Solubilizing compound enzyme and method for synchronously preparing instant protein powder and isolated protein powder by using solubilizing compound enzyme

Abstract

The invention provides a solubilizing compound enzyme and a method for synchronously preparing instant protein powder and isolated protein powder by using the solubilizing compound enzyme, and relates to the technical field of plant natural product extraction. The solubilizing compound enzyme provided by the invention comprises chymotrypsin, and / or nattokinase, and / or elastase; according to the synchronous preparation method of the instant protein powder and the isolated protein provided by the invention, the solubilizing compound enzyme is firstly added to successfully and quickly recover the protein function in the meal powder, and then the purified compound enzyme is added to sequentially prepare the instant protein powder and the isolated protein powder, so that the extraction rate of the instant protein powder is remarkably and synchronously improved; the production efficiency of the protein powder can be improved to the greatest extent, and the implementation process is simple, convenient and economical and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of plant natural product extraction, in particular to a solubilizing composite enzyme and a method for simultaneously preparing instant protein powder and separated protein powder by using the composite enzyme. Background Art

[0002] At present, nuts are one of the important woody oil crops in my country, and the planting area has jumped to the first place in the world. In particular, macadamia nuts are rich in nutrients such as oil and protein, with an oil content of up to 70%. Macadamia nut meal is a by-product of the macadamia nut oil industry and a high-quality source of protein with a protein content of 20-30%. However, it also contains a large amount 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. Among the existing methods for extracting plant proteins, most of them use alkali dissolution and acid precipitation methods, and oils are easy to form emulsification, affecting protein dissolution; 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 process of pressing and oiling macadamia nut meal, protein denaturation leads to partial loss of functionality. Before extracting protein, the cake meal should be pretreated to restore protein function.

[0003] Instant protein powder is mainly obtained by grinding, solubilization modification, and centrifugation 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. However, the water-insoluble protein containing more than 60% in the residue is not well utilized. The isolated protein powder is mainly prepared by alkali dissolution and acid precipitation process. During acid precipitation and water washing, the protein gel is easily dissolved and washed away by water, reducing the protein purity and extraction rate. Generally, after the acid precipitation of macadamia protein, it is produced without water washing process. The protein content in the separated protein powder is 60-65%, and a large amount of hydrogen ions and salts remain. The protein purity is low, the color is dark, and the taste is astringent. It is not suitable for the deep processing of high-quality protein powder. The gluten rich in macadamia protein isolate is water-insoluble, resulting in poor functional properties such as emulsification and foaming, which seriously limits its application in beverages, nutritional proteins and other foods.

[0004] In summary, it is of great significance to develop a composite enzyme suitable for simultaneous extraction of high-purity instant protein and isolated protein powder. Summary of the invention

[0005] The purpose of the present invention is to provide a solubilizing complex enzyme and a method for simultaneously preparing instant protein powder and separated protein powder using the same, so as to solve the technical problems of low protein powder extraction efficiency and high preparation cost in the prior art.

[0006] In order to solve the above technical problems, the present invention provides a solubilizing complex enzyme, which includes chymosin, and / or nattokinase, and / or elastase.

[0007] Furthermore, the contents of the components of the solubilizing complex enzyme are as follows by mass:

[0008] The content of chymosin is 0.1-5 parts;

[0009] And / or, the content of nattokinase is 0.1-5 parts;

[0010] And / or, the content of the elastase is 0.1-10 parts.

[0011] Chymotrypsin can quickly break down denatured proteins. For proteins that have lost their functionality, such as oil pressing residues, chymotrypsin can restore their functionality and thus turn them into protein ingredients.

[0012] Nattokinase can not only destroy cell walls to increase protein dissolution, but also promote protein hydrolysis and increase soluble protein content.

[0013] Elastase is mainly used to cleave the peptide bonds formed by the carboxyl side chains of neutral amino acid residues such as alanine, glycine, and serine and the amino groups of other amino acids. It has protein hydrolysis activity and can also maintain protein color.

