Extraction method and application of fructus cannabis protein

Abstract

The invention discloses an extraction method of fructus cannabis protein and application of the fructus cannabis protein as an emulsifier for embedding fat-soluble functional components or application of the fructus cannabis protein as a protein carrier for loading the fat-soluble functional components. The extraction method comprises the following steps: crushing and sieving degreased fructus cannabis cake meal to obtain fructus cannabis cake meal powder; mixing the fructus cannabis cake meal with water, and stirring to obtain fructus cannabis meal slurry; adding a compound enzyme into the fructus cannabis pulp, and performing enzymolysis to obtain enzymatic hydrolysate; adjusting the pH value of the enzymatic hydrolysate to 8.0, stirring and mixing, then centrifuging, carrying out ultrafiltration on the supernate by using a membrane with the molecular weight cut-off of 10KDa, adjusting the pH value of the obtained cut-off liquid to 7.5, and then carrying out spray drying to obtain the fructus cannabis protein. Crude fibers, phytic acid and protein glutamine in the cake meal are selectively hydrolyzed through the compound enzyme, and the extraction rate, solubility, emulsibility and other characteristics of protein are remarkably improved. The extraction conditions are mild, the method is environment-friendly, and the obtained hemp seed protein can embed or load fat-soluble components.

Description

Technical Field

[0001] The present invention relates to a method for extracting and applying hemp seed protein, belonging to the technical field of plant protein extraction. Background Art

[0002] Hemp seeds are the kernels of the cannabis plant of the Moraceae family. They contain 20% - 25% protein, 25% - 30% oil, 20% - 30% crude fiber, and minerals, etc., and are an important oilseed and functional food raw material. The residue produced after pressing hemp seeds for oil is hemp seed cake meal, which has a high protein content. Especially for the cake meal of dehulled hemp seeds, the protein content can reach 60%. Hemp seed protein is mainly composed of edestin and albumin, and contains all the essential amino acids for the human body, especially the prominent contents of arginine and glutamic acid. Hemp seed protein is a high-quality plant protein with multiple functions, having the effects of enhancing immunity, antioxidation, anti-fatigue, reducing blood sugar, and maintaining the balance of intestinal flora. In China, the output of hemp accounts for 50% of the global total output, and a large amount of hemp seed cake meal is produced every year. However, at present, the development and utilization rate of hemp seed cake meal is low, and most of it is used as low-value animal feed, fertilizer, or directly discarded, which not only causes waste of resources but also leads to environmental pollution problems. Therefore, recovering hemp seed protein from hemp seed cake meal and making effective use of it will help improve the added value of hemp seed cake meal.

[0003] There are various methods for extracting protein from hempseed cake meal. Among them, the alkali extraction (acid precipitation) method is widely used in the extraction of hempseed protein. Prior art 1: Chinese Patent CN201911026365.0 discloses a method for preparing a hempseed protein extract using an alkali method. Prior art 2: Chinese Patent CN202111010411.5 discloses a method for separating and extracting hempseed protein and hempseed oil using the alkali extraction and acid precipitation method. Both prior art 1 and prior art 2 use strong alkali (pH 10 - 12) to dissolve the protein. Among them, prior art 2 further uses acidification (pH 4.5 - 5.0) to the isoelectric point to precipitate the protein on the basis of alkali extraction of protein, and certain extraction rates are achieved. However, the acid and alkali treatments during extraction can cause changes in the protein structure, resulting in protein denaturation, and further deteriorating the solubility, functional properties, and color and flavor of the protein. This method also easily produces high-salt wastewater. Prior art 3: Chinese Patent CN201310068420.9 discloses a method for extracting hempseed protein using a sodium chloride salt solution. This method requires repeated low-temperature sedimentation for desalting to purify the protein, and this process has high energy consumption. Prior art 4: Chinese Patent CN201610335417.2 discloses a processing technology for high-purity hempseed protein, which uses a sodium chloride solution to extract hempseed protein, and the obtained protein has high purity, but salt recovery is also required. Prior art 5: Chinese Patent CN201410174134.5 discloses a method for extracting hempseed protein from defatted hempseed residue using hot water. Since the solubility of protein in pure water is low, the extraction rate is not high. Prior art 6: The literature "Study on the Preparation Process of Hempseed Protein by Hydrolytic Enzyme Method" in "China Brewing" uses 6 kinds of proteases to extract hempseed protein. Proteases can hydrolyze proteins into small peptides, thus promoting the dissolution of proteins. The enzymatic method has the advantages of strong selectivity and mild extraction conditions. The degradation of macromolecular proteins into polypeptides can improve their utilization rate. However, the molecular weight of the extracted protein decreases after degradation, and it is mainly used for the preparation of amino acids or polypeptides, which cannot meet the requirements of food colloids (such as emulsifiers) for high-molecular-weight proteins. Prior art 7: The literature "Study on the Ultrasonic-Assisted Extraction Process of Hempseed Protein" in "Journal of Xinxiang University" discloses a method for ultrasonic-assisted extraction of hempseed protein using n-hexane solvent. Ultrasonic treatment improves the protein extraction rate, but ultrasonic treatment has high energy consumption, and high-frequency vibration easily causes protein thermal denaturation. These above-mentioned technologies can successfully extract hempseed protein through different methods. However, in the application process of the protein, it is found that the solubility and functionality of the protein obtained by these extraction methods are not ideal. Therefore, in order to solve the problems of low extraction rate, low solubility, and poor functionality of hempseed protein in the prior art, it is necessary to develop a hempseed protein extraction method that can improve the protein extraction rate, solubility, and functionality. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: the problems of low extraction rate of hemp seed protein, low solubility of the extracted protein and poor functionality in the prior art.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] A method for extracting hemp seed protein, comprising the following steps:

