Biological compound seasoning and blending method thereof

Abstract

The invention relates to the field of walnut protein biological active peptides, and discloses a biological compound seasoning and a blending method thereof.The biological compound seasoning comprises a basic seasoning agent and biological active peptides with oxidation resistance, and the biological active peptides are walnut peptides prepared through the following steps that S1, walnut protein is used as a raw material and mixed with the basic seasoning agent; carrying out two-step enzymolysis on the walnut protein by using single thermolysin and compound enzyme in sequence to obtain enzymatic hydrolysate; and S2, separating and purifying the enzymatic hydrolysate which is sequentially subjected to the enzymolysis of the single thermolysin and the compound enzyme in the step S1 through molecular sieve chromatography and ion exchange chromatography, and drying to obtain the walnut peptide. Compared with the prior art, the feed additive is large in small peptide proportion, easy to digest and absorb and high in oxidation resistance.

Description

Technical Field

[0001] The invention relates to the field of seasonings, and in particular to a biological composite seasoning and a preparation method thereof. Background Art

[0002] Active peptide bio-complex seasoning with antioxidant properties is a product that combines the functions of bioactive peptides and seasonings. It aims to improve the flavor of food while providing health benefits by ingesting active peptides with antioxidant properties. The commonly used active peptide is walnut peptide.

[0003] Walnut protein contains 4 kinds of proteins, namely albumin, globulin, alcohol-soluble protein and gluten, accounting for about 7%, 18%, 5% and 70% of the total walnut protein respectively. Walnut protein is a high-quality protein due to its high digestibility and net protein ratio, and has a high value for human utilization. Walnut protein contains 18 kinds of amino acids, and the content of 8 essential amino acids required by the human body is reasonable. Walnut protein also contains more brain-boosting substances - aspartic acid, glutamic acid, arginine, etc. Due to its special composition, it has a series of nutritional and health benefits.

[0004] In recent years, scientific research has found that after the body digests protein, it is not absorbed mainly in the form of amino acids, but in the form of peptides. At the same time, some peptides can not only provide nutrients needed for human growth and development, but also have a variety of physiological functions. Active peptides have the advantages of small molecular weight, no antigenicity, easy absorption, avoidance of drug resistance, and no side effects after entering the human body through the digestive tract. At present, the research on bioactive peptides is developing rapidly.

[0005] There are a large number of methods for preparing different walnut protein bioactive peptides by enzymatic hydrolysis technology in the prior art, such as CN112813125A, CN115505621A, CN108796016B, etc. The walnut peptides prepared by the prior art are basically low in small peptide content and weak in antioxidant capacity. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the walnut peptides prepared in the prior art when preparing biological composite seasonings basically have low small peptide content and weak antioxidant capacity. The purpose is to provide a biological composite seasoning and a preparation method thereof to solve the above problems.

[0007] The present invention is achieved through the following technical solutions:

[0008] A biological composite seasoning comprises a basic seasoning and a bioactive peptide with antioxidant properties, wherein the bioactive peptide is a walnut peptide prepared by the following steps:

[0009] S1, using walnut protein as raw material, sequentially using a single thermophilic protease and a composite enzyme to perform two-step enzymolysis on the walnut protein to obtain an enzymolysis solution;

[0010] S2, separating and purifying the enzymatic hydrolysate obtained by single thermophilic protease and composite enzyme enzymolysis in step S1 by molecular sieve chromatography and ion exchange chromatography, and obtaining walnut peptide after drying;

[0011] In step S1, the complex enzyme includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 3:1.5-2 in terms of mass.

[0012] In the present invention, the thermophilic protease must be used to perform the first enzymatic hydrolysis on the walnut protein, and then the composite enzyme of alkaline protease and trypsin is used. The enzymes used in the two-step enzymatic hydrolysis cannot be exchanged, because the enzymatic hydrolysis temperature of the thermophilic protease is 65-85°C, and the thermal denaturation of the walnut protein occurs at about 60°C. After the walnut protein is denatured, the spatial structure is destroyed, so that the amino acids are fully exposed and are more easily enzymatically hydrolyzed. Therefore, the thermophilic protease has a good effect on the enzymatic hydrolysis of the walnut protein. After the thermophilic protease is enzymatically hydrolyzed, the walnut protein has undergone irreversible denaturation and is easily enzymatically hydrolyzed. When the composite enzyme is used for the second step enzymatic hydrolysis, the enzymatic hydrolysis effect will be better.