[0014] On the other hand, the present invention also provides a method for simultaneously preparing instant protein powder and isolated protein powder, wherein the method uses the above-mentioned solubilizing complex enzyme, and the specific steps are as follows:

[0015] Step 1, adding 100 parts of defatted meal powder into a first protein extraction tank, adding 100-1000 parts of softened water, adjusting the pH to 6-9, and obtaining a defatted meal powder suspension;

[0016] Step 2, adding 1-5 parts of the solubilizing complex enzyme to the defatted meal suspension for enzymolysis, evacuating the tank and maintaining negative pressure for stirring for 1-8 hours, and centrifuging to obtain separated protein precipitate and instant protein solution;

[0017] By adding solubilizing compound enzymes, the protein function in defatted meal can be quickly restored, denatured proteins can be decomposed, and hydrophilic amino acid residues can be increased, which is beneficial to increasing the dissolution rate of water-soluble proteins. The mixed use of multiple decomposing enzymes is more conducive to accelerating the enzymatic hydrolysis efficiency and improving the protein extraction rate.

[0018] Step 3, after the instant protein solution is inactivated, the first supernatant is added to the first acid precipitation tank and the pH is adjusted to 3-7;

[0019] Add 1-8 parts of purified complex enzyme, evacuate the tank to perform negative pressure enzymolysis for 1-5 hours, perform static acid precipitation enrichment for 0.1-2 hours, and remove the second supernatant by centrifugation to obtain instant protein gel;

[0020] After the enzyme is inactivated, ultrafiltration is performed to remove salt, and the mixture is concentrated and spray-dried to obtain instant protein powder;

[0021] For instant protein gel, the water washing method is used to remove salt, which easily causes the protein to dissolve. Therefore, the present invention adopts the method of ultrafiltration to remove salt, so as to obtain high-purity instant protein powder.

[0022] Step 4, taking 50 parts of the isolated protein precipitate and adding it to the second protein extraction tank, and adding 50-500 parts of softened water, adjusting the pH to 7-11, evacuating the tank, performing negative pressure alkaline dissolution for 1-5 hours, the reaction temperature is 40-60° C., and centrifuging to obtain a third supernatant as the isolated protein liquid;

[0023] Step 5, adding the protein isolate solution into a second acid precipitation tank and adjusting the pH to 3-7;

[0024] Add 0.5-4 parts of purified complex enzyme, evacuate the tank to perform negative pressure enzymolysis for 1-5 hours, and then perform static acid precipitation enrichment for 0.1-2 hours. Centrifuge to obtain separated protein gel;

[0025] After the enzyme is inactivated, the product is washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain isolated protein powder;

[0026] The purified complex enzyme includes glutamine aminotransferase, glycoside hydrolase and tannase;

[0027] Adding an appropriate amount of purified complex enzyme in step 3 and step 5 can remove polyphenols and polysaccharide insoluble substances in the protein gel, which is beneficial to improving the purity and color of instant protein powder and isolated protein powder.

[0028] The above components are all calculated by mass.

[0029] Furthermore, the preparation steps of the defatted meal in step 1 are as follows:

[0030] Step S1, selecting nut meal, crushing and sieving to obtain meal powder;

[0031] Step S2, taking 100 parts of meal and adding them into the residual oil extraction tank, heating to 45-65° C. and stirring for 5-30 minutes to obtain preheated meal;

[0032] Step S3, adding 2-10 parts of hot water at 60-100° C. to the preheated meal, evacuating the tank, reacting under negative pressure for 10-60 minutes, continuously stirring, and centrifuging to remove upper fat to obtain defatted meal;

[0033] The above components are all measured by mass.

[0034] Using the principle of water replacement, the meal is heated and stirred and hot water of similar temperature is added to rupture the cells, dissolve the residual oil in the meal, and absorb the water preferentially by the protein in the meal. This reduces the amount of residual oil in the meal and obtains defatted meal.

[0035] Preferably, the mass ratio of the solubilizing complex enzyme is chymosin: nattokinase: elastase = (0.1-5): (0.1-5): (0.1-10).

