used for nanofiltration to separate small molecular impurities to obtain a retentate, which is freeze-dried to obtain papain.

[0033] Furthermore, the freeze drying is carried out at -10°C to -40°C for 8 to 12 hours.

[0034] Preferably, the freeze-drying process is performed at -30°C for 12 hours.

[0035] On the other hand, the present invention provides a use of a low eutectic solvent for extracting papain, wherein the low eutectic solvent is composed of a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond acceptor is one of choline chloride and an amino acid, and the hydrogen bond donor is one or more of glucose, glycerol, polyethylene glycol, ethylene glycol, n-propanol, propylene glycol, and butanediol; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-4); relative to 1g of papaya peel sample, the amount of the low eutectic solvent used is 10-60mL.

[0036] On the other hand, the present invention provides a use of low-temperature plasma for extracting papain, wherein the low-temperature plasma is used to assist the extraction of papain by a low eutectic solvent. The working gas of the low-temperature plasma is selected from one of argon, helium, and air, or a mixed gas of helium / argon and oxygen in any proportion; the discharge form of the low-temperature plasma is selected from one of glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge; the working voltage of the low-temperature plasma is 15-45V, and the processing time is 1-4min.

[0037] The beneficial effects of the present invention include:

[0038] 1. Low-temperature plasma-assisted low eutectic solvent extraction of papain from papaya peel can obtain high-quality and high-activity protease, increase the utilization value of papaya, promote the comprehensive utilization of papaya processing waste, reduce environmental pollution, avoid resource waste, be suitable for large-scale industrial application, and improve economic benefits at multiple levels.

[0039] 2. The present invention uses a low eutectic solvent mixed with papaya pulp, and the low eutectic solvent has good solubility for protein, avoiding the use of a large amount of organic solvents in the traditional method. The low eutectic solvent composed of a hydrogen bond acceptor and a hydrogen bond donor used in the present invention is a new type of solvent, which is simple and fast to synthesize, green and pollution-free, biodegradable, and inexpensive. It can achieve the effect of green environmental protection while reducing production costs.

[0040] 3. The present invention utilizes low temperature plasma to assist the extraction of papain by a low eutectic solvent. Compared with the single extraction method using a low eutectic solvent, the present invention uses low temperature plasma treatment to bombard biological cells with high energy particles, causing etching on the surface, causing cell wall rupture in a relatively short time, increasing the contact area between protein and the low eutectic solvent, increasing the rate at which the low eutectic solvent dissolves protein, shortening the extraction time, and having the characteristics of low temperature, rapid and no residue, simple operation, high efficiency and environmental protection. In addition, treatment at low temperature avoids the denaturation of papain at high temperature.

[0041] 4. Using low-temperature plasma to assist in the extraction of papain can increase the enzyme yield and enzyme activity; the process steps are simple, suitable for large-scale industrial production, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Flow chart of the method for extracting papain using low-temperature plasma-assisted deep eutectic solvent

[0043] Figure 2 Schematic diagram of dielectric barrier discharge plasma structure DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The flow chart of the method for extracting papain with low-temperature plasma assisted deep eutectic solvent in the following embodiment is as follows Figure 1 As shown, the dielectric barrier discharge plasma structure is as follows Figure 2 The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0045] Example 1 Comparison of different extraction methods of papain

[0046] The main existing papain extraction processes and their advantages and disadvantages are as follows:

[0047] (1) Salting-out precipitation method: Add a certain concentration of sodium chloride solution to papaya latex to cause the papain colloids to condense and precipitate due to water loss, then use a certain concentration of ammonium sulfate solution to salt out again, then adjust the pH, centrifuge and precipitate, and dry to obtain the enzyme product.

[0048] Advantages: The process is mature and can produce crude enzyme, refined enzyme and purified enzyme.

[0049] Disadvantages: The product contains the foul smell of ammonium sulfate and cannot meet the requirements of food-grade enzymes without desalination.

