Coagulation-increasing compound enzyme and application thereof in preparation of walnut protein powder

Abstract

The invention provides a coagulation-increasing compound enzyme and application thereof in preparation of walnut protein powder, and relates to the technical field of plant natural product extraction. The invention provides a coagulation-increasing compound enzyme. The coagulation-increasing compound enzyme is a mixture of at least two biological enzymes selected from glutamine transaminase, glutamyl endopeptidase, tannase and glucoside hydrolase. The coagulation-increasing compound enzyme is adopted in the extraction process of the instant protein and the extraction process of the isolated protein, forming of stronger protein gel is facilitated, higher-quality protein powder is obtained, impurities are fewer, the color is whiter, the preparation method is simple in step and easy to operate, the protein extraction rate is higher, and the method is suitable for industrial production. The synchronous preparation of the high-purity instant protein powder and the isolated protein powder is realized, and the production efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant natural product extraction, in particular to a coagulant composite enzyme and application thereof in the preparation of walnut protein powder. Background Art

[0002] Walnut meal is a by-product of the walnut oil industry. The protein content in the meal is as high as 50%, which is a high-quality source of protein. In addition, walnut meal also contains 10-20% of oil, carbohydrates and polyphenols, which interact with proteins and are difficult to separate, affecting the extraction rate, purity, quality and physical and chemical properties of the protein. Therefore, defatting, desugaring and dephenolization are three important links in the extraction of walnut protein. However, most of the existing methods for extracting plant protein use alkali dissolution and acid precipitation methods. The oil is easy to form emulsification and affect the dissolution of protein; carbohydrates such as polysaccharides and proteins co-precipitate, and proteins and polysaccharides cannot be effectively separated; polyphenols are easily attached to proteins, causing changes in the color, flavor and functional properties of plant proteins. In addition, during the oil pressing process of walnut meal, protein denaturation leads to partial loss of functionality. Before extracting protein, the cake should be pretreated to restore protein function. Walnut protein is rich in 60-70% gluten and is water-insoluble, resulting in poor functional properties such as solubility, emulsification and foaming, which seriously limits its application in beverages, nutritional proteins and other foods.

[0003] Instant protein powder is mainly obtained by grinding, solubilizing, modifying and centrifuging under neutral conditions to obtain the supernatant, without acid precipitation and water washing process, and directly spraying. The protein content is 20-40%, and the protein purity is extremely low. In addition, in the process of extracting instant protein from walnuts, the water-soluble protein extraction effect is good, but the residue contains more than 60% of water-insoluble protein (gluten) which is not well utilized. Walnut protein isolate powder is mainly produced by alkali dissolution and acid precipitation. A large amount of hydrogen ions and salts remain in the protein curd separated by acid precipitation, which can be removed by water washing. The protein content of the prepared walnut protein isolate powder is 70-80%, the protein purity is low, the color is dark, and the taste is astringent, which is not suitable for the deep processing of high-quality protein powder. Therefore, the development of a biocoagulant composite enzyme to improve the protein extraction efficiency is of great significance to improve the color and purity of instant protein and protein isolate. Summary of the invention

[0004] The purpose of the present invention is to provide a coagulant complex enzyme and its application in the preparation of protein powder, so as to solve the technical problems of poor protein solubility, low protein purity and dark color existing in the prior art.

[0005] In order to solve the above technical problems, the present invention provides a coagulant complex enzyme, which is a mixture of at least two biological enzymes selected from transglutaminase, glutamyl endopeptidase, tannase, and glycoside hydrolase.

[0006] Furthermore, the amount of the glutamine transaminase added is 0.1-5 parts by weight; and / or, the amount of glutamyl endopeptidase added is 0.1-8 parts; and / or, the glycoside hydrolase is added in an amount of 0.1-10 parts; And / or, the added amount of the tannase is 0.1-10 parts.

[0007] Glutamine transaminase, enzyme-mediated molecular cross-linking can improve the thermal stability and water holding capacity of proteins, help form strong gels, and improve the quality of protein products.