[0036] Preferably, the mass ratio of the purified complex enzyme is glutamine aminotransferase:glycoside hydrolase:tannase=(0.1-5):(0.1-10):(0.1-10).

[0037] Preferably, the enzymatic hydrolysis temperature is 40-60°C.

[0038] Preferably, the negative pressure is -0.01 to -5 MPa. The extraction process is carried out under negative pressure, which can further improve the extraction rate and yield of protein and is more suitable for large-scale industrial production.

[0039] Furthermore, the molecular weight cut-off range of the ultrafiltration membrane selected for the ultrafiltration desalination process in step 3 is 0-10 kD.

[0040] Furthermore, the stirring speed is 40-80 r / min.

[0041] Furthermore, the sieving mesh number in step S1 is 30-200 meshes.

[0042] By adopting the above technical solution, the present invention has the following beneficial effects:

[0043] The invention provides a solubilizing complex enzyme and a method for simultaneously preparing instant protein powder and separated protein powder using the same. The invention creatively adds a solubilizing complex enzyme prepared by mixing a plurality of decomposing enzymes according to a certain ratio in the process of preparing the instant protein powder, thereby increasing the protein extraction rate of the instant protein powder to 46.1%, and increasing the protein content from 36.4% to more than 89%. On the other hand, the preparation method provided by the invention improves the extraction rate of the instant protein powder while ensuring the extraction efficiency of the separated protein powder, thereby maximally improving the production efficiency of the protein powder, and the implementation process is simple and economical, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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.

[0045] FIG. 1( a ) is a schematic diagram of the instant protein powder prepared in Example 4;

[0046] Figure 1(b) is a schematic diagram of the isolated protein powder prepared in Example 4;

[0047] FIG2( a) is a schematic diagram of the instant protein powder prepared in Comparative Example 1;

[0048] Figure 2(b) is a schematic diagram of the isolated protein powder prepared in Comparative Example 1;

[0049] Figure 3 This is a curve chart of protein content in the instant protein powder obtained by using a combination of five solubilizing complex enzymes in Example 5. DETAILED DESCRIPTION

[0050] 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.

[0051] The present invention is further explained below in conjunction with specific implementation modes.

[0052] The method for simultaneously preparing instant protein powder and isolated protein powder provided in this embodiment comprises the following specific steps:

[0053] Step 1, selecting nut meal, crushing and sieving with a 30-200 mesh filter to obtain meal powder; taking 100 parts of meal powder and adding it to a residual oil extraction tank, heating it to 45-65° C. and stirring it for 5-30 minutes to obtain preheated meal powder; adding 2-10 parts of 60-100° C. hot water to the preheated meal powder, evacuating the tank body, maintaining a negative pressure of -0.01 to -5 MPa, reacting for 10-60 minutes, continuously stirring, and centrifuging to remove upper grease to obtain defatted meal powder;

[0054] Step 2, taking 100 parts of defatted meal powder, adding them to the first protein extraction tank, adding 100-1000 parts of softened water, and adjusting the pH to 6-9 to obtain a defatted meal powder suspension;

[0055] Add 1-5 parts of solubilizing complex enzyme to the defatted meal suspension and perform enzymolysis at 40-60° C., evacuate the tank and maintain a negative pressure of -0.01 to -5 MPa, stir for 1-8 hours, and centrifuge to obtain separated protein precipitate and instant protein solution;

[0056] After the instant protein solution is inactivated, the first supernatant is added to the first acid precipitation tank, and the pH is adjusted to 3-7; 1-8 parts of purified complex enzyme are added, the tank is evacuated for negative pressure enzymolysis for 1-5 hours, static acid precipitation is performed for enrichment for 0.1-2 hours, and the second supernatant is removed by centrifugation to obtain an instant protein gel;

[0057] After the enzyme is inactivated, ultrafiltration is performed to remove salt using an ultrafiltration membrane with a molecular weight cutoff range of 0-10KD, and then concentrated and spray-dried to obtain instant protein powder;