[0050] (2) Organic precipitation method: Use a certain concentration of ethanol to extract papain under different conditions.

[0051] Advantages: High resolution, can be used for protease purification Disadvantages: Many process steps, organic solvents can destroy the hydrogen bonds of proteins, easily cause enzyme denaturation and inactivation, and pollute the environment

[0052] (3) Affinity chromatography: separation and purification of papain by using the affinity of the affinity ligand coupled to the solid phase carrier to the specific macromolecule of papain.

[0053] Advantages: Efficient and rapid separation and purification of proteins

[0054] Disadvantages: complicated operation, not easy to scale up industrial production

[0055] (4) Aqueous two-phase extraction: It utilizes the low interfacial tension of the two phases to maintain biological activity and enhance mass transfer between the two phases. It has been widely used in the extraction of various proteins and enzymes.

[0056] Advantages: Continuous operation, suitable for separation and purification of biological substances

[0057] Disadvantages: Non-degradable, proven to have low ecotoxicity

[0058] We compared the above affinity chromatography method, aqueous two-phase extraction method and the low-temperature plasma-assisted low eutectic solvent extraction method of papain proposed in the present invention, and tested the enzyme yield and enzyme activity respectively.

[0059] Papain is a sulfhydryl (-SH) endopeptidase with protease and esterase activity, broad specificity, and strong hydrolysis ability for plant and animal proteins, peptides, esters, amides, etc. Therefore, we can use bovine serum albumin as a standard and use the Bradford method to determine the protein mass fraction. Casein is used as a substrate to determine the activity of papain. The calculation of total enzyme activity, enzyme activity recovery rate, relative enzyme activity and purification multiple is shown in the following formula (1):

[0060] EA=SAXm

[0061]

[0062]

[0063]

[0064]

[0065] Where: EA is the total enzyme activity added to the system / U; SA is the specific activity of the enzyme / (U / mg); m is the mass of the enzyme / mg. AR is the enzyme activity recovery rate / %; EA T EA is the enzyme activity of the upper phase / (U / mL); VT is the volume of the upper phase / mL. solv is the enzyme activity in the presence of the reagent / (U / mL); EA water is the enzyme activity of the blank control / (U / mL); PF is the purification factor; SA i is the specific activity of the initial enzyme / (U / mg).

[0066] The affinity chromatography method is as follows:

[0067] 1) Agarose activation

[0068] Take 20ml of Sepharose 4B and place it in a Buchner funnel to drain, add a small amount of 0.1Mol / L pH 9.0NaHCO3 solution to wash, immediately transfer to a 100ml beaker, and place it on an ice bath on a magnetic stirrer; weigh 2g of cyanogen bromide in a fume hood, add 20ml of water to dissolve, then pour it into the agarose, carefully add 2Mol / L NaOH to keep the pH at around 11, and react for 10min. Rapidly adjust the pH to 8 within 1min to 2min and maintain it for 10min; quickly pour the activated agarose into a Buchner funnel, wash it with ice water to neutralize it, and then quickly wash it with 250ml of cold 0.1Mol / L pH 9.0NaHCO3.

[0069] 2) Coupling protein

[0070] In advance, place papaya peel in 0.1Mol / L pH 9.0 NaHCO3 solution for dialyzing for several hours (generally the coupling amount is 10-30mg / g carrier) to obtain papain extract; quickly pour the activated agarose into the extract (within 1.5min, from extraction to coupling), and slowly stir overnight at 4℃ to allow papain to combine with the activated agar.

[0071] 3) Column Packing

[0072] Column selection: It should not be too large. Generally, a 1.5×1.5 cm chromatography column can hold about 30 ml of protein-coupled agarose.

[0073] Column loading: Load the protein-coupled agarose into the chromatography column, tighten the lower clamp, let it sink, and loosen the lower clamp after a few minutes to allow the solution to flow out at a rate of about 1 ml / min.

