Walnut protein and extraction method thereof

Abstract

The invention provides walnut protein and an extraction method thereof, and belongs to the technical field of protein processing, and the extraction method comprises the following steps: (1) carrying out extrusion puffing on walnuts, crushing, mixing with a salt solution, and carrying out pressurized soaking; (2) adding a compound enzyme for enzymolysis, and centrifuging to obtain a middle-layer clear liquid 1 and a bottom-layer precipitate 1; (3) washing residues, centrifuging, collecting middle-layer clear liquid 2, and mixing with the middle-layer clear liquid 1; (4) adding saccharifying enzyme to carry out secondary enzymolysis, deactivating enzyme, adjusting the pH value, and centrifuging to obtain a protein crude extract; and (5) water is added for redissolution, then a hydrogen peroxide solution is added for a reaction, freeze drying is carried out, walnut protein can be obtained, and the compound enzyme is composed of bromelain, bacillus subtilis neutral protease, papain and compound flavor protease. According to the extraction method of the walnut protein, the extraction rate and functionality of the walnut protein can be effectively improved, the color of the walnut protein is improved, and the availability of the walnut protein is improved.

Description

Technical Field

[0001] The invention relates to the technical field of protein processing, in particular to walnut protein and an extraction method thereof. Background Art

[0002] Walnut, also known as walnut and Qiang peach, is rich in high-quality fat, protein, carbohydrates, vitamins, trace elements, etc. It has the effects of brain-boosting, blood lipid-lowering, anti-oxidation, cancer prevention, and weight regulation. Walnut can be processed into walnut oil, walnut leaf tea, walnut protein beverage, walnut meal replacement powder, walnut health wine, walnut heartwood health tea, etc. However, at present, walnuts are mostly used to produce walnut oil except for direct consumption or a small amount of processed food sales, and the by-product, walnut meal, still contains the main nutrients of walnut kernels, rich in high-quality protein, including leucine, valine, phenylalanine, isoleucine, threonine, lysine, methionine, tryptophan and other 8 essential amino acids for the human body, with a complete range and reasonable content, suitable for human absorption. However, the existing walnut protein extraction method has low purity, low extraction rate, low availability, and dark color, which limits the further application of walnut protein.

[0003] Therefore, it is hoped to provide a walnut protein extraction method to improve the extraction rate and functional properties of walnut protein and improve the color of walnut protein. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting walnut protein, improve the extraction rate and functionality of walnut protein, improve the color of walnut protein, and improve the availability of walnut protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for extracting walnut protein, comprising the following steps:

[0007] (1) extruding and puffing walnuts, then crushing and mixing with a salt solution, and soaking under pressure to obtain a solution 1;

[0008] (2) adding a composite enzyme to solution 1 for the first enzymatic hydrolysis to obtain solution 2, centrifuging to obtain a middle layer clear liquid 1 and a bottom layer precipitate 1, and discarding the upper layer emulsion;

[0009] (3) adding water to the bottom precipitate 1, stirring and washing the residue, centrifuging, collecting the middle layer clear liquid 2, and mixing it with the middle layer clear liquid 1 to obtain solution 3;

[0010] (4) adding saccharifying enzyme to solution 3 for a second enzymatic hydrolysis, inactivating the enzyme, adjusting the pH, and centrifuging to obtain a bottom precipitate 2 which is a crude protein extract;

[0011] (5) adding water to redissolve the bottom precipitate 2 to obtain a solution 4, adding a hydrogen peroxide solution to the solution 4 to react, and drying to obtain walnut protein;

[0012] The composite enzyme consists of bromelain, bacillus subtilis neutral protease, papain and composite flavor protease.

[0013] Preferably, the initial moisture content of the walnuts extruded and puffed in step (1) is 12-15% (w / v); the extrusion and puffing conditions are: the die hole diameter is 8-16 mm, the screw speed is 95-100 r / min, the sleeve temperature is 73-87° C.; and the crushed particle size is 25-50 μm.

[0014] Preferably, the salt solution in step (1) is a sodium chloride solution with a concentration of 1 to 1.5 mol / L, and the amount added is 1 to 3 times the total volume of the puffed walnuts.

