Method for extracting vegetable protein by high hydrostatic pressure assisted deep eutectic solvent

Abstract

The invention belongs to the technical field of extraction, and particularly relates to a high hydrostatic pressure assisted deep eutectic solvent extraction method for plant protein, which comprises the following steps: S1, mixing a plant raw material and a deep eutectic solvent to obtain a mixed material liquid; and S2, carrying out high hydrostatic pressure treatment on the mixed material liquid to obtain an extracting solution containing the vegetable protein. According to the extraction method disclosed by the invention, the plant raw material and the deep eutectic solvent are firstly mixed, and then the plant protein in the plant raw material is extracted by assisting high hydrostatic pressure treatment, so that the extraction method has the advantages of high extraction efficiency of the plant protein, reduction of degradation of heat-sensitive components, short extraction time, simplicity, small influence on the environment and the like; the vegetable protein obtained by the extraction method has the advantages of high digestibility, good emulsibility and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of extraction technology, and in particular relates to a method for extracting plant protein with the assistance of a high hydrostatic pressure deep eutectic solvent. Background Art

[0002] Traditional methods for extracting plant proteins primarily include physical disruption, chemical solvent extraction, and enzymatic hydrolysis. While these methods are effective to a certain extent, they each have limitations. Physical disruption often consumes a lot of energy, is inefficient, and can damage the structure and function of proteins. Chemical solvent extraction can lead to residual solvents, posing potential risks to the environment and human health. Enzymatic hydrolysis, however, is limited by the specificity and action conditions of enzymes, limiting its efficiency and scope of application.

[0003] Therefore, there is an urgent need to develop a plant protein extraction method that is efficient, environmentally friendly, low in application cost and can maintain protein activity. Summary of the Invention

[0004] The present invention aims to address, at least to a certain extent, at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for extracting plant protein using a high hydrostatic pressure-assisted deep eutectic solvent. This method of extracting plant protein has the advantages of high plant protein extraction efficiency, reduced degradation of heat-sensitive components, short extraction time, simplicity, and minimal environmental impact. The plant protein obtained using this method has advantages such as high digestibility and good emulsification properties, and has broad application prospects.

[0005] The present invention proposes a method for extracting plant protein. According to an embodiment of the present invention, the method comprises: S1: mixing plant raw materials and deep eutectic solvents to obtain a mixed liquid; S2: subjecting the mixed liquid to high hydrostatic pressure treatment to obtain an extract containing plant protein. According to the method for extracting plant protein of an embodiment of the present invention, the plant raw materials and deep eutectic solvents are first mixed, and then high hydrostatic pressure treatment is performed to extract the plant protein from the plant raw materials. The extraction method has the advantages of high plant protein extraction efficiency, reduced degradation of heat-sensitive components, short extraction time, simple method and small impact on the environment. The plant protein obtained by the extraction method of the present invention has the advantages of high digestibility, good emulsification and the like, and has broad application prospects.

[0006] According to an embodiment of the present invention, the above method for extracting plant protein may also have the following additional technical features:

[0007] According to an embodiment of the present invention, the pressure of the high hydrostatic pressure treatment is 100 to 500 MPa, and the time of the high hydrostatic pressure treatment is 5 to 25 minutes.

[0008] According to an embodiment of the present invention, the hydrogen bond acceptor of the deep eutectic solvent includes one or more of choline chloride, betaine and L-proline.

[0009] According to an embodiment of the present invention, the hydrogen bond donor of the deep eutectic solvent includes one or more of urea, 1,4-butanediol and glycerol.

[0010] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is (1-5):(1-5).

[0011] According to a preferred embodiment of the present invention, the hydrogen bond acceptor of the deep eutectic solvent is choline chloride, the hydrogen bond donor is urea, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-3).

[0012] According to an embodiment of the present invention, the preparation method of the deep eutectic solvent includes: mixing a hydrogen bond acceptor and a hydrogen bond donor and performing a heat treatment; the heating temperature is 60 to 100° C. and the time is 1 to 60 minutes.

[0013] According to an embodiment of the present invention, the water content of the deep eutectic solvent is 0% to 40%.

[0014] According to an embodiment of the present invention, the material-liquid ratio of the plant raw material to the deep eutectic solvent is 1:(15-35), and the unit is g / mL.

[0015] According to an embodiment of the present invention, the plant raw material includes one or more of broccoli, cauliflower, wheat, peas, soybeans and almonds.

[0016] According to an embodiment of the present invention, the particle size of the plant raw material is ≤1 mm.

[0017] According to an embodiment of the present invention, the method further comprises: separating the extract containing the plant protein to obtain a plant protein sample.

[0018] According to an embodiment of the present invention, the separation treatment is performed under the condition of retaining plant proteins larger than 1000Da in the extract containing plant proteins.

[0019] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0021] Figure 1Graphs showing the single-factor experimental results of high hydrostatic pressure-assisted deep eutectic solvent extraction of broccoli plant protein in Examples 4 to 27 of the present invention, wherein (A) is a graph showing the single-factor experimental results of different HBDs, (B) is a graph showing the single-factor experimental results of different molar ratios of HBA to HBD, (C) is a graph showing the single-factor experimental results of different water contents of DES, (D) is a graph showing the single-factor experimental results of different material-liquid ratios, (E) is a graph showing the single-factor experimental results of different high hydrostatic pressures, and (F) is a graph showing the single-factor experimental results of different high hydrostatic pressure treatment times. a, b, and c are used to indicate significance, with a being the highest, b being significantly different from the highest value, and c being the lowest and significantly different from the highest and second-highest values.