[0007] Step 1): Crushing and sieving defatted hemp seed cake meal to obtain hemp seed cake meal powder;

[0008] Step 2): Mixing the hemp seed cake meal powder with water and stirring to obtain a hemp seed meal slurry;

[0009] Step 3): Adding a composite enzyme to the hemp seed meal slurry and enzymatically hydrolyzing to obtain an enzymatic hydrolysate;

[0010] Step 4): Adjusting the pH of the enzymatic hydrolysate to 8.0, stirring and mixing, then centrifuging, and ultrafiltering the supernatant with a membrane having a molecular weight cut-off of 10 kDa. Adjust the pH of the retentate to 7.5, and then spray-dry to obtain hemp seed protein.

[0011] Preferably, in the step 1), the defatted hemp seed cake meal is crushed and sieved through a 40-80 mesh sieve.

[0012] Preferably, in the step 2), the mass-volume ratio of the hemp seed cake meal powder to water is 1 g: 15-25 mL.

[0013] Preferably, in the step 3), a composite enzyme is added to the hemp seed meal slurry, and enzymatic hydrolysis is carried out at 40 °C - 60 °C and pH 5-7 for 5-8 h to obtain an enzymatic hydrolysate.

[0014] Preferably, in the step 3), the composite enzyme includes cellulase, pectinase, protein glutaminase and phytase.

[0015] More preferably, in the composite enzyme, the enzyme activity of cellulase ≥ 80,000 U / g, the enzyme activity of pectinase ≥ 300,000 U / g, the enzyme activity of protein glutaminase is 400 U / g, and the enzyme activity of phytase is 8 U / g.

[0016] More preferably, in the composite enzyme, the addition amounts of cellulase, pectinase, protein glutaminase and phytase are respectively 0.1-0.5%, 0.05-0.2%, 0.5-1% and 0.01-0.05% of the mass of the defatted hemp seed cake meal.

[0017] Preferably, in the step 4), the temperature of stirring and mixing is 40-60 °C and the time is 1-2 h.

[0018] Preferably, in step 4), the centrifugation speed is 4500 - 5500 rpm, and the time is 10 - 20 min. More preferably, it is centrifuged at 5000 rpm for 15 min.

[0019] Preferably, in step 4), the pH of the retentate is adjusted to 7.5 with hydrochloric acid.

[0020] The present invention also provides the hemp seed protein extracted by the above - mentioned hemp seed protein extraction method as an emulsifier for encapsulating fat - soluble functional components or as a protein carrier for loading fat - soluble functional components.

[0021] Preferably, the present invention also provides the application of the above - mentioned hemp seed protein in encapsulating curcumin.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) Substances such as crude fiber in hemp seed cake meal form obstacles to the dissolution of protein in space, affecting protein extraction. The present invention uses cellulase and pectinase to degrade the crude fiber in the cake meal, promoting the release and dissolution of the protein wrapped in tissue cells and improving the extraction rate of hemp seed protein.