[0074] Wash the column: Wash with 0.2 Mol / L pH 9.0 NaHCO3 (containing 0.1 Mol / L NaCl) until the OD280 of the eluate is less than 0.02.

[0075] Collect all the eluates, and the measured OD280 value × the total ml of the eluate is the content of uncoupled enzyme protein, from which the coupling rate can be calculated.

[0076] 4) Adsorption and desorption

[0077] Add sample at 1 / 10 of the bed volume, with a concentration of 1% to 2%, slowly add the enzyme to be purified, and elute with 0.01Mol / L pH7.4 PBS solution at a flow rate of 1ml / min, until the OD280 of the eluate is less than 0.02; add 0.1Mol / L pH2.4 glycine-HCl buffer at a flow rate of 1ml / min, collect the desorbed components, and immediately neutralize with 1Mol / L NaHCO3 to prevent enzyme protein denaturation.

[0078] 5) Wash with twice the volume of 7Mol / L urea, then wash with 0.01Mol / L pH 7.4 PB solution or saline. After equilibration, it can be used again. For storage, add 0.02% NaN3 and store at 4°C to prevent freezing and cracking.

[0079] 6) Result determination

[0080] The desorbed protein is subjected to disc electrophoresis or two-phase agarose electrophoresis for purity identification; the eluates from each tube with good purity are combined, dialyzed with physiological saline, and then concentrated or freeze-dried for storage to obtain the enzyme product.

[0081] The steps of the aqueous two-phase extraction method are as follows:

[0082] 1) crushing the immature green-skinned papaya, and adding the crushed papaya into a solution consisting of ethylenediaminetetraacetic acid, sodium chloride and cysteine ​​hydrochloride to mix them thoroughly;

[0083] 2) filtering out the insoluble skin residue in the mixed solution, centrifuging the filtrate obtained after filtration at 3-10° C., and freeze-drying to obtain the papaya latex crude enzyme;

[0084] 3) adding the crude enzyme extracted from papaya latex to a quaternary two-phase aqueous system consisting of a phase forming agent and an extraction auxiliary agent, mixing thoroughly, and allowing the mixture to stand until distinct upper and lower phases are formed, wherein the enzyme-containing extract is located in the upper phase, and recovering the upper phase;

[0085] 4) Concentrating the recovered enzyme-containing extract; freeze-drying the concentrate to obtain pure papain;

[0086] The low temperature plasma assisted deep eutectic solvent extraction method is as follows:

[0087] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0088] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 65° C. for 1.5 h to form a homogeneous phase, thereby obtaining a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0089] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber and treated at a high voltage of 35 kV for 2 min to obtain an extract.

[0090] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0091] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0092] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0093] The enzyme yield and enzyme activity of papain were determined according to the above method. The results are shown in Table 1.

[0094] Table 1: Enzyme yield and activity of papain extracted by affinity chromatography, aqueous two-phase extraction, and low-temperature plasma-assisted deep eutectic solvent extraction

[0095]

[0096] The results showed that the enzyme activity of papain extracted by low-temperature plasma-assisted deep eutectic solvent extraction was higher than that by affinity chromatography and aqueous two-phase extraction; the enzyme yield of papain extracted by low-temperature plasma-assisted deep eutectic solvent extraction was higher than that by affinity chromatography and aqueous two-phase extraction.

[0097] Example 2 Comparison of deep eutectic solvents and common solvents

[0098] Common solvents for extracting papain are organic solvents, wherein the existing process uses ethanol and acetone for organic extraction. This example uses the method for determining the enzyme yield and enzyme activity in Example 1 to determine the enzyme yield and enzyme activity of papain extracted by a low eutectic solvent and an organic solvent.