[0013] In the present invention, after the thermophilic protease performs the first step of enzymolysis on walnut protein, no enzyme inactivation treatment is performed. When the composite enzyme is used for the second step of enzymolysis, the thermophilic protease can still continue to produce an enzymolysis effect, but the enzymolysis intensity of the thermophilic protease is relatively weak in the second step of enzymolysis. Then, the second step of enzymolysis is equivalent to the synergistic effect of thermophilic protease, alkaline protease and trypsin to continuously hydrolyze the walnut protein. The polypeptide content shows a trend of first increasing and then decreasing with the increase of enzymolysis time. When the enzymolysis time is extended to a certain extent, the polypeptide amount will increase significantly. After multiple experiments, it can be known that the thermophilic protease enzymolysis of walnut protein obtained Walnut peptides contain a large amount of antioxidant active peptides, strong antioxidant capacity, and a large proportion of small peptides after thermolysin enzymolysis, but the suitable temperature for thermolysin enzymolysis is 65-85 ° C. Walnut protein will be over-denatured when it is at this temperature for a long time. Excessive denaturation may breed new harmful substances or cause a large loss of nutrients. The present invention shortens the thermolysin enzymolysis time, and uses a composite enzyme with an enzymolysis temperature lower than the denaturation temperature of walnut protease to cooperate with thermolysin, which significantly improves the effect of enzymolysis, and the obtained walnut peptide has superior performance, and the antioxidant performance of the biological composite seasoning is better. The preparation method of the composite enzyme in the present invention is a prior art, and no further elaboration is made here.

[0014] Preferably, the complex enzyme in step S1 includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 5:3 in terms of mass. The specific steps of S1 are as follows:

[0015] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0016] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0017] c. Rapidly cool the enzymolysis solution A, keep the temperature constant after cooling to 55°C, adjust the pH of the enzymolysis solution A with a pH regulator so that the pH of the enzymolysis solution A is maintained at 8, then add the complex enzyme, perform enzymolysis for 3 hours, obtain the enzymolysis solution B, and perform the operation of step S2 on the enzymolysis solution B; in units of mass, the amount of the complex enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution. The peptide content (on a dry basis) in the walnut peptide reaches 88.73%, and the relative molecular mass of all peptide segments is within 1300Da, wherein the peptide segment content of 1000-180Da in the relative molecular mass is greater than 95%, and the peptide segment content of 500-180Da in the peptide segment of 1000-180Da accounts for 65%.

[0018] Compared with the prior art, the walnut peptide obtained by the present invention has a large proportion of small peptides, is easy to digest and absorb, and has strong antioxidant capacity. In the present invention, the time for enzymolysis by a single thermolysin is 2 hours. The walnut protein will not be over-denatured when it is at 75°C for 2 hours, but when it exceeds 2 hours and approaches 3 hours, a small amount of flocculent precipitation will appear. Therefore, the present invention determines the time for enzymolysis by a single thermolysin to be 2 hours. When walnut protein is over-denatured, flocculent precipitation or turbid liquid will appear, and this symptom is visible to the naked eye.

[0019] Furthermore, the enzymatic hydrolyzate which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 is heated to 100° C. to inactivate the enzyme, and then separated and purified by molecular sieve chromatography and ion exchange chromatography, and finally dried to obtain the walnut peptide product.

[0020] Furthermore, the basic seasoning includes salty agents, sweeteners, vitamin E, tea polyphenols, and spices.

[0021] Furthermore, the method for preparing the biological composite seasoning comprises the following steps:

[0022] A. Preparation of bioactive peptides with antioxidant properties:

[0023] S1, using walnut protein as raw material, sequentially using a single thermophilic protease and a composite enzyme to perform two-step enzymolysis on the walnut protein to obtain an enzymolysis solution;

[0024] S2, separating and purifying the enzymatic hydrolysate obtained by single thermophilic protease and composite enzyme enzymolysis in step S1 by molecular sieve chromatography and ion exchange chromatography, and obtaining walnut peptide after drying;

[0025] In step S1, the complex enzyme includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 3:1.5-2 in terms of mass.