[0024] (2) The present invention innovatively provides a method that combines the extraction and modification of hemp seed protein, which not only improves the extraction rate of hemp seed protein but also increases its solubility. Hemp seed protein contains a large amount of glutamine, resulting in low solubility. The composite enzyme used in the present invention contains protein glutaminase, which can specifically remove the amino group on glutamine, increasing the number of carboxyl groups on the protein molecule and promoting protein dissolution. Phytic acid in hemp seeds forms an insoluble complex with protein, and phytase removes the phytic acid bound to the protein molecule through hydrolysis, promoting protein dissolution.

[0025] (3) The hemp seed protein extracted by this invention has its surface charge changed due to the removal of the amino group on protein glutamine during extraction, resulting in a change in its spatial structure, increased solubility and emulsifying property, enhanced functionality, and can be used to load fat - soluble food components, improving the loading rate and stability, providing high - quality raw materials for the development of functional foods and drug carriers.

[0026] (4) The composite enzyme used in this invention is selective, does not damage the protein structure, and the extraction conditions are mild. Description of the Drawings

[0027] Figure 1 It is the encapsulation rate of the emulsion encapsulating curcumin prepared with hemp seed protein provided in Example 3 and Comparative Example 5;

[0028] Figure 2 It is the appearance of the emulsion prepared with hemp seed protein provided in Example 3 and Comparative Example 5;

[0029] Figure 3 To provide the stability of the emulsion-embedded curcumin emulsion prepared from hemp seed protein for Example 3 and Comparative Example 5;

[0030] Figure 4 To provide the loading rate of curcumin in the hemp seed protein-curcumin complex prepared from hemp seed protein for Example 3 and Comparative Example 5. Detailed implementation manners

[0031] To make the present invention more obvious and understandable, preferred embodiments are provided below in conjunction with the accompanying drawings for detailed description.

[0032] In the following examples and comparative examples, defatted hemp seed cake meal is a commercially available product. Cellulase, pectinase, and phytase are all neutral enzymes sold by Xiasheng Group. Protein glutaminase is purchased from Amano Corporation of Japan.

[0033] The present invention provides a method for extracting hemp seed protein and the application of the protein. The specific technical solution is as follows:

[0034] The present invention provides a method for extracting hemp seed protein, comprising the following steps:

[0035] (1) Crushing defatted hemp seed cake meal and sieving it to obtain hemp seed cake meal powder;

[0036] (2) Mixing the hemp seed meal powder with water and stirring to obtain a hemp seed meal slurry.

[0037] (3) Adding a composite enzyme to the hemp seed meal slurry for enzymatic hydrolysis to obtain an enzymatic hydrolysate.

[0038] (4) Adjusting the pH of the enzymatic hydrolysate to 8.0 with 1M NaOH solution, stirring and mixing, then centrifuging, and taking the supernatant for ultrafiltration using a membrane with a molecular weight cut-off of 10KDa. The retained liquid is adjusted to pH 7.5 with hydrochloric acid and then spray-dried to obtain hemp seed protein.

[0039] The preparation method of the present invention uses a composite enzyme to hydrolyze crude fiber, phytic acid, and protein glutamine in the cake meal, effectively removing the factors that hinder the dissolution and solution of proteins, improving the extraction rate and solubility of hemp seed protein, while improving the functionality of hemp seed protein and enhancing its application value.

[0040] Preferably, the mesh number of sieving in step (1) is preferably 40 - 80 meshes, and more preferably 60 meshes.

[0041] Preferably, the mass-volume ratio of defatted hemp seed cake meal powder to water in step (2) is preferably 1:15 - 25 g / mL, and more preferably 1:20 g / mL.

[0042] Preferably, the complex enzyme described in step (3) includes cellulase, pectinase, protein glutaminase, and phytase.

[0043] Preferably, the addition amount of cellulase is 0.1 - 0.5% (w / w) of defatted hemp seed cake meal, the addition amount of pectinase is 0.05 - 0.2% (w / w) of defatted hemp seed cake meal, the addition amount of protein glutaminase is 0.5 - 1% (w / w) of defatted hemp seed cake meal, and the addition amount of phytase is 0.01 - 0.05% (w / w) of the weight of defatted hemp seed cake meal.