[0022] Figure 2 2 is a graph showing the digestibility measurement results of broccoli plant protein obtained under different extraction conditions in Example 28 of the present invention, wherein ALK-BPI is a graph showing the digestibility measurement results of broccoli protein extracted by method 1, DES-BPI is a graph showing the digestibility measurement results of broccoli protein extracted by method 2, and HDES-BPI is a graph showing the digestibility measurement results of broccoli protein extracted by method 3;

[0023] Figure 3 2 is a graph showing the particle size distribution of broccoli protein obtained under different extraction conditions in Example 28 of the present invention, wherein ALK-BPI is a graph showing the particle size distribution of broccoli protein extracted by method 1, DES-BPI is a graph showing the particle size distribution of broccoli protein extracted by method 2, and HDES-BPI is a graph showing the particle size distribution of broccoli protein extracted by method 3. a, b, and c are used to indicate significance, with a being the highest, b being significantly different from the highest value, and c being the lowest and significantly different from the highest and second-highest values.

[0024] Figure 4 The figure is an electrophoresis result diagram of broccoli plant protein obtained under different extraction conditions in Example 28 of the present invention, wherein BP is unextracted broccoli powder, ALK-BPI is an electrophoresis result diagram of broccoli protein extracted by method 1, DES-BPI is an electrophoresis result diagram of broccoli protein extracted by method 2, and HDES-BPI is an electrophoresis result diagram of broccoli protein extracted by method 3;

[0025] Figure 5 2 are graphs showing the emulsification test results of broccoli plant protein obtained under different extraction conditions in Example 28 of the present invention, wherein ALK-BPI is a graph showing the emulsification test results of broccoli protein extracted by method 1, DES-BPI is a graph showing the emulsification test results of broccoli protein extracted by method 2, and HDES-BPI is a graph showing the emulsification test results of broccoli protein extracted by method 3. Value a is the highest, value b is significantly different from the highest value, and value c is the lowest and is significantly different from the highest and second highest values. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0027] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0028] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0029] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0030] The present invention proposes a method for extracting plant protein. According to an embodiment of the present invention, the method comprises: S1: mixing plant raw materials and deep eutectic solvents to obtain a mixed liquid; S2: subjecting the mixed liquid to high hydrostatic pressure treatment to obtain an extract containing plant protein. According to the method for extracting plant protein of an embodiment of the present invention, the plant raw materials and deep eutectic solvents are first mixed, and then high hydrostatic pressure treatment is performed to extract the plant protein from the plant raw materials. The extraction method has the advantages of high plant protein extraction efficiency, reduced degradation of heat-sensitive components, short extraction time, simple method and small impact on the environment. The plant protein obtained by the extraction method of the present invention has the advantages of high digestibility, good emulsification and the like, and has broad application prospects.

[0031] According to an embodiment of the present invention, the pressure of the high hydrostatic pressure treatment is 300-700 MPa, and the time of the high hydrostatic pressure treatment is 5-30 min. Thus, by performing high hydrostatic pressure treatment under appropriate pressure and time, the plant cell walls and cell membranes of the plant raw materials can be effectively destroyed, and the extraction efficiency of plant protein can be improved while maintaining the activity and function of the protein. Exemplarily, the pressure of the high hydrostatic pressure treatment is 100 MPa, 200 MPa, 300 MPa, 400 MPa, 500 MPa, preferably 300-500 MPa, more preferably 400 MPa; the time of the high hydrostatic pressure treatment is 5 min, 10 min, 15 min, 20 min, 25 min, preferably 10-20 min, more preferably 10 min.

[0032] According to an embodiment of the present invention, the hydrogen bond acceptor of the deep eutectic solvent includes one or more of choline chloride, betaine, and L-proline. Thus, through the interaction with hydrogen bonds in proteins, the release of proteins is promoted, further improving the extraction efficiency of plant proteins.

[0033] According to an embodiment of the present invention, the hydrogen bond donor of the deep eutectic solvent includes one or more of urea, lactic acid, maltose, D-glucose, and glycerol. This allows for selection of the most suitable donor based on the properties of different plant proteins to optimize extraction efficiency and selectivity, further improving the extraction efficiency of different plant proteins.

[0034] According to an embodiment of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is 1:(1-3). Thus, by adjusting the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor, a better plant protein extraction effect can be achieved. Exemplarily, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is 1:1, 1:1.5, 1:2, 1:2.5, 1:3, preferably 1:2.

[0035] According to a preferred embodiment of the present invention, the hydrogen bond acceptor of the deep eutectic solvent is choline chloride, the hydrogen bond donor is urea, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: (1 to 3). Thus, when broccoli plant protein is extracted, the hydrogen bond acceptor of the deep eutectic solvent is choline chloride, the hydrogen bond donor is urea, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: (1 to 3), the broccoli plant protein extraction efficiency is higher. Exemplarily, when the hydrogen bond acceptor of the deep eutectic solvent is choline chloride and the hydrogen bond donor is urea, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: 1, 1: 1.5, 1: 2, 1: 2.5, 1: 3, preferably 1: 2.

[0036] According to an embodiment of the present invention, the preparation method of the deep eutectic solvent comprises: mixing and heating a hydrogen bond acceptor and a hydrogen bond donor; the temperature of the heating treatment is 60 to 100°C, and the time is 1 to 60 minutes. Thus, a stable deep eutectic solvent is obtained, which can be used for the extraction of plant protein. Exemplarily, the temperature of the heating treatment is 60°C, 70°C, 80°C, 90°C, 100°C, preferably 80 to 100°C, more preferably 90°C; the time of the heating treatment is 1 minute, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, preferably 10 to 60 minutes, more preferably 60 minutes.

[0037] According to an embodiment of the present invention, the water content of the deep eutectic solvent is 0% to 40%. Thus, by adding water after the deep eutectic solvent is prepared, the fluidity of the deep eutectic solvent is enhanced, thereby further improving the extraction efficiency of plant protein. Exemplarily, the water content of the deep eutectic solvent is 0%, 10%, 20%, 30%, or 40%, preferably 20-40%, and more preferably 20%.

[0038] According to an embodiment of the present invention, the solid-liquid ratio of the plant raw material to the deep eutectic solvent is 1:(15-35), expressed in g / mL. Thus, the plant raw material and the deep eutectic solvent are fully mixed to further improve the extraction efficiency of plant protein. Exemplarily, the solid-liquid ratio of the plant raw material to the deep eutectic solvent is 1:15, 1:20, 1:25, 1:30, 1:35, preferably 1:(25-1:35), and more preferably 1:25.