[0015] Preferably, the pressurized soaking condition in step (1) is 250-300 MPa for 2-3 times, each pressurized treatment lasts for 8-12 minutes, the interval between each pressurized treatment is 20-30 minutes, and the temperature is 35-55°C.

[0016] Preferably, in step (2), before the first enzymatic hydrolysis, the initial pH of solution 1 is adjusted to 8.5-9.0, the enzymatic hydrolysis temperature is 50-55° C., and the enzymatic hydrolysis time is 3.5-4.5 h;

[0017] The added amount of the complex enzyme is 1100-1200 U / g walnut;

[0018] The centrifugal conditions are: 4000-5000 rpm, 10-15 min;

[0019] The mass ratio of bromelain, bacillus subtilis neutral protease, papain and compound flavor protease is 1-2:1-2:1-2:1-2.

[0020] Preferably, in step (3), the volume ratio of water to bottom sediment 1 is 2 to 3:1;

[0021] The stirring speed is 200-300 rpm and the time is 30-40 min;

[0022] The centrifugal conditions are: 3000-3500 rpm, 10-15 min.

[0023] Preferably, the initial pH value of the second enzymatic hydrolysis in step (4) is 4.3-4.5, and the enzymatic hydrolysis conditions are: 40-55° C., 1-2 h;

[0024] The saccharifying enzyme is α-amylase, and the added amount is 300-400 U / mL solution 3.

[0025] Preferably, the conditions for inactivating the enzyme in step (4) are: 90-95°C, water bath for 15-20 min; after inactivating the enzyme, the pH is adjusted to 3.8-4.0;

[0026] The centrifugal conditions are 5000-6000 rpm, 5-10 min.

[0027] Preferably, in step (5), the ratio of water to bottom precipitate 2 is: 2-3 mL: 1 g;

[0028] The volume fraction of the hydrogen peroxide is 2.8-3.4%, the volume ratio of the hydrogen peroxide to the solution 4 is 0.6-0.8:1, and the reaction time is 1.0-2.0h.

[0029] The invention also provides walnut protein prepared by the preparation method.

[0030] The present invention improves the dissolution rate of protein by extrusion puffing pretreatment, combined with salt solution soaking and pressure treatment; improves the enzymolysis efficiency of walnut protein by a specific composite enzyme, improves its functional properties, improves the utilization rate, removes lipids and polysaccharides in walnut protein by centrifugation and the use of amylase, improves protein purity, and uses hydrogen peroxide for decolorization, which can effectively improve protein whiteness and also help improve the functional properties of protein. In summary, the extraction method of walnut protein provided by the present invention can effectively improve the extraction rate and functionality of walnut protein, improve the color of walnut protein, and improve the utilization rate of walnut protein. DETAILED DESCRIPTION

[0031] In the present invention, bromelain, Bacillus subtilis neutral protease, papain and compound flavor protease were purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd. with an activity of 20,000 U / g, and α-amylase was purchased from Shandong Yousuo Chemical Technology Co., Ltd. with an activity of 2000 U / g.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] A method for extracting walnut protein comprises the following steps:

[0035] (1) Peel the walnuts, crush them through a 60-mesh sieve, adjust the initial moisture content of the walnuts to 12% (w / v), extrusion puff (die hole diameter of 12 mm, screw speed of 96 r / min, sleeve temperature of 78° C.), crush them to 50 μm, add a 1 mol / L sodium chloride solution in an amount twice the total volume of the puffed walnuts, and pressurize them twice at 50° C. and 280 MPa, each pressurization treatment for 10 min, with an interval of 25 min between each pressurization treatment, to obtain a solution 1;

[0036] (2) adjusting the pH of solution 1 to 9, adding a composite enzyme (composed of bromelain, Bacillus subtilis neutral protease, papain, and composite flavor protease in a mass ratio of 1:1:1:1) at a ratio of 1200 U / g substrate (walnut), performing enzymolysis at 55° C. for 3.5 h to obtain solution 2, centrifuging at 4500 rpm for 10 min, obtaining a middle layer clear liquid 1 and a bottom layer precipitate 1, and discarding the upper layer emulsion;