[0044] Preferably, the enzymatic hydrolysis conditions described in step (3) are preferably 40°C - 60°C, pH 5 - 7, and the reaction is carried out for 5 - 8 h, more preferably at 50°C, pH 6 for 6 h. The pH regulator used is 1M NaOH or 1M HCl.

[0045] Preferably, the enzyme activity of cellulase in the complex enzyme is ≥80,000 U / g, the enzyme activity of pectinase is ≥300,000 U / g, the enzyme activity of protein glutaminase is 400 U / g, and the enzyme activity of phytase is 8 U / g.

[0046] Preferably, the stirring and mixing conditions described in step (4) are stirring and mixing at 40 - 60°C for 1 - 2 h, more preferably stirring and mixing at 50°C for 1.5 h.

[0047] Preferably, the centrifugation conditions described in step (4) are 4500 rpm - 5500 rpm, 10 - 20 min, more preferably centrifugation at 5000 rpm for 15 min.

[0048] Example 1

[0049] (1) The defatted hemp seed cake meal is pulverized and passed through a 60 - mesh sieve to obtain hemp seed cake meal powder;

[0050] (2) The hemp seed cake meal powder is mixed with water in a ratio of 1:20 (g / mL) and stirred to obtain a hemp seed meal slurry;

[0051] (3) 0.5% cellulase, 0.1% pectinase, 0.5% protein glutaminase, and 0.02% phytase are added to the hemp seed meal slurry, and enzymatic hydrolysis is carried out at 50°C and pH 6 for 6 h.

[0052] (4) The pH of the enzymatic hydrolysate is adjusted to 8.0, and it is stirred and mixed at 50°C for 1.5 h. Then it is centrifuged at 5000 rpm for 15 min, and the supernatant is ultrafiltered using a membrane with a molecular cut - off of 10 KDa. The retentate is adjusted to pH 7.5 with hydrochloric acid and then spray - dried to obtain hemp seed protein.

[0053] Example 2

[0054] (1) The defatted hemp seed cake meal is pulverized and passed through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0055] (2) The hemp seed cake meal powder is mixed with water in a ratio of 1:20 (g / mL) and stirred to obtain a hemp seed cake meal slurry;

[0056] (3) 0.3% cellulase, 0.2% pectinase, 0.6% protein glutaminase, and 0.03% phytase are added to the hemp seed cake meal slurry, and enzymatic hydrolysis is carried out at 55 °C and pH 6 for 6 h.

[0057] (4) The pH of the enzymatic hydrolysate is adjusted to 8.0, and it is stirred and mixed at 50 °C for 1.5 h. Then, it is centrifuged at 5000 rpm for 15 min, and the supernatant is ultrafiltered using a membrane with a molecular cut-off of 10 KDa. The obtained retentate is adjusted to pH 7.5 with hydrochloric acid and then spray-dried to obtain hemp seed protein.

[0058] Example 3

[0059] (1) The defatted hemp seed cake meal is pulverized and passed through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0060] (2) The hemp seed cake meal powder is mixed with water in a ratio of 1:20 (g / mL) and stirred to obtain a hemp seed cake meal slurry;

[0061] (3) 0.4% cellulase, 0.1% pectinase, 1% protein glutaminase, and 0.04% phytase are added to the hemp seed cake meal slurry, and enzymatic hydrolysis is carried out at 50 °C and pH 6 for 6 h;

[0062] (4) The pH of the enzymatic hydrolysate is adjusted to 8.0, and it is stirred and mixed at 50 °C for 1.5 h. Then, it is centrifuged at 5000 rpm for 15 min, and the supernatant is ultrafiltered using a membrane with a molecular cut-off of 10 KDa. The obtained retentate is adjusted to pH 7.5 with hydrochloric acid and then spray-dried to obtain hemp seed protein.

[0063] Example 4

[0064] (1) The defatted hemp seed cake meal is pulverized and passed through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0065] (2) The hemp seed cake meal powder is mixed with water in a ratio of 1:20 (g / mL) and stirred to obtain a hemp seed cake meal slurry;

[0066] (3) 0.2% cellulase, 0.2% pectinase, 0.8% protein glutaminase, and 0.05% phytase are added to the hemp seed cake meal slurry, and enzymatic hydrolysis is carried out at 50 °C and pH 7 for 6 h;

[0067] (4) Adjust the pH of the enzymatic hydrolysate to 8.0, stir and mix at 50 °C for 1.5 h. Then centrifuge at 5000 rpm for 15 min, take the supernatant and ultrafilter it with a membrane with a molecular cut-off of 10 KDa. Adjust the pH of the retentate to 7.5 with hydrochloric acid, and then spray-dry to obtain hemp seed protein.