[0099] Among them, a total of six groups of experiments were set up, namely ethanol extraction, acetone extraction, deep eutectic solvent extraction, low-temperature plasma-assisted ethanol extraction, low-temperature plasma-assisted acetone extraction, and low-temperature plasma-assisted deep eutectic solvent extraction. The above organic solvents were all used at a concentration of 60%.

[0100] The specific method is as follows:

[0101] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0102] 2) Preparation of low eutectic solvent: Mix choline chloride and glycerol in a molar ratio of 1:3, heat and stir at 65°C for 1.5h to form a homogeneous phase to obtain a low eutectic solvent, and prepare an extract with a volume fraction of 60% of the low eutectic solvent. (This step is not performed for the organic solvent group)

[0103] 3) Low-temperature plasma treatment: Add 1g of the sample in step (1) to 40mL of a low eutectic solvent (for the organic solvent group, add a 60% organic solvent, stir evenly, and place in a low-temperature refrigerator for 30 minutes), and place them together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treat at 35kV for 2 minutes to obtain an extract. (This step is not performed for the single solvent extraction group)

[0104] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0105] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0106] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0107] The enzyme yield and activity of papain extracted by ethanol, acetone, deep eutectic solvent, low-temperature plasma-assisted ethanol, low-temperature plasma-assisted acetone, and low-temperature plasma-assisted deep eutectic solvent were measured respectively. The results are shown in Table 2.

[0108] Table 2: Enzyme yield and activity of papain extracted from ethanol, acetone, and acetylcholine-glycerol

[0109]

[0110]

[0111] The results showed that organic solvents can destroy the hydrogen bonds of proteins and easily cause denaturation and inactivation, while low eutectic solvents can better protect enzyme activity; low-temperature plasma can significantly improve the extraction rate of papain by low eutectic solvents; the enzyme yield and activity of papain extracted by low eutectic solvents composed of acetylcholine-glycerol are much higher than those extracted by organic solvents; the extraction rate and enzyme activity of papain by low eutectic solvents are higher than those by organic solvents.

[0112] Example 3 Different physical fields assisted extraction of papain by deep eutectic solvents

[0113] The papain extraction method provided by the present invention introduces the use of low-temperature plasma. Compared with the single use of low eutectic solvent extraction method, the low-temperature plasma treatment allows high-energy particles to bombard biological cells, causing etching on their surfaces, resulting in cell wall rupture in a relatively short time, increasing the contact area between protein and low eutectic solvent, increasing the rate at which low eutectic solvent dissolves protein, shortening the extraction time, and having the characteristics of low temperature, rapid and no residue, simple operation, high efficiency and environmental protection. And it can be seen from Example 2 that low-temperature plasma can significantly improve the extraction rate of papain.

[0114] Considering that enzymes are prone to denaturation at high temperatures, this example introduces three low-temperature physical fields: ultrahigh pressure, pulsed electric field, and ultrasound. A total of five groups of experiments are set up: blank control (no physical field), ultrahigh pressure, pulsed electric field, ultrasound, and low-temperature plasma. This is to study the different physical fields to assist the extraction of papain by low eutectic solvents and screen the best auxiliary physical field.

[0115] The specific method is as follows:

[0116] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0117] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 65° C. for 1.5 h to form a homogeneous phase, thereby obtaining a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0118] 3) Physical field treatment: 1 g of the sample in step (1) was added to 40 mL of a deep eutectic solvent, and then one of the following treatments was performed according to the experimental group:

[0119] a) Low-temperature plasma treatment: Put them together in a dielectric barrier discharge low-temperature plasma reaction chamber, treat them at a high voltage of 35kV and a frequency of 50Hz for 2 minutes to obtain an extract. (The control group does not undergo this treatment)

[0120] b) Ultrahigh pressure treatment: put them together into the sample hole of the ultrahigh pressure instrument, fix the pressure holding time to 4 minutes, set the extraction pressure to 500 MPa and perform ultrahigh pressure treatment to obtain the extract.