[0008] Glutamyl endopeptidase can hydrolyze proteins to form gels with higher whiteness. It can cleave the peptide bond formed by glutamic acid and the α-carboxyl group of aspartic acid residues at the C-terminus of the protein polypeptide chain, and play a hydrophobic modification role on plant proteins, thereby promoting the coagulation of plant proteins. In the presence of salt ions, the gel formed has higher water retention and viscosity, and can be used in the preparation of high-quality protein powder.

[0009] Tannin can effectively cleave the covalent bonds formed by protein and polyphenols, freeing the protein and polyphenols. The freed protein is then enriched through subsequent acid precipitation, thereby improving the purity of the protein. Walnuts contain a large amount of polyphenols. When the protein is acid precipitated, they are condensed into water-insoluble substances under the action of strong acid, affecting the purity and color of the protein powder. The polyphenol impurities in the protein gel can only be removed by tannin.

[0010] Glycoside hydrolases are a type of enzyme that hydrolyzes glycosidic bonds. They are mainly used to remove insoluble polysaccharides in protein gels, thereby purifying proteins.

[0011] The invention also provides a method for synchronously preparing walnut instant protein powder and isolated protein powder, and the coagulant complex enzyme is used to purify the protein.

[0012] Furthermore, the above preparation method comprises the following specific steps: Step 1, after crushing and sieving the walnut meal raw material, adding it to the residual oil extraction tank, adding 60-100° C. hot water, reacting for 10-60 minutes under negative pressure, and centrifuging to remove the upper layer of fat to obtain walnut defatted meal powder; The mass ratio of hot water to meal is (2-10):100; Step 2, taking 100 parts of the defatted walnut meal, adding 100-1000 parts of softened water to mix, adjusting the pH to 6-9; adding 1-5 parts of chymosin, stirring continuously for 1-8 hours under negative pressure, centrifuging to remove the separated protein precipitate, and obtaining a first supernatant as a walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to acidic; 1-8 parts of the coagulant complex enzyme are added, and enzymolysis is performed under negative pressure for 1-5 hours; then the solution is allowed to stand for acid precipitation enrichment for 0.1-2 hours, and a walnut instant protein gel is obtained after centrifugation; after the enzyme is inactivated, the walnut instant protein gel is ultrafiltered to remove salt, concentrated, and dried to obtain an instant protein powder; Step 4, taking the isolated protein precipitate described in step 2, adding softened water and adjusting the pH to 7-11; alkali dissolving for 1-5 hours under negative pressure, continuously stirring, and centrifuging to obtain a third supernatant; taking the third supernatant and adjusting the pH to acidic; adding the coagulant complex enzyme, after enzymolysis for 1-5 hours under negative pressure, standing for acid precipitation enrichment for 0.1-2 hours, and obtaining a walnut protein isolate gel after centrifugation; The mass ratio of the isolated protein precipitate: softened water: coagulant complex enzyme is 100: (100-1000): (1-8); Adding a coagulant complex enzyme during the extraction and separation of protein gel can enhance the strength and whiteness of the generated protein gel, increase the hydrophobic amino acid residues, and facilitate subsequent water washing and desalting.

[0013] Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain protein isolate powder; The coagulant complex enzyme is a mixture of at least two biological enzymes selected from transglutaminase, glutamyl endopeptidase, tannase, and glycoside hydrolase; The above components are all calculated by mass.

[0014] Furthermore, the mesh size of the sieving in step 1 is 30-200 meshes.

[0015] Furthermore, the amount of hot water added in step 1 is 2-10% of the mass of the meal.

[0016] Preferably, in step 3, the molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration desalination process is 0-10 kD.

[0017] Furthermore, the addition amount of each component in the coagulant complex enzyme is as follows by weight: The amount of glutamine transaminase added is 0.1-5 parts; and / or, the amount of glutamyl endopeptidase added is 0.1-8 parts; and / or, the glycoside hydrolase is added in an amount of 0.1-10 parts; And / or, the added amount of the tannase is 0.1-10 parts.

[0018] Further, the temperature of the enzymatic hydrolysis is 40-70°C; The pH value of the enzymatic hydrolysis of the coagulant complex enzyme is 3-7.