[0058] Step 3, taking 50 parts of the isolated protein precipitate and adding it to a second protein extraction tank, and adding 50-500 parts of softened water, adjusting the pH to 7-11, evacuating the tank, alkali dissolving for 1-5 hours under a negative pressure of -0.01 to -5 MPa, and centrifuging to obtain a third supernatant as an isolated protein liquid;

[0059] The separated protein liquid is added to the second acid precipitation tank, and after adjusting the pH to 3-7, 0.5-4 parts of purified complex enzyme are added, the tank body is evacuated, and enzymolysis is carried out at a negative pressure of -0.01 to -5Mpa for 1-5 hours, and static acid precipitation is carried out for enrichment for 0.1-2 hours, and separated protein gel is obtained by centrifugation; after the enzyme is inactivated, it is washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain separated protein powder.

[0060] Wherein, the solubilizing complex enzyme is a mixture of at least two biological enzymes selected from 0.1-5 parts of chymosin, 0.1-5 parts of nattokinase, and 0.1-10 parts of elastase;

[0061] The mass ratio of the purified complex enzyme is aminotransferase:glycoside hydrolase:tannase=(0.1-5):(0.1-10):(0.1-10).

[0062] The above components are all calculated by mass.

[0063] Determination methods of protein content, protein yield and protein extraction rate:

[0064] With reference to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food", the protein mass fraction was determined using a fully automatic Kjeldahl nitrogen analyzer. The calculation formula for protein content (X1) is shown in formula (1), the calculation formula for protein extraction rate (X2) is shown in formula (2), and the calculation formula for protein yield (X3) is shown in formula (3).

[0065] X1=M1 / M2×100% (1)

[0066] X2=M3 / M4×100% (2)

[0067] X3=M5 / M6×100% (3)

[0068] In the formula: M1 is the protein mass of protein powder; M2 is the mass of protein powder; M3 is the total protein content of protein powder; M4 is the total protein content of meal raw material; M5 is the weight of protein powder; M6 is the weight of meal raw material.

[0069] Example 1

[0070] The instant protein powder and the separated protein powder are prepared synchronously by the preparation method provided by the present invention, wherein the formula of the solubilizing complex enzyme is that the mass ratio of chymosin:nattokinase is 1:2.

[0071] Step 1, selecting nut meal and crushing it, and sieving it through a 30-200 mesh filter to obtain meal powder; taking 1000g of meal powder and adding it to a residual oil extraction tank, heating it to 50°C and stirring it for 15min, then adding 50g of 75°C hot water, evacuating the tank, maintaining a negative pressure of -0.08MPa, reacting for 20min, continuously stirring at a speed of 55r / min, and centrifuging to remove upper grease to obtain defatted meal powder;

[0072] Step 2, taking 1000 g of defatted meal, adding it to the first protein extraction tank, adding 8000 g of softened water, and adjusting the pH to 7 to obtain a defatted meal suspension;

[0073] 15 g of a solubilizing compound enzyme is added to the defatted meal suspension, wherein the formula of the solubilizing compound enzyme is a mass ratio of chymosin to nattokinase of 1:2, and enzymolysis is performed. The tank is evacuated and maintained at a negative pressure of -0.08 MPa, and the mixture is stirred at a rate of 60 r / min for 2 h. The separated protein precipitate and the instant protein solution are obtained by centrifugation.

[0074] After the instant protein liquid is inactivated, the supernatant is added to the first acid precipitation tank, and the pH is adjusted to 4.5; 20g of purified complex enzyme is added, wherein the formula of the purified complex enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, and static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain an instant protein gel;

[0075] After the enzyme is inactivated, ultrafiltration is performed to remove salt using an ultrafiltration membrane with a molecular weight cutoff range of 0-10KD, and then concentrated and spray-dried to obtain instant protein powder;

[0076] Step 3, taking 500 g of the isolated protein precipitate and adding it to a second protein extraction tank, and adding 4000 g of softened water, adjusting the pH to 10, evacuating the tank, and performing alkaline dissolution under a negative pressure of -0.08 MPa for 1 h, the reaction temperature is 45° C., the stirring speed is 60 r / min, and centrifuging to obtain a supernatant as an isolated protein liquid;

[0077] The obtained separated protein liquid is added to the second acid precipitation tank, and after adjusting the pH to 5, 10g of purified complex enzyme is added, wherein the formula of the purified complex enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank body is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, and static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain separated protein gel; after the enzyme is inactivated, it is washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain separated protein powder.