[0068] Comparative Example 1

[0069] (1) Crush defatted hemp seed cake meal, sieve it through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0070] (2) Mix the hemp seed cake meal powder and water in a ratio of 1:20 (g / mL) and stir to obtain hemp seed meal slurry;

[0071] (3) Add 0.1% pectinase, 0.5% protein glutaminase, and 0.02% phytase to the hemp seed meal slurry, and enzymatically hydrolyze at 50 °C and pH 6 for 6 h;

[0072] (4) Adjust the pH of the enzymatic hydrolysate to 8.0, stir and mix at 50 °C for 1.5 h. Then centrifuge at 5000 rpm for 15 min, take the supernatant and ultrafilter it with a membrane with a molecular cut-off of 10 KDa. Adjust the pH of the retentate to 7.5 with hydrochloric acid, and then spray-dry to obtain hemp seed protein.

[0073] Comparative Example 2

[0074] (1) Crush defatted hemp seed cake meal, sieve it through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0075] (2) Mix the hemp seed cake meal powder and water in a ratio of 1:20 (g / mL) and stir to obtain hemp seed meal slurry;

[0076] (3) Add 0.5% cellulase, 0.5% protein glutaminase, and 0.02% phytase to the hemp seed meal slurry, and enzymatically hydrolyze at 50 °C and pH 6 for 6 h;

[0077] (4) Adjust the pH of the enzymatic hydrolysate to 8.0, stir and mix at 50 °C for 1.5 h. Then centrifuge at 5000 rpm for 15 min, take the supernatant and ultrafilter it with a membrane with a molecular cut-off of 10 KDa. Adjust the pH of the retentate to 7.5 with hydrochloric acid, and then spray-dry to obtain hemp seed protein.

[0078] Comparative Example 3

[0079] (1) Crush defatted hemp seed cake meal, sieve it through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0080] (2) Mix the hemp seed cake meal powder and water in a ratio of 1:20 (g / mL) and stir to obtain hemp seed meal slurry;

[0081] (3) Add 0.5% cellulase, 0.1% pectinase, and 0.02% phytase to the hemp seed meal slurry, and perform enzymatic hydrolysis at 50 °C and pH 6 for 6 h.

[0082] (4) Adjust the pH of the enzymatic hydrolysate to 8.0, and stir and mix at 50 °C for 1.5 h. Then centrifuge at 5000 rpm for 15 min, and take the supernatant for ultrafiltration using a membrane with a molecular cut-off of 10 KDa. Adjust the pH of the retentate to 7.5 with hydrochloric acid, and then spray-dry to obtain hemp seed protein.

[0083] Comparative Example 4

[0084] (1) Crush the defatted hemp seed cake meal and pass through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0085] (2) Mix the hemp seed cake meal powder and water at a ratio of 1:20 (g / mL) and stir to obtain hemp seed meal slurry;

[0086] (3) Add 0.5% cellulase, 0.1% pectinase, and 0.5% protein glutaminase to the hemp seed meal slurry, and perform enzymatic hydrolysis at 50 °C and pH 6 for 6 h.

[0087] (4) Adjust the pH of the enzymatic hydrolysate to 8.0, and stir and mix at 50 °C for 1.5 h. Then centrifuge at 5000 rpm for 15 min, and take the supernatant for ultrafiltration using a membrane with a molecular cut-off of 10 KDa. Adjust the pH of the retentate to 7.5 with hydrochloric acid, and then spray-dry to obtain hemp seed protein.

[0088] Comparative Example 5

[0089] (1) Crush the defatted hemp seed cake meal and pass through a 60-mesh sieve to obtain hemp seed cake meal powder;

[0090] (2) Mix the hemp seed cake meal powder and water at a ratio of 1:20 (g / mL) and stir to obtain hemp seed meal slurry;

[0091] (3) Adjust the pH of the hemp seed meal slurry to 10 with 2M NaOH, extract at 50 °C for 1.5 h, then adjust the pH to 5 with 2M HCl, centrifuge at 8000 rpm for 20 min, wash the obtained precipitate 3 times with water, adjust the pH to 7.0, redissolve in deionized water, and spray-dry to obtain hemp seed protein.