[0019] Further, the negative pressure is -0.01 to -5 MPa; The stirring rate is 40-80r / min; By adopting the above technical solution, the present invention has the following beneficial effects: The present invention provides a coagulant complex enzyme and its application in the preparation of protein powder. In the extraction process of instant protein powder and separated protein powder, a coagulant complex enzyme mixed with multiple biological enzymes is used, which is conducive to the formation of a stronger protein gel, so that the obtained protein powder is higher in quality and has fewer impurities. The preparation method is simple and easy to operate, which is conducive to the simultaneous high-purity extraction of instant protein powder and separated protein powder, thereby improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 A flowchart of walnut protein powder extraction provided by the present invention; Figure 2 This is a comparison diagram of the appearance of the walnut instant protein powder prepared in Example 1 and the walnut instant protein powder prepared in Comparative Example 1; Figure 3 This is a comparison diagram of the appearance of the walnut protein isolate powder prepared in Example 1 and the walnut protein isolate powder prepared in Comparative Example 1. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The present invention is further explained below in conjunction with specific implementation modes.

[0024] The method provided by the present invention for simultaneously preparing walnut instant protein powder and isolated protein powder by using a coagulant complex enzyme comprises the following specific steps: Step 1, crushing the walnut meal raw material and filtering it through a 30-200 mesh filter, adding it to a residual oil extraction tank, first heating it to 45° C. to 65° C., and stirring it for 5-30 min; then adding 60-100° C. hot water, the mass of which is 2-10% of the mass of the walnut meal, evacuating the tank, reacting it at a negative pressure of -0.01 to -5 MPa for 10 to 60 min, and centrifuging to remove the upper layer of fat to obtain walnut defatted meal powder; Step 2, taking 100 parts of defatted walnut meal, adding 100 to 1000 parts of softened water to mix, adjusting the pH to 6 to 9; adding 1 to 5 parts of chymosin, stirring continuously for 1 to 8 hours under a negative pressure of -0.01 to -5 MPa, the stirring speed is 40 to 80 r / min, centrifuging to remove the separated protein precipitate for standby use, and obtaining a first supernatant as a walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to 3-7; 1-8 parts of a coagulant complex enzyme are added, and enzymolysis is performed for 1-5 hours under a negative pressure of -0.01 to -5 MPa; then the walnut instant protein gel is obtained by standing for acid precipitation enrichment for 0.1-2 hours, and after centrifugation, the walnut instant protein gel is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 0-10 kD after the enzyme is inactivated, and concentrated and dried to obtain instant protein powder; Step 4, taking the separated protein precipitate in step 2, adding softened water and adjusting the pH to 7-11; performing alkaline dissolution for 1-5 hours at a negative pressure of -0.01 to -5 MPa, the reaction temperature is 40 to 60° C., stirring is continued at a speed of 40 to 80 r / min, and centrifugation is performed to obtain a third supernatant; the third supernatant is added to an acid precipitation tank and the pH is adjusted to 3-7; a coagulant complex enzyme is added, and after enzymolysis for 1-5 hours at a negative pressure of -0.01 to -5 MPa, the acid precipitation is allowed to stand for enrichment for 0.1 to 2 hours, and a walnut protein isolate gel is obtained after centrifugation; Wherein, the separated protein precipitate, softened water, and coagulant complex enzyme can be added in the order of a mass ratio of 100: (100-1000): (1-8); The added amount of each component in the coagulant complex enzyme is calculated as follows by weight: the added amount of the glutamine aminotransferase is 0.1-5 parts, and / or the added amount of the glutamyl endopeptidase is 0.1-8 parts, and / or the added amount of the glycoside hydrolase is 0.1-10 parts, and / or the added amount of the tannase is 0.1-10 parts.

[0025] Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain isolated protein powder; Example 1 like Figure 1 As shown, in this embodiment, according to the preparation method provided by the present invention, walnut instant protein powder and isolated protein powder are prepared simultaneously using a coagulant complex enzyme. The specific formula of the coagulant complex enzyme is a mass ratio of glutamine transaminase: glutamyl endopeptidase: tannase of 1:1:2.