[0039] According to an embodiment of the present invention, the plant raw material comprises one or more of broccoli, cauliflower, wheat, peas, soybeans and almonds. Thus, the method for extracting plant protein according to the present invention is applicable to different plant raw materials.

[0040] According to an embodiment of the present invention, the particle size of the plant material is ≤1 mm. Thus, the plant material is fully in contact with the deep eutectic solvent, so that the deep eutectic solvent fully acts on the plant material, further improving the extraction efficiency of the plant protein.

[0041] According to an embodiment of the present invention, the method further comprises: separating the plant protein-containing extract to obtain a plant protein sample. Thus, through separation treatments such as dialysis, a plant protein sample of higher purity is obtained from the plant protein-containing extract, further improving the extraction efficiency of the plant protein.

[0042] According to an embodiment of the present invention, the separation treatment is performed under conditions such as to retain plant proteins larger than 1000 Da in the plant protein-containing extract, thereby separating plant proteins within a specific size range by controlling the molecular weight cut-off of the separation membrane.

[0043] In the embodiment of the present invention, before extracting the plant protein from broccoli, a stable deep eutectic solvent must be prepared. The preparation of the deep eutectic solvent requires two parts: hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). The naming and preparation method of the deep eutectic solvent are as follows:

[0044] Take hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs), mix them in a certain molar ratio, heat them at 80 ° C under magnetic stirring and stir them vigorously to form a uniform transparent liquid deep eutectic solvent. The deep eutectic solvent is named according to the specific names and molar ratios of hydrogen bond acceptors and hydrogen bond donors. 氢键受体名称-氢键供体名称(氢键受体与氢键供体的摩尔比)After cooling, the deep eutectic solvent 氢键受体名称-氢键供体名称(氢键受体与氢键供体的摩尔比) Stored at room temperature for later use.

[0045] The schemes of the present application will be explained below with examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained commercially.

[0046] Example 1: Screening of deep eutectic solvents for extracting broccoli plant protein

[0047] 1. Preparation of deep eutectic solvent

[0048] Based on the literature review, the inventors preliminarily judged that the following deep eutectic solvents could be used for the extraction of broccoli plant protein after preparation. The systems involved in the preparation of deep eutectic solvents are shown in Table 1.

[0049] Table 1: Preparation and name of deep eutectic solvents

[0050]

[0051] 2. Pretreatment of broccoli raw material

[0052] Fresh broccoli was washed to remove dirt, freeze-dried, broken by a cell crusher, and sieved to obtain broccoli powder for later use.

[0053] 3. Extraction of broccoli plant protein

[0054] Three 1g portions of broccoli powder were accurately weighed and immersed in 25mL of the above candidate deep eutectic solvents, respectively. The mixture was vortexed for 30 seconds until a uniform thick mixture was obtained. The uniform thick mixture was subjected to high hydrostatic pressure assisted extraction treatment, with a high hydrostatic pressure of 400MPa and a high hydrostatic pressure treatment time of 10min, to obtain a broccoli mixture. The mixture was magnetically stirred at 600rpm at 25℃ for 1h, and then centrifuged at 4℃ at a speed of 6000rpm for 5min. The supernatant was collected to obtain a broccoli plant protein extract. The broccoli mixture was centrifuged at 4℃ at a speed of 6000rpm for 5min, and the supernatant was collected to obtain a broccoli plant protein extract. The broccoli plant protein extract was subjected to dialysis treatment (with a cutoff range of 1000Da or above) for 3 days, and then freeze-dried to obtain a crude broccoli protein extract.

[0055] 4. Determination and calculation of broccoli plant protein content

[0056] The method for determining the content of plant protein in broccoli is the BCA method. 0.1 mL of broccoli protein extract is used to calculate the content of plant protein in the protein extract sample using the BCA method. The parallel determination and calculation are performed three times, which are recorded as A1, A2, and A3. The average value is recorded as A 平均 , and then calculate the broccoli plant protein content in the broccoli protein extract based on the following formula:

[0057] A=m1 / m

[0058] Wherein, A is the broccoli plant protein content in the crude broccoli protein extract, in mg / g; m1 is the broccoli plant protein content in the crude protein extract sample calculated by the BCA method, in mg; and m is the mass of broccoli powder, in g.

[0059] The extraction results of broccoli plant protein using different deep eutectic solvent extraction systems are shown in Table 2.

[0060] Table 2 Extraction results of broccoli plant protein in different deep eutectic solvent extraction systems

[0061] serial number Name of deep eutectic solvent <![CDATA[A1]]> [A2] [A3] <![CDATA[A 平均 ]]> Total plant protein content (mg / g) 1 <![CDATA[DES 氯化胆碱-尿素(1:2) ]]> 12.71 13.31 12.88 12.97 129.68 2 <![CDATA[DES 氯化胆碱-1,4-丁二醇(1:2) ]]> 8.52 7.22 7.18 7.64 76.39 3 DES 氯化胆碱-甘油(1:2) ]]> 10.29 9.17 9.90 9.79 97.89

[0062] The experimental results show that among the candidate deep eutectic solvents, when the hydrogen bond acceptor is choline chloride and the hydrogen bond donor is urea, the total plant protein extraction content is higher; among them, the deep eutectic solvent DES is selected 氯化胆碱-尿素(1:2) , the total plant protein extraction content was the highest, at 129.68 mg / g, which was much higher than the total plant protein content level of broccoli plant protein extracted using other deep eutectic solvent systems.

[0063] Example 2: Screening of methods for extracting broccoli plant protein using assisted deep eutectic solvents

[0064] In order to improve the extraction efficiency of broccoli plant protein by deep eutectic solvent, the inventors used deep eutectic solvent DES 氯化胆碱-尿素(1:2) Taking the extraction system as an example, the methods of assisting deep eutectic solvents in extracting broccoli plant protein were screened. The steps are as follows:

[0065] 1. Pretreatment of broccoli raw materials

[0066] Refer to step 2 in Example 1.