[0026] B. Mixing the bioactive peptides with basic seasonings: Mix salting agents, sweeteners, vitamin E, tea polyphenols, and spices to obtain basic seasoning powder, and then evenly mix the walnut peptides in step S2 with the basic seasoning powder in proportion to obtain a biocomposite seasoning.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] 1. Compared with the prior art, the biological composite seasoning of the present invention has a large proportion of small peptides, is easy to digest and absorb, and has strong antioxidant capacity;

[0029] 2. In the preparation process of a biological composite seasoning of the present invention, the enzymatic hydrolysis time of thermophilic bacteria protease is shortened, and a composite enzyme with an enzymatic hydrolysis temperature lower than the denaturation temperature of walnut protease is used in combination with thermophilic bacteria protease, which significantly improves the effect of enzymatic hydrolysis. The obtained walnut peptide has superior performance, and thus the antioxidant property of the biological composite seasoning is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0031] Figure 1 This is a result diagram of Test Example 1 of the present invention;

[0032] Figure 2 This is a result diagram of Test Example 2 of the present invention;

[0033] Figure 3 This is a result diagram of Test Example 3 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0035] Example 1

[0036] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0037] S1. Walnut protein was used as raw material, and the walnut protein was hydrolyzed in two steps using a single thermophilic protease and a composite enzyme in sequence to obtain an enzymatic solution; in units of mass, the usage ratio of alkaline protease to trypsin was 2:1 (3:1.5).

[0038] The specific steps of S1 are as follows:

[0039] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0040] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0041] c. Rapidly cool the enzymatic solution A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic solution A so that the pH of the enzymatic solution A is maintained at 8. Then add the composite enzyme and perform enzymolysis for 3 hours to obtain the enzymatic solution B. The enzymatic solution B is subjected to the operation of step S2. In units of mass, the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution.

[0042] S2, heating the enzymatic hydrolyzate which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0043] Using the polypeptide determination method in Appendix B of GB / T 22492-2008 and the peptide relative molecular mass distribution determination method in Appendix A of GB / T 22492-2008, the peptide content (on a dry basis) in walnut peptides reached 87.7%, and the relative molecular masses of all peptide segments were within 1300Da, of which the content of peptide segments with a relative molecular mass of 1000-180Da was greater than 95%, and the content of peptide segments of 500-180Da in the peptide segments of 1000-180Da accounted for 60%.

[0044] A method for preparing a biological composite seasoning comprises the following steps:

[0045] A. Preparation of bioactive peptides with antioxidant properties:

[0046] S1, using walnut protein as raw material, sequentially using a single thermophilic protease and a composite enzyme to perform two-step enzymolysis on the walnut protein to obtain an enzymolysis solution;

[0047] S2, separating and purifying the enzymatic hydrolysate obtained by single thermophilic protease and composite enzyme enzymolysis in step S1 by molecular sieve chromatography and ion exchange chromatography, and obtaining walnut peptide after drying;

[0048] In step S1, the complex enzyme includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 3:1.5-2 in terms of mass.

[0049] B. Mixing the bioactive peptides with basic seasonings: Mix salting agents, sweeteners, vitamin E, tea polyphenols, and spices to obtain basic seasoning powder, and then evenly mix the walnut peptides in step S2 with the basic seasoning powder in proportion to obtain a biocomposite seasoning.

[0050] Example 2

[0051] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0052] S1. Walnut protein was used as raw material, and the walnut protein was hydrolyzed in two steps using a single thermophilic protease and a composite enzyme in sequence to obtain an enzymatic hydrolyzate; in units of mass, the usage ratio of alkaline protease to trypsin was 5:3 (3:1.8).

[0053] The specific steps of S1 are as follows:

[0054] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0055] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0056] c. Rapidly cool the enzymatic solution A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic solution A so that the pH of the enzymatic solution A is maintained at 8. Then add the composite enzyme and perform enzymolysis for 3 hours to obtain the enzymatic solution B. The enzymatic solution B is subjected to the operation of step S2. In units of mass, the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution.

[0057] S2, heating the enzymatic hydrolyzate which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0058] Using the polypeptide determination method in Appendix B of GB / T 22492-2008 and the peptide relative molecular mass distribution determination method in Appendix A of GB / T 22492-2008, the peptide content (on a dry basis) in walnut peptides reached 88.73%, and the relative molecular masses of all peptide segments were within 1300Da, of which the content of peptide segments in the relative molecular mass range of 1000-180Da was greater than 95%, and the content of peptide segments in the range of 500-180Da accounted for 65% of the peptide segments in the range of 1000-180Da.

[0059] Example 3

[0060] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0061] S1. Using walnut protein as raw material, the walnut protein is hydrolyzed in two steps using a single thermophilic protease and a composite enzyme in sequence to obtain an enzymatic hydrolyzate; in terms of mass, the amount ratio of alkaline protease to trypsin is 3:2;

[0062] The specific steps of S1 are as follows:

[0063] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0064] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0065] c. Rapidly cool the enzymatic solution A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic solution A so that the pH of the enzymatic solution A is maintained at 8. Then add the composite enzyme and perform enzymolysis for 3 hours to obtain the enzymatic solution B. The enzymatic solution B is subjected to the operation of step S2. In units of mass, the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution.