[0026] Step 1, 100g of walnut meal raw material is crushed and filtered through a 200-mesh filter, and then added to a residual oil extraction tank, first heated to 50°C, and stirred for 15 minutes; then 5g of 75°C hot water is added, the tank is evacuated, and the reaction is carried out at a negative pressure of -0.08MPa for 20 minutes, the stirring rate is 55r / min, and the upper layer of fat is removed by centrifugation to obtain walnut defatted meal; Step 2, take 100g of defatted walnut meal, add 600g of softened water to mix, adjust the pH to 7; add 2g of chymosin, stir continuously for 2h under a negative pressure of -0.08MPa, the stirring speed is 60r / min, centrifuge to remove the separated protein precipitate, and obtain the first supernatant as walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to 4.5; 2g of a coagulant complex enzyme is added, wherein the mass ratio of glutamine transaminase: glutamyl endopeptidase: tannase is 1:1:2, and enzymolysis is performed under a negative pressure of -0.08MPa for 2h; then the liquid is allowed to stand for acid precipitation enrichment for 30min, and a walnut instant protein gel is obtained after centrifugation; after the enzyme is inactivated, the walnut instant protein gel is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 0-10kD, and concentrated and dried to obtain an instant protein powder; Step 4, taking 50g of the separated protein precipitate in step 2, adding 250g of softened water and adjusting the pH to 10; alkali dissolving for 1h under a negative pressure of -0.08MPa, the reaction temperature is 45°C, stirring is continued at a speed of 60r / min, and centrifugation is performed to obtain a third supernatant; the third supernatant is added to an acid precipitation tank and the pH is adjusted to 4.5; 1g of a coagulant complex enzyme is added, wherein the mass ratio of glutamine transaminase: glutamyl endopeptidase: tannase is 1:1:2, after enzymolysis for 2h under a negative pressure of -0.08MPa, the solution is allowed to stand for acid precipitation enrichment for 30min, and the walnut protein isolate gel is obtained after centrifugation; Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain isolated protein powder; Example 2 In this embodiment, according to the preparation method provided by the present invention, glutamine aminotransferase, glutamyl endopeptidase and glycoside hydrolase are mixed in a mass ratio of 1:1:3 as a coagulant complex enzyme to simultaneously prepare walnut instant protein powder and isolated protein powder.

[0027] Step 1, 100g of walnut meal raw material is crushed and filtered through a 200-mesh filter, and then added to a residual oil extraction tank, first heated to 45°C, and stirred for 25 minutes; then 5g of 60°C hot water is added, the tank is evacuated, and the reaction is carried out at a negative pressure of -0.08MPa for 20 minutes, the stirring rate is 55r / min, and the upper layer of fat is removed by centrifugation to obtain walnut defatted meal powder; Step 2, take 100g of defatted walnut meal, add 600g of softened water to mix, adjust the pH to 7; add 2g of chymosin, stir continuously for 4h under a negative pressure of -0.08MPa, the stirring speed is 60r / min, centrifuge to remove the separated protein precipitate, and obtain the first supernatant as walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to 5; 2g of a coagulant complex enzyme is added, wherein the specific formula of the coagulant complex enzyme is glutamine aminotransferase, glutamyl endopeptidase, and glycoside hydrolase, and the mass ratio is 1:1:3, and enzymolysis is performed under a negative pressure of -0.08MPa for 3h; then the solution is allowed to stand for acid precipitation enrichment for 1h, and a walnut instant protein gel is obtained after centrifugation; after the enzyme is inactivated, the walnut instant protein gel is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 0-10kD, and concentrated and dried to obtain an instant protein powder; Step 4, taking 50g of the isolated protein precipitate in step 2, adding 250g of softened water and adjusting the pH to 10; alkali dissolving for 1h under a negative pressure of -0.08MPa, the reaction temperature is 55°C, stirring is continued at a speed of 60r / min, and centrifugation is performed to obtain a third supernatant; the third supernatant is added to an acid precipitation tank and the pH is adjusted to 5; 1g of a coagulant complex enzyme is added, wherein glutamine transaminase, glutamyl endopeptidase, and glycoside hydrolase are included in a mass ratio of 1:1:3, and after enzymatic hydrolysis for 3h under a negative pressure of -0.08MPa, the acid precipitation is allowed to stand for 1h, and the walnut protein isolate gel is obtained after centrifugation; Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain isolated protein powder; Example 3 In this embodiment, according to the preparation method provided by the present invention, tannase and glycoside hydrolase in a mass ratio of 1:1.5 are mixed as coagulant complex enzymes to simultaneously prepare walnut instant protein powder and isolated protein powder.