[0121] c) Pulse electric field treatment: Put them together in a pulse electric field treatment chamber and treat them at a high voltage of 35kV for 2 minutes to obtain an extract

[0122] d) Ultrasonic treatment: put them together in an ultrasonic chamber, set the ultrasonic temperature to 25°C, and the ultrasonic time to 15 minutes to obtain the extract.

[0123] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0124] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to obtain filtrate to remove colloids, polypeptides and other macromolecular organic matter, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain retentate.

[0125] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0126] The extraction rate of papain by the deep eutectic solvents treated by different physical fields was measured, and the results are shown in Table 3.

[0127] Table 3: Extraction rate of papain by deep eutectic solvents assisted by different physical fields

[0128]

[0129] The results showed that compared with ultra-high voltage, pulsed electric field and ultrasound, low-temperature plasma treatment could improve the extraction rate of papain by low eutectic solvent to a greater extent; compared with the control group, low-temperature plasma treatment significantly improved the extraction rate of papain by low eutectic solvent.

[0130] Example 4 Screening for the Optimal Volume Fraction of the Deep Eutectic Solvent

[0131] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0132] 2) Preparation of low eutectic solvent: choline chloride was mixed with glucose, glycerol, polyethylene glycol, n-propanol and propylene glycol at a molar ratio of 1:3, respectively, and heated and stirred at 70°C for 1.5 h to form a homogeneous phase to obtain a low eutectic solvent, which was diluted with water to prepare extracts with volume fractions of 20%, 40%, 60%, 80% and 100% of the low eutectic solvent, respectively.

[0133] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treated at a high voltage of 30 kV and a frequency of 50 Hz for 1 min to obtain an extract.

[0134] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0135] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to obtain filtrate to remove colloids, polypeptides and other macromolecular organic matter, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain retentate.

[0136] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0137] The extraction rate of papain by low-temperature plasma-assisted extraction was determined at different volume fractions of deep eutectic solvents, and the results are shown in Table 4.

[0138] Table 4: Extraction rate of papain assisted by low-temperature plasma at different volume fractions of deep eutectic solvents

[0139]

[0140] The results showed that when the volume fraction of the deep eutectic solvent was 100%, the extraction rate of papain reached the highest.

[0141] Example 5 Screening for the Optimal Hydrogen Bond Acceptor

[0142] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0143] 2) Preparation of low eutectic solvent: Choline chloride, L-proline, betaine and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 80° C. for 1.5 h to form a homogeneous phase to obtain a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0144] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treated at a high voltage of 35 kV and a frequency of 50 Hz for 1 min to obtain an extract.

[0145] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0146] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0147] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0148] The extraction rate of papain extracted by low eutectic solvents prepared by different hydrogen bond acceptors was measured, and the results are shown in Table 5.

[0149] Table 5: Extraction efficiency of papain extracted from deep eutectic solvents prepared with different hydrogen bond acceptors

[0150]

[0151] The results showed that when the hydrogen bond acceptor of the deep eutectic solvent was choline chloride, the extraction rate of papain was the highest.

[0152] In addition, choline chloride has the following advantages:

[0153] (1) Low cost. According to the synthesis mechanism of DESs and the principles of green chemistry, cheap, readily available and environmentally friendly green materials are preferred. They are quite cheap and are often used as hydrogen bond acceptors in the synthesis of DESs.

[0154] (2) Choline chloride-based low eutectic solvents have a good protective effect on proteins.

[0155] (3) DESs based on choline chloride have a large number of hydrogen bonds, which is beneficial for protein extraction.

[0156] (4) Reusable. It can be reused for more than 5 cycles without any significant loss.

[0157] It can be seen that the choline chloride-based low eutectic solvent is not only efficient, but also cheap, easy to obtain, environmentally friendly, conducive to protein extraction and protein protection, and can be reused. Therefore, the low eutectic solvent using choline chloride as a hydrogen bond acceptor is more suitable for the extraction of papain.