[0067] 2. Preparation of deep eutectic solvent DES 氯化胆碱-尿素(1:2)

[0068] The deep eutectic solvent DES was carried out with choline chloride as hydrogen bond acceptor (HBAs) and urea as hydrogen bond donor (HBDs) at a molar ratio of choline chloride to urea of ​​1:2. 氯化胆碱-尿素(1:2) Preparation.

[0069] 3. Treatment group settings

[0070] Take 1g of broccoli powder and add deep eutectic solvent DES 氯化胆碱-尿素(1:2) In addition, high hydrostatic pressure treatment, ~ ~ for DES 氯化胆碱-尿素(1:2) The extraction system was processed, and the specific parameter settings are shown in Table 3.

[0071] Treatment group 1: high hydrostatic pressure treatment (400 MPa, 10 min).

[0072] Treatment group 2: Same as treatment group 1, except that high hydrostatic pressure treatment was not performed.

[0073] Table 3 Auxiliary means condition parameter settings

[0074] Group Extraction auxiliary means and parameters 1 High hydrostatic pressure treatment (400MPa, 10min) 2 No high hydrostatic pressure treatment

[0075] 4. Determination and calculation of plant protein content in broccoli

[0076] The protein content of the crude broccoli protein extract obtained from each treatment group was determined. The specific determination and calculation methods refer to step 4 of Example 1.

[0077] The results of protein content determination in each treatment group are shown in Table 4.

[0078] Table 4 Effects of different auxiliary extraction methods on DES 氯化胆碱-尿素(1:1) Effect of extraction system on plant protein extraction efficiency

[0079]

[0080]

[0081] The results showed that the total plant protein content was ranked as 1>2.

[0082] The above results show that the high hydrostatic pressure-assisted deep eutectic solvent extraction system is used to extract broccoli plant protein, and the plant protein extraction effect is better.

[0083] Example 3: Extraction of plant protein from broccoli using high hydrostatic pressure-assisted deep eutectic solvent

[0084] 1. Preparation and processing of broccoli

[0085] Refer to step 2 in Example 1.

[0086] 2. Preparation of deep eutectic solvent

[0087] The deep eutectic solvent DES was carried out with choline chloride as hydrogen bond acceptor (HBAs) and urea as hydrogen bond donor (HBDs) at a molar ratio of choline chloride to urea of ​​1:2. 氯化胆碱-尿素(1:2) Preparation.

[0088] By adding water to the prepared deep eutectic solvent, the viscosity of the deep eutectic solvent can be significantly reduced, thereby enhancing the flowability of the deep eutectic solvent to ensure the efficiency of the deep eutectic solvent in extracting the broccoli plant protein. Here, the required water content of the deep eutectic solvent is 20%, so 20% water is added after the preparation of the deep eutectic solvent.

[0089] 3. Extraction of broccoli plant protein

[0090] According to the solid-liquid ratio of 1:25 (Note: solid-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1 g of broccoli powder was accurately weighed and immersed in 25 mL of the deep eutectic solvent DES prepared in step 2, and vortexed for 30 seconds until a uniform thick mixture was obtained; the uniform thick mixture was subjected to high hydrostatic pressure assisted extraction treatment, the high hydrostatic pressure was 400 MPa, and the high hydrostatic pressure treatment time was 10 min, to obtain a broccoli mixture; the mixture was subjected to magnetic stirring at 600 rpm at 25°C for 1 h, and the broccoli mixture was centrifuged at 6000 rpm at 4°C for 5 min, and the supernatant was collected to obtain a broccoli plant protein extract; the broccoli plant protein extract was subjected to dialysis treatment (with a cutoff range of 1000 Da or more) for 3 days, and then freeze-dried to obtain a crude broccoli protein extract. 氯化胆碱-尿素(1:2)

[0091] 4. Determination and calculation of the content of broccoli plant protein

[0092] Reference Example 1, step 4.

[0093] Examples 4-27: Optimization of the extraction conditions for the high hydrostatic pressure assisted deep eutectic solvent extraction of plant protein from broccoli

[0094] Based on the selection of choline chloride and urea as the HBA and HBD, respectively, of the deep eutectic solvent for extracting broccoli plant protein, the inventors further investigated the effects of the molar ratio of HBD to HBA, the water content of the deep eutectic solvent, the solid-liquid ratio, the high hydrostatic pressure, and the high hydrostatic pressure time on the extraction content of broccoli plant protein by single-factor experiments.

[0095] The differences between Examples 4-27 and Example 3 are shown in Table 5, wherein:

[0096] 1. Optimization of the molar ratio of HBD to HBA

[0097] In Examples 3-7, the inventors optimized the molar ratio of HBD to HBA. The specific differences between Examples 4-8 and Example 3 are shown in step 2, as follows:

[0098] ​In Example 4, the hydrogen bond acceptor (HBAs) was choline chloride, the hydrogen bond donor (HBDs) was urea, and the molar ratio of choline chloride to urea was 1:1.5. 氯化胆碱-尿素(1:1) The water content of the deep eutectic solvent is 0%, and 20% water is added after the deep eutectic solvent is prepared.

[0099] In Example 5, the hydrogen bond acceptor (HBAs) is choline chloride, the hydrogen bond donor (HBDs) is urea, and the molar ratio of choline chloride to urea is 1:2. 氯化胆碱-尿素(1:1.5) The water content of the deep eutectic solvent is 0%, and 20% water is added after the deep eutectic solvent is prepared.

[0100] In Example 6, the hydrogen bond acceptor (HBAs) was choline chloride, the hydrogen bond donor (HBDs) was urea, and the molar ratio of choline chloride to urea was 1:2.5. 氯化胆碱-尿素(1:2.5) The water content of the deep eutectic solvent is 0%, and 20% water is added after the deep eutectic solvent is prepared.