[0037] (3) Add 2 times the volume of water to the bottom sediment 1, stir at 300 rpm for 30 min to wash the residue, then centrifuge at 3500 rpm for 10 min, collect the middle layer clear liquid 2, and mix it with the middle layer clear liquid 1 to obtain solution 3;

[0038] (4) adjusting the pH value of solution 3 to 4.5, adding α-amylase at a ratio of 300 U / mL of solution 3, performing enzymatic hydrolysis at 55°C for 1 h, inactivating the enzyme in a boiling water bath at 95°C for 20 min, adjusting the pH to 3.8, and centrifuging at 5500 rpm for 8 min. The bottom precipitate 2 obtained is the crude protein extract;

[0039] (5) Add water at a ratio of 2 mL: 1 g to redissolve the bottom precipitate 2 to obtain solution 4, add a 2.8% volume fraction hydrogen peroxide solution to solution 4, and the volume ratio of solution 4 to hydrogen peroxide solution is 1:0.6. The reaction is carried out for 1.0 h, and freeze-dried (vacuum degree of 25 Pa, temperature of -50 ° C, 24 h) to obtain walnut protein.

[0040] Example 2

[0041] A method for extracting walnut protein comprises the following steps:

[0042] (1) Peel the walnuts, crush them through a 60-mesh sieve, adjust the initial moisture content of the walnuts to 14% (w / v), extrusion puff (die hole diameter of 12 mm, screw speed of 96 r / min, sleeve temperature of 78° C.), crush them to 25 μm, add a 1.5 mol / L sodium chloride solution in an amount that is twice the total volume of the puffed walnuts, and then pressurize them at 55° C. and 280 MPa for 3 times, each pressurization time of 10 min, and each interval of 20 min to obtain a solution 1;

[0043] (2) adjusting the pH of solution 1 to 8.5, adding a composite enzyme (composed of bromelain, Bacillus subtilis neutral protease, papain, and composite flavor protease in a mass ratio of 1:1:1:1) at a ratio of 1200 U / g substrate (walnut), performing enzymolysis at 45° C. for 4 h to obtain solution 2, centrifuging at 4500 rpm for 10 min, obtaining a middle layer clear liquid 1 and a bottom layer precipitate 1, and discarding the upper layer emulsion;

[0044] (3) Add 2 times the volume of water to the bottom precipitate 1, stir at 250 rpm for 40 min to wash the residue, centrifuge at 3000 rpm for 15 min, collect the middle layer clear liquid 2, and mix it with the middle layer clear liquid 1 to obtain solution 3;

[0045] (4) adjusting the pH value of solution 3 to 4.5, adding α-amylase at a ratio of 350 U / mL of solution 3, performing enzymatic hydrolysis at 55°C for 1.5 h, inactivating the enzyme in a boiling water bath at 95°C for 20 min, adjusting the pH to 3.8, centrifuging at 5000 rpm for 10 min, and obtaining the bottom precipitate 2, which is the crude protein extract;

[0046] (5) Add water at a ratio of 2 mL: 1 g to redissolve the bottom precipitate 2 to obtain solution 4, add a 3% volume fraction of hydrogen peroxide solution to solution 4, and the volume ratio of solution 4 to hydrogen peroxide solution is 1:0.7. The reaction is carried out for 1.5 h, and freeze-dried (vacuum degree of 25 Pa, temperature of -50 ° C, 24 h) to obtain walnut protein.