[0066] S2, heating the enzymatic hydrolyzate which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0067] Using the polypeptide determination method in Appendix B of GB / T 22492-2008 and the peptide relative molecular mass distribution determination method in Appendix A of GB / T 22492-2008, the peptide content (on a dry basis) in walnut peptides reached 88.72%, and the relative molecular masses of all peptide segments were within 1300Da, of which the content of peptide segments with a relative molecular mass of 1000-180Da was greater than 95%, and the content of peptide segments of 500-180Da in the peptide segments of 1000-180Da accounted for 63%.

[0068] Comparative Example 1

[0069] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0070] S1. Walnut protein was used as raw material, and the walnut protein was hydrolyzed in two steps using a single thermophilic protease and a composite enzyme in sequence to obtain an enzymatic hydrolyzate; in units of mass, the usage ratio of alkaline protease to trypsin was 3:1.

[0071] The specific steps of S1 are as follows:

[0072] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0073] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0074] c. Rapidly cool the enzymatic solution A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic solution A so that the pH of the enzymatic solution A is maintained at 8. Then add the composite enzyme and perform enzymolysis for 3 hours to obtain the enzymatic solution B. The enzymatic solution B is subjected to the operation of step S2. In units of mass, the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution.

[0075] S2, heating the enzymatic solution which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide

[0076] The difference from Example 2 is that the usage ratio of alkaline protease to trypsin is 3:1 in terms of mass.

[0077] Comparative Example 2

[0078] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0079] S1. Walnut protein was used as raw material, and the walnut protein was hydrolyzed in two steps using a single thermophilic protease and a composite enzyme in sequence to obtain an enzymatic hydrolyzate; in units of mass, the usage ratio of alkaline protease to trypsin was 6:5 (3:2.5).

[0080] The specific steps of S1 are as follows:

[0081] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0082] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0083] c. Rapidly cool the enzymatic solution A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic solution A so that the pH of the enzymatic solution A is maintained at 8. Then add the composite enzyme and perform enzymolysis for 3 hours to obtain the enzymatic solution B. The enzymatic solution B is subjected to the operation of step S2. In units of mass, the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution.

[0084] S2, heating the enzymatic solution which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide

[0085] The difference from Example 2 is that the usage ratio of alkaline protease to trypsin is 3:2.5 in terms of mass.

[0086] Comparative Example 3

[0087] The difference from Example 2 is that, in terms of mass, the added amount of the complex enzyme accounts for 0.8% of the amount of walnut protein in the walnut protein aqueous solution.

[0088] Comparative Example 4

[0089] The difference from Example 2 is that, in terms of mass, the added amount of the complex enzyme accounts for 1.2% of the amount of walnut protein in the walnut protein aqueous solution.

[0090] Comparative Example 5

[0091] A preparation method of walnut peptide disclosed in Patent No. CN112813125A is used as the prior art for comparison with the present invention.

[0092] Comparative Example 6

[0093] A walnut oligopeptide powder disclosed in Patent No. CN105567774B and its preparation method and use are compared with the present invention as the prior art.

[0094] Comparative Example 7

[0095] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0096] S1. Using walnut protein as raw material, single thermophilic protease is used to enzymolyze the walnut protein to obtain enzymolyzate.

[0097] The specific steps of S1 are as follows:

[0098] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0099] b. The walnut protein is configured into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 5 hours to obtain an enzymolysis solution;

[0100] S2, heating the enzymatic hydrolyzate which has been enzymatically hydrolyzed by a single thermophilic protease in step S1 to 100°C to inactivate the enzyme, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0101] The enzyme used was a single thermolysin.

[0102] Comparative Example 8

[0103] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0104] S1. Walnut protein is used as raw material, and a composite enzyme is used to enzymolyze the walnut protein to obtain an enzymolyzate. The usage ratio of alkaline protease to trypsin is 3:1.8 in terms of mass.

[0105] The specific steps of S1 are as follows:

[0106] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0107] b. The walnut protein is configured into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 55° C. and then maintained at a constant temperature of 55° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then a composite enzyme is added and enzymolyzed for 5 hours to obtain an enzymolysis solution, wherein the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution in terms of mass;

[0108] S2, heating the enzymatic hydrolyzate which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0109] The enzyme used is a complex enzyme.

[0110] Comparative Example 9

[0111] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0112] S1. Using walnut protein as raw material, the walnut protein is hydrolyzed in two steps using thermophilic protease and alkaline protease in sequence to obtain an enzymatic hydrolyzate.