[0092] Experimental Example 1

[0093] Determine the protein recovery rate, protein content, and protein solubility of Examples 1-4 and Comparative Examples 1-5 of the present invention.

[0094] Determination of protein content: The protein content was determined by the micro-Kjeldahl method, and the protein conversion coefficient was 6.25. The protein contents in the extracted protein powder and defatted hemp seed cake were determined respectively. The calculation formula for protein recovery rate is as follows:

[0095]

[0096] Determination of solubility: Weigh hemp seed protein powder, add deionized water to make the concentration of the protein powder 10 mg / mL, stir magnetically for 30 min, hydrate overnight, centrifuge at 4000 rpm for 10 min, and use the Bradford kit to determine the protein concentration in the supernatant. Use bovine serum albumin to make a standard curve for protein concentration. The calculation formula for solubility is as follows:

[0097]

[0098] Table 1 shows the protein recovery rate, protein solubility and protein content of Examples 1-4 and Comparative Examples 1-5.

[0099] Table 1

[0100]

[0101]

[0102] As can be seen from the results in Table 1, the protein recovery rate and protein solubility of the hemp seed protein extracted by Examples 1-4 of the present invention are both higher than those of the preparation methods described in Comparative Examples 1-5. In Comparative Example 1, due to the lack of degradation of crude fiber by cellulase, the protein recovery rate is low, but it does not affect the protein solubility. In Comparative Example 2, due to the lack of degradation of crude fiber by pectinase, the protein recovery rate is low, but it does not affect the protein solubility. In Comparative Example 3, due to the lack of deamidation of protein by protein glutaminase, the protein extraction rate is slightly reduced and the protein solubility is poor. In Comparative Example 4, due to the lack of phytase, the protein extraction rate and solubility are reduced. In Comparative Example 5, the alkali solution acid precipitation method is used for extraction, and the obtained protein extraction rate and solubility are low. It shows that the synergistic effect of the composite enzyme of the present invention is beneficial to the extraction and dissolution of proteins.

[0103] Experimental Example 2

[0104] Measure the emulsifying activity, emulsifying stability, particle size and zeta potential of the emulsions of Examples 1-4 and Comparative Examples 1-5 of the present invention. Measurement of emulsifying activity: Prepare a 10 mg / mL hemp seed protein solution and hydrate it at 4 °C for 12 h. Mix the hemp seed protein solution and olive oil solution at a ratio of 3:1, homogenize it with a high-speed disperser at 20000 r / min for 2 min, and then sonicate it at 400 w for 10 min (working for 2 s and stopping for 2 s) to prepare an emulsion. Take 50 μL each of the freshly prepared emulsion and the emulsion after standing for 10 min, add them to 5 mL of 0.1% SDS for dilution, mix well by shaking, and measure the absorbance at 500 nm with a UV spectrophotometer, denoted as A0 and A10 respectively. Using the 0.1% SDS solution as a blank, calculate the emulsifying activity and emulsifying stability according to the following formula:

[0105]

[0106] In the formula: N represents the dilution factor; C represents the protein concentration; A0 and A10 represent the absorbance values at 0 min and 10 min respectively; t represents the standing time; L represents the optical path of the cuvette; φ represents the volume fraction of the oil phase.

[0107] The particle size and zeta potential of the emulsion were measured using an S3500 laser particle size analyzer.

[0108] Table 2 shows the emulsifying activity, emulsifying stability, particle size and zeta potential of the emulsions of Examples 1-4 and Comparative Examples 1-5.

[0109] Table 2

[0110] Group <![CDATA[Emulsifying activity (m 2 / g)]]> Emulsion stability (min) Emulsion particle size (μm) Emulsion potential (mV) Example 1 341.7±3.5 2610±278 1.25±0.03 -10.09±0.30 Example 2 323.5±4.9 2745±316 1.09±0.05 -9.86±0.55 Example 3 349.3±10.5 2814±214 1.02±0.01 -10.84±0.57 Example 4 331.1±5.1 2702±126 1.5±0.49 -9.73±0.85 Comparative Example 1 322.4±6.4 2696±83 1.32±0.31 -11.14±0.49 Comparative Example 2 325.2±9.1 2759±11 1.19±0.22 -9.05±0.62 Comparative Example 3 154.6±5.3 692±20 4.64±0.12 -5.23±0.43 Comparative Example 4 328.9±8.7 2630±166 1.27±0.15 -10.59±0.76 Comparative Example 5 142.6±4.2 579±326 6.86±0.46 -3.17±0.63