[0047] Example 3

[0048] A method for extracting walnut protein comprises the following steps:

[0049] (1) Peel the walnuts, crush them through a 60-mesh sieve, adjust the initial moisture content of the walnuts to 15% (w / v), extrusion puff (die hole diameter of 12 mm, screw speed of 96 r / min, sleeve temperature of 78° C.), crush them to 25 μm, add a 1.5 mol / L sodium chloride solution in an amount twice the total volume of the puffed walnuts, and pressurize them twice at 50° C. and 300 MPa, each pressurization time of 12 min and an interval of 30 min to obtain a solution 1;

[0050] (2) adjusting the pH of solution 1 to 9.5, adding a composite enzyme (bromelain, Bacillus subtilis neutral protease, papain, and composite flavor protease in a mass ratio of 1:1:1:1) at a ratio of 1200 U / g substrate (walnut), performing enzymolysis at 50° C. for 4 h to obtain solution 2, centrifuging at 5000 rpm for 10 min, obtaining a middle layer clear liquid 1 and a bottom layer precipitate 1, and discarding the upper layer emulsion;

[0051] (3) Add 3 times the volume of water to the bottom sediment 1, stir at 300 rpm to wash the residue for 35 min, centrifuge at 3500 rpm for 13 min, collect the middle layer clear liquid 2, and mix it with the middle layer clear liquid 1 to obtain solution 3;

[0052] (4) adjusting the pH value of solution 3 to 4.5, adding α-amylase at a ratio of 400 U / mL of solution 3, performing enzymatic hydrolysis at 50°C for 2 h, inactivating the enzyme in a boiling water bath at 95°C for 20 min, adjusting the pH to 4.0, and centrifuging at 6000 rpm for 10 min. The bottom precipitate 2 obtained is the crude protein extract;

[0053] (5) Add water at a ratio of 2 mL: 1 g to redissolve the bottom precipitate 2 to obtain solution 4, add 0.8 times hydrogen peroxide solution (volume fraction 3.4%) to solution 4 and react for 2 h, and freeze-dry (vacuum degree 25 Pa, temperature -50 ° C, 24 h) to obtain walnut protein.

[0054] Comparative Example 1

[0055] Different from Example 3, step (1) of this comparative example is to peel the walnuts and grind them into 25 μm, add 2 times the volume of water, and soak them at 50° C. for 24 hours to prepare solution 1.

[0056] Comparative Example 2

[0057] Different from Example 3, the composition of the composite enzyme in step (2) of this comparative example is: bromelain, Bacillus subtilis neutral protease, papain and Protamex (activity is 100 U / mg), and the mass ratio is 1:1:1:1.

[0058] Comparative Example 3

[0059] Different from Example 3, in step (5) of this comparative example, activated carbon is added after the crude protein extract is reconstituted with water, decolorized for 5 hours, the activated carbon is separated, and then freeze-dried to obtain walnut protein.

[0060] Test Example 1

[0061] Take the walnut protein powder prepared in Examples 1 to 3 and Comparative Examples 1 to 3:

[0062] (1) The protein content (determined by the Kjeldahl method in GB5009.5-2016), fat content (determined by the Soxhlet extraction method in GB5009.6-2016) and whiteness (WSB-IV intelligent whiteness measuring instrument) were measured. The results are shown in Table 1:

[0063] Table 1 Chemical composition content and whiteness

[0064] Group Protein content / % Fat content / % Whiteness / % Example 1 80.14 0.97 53.14 Example 2 82.45 1.04 52.19 Example 3 81.59 0.97 52.87 Comparative Example 1 62.34 1.65 48.67 Comparative Example 2 63.17 1.45 48.00 Comparative Example 3 66.13 1.25 38.43

[0065] (2) Functional evaluation of walnut protein

[0066] Solubility: Solubility determination method: add 250g sample into 100mL, 0.05mol / L phosphate buffer (pH7.0), stir at 25℃ for 1h, centrifuge at 4000r / min for 20min, and determine the soluble protein content of the supernatant by the biuret method.

[0067] Solubility (%) = (soluble protein amount / protein amount of sample) × 100

[0068] Water holding capacity: WHC determination method, take a 50mL centrifuge tube (m1), put a certain amount of protein sample accurately weighed into it, add water and stir evenly, adjust pH to 7.0, place in a 60℃ water bath for 30min, then cool and stand for 1h, centrifuge at 25℃ 4000r / min for 15min, discard the supernatant, and weigh the mass of the centrifuge tube (m2)

[0069] Water holding capacity = (m2-m1) sample mass

[0070] Oil retention: 5.00 g sample was dispersed in 95 ml oil, stirred at 25 °C for 15 min, then allowed to stand for 2 h, centrifuged at 4000 r / min for 15 mm, the supernatant was discarded, and the precipitate was weighed.