[0113] The specific steps of S1 are as follows:

[0114] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0115] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0116] c. Rapidly cool the enzymatic hydrolysate A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic hydrolysate A so that the pH of the enzymatic hydrolysate A is maintained at 8. Then, add alkaline protease and perform enzymolysis for 3 hours to obtain an enzymatic hydrolysate B. The enzymatic hydrolysate B is subjected to step S2.

[0117] S2, heating the enzymatic hydrolyzate which has been enzymatically hydrolyzed by the single thermophilic protease and the composite enzyme in step S1 to 100°C for enzyme inactivation, and then separating and purifying it by molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0118] The enzymes used are thermolysin and alkaline protease.

[0119] Comparative Example 10

[0120] A biological composite seasoning includes walnut peptides prepared by the following steps:

[0121] S1. Using walnut protein as raw material, the walnut protein is hydrolyzed in two steps using thermolysin and trypsin in sequence to obtain an enzymatic hydrolyzate.

[0122] The specific steps of S1 are as follows:

[0123] a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein;

[0124] b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A;

[0125] c. Rapidly cool the enzymatic hydrolysate A to 45° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymatic hydrolysate A so that the pH of the enzymatic hydrolysate A is maintained at 7.0. Then, add trypsin and perform enzymolysis for 4 hours to obtain an enzymatic hydrolysate B. The enzymatic hydrolysate B is subjected to step S2.

[0126] S2, heating the enzymatic hydrolysate which has been subjected to single thermophilic protease and trypsin in step S1 to 100°C to inactivate the enzymes, and then separating and purifying it through molecular sieve chromatography and ion exchange chromatography, and finally drying it to obtain walnut peptide.

[0127] The enzymes used were thermolysin and trypsin.

[0128] Comparative Example 11

[0129] A biological composite seasoning comprises enzymolysis of walnut protein with a single alkaline protease, wherein the enzymolysis conditions are: enzymolysis temperature of 55 DEG C, pH of 8, substrate concentration (walnut protein aqueous solution) of 3%, enzyme addition amount of 4000U / g, and enzymolysis for 4 hours.

[0130] Comparative Example 12

[0131] A biological composite seasoning comprises enzymolysis of walnut protein with single trypsin, wherein the enzymolysis conditions are as follows: enzymolysis pH is 7.0, enzymolysis temperature is 45 DEG C, substrate concentration (walnut protein aqueous solution) is 3%, enzyme addition amount is 5000U / g, and enzymolysis is carried out for 4 hours.

[0132] Test Example 1

[0133] To determine the influence of substrate concentration (walnut protein aqueous solution) and enzyme addition amount on polypeptide content, walnut protein bioactive peptides were prepared by enzymatic hydrolysis of walnut protein aqueous solution with concentrations of 3%, 4%, 5%, 6%, 7%, and 8% according to the method of Example 2, and then the polypeptide content of walnut protein bioactive peptides with different substrate concentrations was determined by the polypeptide content detection method in Appendix B of GB / T22492-2008; the determination results are shown in Figure 1 As the substrate concentration, i.e., protein concentration, increases, the polypeptide content also increases, and then becomes relatively stable; the peptide content is higher when the substrate concentration is 6%, and then becomes relatively stable. The higher the substrate concentration, the more enzyme is required, and the greater the consumption. In order to meet energy conservation and environmental protection requirements, the present invention uses 6% as the optimal substrate concentration.

[0134] Test Example 2

[0135] Using Examples 1-3 and Comparative Examples 1-2 as the test group, the effects of different ratios of complex enzymes on the polypeptide content of walnut protein bioactive peptides were tested; the test results are shown in Figure 2 ; The polypeptide content detection method adopts the polypeptide content detection method in Appendix B of GB / T 22492-2008. The enzyme ratio of the composite enzyme (alkaline protease: trypsin) is 3:1, 3:1.5, 3:1.8, 3:2, and 3:2.5; different composite enzyme ratios have a great influence on the polypeptide content. When the mass ratio of alkaline protease to trypsin is 3:1.8, the polypeptide content reaches the highest, which is 88.73% at the highest. Alkaline protease and trypsin are commonly used proteases in walnut protein hydrolysis, and have high enzymolysis efficiency. Therefore, the present invention uses alkaline protease and trypsin as components of the composite enzyme of the present invention. The dosage of alkaline protease and trypsin will be affected by the first step of thermolysin enzymolysis during use. Therefore, the dosage of alkaline protease and trypsin does not change linearly with the peptide content, and the optimal ratio of alkaline protease to trypsin cannot be directly inferred. Under the condition of determining the usage amount of thermolysin, the present invention continuously screens the enzyme ratio of the composite enzyme, takes the polypeptide content as a reference, and optimizes the enzyme ratio of the composite enzyme with a polypeptide content significantly increased compared with the prior art.