[0101] In Example 7, the hydrogen bond acceptor (HBAs) is choline chloride, the hydrogen bond donor (HBDs) is urea, and the molar ratio of choline chloride to urea is 1:3. 氯化胆碱-尿素(1:3) The water content of the deep eutectic solvent is 0%, and 20% water is added after the deep eutectic solvent is prepared.

[0102] 2. Optimization of water content in deep eutectic solvents

[0103] In Examples 8 to 12, the inventors optimized the water content of the deep eutectic solvent. The specific difference between Examples 8 to 12 and Example 3 lies in step 2, as follows:

[0104] In Examples 8 to 12, the hydrogen bond acceptor (HBAs) was choline chloride, the hydrogen bond donor (HBDs) was urea, and the molar ratio of choline chloride to urea was 1:2. 氯化胆碱-尿素(1:2) Preparation.

[0105] In Example 8, the water content of the deep eutectic solvent required by the deep eutectic solvent is 0%, so after the deep eutectic solvent is prepared, no water addition operation is performed.

[0106] In Example 9, the deep eutectic solvent required a water content of 10%. Therefore, after the deep eutectic solvent was prepared, water was added according to the required water content of 10% of the deep eutectic solvent. 氯化胆碱-尿素(1:2) =1:9 add water, shake thoroughly, make the deep eutectic solvent DES 氯化胆碱-尿素(1:2) Mix well with water.

[0107] Example 10 is the same as Example 3.

[0108] In Example 11, the deep eutectic solvent required a water content of 30%. Therefore, after the deep eutectic solvent was prepared, water was added according to the required water content of 30% of the deep eutectic solvent. 氯化胆碱-尿素(1:2) =3:7 Add water and shake thoroughly to make the deep eutectic solvent DES 氯化胆碱-尿素(1:2) Mix well with water.

[0109] In Example 12, the deep eutectic solvent required a water content of 40%. Therefore, after the deep eutectic solvent was prepared, water was added according to the required water content of 40% of the deep eutectic solvent. The water: deep eutectic solvent DES 氯化胆碱-尿素(1:2) =4:6 Add water and shake thoroughly to make the deep eutectic solvent DES 氯化胆碱-尿素(1:2) Mix well with water.

[0110] 3. Optimization of material-liquid ratio

[0111] In Examples 13 to 17, the inventors optimized the material-liquid ratio. The specific difference between Examples 13 to 17 and Example 3 lies in step 3, as follows:

[0112] In Example 12, according to the material-liquid ratio of 1:15 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1g of broccoli powder was accurately weighed and soaked in 20mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 600 rpm for 1 hour at 25° C., and the broccoli mixture was centrifuged at 6000 rpm for 5 minutes at 4° C. to collect the supernatant to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of 1000 or more) and freeze-dried to obtain a crude broccoli protein extract.

[0113] In Example 14, according to the material-liquid ratio of 1:20 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1g of broccoli powder was accurately weighed and soaked in 25mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2)In step 3, the mixture was vortexed for 30 seconds until a uniform thick mixture was obtained; the uniform thick mixture was subjected to high hydrostatic pressure assisted extraction treatment, the high hydrostatic pressure pressure was 400 MPa, the high hydrostatic pressure treatment time was 10 min, and a broccoli mixture was obtained; the mixture was subjected to magnetic stirring at 600 rpm at 25 °C for 1 h; the broccoli mixture was centrifuged at 4 °C at a speed of 6000 rpm for 5 min, and the supernatant was collected to obtain a broccoli plant protein extract; the broccoli plant protein extract was subjected to dialysis treatment (with a cutoff range of 1000 Da or more) for 3 days, and was subjected to freeze-drying to obtain a crude broccoli extract.

[0114] Example 15 is the same as Example 3.

[0115] In Example 16, 1 g of broccoli powder was accurately weighed according to a solid-liquid ratio of 1:30 (Note: solid-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), and was immersed in 30 mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) In step 3, the mixture was vortexed for 30 seconds until a uniform thick mixture was obtained; the uniform thick mixture was subjected to high hydrostatic pressure assisted extraction treatment, the high hydrostatic pressure pressure was 400 MPa, the high hydrostatic pressure treatment time was 10 min, and a broccoli mixture was obtained; the mixture was subjected to magnetic stirring at 600 rpm at 25 °C for 1 h; the broccoli mixture was centrifuged at 4 °C at a speed of 6000 rpm for 5 min, and the supernatant was collected to obtain a broccoli plant protein extract; the broccoli plant protein extract was subjected to dialysis treatment (with a cutoff range of 1000 Da or more) for 3 days, and was subjected to freeze-drying to obtain a crude broccoli extract.

[0116] In Example 17, 1 g of broccoli powder was accurately weighed according to a solid-liquid ratio of 1:35 (Note: solid-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), and was immersed in 35 mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) In step 3, the mixture was vortexed for 30 seconds until a uniform thick mixture was obtained; the uniform thick mixture was subjected to high hydrostatic pressure assisted extraction treatment, the high hydrostatic pressure pressure was 400 MPa, the high hydrostatic pressure treatment time was 10 min, and a broccoli mixture was obtained; the mixture was subjected to magnetic stirring at 600 rpm at 25 °C for 1 h; the broccoli mixture was centrifuged at 4 °C at a speed of 6000 rpm for 5 min, and the supernatant was collected to obtain a broccoli plant protein extract; the broccoli plant protein extract was subjected to dialysis treatment (with a cutoff range of 1000 Da or more) for 3 days, and was subjected to freeze-drying to obtain a crude broccoli extract.

[0117] 4. Optimization of high hydrostatic pressure pressure

[0118] In Examples 18 to 22, the inventors optimized the high hydrostatic pressure. The specific difference between Examples 18 to 22 and Example 3 lies in step 3, as follows:

[0119] In Example 18, according to the material-liquid ratio of 1:25 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1g of broccoli powder was accurately weighed and soaked in 25mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 25°C and 600 rpm for 1 hour, and the broccoli mixture was centrifuged at 4°C and 6000 rpm for 5 minutes, and the supernatant was collected to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of 1000 Da or more) and freeze-dried to obtain a crude broccoli protein extract.