[0158] Example 6 Screening for the Optimal Hydrogen Bond Donor

[0159] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0160] 2) Preparation of low eutectic solvent: choline chloride was mixed with glucose, glycerol, polyethylene glycol, n-propanol and propylene glycol in molar ratios of 1:1, 1:2, 1:3 and 1:4, respectively, and heated and stirred at 80°C for 1.5h to form a homogeneous phase to obtain a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0161] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treated at a high voltage of 35 kV and a frequency of 50 Hz for 1 min to obtain an extract.

[0162] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0163] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0164] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0165] The extraction rate of papain extracted by low eutectic solvents prepared by different hydrogen bond donors was measured, and the results are shown in Table 6.

[0166] Table 6: Extraction efficiency of papain assisted by low temperature plasma at different HBA / HBD molar ratios

[0167]

[0168] The results showed that the low eutectic solvent composition was a choline chloride-glycerol molar ratio of 1:3, and the extraction rate reached the highest, which was 92%.

[0169] Example 7 Screening for the Optimal Preparation Temperature of a Low Eutectic Solvent

[0170] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0171] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 60, 70, 80, 90, and 100° C. for 1.5 h to form a homogeneous phase to obtain a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0172] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treated at a high voltage of 35 kV and a frequency of 50 Hz for 1 min to obtain an extract.

[0173] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0174] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0175] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0176] The effects of low eutectic solvents prepared at different temperatures on the extraction rate of papain were determined, and the results are shown in Table 7.

[0177] Table 7: Extraction rate of papain by low temperature plasma assisted deep eutectic solvent extraction at different temperatures

[0178]

[0179] The results showed that the extraction effect of choline chloride-glycerol was best when stirred at 65℃ for 1.5h, and the extraction rate was 94%.

[0180] Example 8 Screening for the Optimal Low-Temperature Plasma

[0181] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0182] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 65° C. for 1.5 h to form a homogeneous phase, thereby obtaining a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0183] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and placed together in a glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, or jet discharge low-temperature plasma reaction chamber, and treated for 1 min respectively to obtain an extract.

[0184] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0185] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0186] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0187] The extraction rate of papain under different low-temperature plasma working conditions was determined, and the results are shown in Table 8.

[0188] Table 8: Extraction rate of papain after 1 min of different low-temperature plasma treatments

[0189]

[0190] The results showed that the extraction rate of papain was the highest under the action of dielectric barrier discharge low-temperature plasma.

[0191] Example 9 Screening the Optimal Low-Temperature Plasma Working Gas

[0192] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0193] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 65° C. for 1.5 h to form a homogeneous phase, thereby obtaining a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0194] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, wherein the working gases were argon, helium, air, a mixture of helium and oxygen, and a mixture of argon and oxygen, respectively, and the treatment was performed for 1 min to obtain an extract.

[0195] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0196] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0197] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0198] The extraction rate of papain under different working gases of low-temperature plasma was measured, and the results are shown in Table 9.

[0199] Table 9: Extraction rate of papain after low-temperature plasma treatment with different working gases for 1 min

[0200]

[0201] The results showed that the extraction rate of papain was the highest when air was used as the working gas of low-temperature plasma.

[0202] Example 10 Screening for the Optimal Low-Temperature Plasma Operating Voltage

[0203] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0204] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 65° C. for 1.5 h to form a homogeneous phase, thereby obtaining a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0205] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treated at high voltages of 15, 20, 25, 30, 35, 40, and 45 kV and a frequency of 50 Hz for 1 min to obtain an extract.

[0206] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0207] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0208] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0209] The extraction rate of papain under different working voltages of low-temperature plasma was determined, and the results are shown in Table 10.