[0078] Example 2

[0079] The preparation method provided by the invention is adopted to simultaneously prepare instant protein powder and isolated protein powder, wherein the formula of the solubilizing complex enzyme is that the mass ratio of nattokinase to elastase is 1:2.

[0080] Step 1, selecting nut meal and crushing it, and sieving it through a 30-200 mesh filter to obtain meal powder; taking 1000g of meal powder and adding it to a residual oil extraction tank, heating it to 50°C and stirring it for 15min, then adding 50g of 75°C hot water, evacuating the tank, maintaining a negative pressure of -0.08MPa, reacting for 20min, continuously stirring at a speed of 55r / min, and centrifuging to remove upper grease to obtain defatted meal powder;

[0081] Step 2, taking 1000 g of defatted meal, adding it to the first protein extraction tank, adding 8000 g of softened water, and adjusting the pH to 7 to obtain a defatted meal suspension;

[0082] 15 g of a solubilizing compound enzyme was added to the defatted meal suspension, wherein the formula of the solubilizing compound enzyme was nattokinase: elastase in a mass ratio of 1:2, and enzymolysis was performed. The tank was evacuated and maintained at a negative pressure of -0.08 MPa, and the mixture was stirred at a rate of 60 r / min for 2 h. The separated protein precipitate and the instant protein solution were obtained by centrifugation.

[0083] After the instant protein liquid is inactivated, the supernatant is added to the first acid precipitation tank, and the pH is adjusted to 4.5; 20g of purified complex enzyme is added, wherein the formula of the purified complex enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, and static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain an instant protein gel;

[0084] After the enzyme is inactivated, ultrafiltration is performed to remove salt using an ultrafiltration membrane with a molecular weight cutoff range of 0-10KD, and then concentrated and spray-dried to obtain instant protein powder;

[0085] Step 3, taking 500 g of the isolated protein precipitate and adding it to a second protein extraction tank, and adding 4000 g of softened water, adjusting the pH to 10, evacuating the tank, and performing alkaline dissolution under a negative pressure of -0.08 MPa for 1 h, the reaction temperature is 45° C., the stirring speed is 60 r / min, and centrifuging to obtain a supernatant as an isolated protein liquid;

[0086] The obtained separated protein liquid is added to the second acid precipitation tank, and after adjusting the pH to 5, 10g of purified complex enzyme is added, wherein the formula of the purified complex enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank body is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, and static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain separated protein gel; after the enzyme is inactivated, it is washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain separated protein powder.

[0087] Example 3

[0088] The instant protein powder and the separated protein powder are prepared synchronously by the preparation method provided by the invention, wherein the formula of the solubilizing complex enzyme is that the mass ratio of chymosin: elastase is 1:2.

[0089] Step 1, selecting nut meal and crushing it, and sieving it through a 30-200 mesh filter to obtain meal powder; taking 1000g of meal powder and adding it to a residual oil extraction tank, heating it to 50°C and stirring it for 15min, then adding 50g of 75°C hot water, evacuating the tank, maintaining a negative pressure of -0.08MPa, reacting for 20min, continuously stirring at a speed of 55r / min, and centrifuging to remove upper grease to obtain defatted meal powder;

[0090] Step 2, taking 1000 g of defatted meal, adding it to the first protein extraction tank, adding 8000 g of softened water, and adjusting the pH to 7 to obtain a defatted meal suspension;

[0091] 15 g of a solubilizing compound enzyme is added to the defatted meal suspension, wherein the formula of the solubilizing compound enzyme is a mass ratio of chymosin to elastase of 1:2, and enzymolysis is performed. The tank is evacuated and maintained at a negative pressure of -0.08 MPa, and the mixture is stirred at a rate of 60 r / min for 2 h. The separated protein precipitate and the instant protein solution are obtained by centrifugation.