[0028] Step 1, 100g of walnut meal raw material is crushed and filtered through a 200-mesh filter, and then added to a residual oil extraction tank, first heated to 60°C, and stirred for 10 minutes; then 5g of 80°C hot water is added, the tank body is evacuated, and the reaction is carried out at a negative pressure of -0.08MPa for 20 minutes, the stirring rate is 55r / min, and the upper layer of fat is removed by centrifugation to obtain walnut defatted meal; Step 2, take 100g of defatted walnut meal, add 600g of softened water to mix, adjust the pH to 7; add 2g of chymosin, stir continuously for 2h under a negative pressure of -0.08MPa, the stirring speed is 60r / min, centrifuge to remove the separated protein precipitate, and obtain the first supernatant as walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to 5; 2g of a coagulant compound enzyme is added, wherein the specific formula of the coagulant compound enzyme is tannase:glycoside hydrolase with a mass ratio of 1:1.5, and enzymolysis is performed under a negative pressure of -0.08MPa for 3h; then the mixture is allowed to stand for acid precipitation enrichment for 1h, and a walnut instant protein gel is obtained after centrifugation; after the enzyme is inactivated, the walnut instant protein gel is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 0-10kD, and concentrated and dried to obtain an instant protein powder; Step 4, taking 50g of the separated protein precipitate in step 2, adding 250g of softened water and adjusting the pH to 10; alkali dissolving for 1h under a negative pressure of -0.08MPa, the reaction temperature is 45°C, stirring is continued at a speed of 60r / min, and centrifugation is performed to obtain a third supernatant; the third supernatant is added to an acid precipitation tank and the pH is adjusted to 5; 1g of a coagulant complex enzyme is added, wherein the specific formula of the coagulant complex enzyme is a mass ratio of tannase: glycoside hydrolase of 1:1.5, after enzymolysis for 4h under a negative pressure of -0.08MPa, the solution is allowed to stand for acid precipitation enrichment for 1h, and the walnut protein isolate gel is obtained after centrifugation; Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain isolated protein powder; Example 4 In step 3 and step 4, the specific formula of the coagulant complex enzyme is that the mass ratio of glutamine transaminase: glutamyl endopeptidase: tannase: glycoside hydrolase is 1:1:3:2.

[0029] Step 1, 100g of walnut meal raw material is crushed and filtered through a 200-mesh filter, and then added to a residual oil extraction tank, first heated to 60°C, and stirred for 10 minutes; then 5g of 80°C hot water is added, the tank body is evacuated, and the reaction is carried out at a negative pressure of -0.08MPa for 20 minutes, the stirring rate is 55r / min, and the upper layer of fat is removed by centrifugation to obtain walnut defatted meal; Step 2, take 100g of defatted walnut meal, add 600g of softened water to mix, adjust the pH to 7; add 2g of chymosin, stir continuously for 2h under a negative pressure of -0.08MPa, the stirring speed is 60r / min, centrifuge to remove the separated protein precipitate, and obtain the first supernatant as walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to 5; 2g of a coagulant complex enzyme is added, wherein the specific formula of the coagulant complex enzyme is a mass ratio of glutamine transaminase: glutamyl endopeptidase: tannase: glycoside hydrolase of 1:1:3:2, and enzymolysis is performed under a negative pressure of -0.08MPa for 3h; then the solution is allowed to stand for acid precipitation enrichment for 1h, and a walnut instant protein gel is obtained after centrifugation; after the enzyme is inactivated, the walnut instant protein gel is ultrafiltered and desalted using an ultrafiltration membrane with a molecular weight cutoff of 0-10kD, and concentrated and dried to obtain an instant protein powder; Step 4, taking 50g of the separated protein precipitate in step 2, adding 250g of softened water and adjusting the pH to 10; alkali dissolving for 1h under a negative pressure of -0.08MPa, the reaction temperature is 45°C, stirring is continued at a speed of 60r / min, and centrifugation is performed to obtain a third supernatant; the third supernatant is added to an acid precipitation tank and the pH is adjusted to 5; 1g of a coagulant complex enzyme is added, wherein the specific formula of the coagulant complex enzyme is a mass ratio of glutamine transaminase: glutamyl endopeptidase: tannase: glycoside hydrolase of 1:1:3:2, after enzymatic hydrolysis for 4h under a negative pressure of -0.08MPa, the solution is allowed to stand for acid precipitation enrichment for 1h, and the walnut protein isolate gel is obtained after centrifugation; Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain isolated protein powder; Comparative Example 1 The preparation method of this comparative example is basically the same as that of Example 1, except that no coagulant complex enzyme is added in step 2 and step 3.