[0210] Table 10: Extraction rate of papain after low-temperature plasma treatment at different voltages for 1 min

[0211]

[0212] The results showed that the extraction rate of papain was the highest when the low-temperature plasma voltage was 30 kV and the treatment time was 1 min, and the extraction rate of papain was 95%.

[0213] Example 11 Screening for the Optimal Low-Temperature Plasma Treatment Time

[0214] 1) Raw material processing: Take fresh papaya, wash and peel it, and cut the papaya peel into small pieces.

[0215] 2) Preparation of low eutectic solvent: Choline chloride and glycerol were mixed in a molar ratio of 1:3, heated and stirred at 80° C. for 1.5 h to form a homogeneous phase, thereby obtaining a low eutectic solvent, and an extract having a volume fraction of 100% of the low eutectic solvent was prepared.

[0216] 3) Low-temperature plasma treatment: 1 g of the sample in step (1) was added to 40 mL of a low eutectic solvent and then placed together in a dielectric barrier discharge low-temperature plasma reaction chamber, and treated at a high voltage of 35 kV and a frequency of 50 Hz for 1, 2, 3, and 4 min, respectively, to obtain an extract.

[0217] 4) Cysteine ​​salt is added as a protective agent, and the concentration of cysteine ​​salt in the papain solution is 0.05 mol / L.

[0218] 5) Multi-stage membrane separation: Use 0.02 micron ceramic membrane microfiltration to remove impurities and insoluble components in the extract, use 14 nanometer ceramic membrane ultrafiltration to remove colloids, peptides and other macromolecular organic matter to obtain the filtrate, and finally use 10 nanometer ceramic membrane nanofiltration to separate small molecular impurities to obtain the retentate

[0219] 6) Freeze drying: set the process parameters to -30°C for 12 hours to obtain papain.

[0220] The extraction rate of papain under different treatment times was determined, and the results are shown in Table 11.

[0221] Table 11 Extraction rate of papain after treatment at different times under low temperature plasma 35kV

[0222]

[0223] The results showed that when the low-temperature plasma voltage was 35 kV and the processing time was 2 min, the extraction rate of papain was the highest, and the extraction rate of papain was 92%.

[0224] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting papain using a low temperature plasma assisted deep eutectic solvent, characterized in that: Papaya peel and a low eutectic solvent aqueous solution are mixed, low-temperature plasma extraction is performed to obtain an extract, a cysteine ​​salt protective agent is added to the extract, and then papain is obtained by multi-stage membrane separation and freeze-drying; wherein the low eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is glycerol; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the low eutectic solvent is 1:3; the preparation method of the low eutectic solvent is that the hydrogen bond acceptor and the hydrogen bond donor are mixed in a molar ratio of 1:3, and stirred at 60-80°C for 1.5h; the volume fraction of the low eutectic solvent is 100%; the discharge form of the low-temperature plasma is selected as dielectric barrier discharge; the working voltage of the low-temperature plasma is 20-40kv, and the processing time is 2-3min; the working gas of the low-temperature plasma is selected as air.

2. The method for extracting papain using a low temperature plasma-assisted deep eutectic solvent as claimed in claim 1, characterized in that: The stirring temperature for preparing the hydrogen bond acceptor and the hydrogen bond donor is 65°C.

3. The method for extracting papain using a low temperature plasma-assisted deep eutectic solvent as claimed in claim 1, characterized in that: The low-temperature plasma operating voltage is 35 kV.

4. The method for extracting papain using a low temperature plasma assisted deep eutectic solvent as claimed in claim 1, characterized in that: The low-temperature plasma treatment time is 2 minutes.

5. The method for extracting papain using a low temperature plasma assisted deep eutectic solvent as claimed in claim 1, characterized in that: Relative to 1 g of papaya peel sample, the amount of the low eutectic solvent used is 10-60 mL.

6. The method for extracting papain using a low temperature plasma assisted deep eutectic solvent as claimed in claim 5, characterized in that: The amount of the deep eutectic solvent used is 40 mL.

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