[0092] After the instant protein liquid is inactivated, the supernatant is added to the first acid precipitation tank, and the pH is adjusted to 4.5; 20g of purified complex enzyme is added, wherein the formula of the purified complex enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, and static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain an instant protein gel;

[0093] After the enzyme is inactivated, ultrafiltration is performed to remove salt using an ultrafiltration membrane with a molecular weight cutoff range of 0-10KD, and then concentrated and spray-dried to obtain instant protein powder;

[0094] Step 3, taking 500 g of the isolated protein precipitate and adding it to a second protein extraction tank, and adding 6000 g of softened water, adjusting the pH to 10, evacuating the tank, and performing alkaline dissolution under a negative pressure of -0.08 MPa for 1 h, the reaction temperature is 45° C., the stirring speed is 60 r / min, and centrifuging to obtain a supernatant as an isolated protein liquid;

[0095] The obtained separated protein liquid is added to the second acid precipitation tank, and after adjusting the pH to 5, 10g of purified complex enzyme is added, wherein the formula of the purified complex enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank body is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, and static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain separated protein gel; after the enzyme is inactivated, it is washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain separated protein powder.

[0096] Example 4

[0097] like Figure 1(a)-Figure 1(b) As shown, the preparation method provided by the present invention is used to simultaneously prepare instant protein powder and isolated protein powder, wherein the formula of the solubilizing complex enzyme is a mass ratio of chymosin: nattokinase: elastase of 1:1:2.

[0098] Step 1, selecting nut meal and crushing it, and sieving it through a 30-200 mesh filter to obtain meal powder; taking 1000g of meal powder and adding it to a residual oil extraction tank, heating it to 50°C and stirring it for 15min, then adding 50g of 75°C hot water, evacuating the tank, maintaining a negative pressure of -0.08MPa, reacting for 20min, continuously stirring at a speed of 55r / min, and centrifuging to remove upper grease to obtain defatted meal powder;

[0099] Step 2, taking 1000 g of defatted meal, adding it to the first protein extraction tank, adding 8000 g of softened water, and adjusting the pH to 7 to obtain a defatted meal suspension;

[0100] 15 g of a solubilizing compound enzyme is added to the defatted meal suspension, wherein the formula of the solubilizing compound enzyme is a mass ratio of chymosin: nattokinase: elastase of 1:1:2, the tank is evacuated and maintained at a negative pressure of -0.08 MPa, stirred at a rate of 60 r / min, and enzymolysis is performed for 2 h, and centrifuged to obtain a separated protein precipitate and an instant protein solution;

[0101] After the instant protein liquid is inactivated, the supernatant is added to the first acid precipitation tank, and the pH is adjusted to 5; 20g of purified compound enzyme is added, wherein the formula of the purified compound enzyme is glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank is evacuated, and the enzymatic hydrolysis is maintained at a negative pressure of -0.08MPa for 2h, static acid precipitation is enriched for 30min, and the supernatant is removed by centrifugation to obtain an instant protein gel;

[0102] After the enzyme is inactivated, ultrafiltration is performed to remove salt using an ultrafiltration membrane with a molecular weight cutoff in the range of 0-10KD, and then concentrated and spray-dried to obtain an instant protein powder as shown in FIG1(a);

[0103] Step 3, taking 500 g of the isolated protein precipitate and adding it to a second protein extraction tank, and adding 4000 g of softened water, adjusting the pH to 10, evacuating the tank, and performing alkaline dissolution under a negative pressure of -0.08 MPa for 1 h, the reaction temperature is 45° C., the stirring speed is 60 r / min, and centrifuging to obtain a supernatant as an isolated protein liquid;

[0104] The obtained separated protein liquid was added to the second acid precipitation tank, and after adjusting the pH to 5, 10 g of purified complex enzyme was added, wherein the formula of the purified complex enzyme was glutamine aminotransferase: glycoside hydrolase: tannase in a mass ratio of 1:1:1, the tank was evacuated, and the enzymatic hydrolysis was maintained at a negative pressure of -0.08 MPa for 2 hours, and static acid precipitation was performed for 30 minutes. The supernatant was removed by centrifugation to obtain separated protein gel; after the enzyme was inactivated, it was washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain separated protein powder as shown in Figure 1(b).