[0030] Example 5 The protein mass fractions of the instant protein powders and isolated protein powders prepared in Examples 1-4 and Comparative Example 1 were measured using a Kjeldahl nitrogen analyzer to obtain protein content, protein yield, protein extraction rate and solubility data as shown in Table 1 below: Table 1 Comparison of relevant properties of protein powder samples in each group

[0031] As shown in Table 1 above, the appropriate use of the coagulant complex enzyme in the extraction process of instant protein powder and isolated protein greatly increases the protein content. This is because the addition of glutamine aminotransferase promotes enzyme-mediated molecular cross-linking and helps to form a strong gel; at the same time, the addition of tannase and glycoside hydrolase can be seen that the polysaccharide impurities and polyphenol impurities in the protein powder are separated from the gel, and finally the impurities are filtered out by the ultrafiltration membrane, which is manifested as a further increase in protein extraction rate and protein yield. Figure 2It can be seen that after enzymatic hydrolysis by the coagulant complex enzyme, more impurities are separated, and the color of the final purified instant protein powder is obviously whiter than that of the traditional method. Therefore, in the method for simultaneously preparing walnut instant protein powder and isolated protein powder provided by the present invention, in order to give full play to the enzymatic hydrolysis effect of each biological enzyme, the formula of the coagulant complex enzyme is preferably glutamine transaminase: glutamyl endopeptidase: tannase: glycoside hydrolase = 1:1:3:2.

[0032] Example 6 Verify the effect of the amount of added coagulant complex enzyme on the related properties of the obtained isolated protein powder.

[0033] The preparation conditions of this embodiment are basically the same as those of Example 4, except that in step 2, the added amounts of the coagulant complex enzyme are 1%, 2%, 3%, 4%, and 5% of the mass of the defatted walnut meal, respectively; correspondingly, in step 3, the added amounts of the coagulant complex enzyme are 1%, 2%, 3%, 4%, and 5% of the mass of the isolated protein precipitate, respectively.

[0034] The relevant properties of the instant protein powder and the isolated protein powder prepared in Example 6 and Comparative Example 1 were measured using a Kjeldahl nitrogen analyzer, and the results are shown in Table 2 below: Table 2 Comparison of related properties of each group of isolated protein powder samples

[0035] From Table 2 and Figure 3 It can also be seen that, although the protein acid precipitated gel obtained after alkali dissolution already contains fewer impurities after the initial extraction process of instant protein, the coagulant complex enzyme can still further purify the protein gel through the powerful complementary effect of multiple biological enzymes, thereby increasing the purity and whiteness of the isolated protein powder.

[0036] In summary, the present invention creatively introduces a coagulant complex enzyme in the synchronous preparation process of walnut instant protein powder and isolated protein powder, and further purifies the formed protein gel through the complementary action of multiple coagulant enzymes, and hydrolyzes polysaccharide and polyphenol impurities. The participation of the coagulant complex enzyme significantly increases the protein extraction rate and protein content of the obtained protein powder, improves the efficiency of the synchronous preparation of walnut instant protein powder and isolated protein powder, and the protein quality, and the preparation method is simple, the raw material of the coagulant complex enzyme is easy to obtain, and is suitable for industrial production.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A coagulant complex enzyme, characterized in that: The coagulant complex enzyme is selected from a mixture of at least two biological enzymes selected from transglutaminase, glutamyl endopeptidase, tannase and glycoside hydrolase.