[0136] Test Example 3

[0137] Using Examples 1-3 and Comparative Examples 3 and 4 as the test group, the effect of different addition amounts of the complex enzyme on the polypeptide content of the walnut protein bioactive peptide was determined; the results are shown in Figure 3 The polypeptide content detection method adopts the polypeptide content detection method in Appendix B of GB / T 22492-2008. When the added amount of complex enzyme is 1%, the polypeptide content is the highest.

[0138] Test 4

[0139] The antioxidant activity of the walnut protein bioactive peptides prepared in Examples 1-3 and Comparative Examples 1-12 was tested, including testing the scavenging rate of the walnut protein bioactive peptides on DPPH free radicals, the scavenging rate of the ABTS free radicals, and the antioxidant activity of the walnut protein bioactive peptides on DPPH free radicals. + The scavenging rates of free radicals, superoxide anions and hydroxyl radicals (·OH) were shown in Table 1.

[0140] About ABTS + The method for determining the free radical scavenging rate is as follows: take 5 mL of 7 mmol / L ABTS solution, add 88.0 μL of 140 mmol / L potassium persulfate, and place in the dark at room temperature to react for 12 h to 16 h to form an ABTS+ free radical stock solution; at 734 nm, dilute ABTS with 70% ethanol by volume to obtain a free radical scavenging rate of 100%. + The free radical stock solution is prepared until the absorbance is 0.70±0.02 and is ready for use; accurately measure 0.1mL of walnut protein bioactive peptide solution of 0.1, 0.5, 1.0, 1.5, 2.0mg / mL, add 0.5mL ABTS+ free radical solution, mix well, react at room temperature for 6min, and measure the absorbance at 734nm as A1; draw 0.5mL ABTS+ free radical solution, add 0.1mL 70% ethanol solution by volume to measure the absorbance as A2, and use VC as a positive control to calculate the clearance rate, the formula is as follows:

[0141] ABTS + ·Clearance rate / %=(A2-A1) / A2×100.

[0142] The method for determining the DPPH free radical scavenging rate is as follows: weigh 2.56 mg of DPPH standard, dissolve it in anhydrous methanol and make up to 100 mL; take 1 mL of walnut protein bioactive peptide solution of 0.1, 0.5, 1.0, 1.5, 2.0 mg / mL in a test tube, add 1 mL of DPPH solution, react at room temperature in the dark for 30 minutes, measure the absorbance at 517 nm, record it as Ai, take 1 mL of anhydrous methanol in a test tube, add 1 mL of DPPH solution, react at room temperature in the dark for 30 minutes, measure the absorbance at 517 nm, record it as Ac, take 1 mL of polypeptide solution in a test tube, add 1 mL of anhydrous methanol solution, react at room temperature in the dark for 30 minutes, measure the absorbance at 517 nm, record it as Aj, and use VC as a positive control. Calculate the DPPH free radical scavenging rate according to the following formula:

[0143] DPPH free radical scavenging rate / %=[1-(Ai-Aj)] / Ac×100.

[0144] The method for determining the scavenging rate of superoxide anion free radicals is as follows: take 5 mL of 0.05 mol / L Tris-HCl buffer (pH 8.2), place it in a 25°C water bath for preheating for 20 min, add 1 mL of 0.1, 0.5, 1.0, 1.5, 2.0 mg / mL walnut protein bioactive peptide solution and 1 mL of 25 mmol / L pyrogallol solution, mix well, react in a 25°C water bath for 5 min, then add 100 μL of 8% HCl by mass to terminate the reaction, and measure the absorbance A1 at a wavelength of 325 nm; take distilled water instead of pyrogallol solution to measure the absorbance A2; take distilled water instead of polypeptide solution as a blank control to measure the absorbance A3, and use VC as a positive control to calculate O2 - Clearance rate, the formula is as follows:

[0145] O2 - ·Clearance rate / %=[1-(A1-A2)] / A3×100.