[0111] As can be seen from Table 2, the hemp seed proteins prepared in Examples 1-4 have high emulsifying activity and emulsifying stability, small emulsion particle size, and large absolute value of the emulsion zeta potential. The hemp seed protein in Comparative Example 3 has low emulsifying activity and emulsifying stability, large emulsion particle size, and small zeta potential, indicating that treatment with protein glutaminase helps to improve the emulsifying activity and emulsifying stability of the protein. Protein glutaminase changes the amide on the protein glutamine to a carboxyl group, and changes the spatial structure of the protein by changing the surface charge of the protein, thereby affecting its function. The hemp seed protein prepared in Comparative Example 5 also has poor emulsifying activity and emulsifying stability, with a relatively large particle size and a small zeta potential of the emulsion.

[0112] Experimental Example 3

[0113] To verify the application of the proteins extracted in Examples 1-4 of the present invention, the hemp seed proteins extracted in Example 3 and Comparative Example 5 were selected to prepare emulsions for encapsulating curcumin.

[0114] The preparation method is as follows: Prepare a 10 mg / mL aqueous solution of hemp seed protein and hydrate it at 4°C for 12 h. Weigh curcumin and dissolve it in olive oil, and dissolve it in the dark at 60°C for 20 min to obtain an olive oil solution of 1 mg / mL curcumin. The hemp seed protein solution and the curcumin oil solution are mixed at a ratio of 3:1, homogenized at 20,000 r for 2 min, and then sonicated at 400 w for 10 min (working for 2 s and stopping for 2 s) to obtain an emulsion loaded with curcumin.

[0115] Determination of the encapsulation rate of curcumin: Take 3 mL of the prepared emulsion, centrifuge it at 5000 g for 10 min to remove the unencapsulated curcumin at the bottom of the centrifuge tube, then add 4 mL of ethanol to extract curcumin, repeat 2 times, and combine the solutions extracted three times to measure the absorbance at OD425, and convert it to the curcumin content according to the standard curve. The calculation formula for the encapsulation rate of curcumin is as follows:

[0116]

[0117] Curcumin is a polyphenolic compound with a variety of biological activities. However, its poor water solubility and stability limit its application in the food field. The hemp seed protein in this invention is used to prepare an emulsion to improve the solubility of curcumin. The encapsulation rate of curcumin is shown in Figure 1 , the encapsulation rate of curcumin in the emulsion prepared by the hemp seed protein in Example 3 of this invention is 90.8%, while the encapsulation rate of curcumin in the emulsion prepared by the hemp seed protein in Comparative Example 5 is 71.2%, indicating that the product of this invention has good performance in encapsulating curcumin. The appearance of the emulsion encapsulating curcumin is shown in Figure 2 , it can be seen that the encapsulation stability of curcumin in the hemp seed protein emulsion of Example 3 of this invention is good. After standing for 2 weeks, the emulsion is stable and does not stratify, while the encapsulation stability of curcumin in the hemp seed protein emulsion of Comparative Example 5 is poor and has stratified at 2 weeks. The emulsion encapsulating curcumin is stored at room temperature for 2 weeks, and the retention rates of curcumin on the 0th day, 7th day, and 14th day are measured respectively. The change of the encapsulation rate with time is shown in Figure 3 . As the storage time prolongs, the encapsulated amount of curcumin gradually decreases, but the encapsulated amount of curcumin in the hemp seed protein emulsion of Example 3 of this invention is always higher than that in the hemp seed protein emulsion of Comparative Example 5 within 14 days.

[0118] Experimental Example 4

[0119] To verify the application of the proteins extracted in Examples 1-4 of this invention, the hemp seed proteins extracted in Example 3 and Comparative Example 5 are selected to prepare hemp seed protein-curcumin complexes to encapsulate curcumin and improve the solubility of curcumin.