[0120] In Example 19, according to the material-liquid ratio of 1:25 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1g of broccoli powder was accurately weighed and soaked in 25mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 600 rpm for 1 h at 25 ° C, and the broccoli mixture was centrifuged at 6000 rpm for 5 min to obtain a broccoli plant protein extract; the broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of more than 1000 Da), and freeze-dried to obtain a crude broccoli protein extract.

[0121] In Example 20, according to the material-liquid ratio of 1:25 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1g of broccoli powder was accurately weighed and soaked in 25mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2)The broccoli mixture was stirred at 600 rpm for 1 h at 25 ° C, and the broccoli mixture was centrifuged at 6000 rpm for 5 min to obtain a broccoli plant protein extract; the broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of more than 1000 Da), and freeze-dried to obtain a crude broccoli protein extract.

[0122] Example 21 is the same as Example 3.

[0123] In Example 22, according to the material-liquid ratio of 1:25 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 1g of broccoli powder was accurately weighed and soaked in 25mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 600 rpm for 1 h at 25 ° C, and the broccoli mixture was centrifuged at 6000 rpm for 5 min to obtain a broccoli plant protein extract; the broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of more than 1000 Da), and freeze-dried to obtain a crude broccoli protein extract.

[0124] 5. Optimization of extraction time

[0125] In Examples 23 to 27, the inventors optimized the material-liquid ratio. The specific difference between Examples 23 to 27 and Example 3 lies in step 3, as follows:

[0126] In Example 23, according to the material-liquid ratio of 1:15 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 7g of broccoli powder was accurately weighed and soaked in 105mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2)The broccoli mixture was stirred at 600 rpm for 1 hour at 25° C., and the broccoli mixture was centrifuged at 6000 rpm for 5 minutes at 4° C. to collect the supernatant to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of 1000 Da or more) and freeze-dried to obtain a crude broccoli protein extract.

[0127] In Example 24, according to the material-liquid ratio of 1:20 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 5.25g of broccoli powder was accurately weighed and soaked in 105mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 600 rpm for 1 hour at 25° C., and the broccoli mixture was centrifuged at 6000 rpm for 5 minutes at 4° C. to collect the supernatant to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of 1000 Da or more) and freeze-dried to obtain a crude broccoli protein extract.

[0128] Example 25 is the same as Example 3.

[0129] In Example 26, according to the material-liquid ratio of 1:30 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 3.5g of broccoli powder was accurately weighed and soaked in 105mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 600 rpm for 1 hour at 25° C., and the broccoli mixture was centrifuged at 6000 rpm for 5 minutes at 4° C. to collect the supernatant to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of 1000 Da or more) and freeze-dried to obtain a crude broccoli protein extract.

[0130] In Example 27, according to the material-liquid ratio of 1:35 (Note: material-liquid ratio = broccoli powder (g): deep eutectic solvent (including added water) (mL)), 3g of broccoli powder was accurately weighed and soaked in 105mL of the deep eutectic solvent DES prepared in step 2. 氯化胆碱-尿素(1:2) The broccoli mixture was stirred at 600 rpm for 1 h at 25 ° C, and the broccoli mixture was centrifuged at 6000 rpm for 5 min. The supernatant was collected to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of more than 1000 Da) and freeze-dried to obtain a crude broccoli protein extract.

[0131] Table 5 Setting of extraction conditions for broccoli plant protein by high hydrostatic pressure-assisted deep eutectic solvent extraction

[0132]

[0133]

[0134] In Examples 4 to 27, the results of single factor experiments on the extraction of broccoli plant protein using high hydrostatic pressure assisted deep eutectic solvents are shown in Table 1. Figure 1 .

[0135] In Examples 4 to 27, the single-factor experimental results of high hydrostatic pressure-assisted deep eutectic solvent extraction of broccoli plant protein are shown in Table 6.

[0136] Table 6 Extraction results of broccoli plant protein by high hydrostatic pressure assisted deep eutectic solvent extraction

[0137] Examples 3 to 7 are single-factor experiments 1, Examples 8 to 12 are single-factor experiments 2, Examples 13 to 17 are single-factor experiments 3, Examples 18 to 22 are single-factor experiments 4, and Examples 23 to 27 are single-factor group experiments 5. Each group of single-factor experiments is compared within the group and the highest value is selected. Therefore, there is a case where the highest value of each group of results is inconsistent, but only comparison is made within the group.

[0138]

[0139]

[0140] The results show:

[0141] (1) In Examples 3-7, under the same conditions of deep eutectic solvent water content, solid-liquid ratio, high hydrostatic pressure, and extraction time, the extraction effect of broccoli plant protein is better when the molar ratio of HBD to HBA is 1: (1.5-2.5), and the extraction effect is better when the molar ratio is 1:2, which is 132.07 mg / g.

[0142] (2) In Examples 8-12, under the same conditions of molar ratio of HBD to HBA, solid-liquid ratio, high hydrostatic pressure, and extraction time, the extraction effect of broccoli plant protein is better when the deep eutectic solvent water content is 20%-40%, and the extraction effect is better when the deep eutectic solvent water content is 20%, which is 129.31 mg / g.

[0143] (3) In Examples 13-17, under the same conditions of molar ratio of HBD to HBA, deep eutectic solvent water content, high hydrostatic pressure, and extraction time, the extraction effect of broccoli plant protein is better when the solid-liquid ratio is 1: (25-35), and the extraction effect is better when the solid-liquid ratio is 1:25, which is 148.36 mg / g.

[0144] (4) In Examples 18-22, under the same conditions of molar ratio of HBD to HBA, deep eutectic solvent water content, solid-liquid ratio, and extraction time, the extraction effect of broccoli plant protein is better when the high hydrostatic pressure is 300-500 MPa, and the extraction effect is better when the high hydrostatic pressure is 400 MPa, which is 151.60 mg / g.