[0146] The scavenging rate detection method of hydroxyl free radical (·0H) is as follows: take 1mL of walnut protein bioactive peptide solution with concentrations of 0.1, 0.5, 1.0, 1.5, and 2.0mg / mL, add 0.5mL of 9mmol / L salicylic acid-ethanol solution, 0.5mL of 9mmol / L FeS04 solution, and finally add 5mL of 8mmol / L H202 solution, start the reaction, shake and mix, and place in a water bath at 37℃ for 30min. Measure the absorbance at 510nm and record it as A1; take 0.5mL of distilled water instead of ferrous sulfate solution to measure the absorbance A2; take 1mL of distilled water instead of polypeptide solution as a blank control to measure the absorbance A0, and VC is used as a positive control. The calculation formula of ·OH scavenging rate is as follows:

[0147] OH removal rate / % = [1-(A1-A2)] / A0×100.

[0148] Table 1 Test results of antioxidant activity of walnut protein bioactive peptides

[0149]

[0150] From the data in Table 1, it can be seen that different enzymes have different scavenging abilities on the enzymolysis products after walnut protein hydrolysis. The antioxidant active peptides produced after walnut protein hydrolysis by thermophilic protease have strong antioxidant ability. The antioxidant active peptides produced after enzymolysis by different enzymes are different, and the antioxidant abilities of the produced peptides are different. Combining Examples 1-3 and all comparative examples in which the antioxidant activity was detected, it can be seen that the antioxidant ability of the walnut peptide of the present invention is significantly better than that of the prior art.

[0151] The patent document corresponding to Comparative Example 5 does not test the antioxidant activity of walnut peptide, indicating that the antioxidant activity of walnut peptide in Comparative Example 5 has not been significantly improved. By comparing the data in Table 2, it can be seen that the performance of walnut peptide prepared by the present invention is significantly better than that of walnut peptide disclosed in Comparative Example 5; Comparative Example 6 does not disclose data on the scavenging rate of hydroxyl radicals (·OH), indicating that the scavenging rate of hydroxyl radicals (·OH) has not changed significantly compared with other existing technologies. In addition, other scavenging rates of walnut peptide of the present invention are significantly higher than those disclosed in Comparative Example 6. The fact that a certain data is not compared does not affect the comparison of the overall performance. Combined with Table 2, the performance of walnut peptide prepared by the present invention is significantly better than that of walnut peptide disclosed in Comparative Example 6.

[0152] Test Example 5

[0153] The peptide content and relative molecular mass distribution of the walnut protein bioactive peptides in Examples 1-3 and Comparative Examples 1-11 were determined using the polypeptide determination method in Appendix B of GB / T 22492-2008 and the peptide relative molecular mass distribution determination method in Appendix A of GB / T 22492-2008. The results are shown in Table 2.

[0154] Table 2 Peptide content and relative molecular mass distribution of walnut protein bioactive peptides

[0155]

[0156]

[0157] Combined with Table 2, it can be seen that the effect of a single thermolysin on walnut protein hydrolysis is significantly better than that of a single alkaline protease and a single trypsin, and compared with Comparative Examples 9-12, it can be seen that the use of thermolysin and alkaline protease or trypsin compound can significantly improve the enzymatic hydrolysis effect of walnut protein. In other words, thermolysin can significantly improve the enzymatic hydrolysis effect of alkaline protease and trypsin in walnut protein; Comparative Example 8 is compared with Comparative Example 11 and Comparative Example 12. It can be seen that the enzymatic hydrolysis effect of alkaline protease and trypsin in walnut protein after compounding is greater than the enzymatic hydrolysis effect of a single alkaline protease and a single trypsin in walnut protein. Therefore, the present invention uses the composite enzyme of alkaline protease and trypsin and thermolysin for walnut protein hydrolysis. Comparing Examples 1-3 and Comparative Examples 7-12, it can be seen that the enzymatic hydrolysis effect of the present invention is the best, and the amount of small peptides in the walnut peptide accounts for the largest proportion. The more small peptides in the walnut peptide account for, the easier it is to be digested and absorbed, which can greatly reduce the burden on the digestive system.

[0158] In the present invention, the first step of enzymolysis of walnut protein must be performed with thermolysin, and then the composite enzyme of alkaline protease and trypsin is used. The enzymes used in the two-step enzymolysis cannot be exchanged, because the enzymolysis temperature of thermolysin is 65-85°C, and the thermal denaturation of walnut protein occurs at about 60°C. After the denaturation of walnut protein, the spatial structure is destroyed, so that the amino acid is fully exposed and more easily hydrolyzed. Therefore, thermolysin has a good effect on the enzymolysis of walnut protein. After the enzymolysis of thermolysin, walnut protein has been denatured and is easily hydrolyzed. When the composite enzyme is used for the second step of enzymolysis, the enzymolysis effect will be better. At 75°C, the denaturation of walnut protein will not produce harmful substances. When the temperature is too low, the amino acid cannot be fully exposed when the walnut protein is denatured. Too high a temperature will cause the loss of nutrients such as vitamins and proteins therein; therefore, the present invention combines the suitable enzymolysis temperature of thermolysin and the denaturation of walnut protein to optimize the most suitable temperature of thermolysin.