[0120] The preparation method is as follows: Prepare a 10 mg / mL hemp seed protein solution, hydrate it at 4 °C for 12 h, adjust the pH to 12.0 with 2M NaOH solution, and stir magnetically for 30 min. Then weigh out curcumin powder and add it to the protein solution to make the concentration of curcumin 10 mg / mL, stir in the dark for 30 min, then adjust the pH to 7.0 with 2M hydrochloric acid, stir for 1 h, and then centrifuge at 3500 rpm for 20 min to remove the unentrapped curcumin precipitate, obtaining a hemp seed protein-curcumin complex solution, and freeze-dry it to obtain a powder.

[0121] Determination of curcumin loading rate: Mix 0.2 mL of the hemp seed protein-curcumin complex with 1.8 mL of absolute ethanol and shake vigorously, centrifuge at 8000 r / min for 15 min, and take the supernatant to measure the absorbance at 425 nm. The calculation formula for the curcumin loading rate is as follows:

[0122]

[0123] The measurement results are as Figure 4 shown. The loading rate of curcumin in hemp seed protein in Example 3 of the present invention is 71.36%, while the loading rate of curcumin in hemp seed protein in Comparative Example 5 is 35.78%. It shows that the hemp seed protein in Example 3 of the present invention can effectively load curcumin and promote the dissolution of curcumin. In an alkaline environment, the spatial structure of hemp protein unfolds, and after curcumin is deprotonated, it is embedded in the hydrophobic core of the protein through hydrophobic interaction. During the acid neutralization stage, hydrogen bonds and electrostatic attraction are formed when the protein refolds, promoting the binding of curcumin to the protein. The deamidation of the hemp seed protein extracted by complex enzyme increases the carboxyl group and changes the spatial structure of the protein, thus promoting the binding of the protein to curcumin.

Claims

1. A method for extracting hemp seed protein, characterized in that, It includes the following steps: Step 1): Crush the defatted hemp seed cake meal and sieve it to obtain hemp seed cake meal powder; Step 2): Mix the hemp seed cake meal powder with water and stir to obtain a hemp seed meal slurry; Step 3): Add a complex enzyme to the hemp seed meal slurry and carry out enzymatic hydrolysis to obtain an enzymatic hydrolysate; Step 4): Adjust the pH of the enzymatic hydrolysate to 8.0, stir and mix, then centrifuge. The supernatant is ultrafiltered with a membrane having a molecular weight cut-off of 10 KDa. The obtained retentate is adjusted to pH 7.5 and then spray-dried to obtain hemp protein.

2. The extraction method of hemp seed protein according to claim 1, characterized in that, In the said Step 1), after crushing, the defatted hemp seed cake meal is sieved through a 40-80 mesh sieve.

3. The extraction method of hemp seed protein according to claim 1, characterized in that, In the said Step 2), the mass-volume ratio of the hemp seed cake meal powder to water is 1 g: 15-25 mL.

4. The extraction method of hemp seed protein according to claim 1, characterized in that, In the said Step 3), a complex enzyme is added to the hemp seed meal slurry, and enzymatic hydrolysis is carried out at 40°C - 60°C and pH 5 - 7 for 5 - 8 h to obtain an enzymatic hydrolysate.

5. The extraction method of hemp seed protein according to claim 1, wherein, In the said Step 3), the complex enzyme includes cellulase, pectinase, protein glutaminase and phytase.

6. The extraction method of hemp seed protein according to claim 5, characterized in that, In the said complex enzyme, the enzyme activity of cellulase ≥ 80,000 U / g, the enzyme activity of pectinase ≥ 300,000 U / g, the enzyme activity of protein glutaminase is 400 U / g, and the enzyme activity of phytase is 8 U / g.

7. The extraction method of hemp seed protein according to claim 5, characterized in that, In the said complex enzyme, the addition amounts of cellulase, pectinase, protein glutaminase and phytase are respectively 0.1 - 0.5%, 0.05 - 0.2%, 0.5 - 1% and 0.01 - 0.05% of the mass of the defatted hemp seed cake meal.

8. The extraction method of hemp seed protein according to claim 1, characterized in that, In the said Step 4), the temperature of stirring and mixing is 40 - 60°C and the time is 1 - 2 h.

9. The extraction method of hemp seed protein according to claim 1, characterized in that, In the said Step 4), the rotation speed of centrifugation is 4500 - 5500 rpm and the time is 10 - 20 min.

10. The hemp protein extracted by the extraction method of the hemp protein according to any one of claims 1 - 9 is used as an emulsifier for encapsulating fat-soluble functional components or as a protein carrier for loading fat-soluble functional components.

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