[0105] Comparative Example 1

[0106] The preparation steps in the preparation method provided in this comparative example are basically the same as those in Example 4, except that in step 2, no solubilizing complex enzyme and purified complex enzyme are added, and in step 3, no purified complex enzyme is added. The obtained product is as follows: Figure 2(a)-Figure 2(b) shown.

[0107] The components of the instant protein powder obtained in Examples 1-4 and Comparative Example 1 were measured, and the comparison results are shown in Table 1 below:

[0108] Table 1 Comparison of composition determination data of instant protein powder obtained in each group of examples and comparative example 1

[0109]

[0110] As can be seen from Table 1, the simultaneous use of chymotrypsin and elastase has a significant effect on improving the solubility of proteins and restoring the functions of damaged proteins. Nattokinase is mainly used to destroy cell walls to increase protein dissolution and promote protein hydrolysis. Therefore, in the method for synchronously preparing instant protein powder and isolated protein powder provided by the present invention, the formula of the solubilizing complex enzyme is preferably: chymotrypsin: nattokinase: elastase = 1:1:2.

[0111] Example 5

[0112] like Figure 3 As shown, the effect of the amount of added solubilizing complex enzyme on the quality of the obtained instant protein powder was verified.

[0113] The preparation method of this embodiment is basically the same as that of embodiment 4, except that the addition amount of the solubilizing complex enzyme is 1%, 1.5%, 2%, 2.5%, and 3%, respectively. The relevant performance tests of the instant protein powder obtained by each group of solutions are shown in the following Table 2:

[0114] Table 2 Related test results of instant protein powder obtained by adding 5 groups of solubilizing complex enzymes

[0115]

[0116] From Table 2 and Figure 3 It can be seen that when the addition ratio of the solubilizing compound enzyme is 1.5%, the protein content, protein yield, protein extraction rate and solubility of the instant protein powder all reach the highest level; when the addition amount continues to increase, the protein content, protein yield, protein extraction rate and solubility of the instant protein powder obtained all decrease to varying degrees. This is because with the increase in the addition amount of the solubilizing compound enzyme, the contact area between the compound enzyme and the defatted meal gradually increases, and the speed of enzymatic hydrolysis will increase with the increase in the addition amount. From the test results, it is shown that the more the solubilizing compound enzyme is added, the higher the protein content, protein yield and protein extraction rate in the instant protein powder obtained; however, when the addition amount of the solubilizing compound enzyme reaches more than 1.5%, various biological enzymes in the reaction system begin to appear excessively surplus, resulting in competitive inhibition between some biological enzymes with similar functions, and some excess biological enzymes form a complex system with proteins, which inhibits the overall activity of the compound enzyme, which is manifested as the relevant measurement indicators slowly decline with the further increase in the addition amount of the solubilizing compound enzyme. Therefore, in the method for simultaneously preparing instant protein and isolated protein provided by the present invention, the added amount of the solubilizing complex enzyme is preferably 1.5%.

[0117] In summary, the relevant parameters of the instant protein powder obtained in Examples 1-5 are summarized in Table 3:

[0118] Table 3 Effect of the mixing ratio of each component of the solubilizing complex enzyme and the overall addition amount on the quality of the obtained instant protein powder

[0119]

[0120]

[0121] As can be seen from Table 3, when the mass ratio of each component of the solubilizing complex enzyme is chymosin: nattokinase: elastase = 1:1:2, the three solubilizing enzymes interact with each other and the enzymolysis effect is the best; when the addition amount of the solubilizing complex enzyme is 1.5% of the mass of the meal, the various performance parameters of the obtained instant protein powder are the highest. It can be seen that the solubilizing complex enzyme provided by the present invention has a significant effect on the enzymolysis rate and extraction effect of the instant protein, and the protein content of the instant protein powder is increased from 36.4% to more than 89%, the protein yield is increased from 6.8% to 8.4%, and the protein extraction rate is increased from 31.6% to 46.1%.