[0145] (5) In Examples 22-27, under the same conditions of molar ratio of HBD to HBA, deep eutectic solvent water content, solid-liquid ratio, and high hydrostatic pressure, the extraction effect of broccoli plant protein is better when the high hydrostatic pressure time is 10-20 min, and the extraction effect is better when the extraction time is 10 min, which is 151.92 mg / g.

[0146] Example 28: Determination of the Digestibility of Broccoli Plant Protein Obtained Under Different Extraction Conditions

[0147] 1. Broccoli plant protein sample I obtained by traditional protein extraction method (Method 1)

[0148] Accurately weigh 1 g of broccoli powder and soak it in 25 mL of pure water. Vortex it vigorously for 30 seconds until a uniform thick mixture is obtained. Add 6 M NaOH to the mixture to adjust the pH to 10. The mixture is magnetically stirred at 600 rpm at 25 ° C for 1 hour. Centrifuge it at 4 ° C and 6000 rpm for 5 minutes, collect the supernatant to obtain broccoli plant protein alkaline extract; adjust the pH to 4.9 with 6 M HCl, and settle at 4 ° C for 1 hour; centrifuge the broccoli mixture at 4 ° C and 6000 rpm for 5 minutes, collect the precipitate, disperse the precipitate with pure water, dialyze it for 3 days (retention range is above 1000 Da), and freeze-dry it to obtain broccoli plant protein sample I.

[0149] 2. Obtaining Broccoli Plant Protein Sample II Using Deep Eutectic Solvents under Optimized Conditions (Method 2)

[0150] Accurately weigh 1g of broccoli powder and soak it in 25mL of deep eutectic solvent DES 氯化胆碱-尿素(1:2) The broccoli mixture was centrifuged at 4°C and 6000 rpm for 5 minutes, and the supernatant was collected to obtain a broccoli plant protein extract. The broccoli plant protein extract was dialyzed for 3 days (with a cutoff range of more than 1000 Da) and freeze-dried to obtain a broccoli plant protein sample II.

[0151] 3. Obtaining Broccoli Plant Protein Sample III Using High Hydrostatic Pressure-Assisted Deep Eutectic Solvents under Optimized Conditions (Method 3)

[0152] With reference to the processing steps and condition settings of Example 3, broccoli plant protein sample III was obtained.

[0153] 4. Determination of digestibility of broccoli plant protein obtained under different extraction conditions

[0154] (1) Determination of digestibility of broccoli plant protein obtained under different extraction conditions

[0155] Simulated digestion experiment: First, 400 mg of broccoli protein extracted by ALK-BPI method (broccoli plant protein sample I), 400 mg of broccoli protein extracted by DES-BPI method (broccoli plant protein sample II), and 400 mg of broccoli protein extracted by HDES-BPI method (broccoli plant protein sample III) were dispersed in 10 mL of pure water.

[0156] Preparation of simulated gastric fluid (SGF): composed of sodium chloride (94 mmol) and potassium chloride (13 mmol), and adjusted to pH 5.3 using 1 M hydrochloric acid;

[0157] Preparation of simulated intestinal fluid (SIF): composed of sodium chloride (164 mmol), potassium chloride (10 mmol) and sodium bicarbonate (85 mmol), adjusted to pH=7.

[0158] Stomach digestion:

[0159] The protein sample and SGF were mixed at a volume ratio of 50:50, and Pesin (pepsin, 2000 U / mL) extracted from rabbit stomach extract was added thereto to obtain simulated gastric digestive fluid; the protein samples were digested at 37°C and pH = 3 for 120 minutes, and gastric samples were collected for analysis at 0 minutes, 10 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively, and the remaining digest was then subjected to intestinal digestion.

[0160] Intestinal digestion:

[0161] SIF was added at a ratio of 25:75 between SGF and SIF, and the pH was adjusted to 7.0 using NaOH. 90 U / mL of porcine trypsin was added as an intestinal enzyme based on the working concentration of trypsin (16 U / mL), and CaCl2 solution (working concentration was 3 mM) was added. The remaining digest was subjected to intestinal digestion at 37°C and pH 7 for 120 min. Intestinal samples were collected for analysis at 0 min, 10 min, 30 min, 60 min, 90 min, and 120 min.

[0162] Digestibility test:

[0163] First, sample pretreatment is performed, then OPA solution is prepared, and finally sample testing is performed. The steps are as follows:

[0164] (I) Sample pretreatment

[0165] A) Stomach sample: 0.1 mL sample + 0.08 mL 10% cold trichloroacetic acid

[0166] B) Intestinal sample: 0.1 mL sample + 0.2 mL water + 0.24 mL 10% cold trichloroacetic acid

[0167] C) Centrifugation: 10,000 × g, 30 min at room temperature

[0168] D) Collect the supernatant to obtain the sample solution for later use

[0169] (II) Preparation of working reagents

[0170] A) Accurately weigh 3.81 g of sodium tetraborate decahydrate into a 100 mL brown Duran flask and add approximately 80 mL of water.

[0171] B) After sodium tetraborate is dissolved, add 0.088 g dithiothreitol (DTT) and 0.1 g sodium dodecyl sulfate (SDS).

[0172] C) 0.080 g of OPA was dissolved in 3 mL of 96% ethanol, which was then transferred to the solution obtained in step B) and filled up to 100 mL with water to obtain a working reagent.

[0173] (III) Sample testing

[0174] A) Mix blank / standard / sample: working reagent in a ratio of 10 μL:200 μL.

[0175] B) React in the dark at room temperature for 15 minutes

[0176] C) Read the absorbance at 340 nm using a microplate reader (o-phthalaldehyde, a fluorescent reagent, reacts with free amino groups to generate fluorescent products. The fluorescence intensity is then used to quantitatively analyze the free amino group content in the sample).

[0177] (IV) Calculation of digestibility

[0178] A) NH2(final): The concentration of free amino groups in the final hydrolyzate of the digested sample

[0179] B) NH2 (acid): total concentration of free amino groups in the sample itself (after acid hydrolysis in 6 M HCl solution at 110°C for 24 h).