2. The coagulant complex enzyme according to claim 1, characterized in that: The amount of glutamine transaminase added is 0.1-5 parts by weight; and / or, the amount of glutamyl endopeptidase added is 0.1-8 parts; and / or, the glycoside hydrolase is added in an amount of 0.1-10 parts; And / or, the added amount of the tannase is 0.1-10 parts.

3. A method for preparing walnut protein powder, characterized in that, The coagulant complex enzyme according to claim 1 is used for protein purification.

4. The preparation method according to claim 3, characterized in that: The specific steps are as follows: Step 1, after crushing and sieving the walnut meal raw material, adding it to the residual oil extraction tank, adding 60-100° C. hot water, reacting for 10-60 minutes under negative pressure, and centrifuging to remove the upper layer of fat to obtain walnut defatted meal powder; The mass ratio of hot water to meal is (2-10):100; Step 2, taking 100 parts of the defatted walnut meal, adding 100-1000 parts of softened water to mix, adjusting the pH to 6-9; adding 1-5 parts of chymosin, stirring continuously for 1-8 hours under negative pressure, centrifuging to remove the separated protein precipitate for standby use, and obtaining a first supernatant as a walnut instant protein liquid; Step 3, after the walnut instant protein liquid is inactivated, the second supernatant is collected and the pH is adjusted to acidic; 1-8 parts of the coagulant complex enzyme are added, and enzymolysis is performed under negative pressure for 1-5 hours; then the solution is allowed to stand for acid precipitation enrichment for 0.1-2 hours, and a walnut instant protein gel is obtained after centrifugation; after the enzyme is inactivated, the walnut instant protein gel is ultrafiltered to remove salt, concentrated, and dried to obtain an instant protein powder; Step 4, taking the isolated protein precipitate described in step 2, adding softened water and adjusting the pH to 7-11; performing alkaline dissolution under negative pressure for 1-5 hours, continuously stirring, and centrifuging to obtain a third supernatant; taking the third supernatant and adjusting the pH to acidic; adding an appropriate amount of the coagulant complex enzyme, performing enzymatic hydrolysis under negative pressure for 1-5 hours, standing for acid precipitation enrichment for 0.1-2 hours, and obtaining a walnut protein isolate gel after centrifugation; The mass ratio of the isolated protein precipitate: softened water: coagulant complex enzyme is 100: (100-1000): (1-8); Step 5, the walnut protein isolate gel is inactivated, washed to remove salt, and then concentrated, homogenized, and dried to obtain protein isolate powder; The coagulant complex enzyme is a mixture of at least two biological enzymes selected from transglutaminase, glutamyl endopeptidase, tannase, and glycoside hydrolase; The above components are all calculated by mass.

5. The preparation method according to claim 4, characterized in that: The mesh size of the sieving in step 1 is 30-200 meshes.

6. The preparation method according to claim 4, characterized in that: The amount of hot water added in step 1 is 2-10% of the mass of the meal.

7. The preparation method according to claim 4, characterized in that: In step 3, the molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration desalination process is 0-10 kD.

8. The preparation method according to claim 4, characterized in that: The addition amount of each component in the coagulant complex enzyme is as follows by weight: The amount of glutamine transaminase added is 0.1-5 parts; and / or, the amount of glutamyl endopeptidase added is 0.1-8 parts; and / or, the glycoside hydrolase is added in an amount of 0.1-10 parts; And / or, the added amount of the tannase is 0.1-10 parts.

9. The preparation method according to claim 4, characterized in that: The temperature of the enzymatic hydrolysis is 40-70°C; The pH value of the enzymatic hydrolysis of the coagulant complex enzyme is 3-7.

10. The preparation method according to claim 4, characterized in that: The negative pressure is -0.01 to -5 MPa; The stirring rate is 40-80 r / min.

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