[0122] 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 with 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 solubilizing complex enzyme, characterized in that The solubilizing complex enzyme includes chymosin, and / or nattokinase, and / or elastase.

2. The solubilizing complex enzyme according to claim 1, characterized in that The content of chymosin is 0.1-5 parts by weight; And / or, the content of nattokinase is 0.1-5 parts; And / or, the content of the elastase is 0.1-10 parts.

3. A method for synchronously preparing instant protein powder and isolated protein powder, characterized in that: Using the solubilizing complex enzyme according to claim 1 or 2, the specific steps are as follows: Step 1, adding 100 parts of defatted meal powder into a first protein extraction tank, adding 100-1000 parts of softened water, adjusting the pH to 6-9, and obtaining a defatted meal powder suspension; Step 2, adding 1-5 parts of the solubilizing complex enzyme to the defatted meal suspension for enzymolysis, evacuating the tank and maintaining negative pressure for stirring for 1-8 hours, and centrifuging to obtain separated protein precipitate and instant protein solution; Step 3, after the instant protein solution is inactivated, the first supernatant is added to the first acid precipitation tank and the pH is adjusted to 3-7; Add 1-8 parts of purified complex enzyme, evacuate the tank to perform negative pressure enzymolysis for 1-5 hours, perform static acid precipitation enrichment for 0.1-2 hours, and remove the second supernatant by centrifugation to obtain instant protein gel; After the enzyme is inactivated, ultrafiltration is performed to remove salt, and the mixture is concentrated and spray-dried to obtain instant protein powder; Step 4, taking 50 parts of the isolated protein precipitate and adding it to the second protein extraction tank, and adding 50-500 parts of softened water, adjusting the pH to 7-11, evacuating the tank, performing negative pressure alkaline dissolution for 1-5 hours, the reaction temperature is 40-60° C., and centrifuging to obtain a third supernatant as the isolated protein liquid; Step 5, adding the protein isolate solution into a second acid precipitation tank and adjusting the pH to 3-7; Add 0.5-4 parts of purified complex enzyme, evacuate the tank to perform negative pressure enzymolysis for 1-5 hours, and then perform static acid precipitation enrichment for 0.1-2 hours. Centrifuge to obtain separated protein gel; After the enzyme is inactivated, the product is washed with water to remove salt, and then concentrated, homogenized and spray-dried to obtain isolated protein powder; The purified complex enzyme includes glutamine aminotransferase, glycoside hydrolase and tannase; The above components are all calculated by mass.

4. The preparation method according to claim 3, characterized in that: The preparation steps of the defatted meal in step 1 are as follows: Step S1, selecting nut meal, crushing and sieving to obtain meal powder; Step S2, taking 100 parts of meal powder and adding them into the residual oil extraction tank, heating to 45-65° C. and stirring for 5-30 minutes to obtain preheated meal powder; Step S3, adding 2-10 parts of hot water at 60-100° C. to the preheated meal, evacuating the tank, reacting under negative pressure for 10-60 minutes, continuously stirring, and centrifuging to remove upper fat to obtain defatted meal; The above components are all calculated by mass.

5. The preparation method according to claim 3, characterized in that: The mass ratio of the components of the purified complex enzyme is: aminotransferase:glycoside hydrolase:tannase=(0.1-5):(0.1-10):(0.1-10).

6. The preparation method according to claim 3 or 4, characterized in that: The negative pressure is -0.01 to -5 MPa.

7. The preparation method according to claim 3, characterized in that: The enzymatic hydrolysis temperature is 40-60°C.

8. The preparation method according to claim 3, characterized in that: The ultrafiltration membrane used in the ultrafiltration desalination process in step 3 has a molecular weight cutoff range of 0-10 kD.

9. The preparation method according to claim 3, characterized in that: The stirring speed is 40-80 r / min.

10. The preparation method according to claim 2, characterized in that: The sieving mesh number in step S1 is 30-200 meshes.

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