[0180] C) NH2(initial): concentration of free amino groups in the undigested sample (G0).

[0181] D) The degree of protein hydrolysis (digestibility) is calculated as follows:

[0182] Digestibility = ([NH2(final)-NH2(initial)] / [NH2(acid)-NH2(initial)])*100%

[0183] The digestibility of broccoli plant protein obtained under different extraction conditions is shown in Figure 2 .

[0184] The results showed that the digestibility of broccoli plant protein obtained under Method 2 was 8.76% higher than that obtained under Method 1, indicating that the digestibility of broccoli protein extracted by DES was higher; the digestibility of broccoli plant protein obtained under Method 3 was 14.17% higher than that obtained under Method 2, indicating that the high hydrostatic pressure treatment method further improved the digestibility of broccoli protein.

[0185] (2) Determination of particle size distribution of broccoli plant protein obtained under different extraction conditions

[0186] The particle size distribution of broccoli protein samples I to III obtained by different extraction methods was detected using a particle size analyzer

[0187] The particle size distribution of broccoli plant protein obtained under different extraction conditions is shown in Figure 3 .

[0188] The results showed that the particle size distribution concentration point of the broccoli plant protein obtained under the conditions of Method 2 was 0.178 times that of the broccoli plant protein obtained under the conditions of Method 1, indicating that the particle size of the broccoli protein extracted by DES was smaller than that of the broccoli protein extracted by the ALK method; the particle size distribution concentration point of the broccoli plant protein obtained under the conditions of Method 3 was 0.708 times that of the broccoli plant protein obtained under the conditions of Method 2, indicating that the particle size of the broccoli protein extracted by high hydrostatic pressure assisted DES was smaller than that of the broccoli protein extracted by the DES method.

[0189] (3) Electrophoresis of broccoli plant proteins obtained under different extraction conditions

[0190] The broccoli protein obtained by ALK-BPI extraction (broccoli plant protein sample I), the broccoli protein obtained by DES-BPI extraction (broccoli plant protein sample II), and the broccoli protein obtained by HDES-BPI extraction (broccoli plant protein sample III) were respectively dissolved in deionized water, and the supernatant concentration was quantified by the BCA method. 30 μg of protein was loaded on each sample and subjected to SDS-PAGE.

[0191] The electrophoresis results of broccoli plant proteins obtained under different extraction conditions are shown in Figure 4 .

[0192] The results showed that the composition of broccoli protein extracted by method 1 was quite different from that of methods 2 and 3, while the composition of broccoli protein extracted by methods 2 and 3 was not much different.

[0193] (4) Determination of the emulsifying properties (EA) of broccoli plant protein obtained under different extraction conditions

[0194] 100 mg each of broccoli protein extracted by the ALK-BPI method (broccoli plant protein sample I), broccoli protein extracted by the DES-BPI method (broccoli plant protein sample II), and broccoli protein extracted by the HDES-BPI method (broccoli plant protein sample III) were dissolved in 20 mL of purified water, and then 1 mL of soybean oil was added. The mixture was homogenized at 12,000 rpm / min for 2 min to obtain an emulsion. 50 μL of the emulsion was added to 5 mL of 0.1% SDS solution, and the absorbance A was immediately detected at 500 nm using a microplate reader. The emulsification formula is as follows:

[0195] EA=(4.606*A*100) / (5*1*0.25*10000).

[0196] The emulsifying properties of broccoli plant protein obtained under different extraction conditions are shown in Figure 5 .

[0197] The results showed that the emulsifying property of the broccoli plant protein obtained under the conditions of Method 2 was 3.61 times that of the broccoli plant protein obtained under the conditions of Method 1, indicating that the emulsifying property of the broccoli protein extracted by DES was better than that of the broccoli protein extracted by the ALK method; the emulsifying property of the broccoli plant protein obtained under the conditions of Method 3 was 1.316 times that of the broccoli plant protein obtained under the conditions of Method 2, indicating that the emulsifying property of the broccoli protein extracted by DES assisted by high hydrostatic pressure was better than that of the broccoli protein extracted by the DES method.

[0198] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0199] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for extracting plant protein, characterized in that: include: S1: mixing the plant raw material and the deep eutectic solvent to obtain a mixed liquid; S2: subjecting the mixed liquid to high hydrostatic pressure treatment to obtain an extract containing plant protein.

2. The method according to claim 1, characterized in that The pressure of the high hydrostatic pressure treatment is 100 to 500 MPa, and the time of the high hydrostatic pressure treatment is 5 to 25 minutes.

3. The method according to claim 1, characterized in that The hydrogen bond acceptor of the deep eutectic solvent comprises one or more of choline chloride, betaine and L-proline; Optionally, the hydrogen bond donor of the deep eutectic solvent comprises one or more of urea, 1,4-butanediol and glycerol; Optionally, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is (1-5):(1-5); Preferably, the hydrogen bond acceptor of the deep eutectic solvent is choline chloride, the hydrogen bond donor is urea, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-3).

4. The method according to claim 1, wherein The preparation method of the deep eutectic solvent comprises: mixing the hydrogen bond acceptor and the hydrogen bond donor and performing a heating treatment; The temperature of the heating treatment is 60-100° C., and the time is 1-60 minutes.

5. The method according to claim 4, characterized in that The water content of the deep eutectic solvent is 0% to 40%.

6. The method according to claim 1, characterized in that The material-liquid ratio of the plant raw material to the deep eutectic solvent is 1:(15-35), and the unit is g / mL.

7. The method according to claim 1, characterized in that The plant raw materials include one or more of broccoli, cauliflower, wheat, peas, soybeans and almonds; Optionally, the particle size of the plant material is ≤1 mm.

8. The method according to claim 1, characterized in that The method further comprises: Separating the extract containing the plant protein to obtain a plant protein sample; Optionally, the separation treatment is performed under conditions to retain plant proteins larger than 1000 Da in the plant protein-containing extract.

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