[0159] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biological composite seasoning, comprising a basic seasoning and a bioactive peptide with antioxidant properties, characterized in that: The bioactive peptide is a walnut peptide prepared by the following steps: S1, using walnut protein as raw material, sequentially using a single thermophilic protease and a composite enzyme to perform two-step enzymolysis on the walnut protein to obtain an enzymolysis solution; S2, separating and purifying the enzymatic hydrolysate obtained by single thermophilic protease and composite enzyme enzymolysis in step S1 by molecular sieve chromatography and ion exchange chromatography, and obtaining walnut peptide after drying; In step S1, the complex enzyme includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 3:1.5-2 in terms of mass.

2. A biological composite seasoning according to claim 1, characterized in that: In step S1, the complex enzyme includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 5:3 in terms of mass.

3. A biological composite seasoning according to claim 2, characterized in that: The specific steps of S1 are as follows: a. Grind walnut kernels through a 30-mesh sieve to obtain walnut residues, add the walnut residues into water, beat, adjust the pH value to 8.0-9.0, stir at 50-65° C. for 4-5 hours, centrifuge for 5-20 minutes, remove the oil layer, take the supernatant, adjust the pH value of the supernatant to 4-5 with a 2.0 mol / L inorganic acid aqueous solution, centrifuge for 25-30 minutes, and wash with ethanol 3 times to obtain walnut protein; b. Walnut protein is prepared into a 6% walnut protein aqueous solution, the temperature of the walnut protein aqueous solution is heated to 75° C. and then maintained at a constant temperature of 75° C., the pH of the walnut protein aqueous solution is adjusted to 8 with a pH regulator, and then thermolysin is added, the amount of thermolysin added is 6000 U / g, and enzymolysis is performed for 2 hours to obtain enzymolysis solution A; c. Rapidly cool the enzymolysis solution A to 55° C. and then keep the temperature constant. Use a pH regulator to adjust the pH of the enzymolysis solution A so that the pH of the enzymolysis solution A is maintained at 8. Then add the composite enzyme and perform enzymolysis for 3 hours to obtain the enzymolysis solution B. The enzymolysis solution B is subjected to the operation of step S2. In units of mass, the amount of the composite enzyme added accounts for 1% of the amount of walnut protein in the walnut protein aqueous solution.

4. A biological composite seasoning according to claim 3, characterized in that: The peptide content (on a dry basis) in walnut peptides reaches 88.73%, and the relative molecular masses of all peptide segments are within 1300Da, among which the peptide segment content in the relative molecular mass range of 1000-180Da is greater than 95%, and the peptide segment content of 500-180Da accounts for 65% of the peptide segment in the 1000-180Da range.

5. The biological composite seasoning according to claim 1, characterized in that: The enzymatic hydrolyzate obtained by the single thermophilic protease and the composite enzyme in step S1 is heated to 100° C. to inactivate the enzyme, and then separated and purified by molecular sieve chromatography and ion exchange chromatography, and finally dried to obtain a walnut peptide product.

6. The biological composite seasoning according to claim 1, characterized in that: The basic seasoning includes salty agents, sweeteners, vitamin E, tea polyphenols and spices.

7. A method for preparing a biological composite seasoning according to any one of claims 1 to 6, characterized in that: The following steps are involved: A. Preparation of bioactive peptides with antioxidant properties: S1, using walnut protein as raw material, sequentially using a single thermophilic protease and a composite enzyme to perform two-step enzymolysis on the walnut protein to obtain an enzymolysis solution; S2, separating and purifying the enzymatic hydrolysate obtained by single thermophilic protease and composite enzyme enzymolysis in step S1 by molecular sieve chromatography and ion exchange chromatography, and obtaining walnut peptide after drying; In step S1, the composite enzyme includes alkaline protease and trypsin, and the usage ratio of alkaline protease to trypsin is 3:1.5-2 in terms of mass. B. Mixing the bioactive peptides with basic seasonings: Mix salting agents, sweeteners, vitamin E, tea polyphenols, and spices to obtain basic seasoning powder, and then evenly mix the walnut peptides in step S2 with the basic seasoning powder in proportion to obtain a biocomposite seasoning.

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