[0071] Oil retention rate = (precipitate mass / sample mass) × 100

[0072] Emulsification characteristics: Weigh 2.5g of sample, dissolve in 50mL of distilled water, adjust to pH 5.0, pH 7.0, pH 9.0, add 50mL of soybean salad oil, homogenize for 1min (10000r / min), and centrifuge for 5min (3000r / min). Take out the centrifuge tube, observe the emulsification, record the height of the emulsified layer and the total height of the liquid in the tube, and make 2 parallel samples. The calculation formula is as follows

[0073] EA (%) = (height of emulsion layer in centrifuge tube / total height of liquid in centrifuge tube) × 100

[0074] The results are shown in Table 2:

[0075] Table 2 Protein functional properties

[0076] Grouping Solubility Water holding capacity Oil retention Emulsifying properties Example 1 52 4.1 3.8 87.65 Example 2 53 4.1 3.7 86.75 Example 3 52 4.2 3.8 88.48 Comparative Example 1 42 3.5 3.1 70.48 Comparative Example 2 41 3.4 3.0 72.89 Comparative Example 3 45 3.5 3.3 76.14

[0077] As can be seen from the above table, the walnut protein prepared by the present invention has high protein content, low fat content, high whiteness and good color, and significantly improved functional properties. The present invention performs extrusion puffing pretreatment on walnut protein to improve the protein dissolution rate. On this basis, the use of salt solution and pressurized treatment can further promote the melting of protein. The present invention also finds that the enzymolysis effects of different complex enzyme combinations are different, and the present invention provides a complex enzyme combination with an enzymolysis effect that is better than other combinations.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting walnut protein, characterized in that: The following steps are involved: (1) extruding and puffing walnuts, then crushing and mixing with a salt solution, and soaking under pressure to obtain a solution 1; (2) adding a composite enzyme to solution 1 for the first enzymatic hydrolysis to obtain solution 2, centrifuging to obtain a middle layer clear liquid 1 and a bottom layer precipitate 1, and discarding the upper layer emulsion; (3) adding water to the bottom precipitate 1, stirring and washing the residue, centrifuging, collecting the middle layer clear liquid 2, and mixing it with the middle layer clear liquid 1 to obtain solution 3; (4) adding saccharifying enzyme to solution 3 for a second enzymatic hydrolysis, inactivating the enzyme, adjusting the pH, and centrifuging to obtain a bottom precipitate 2 which is a crude protein extract; (5) adding water to redissolve the bottom precipitate 2 to obtain a solution 4, adding a hydrogen peroxide solution to the solution 4 to react, and drying to obtain walnut protein; The composite enzyme consists of bromelain, bacillus subtilis neutral protease, papain and composite flavor protease.

2. The extraction method according to claim 1, characterized in that The initial moisture content of the walnuts during extrusion puffing in step (1) is 12-15% (w / v); the extrusion puffing conditions are: the die hole diameter is 8-16 mm, the screw speed is 95-100 r / min, the sleeve temperature is 73-87° C.; and the crushed particle size is 25-50 μm.

3. The extraction method according to claim 2, characterized in that The salt solution in step (1) is a sodium chloride solution with a concentration of 1 to 1.5 mol / L, and the amount added is 1 to 3 times the total volume of the puffed walnuts.

4. The extraction method according to claim 3, characterized in that The conditions for the pressurized soaking in step (1) are 250-300 MPa pressure treatment 2-3 times, each pressure treatment time is 8-12 minutes, the interval time of each pressure treatment is 20-30 minutes, and the temperature is 35-55°C.

5. The extraction method according to claim 4, characterized in that Before the first enzymatic hydrolysis in step (2), the initial pH of solution 1 is adjusted to 8.5-9.0, the enzymatic hydrolysis temperature is 50-55° C., and the enzymatic hydrolysis time is 3.5-4.5 h; The added amount of the complex enzyme is 1100-1200 U / g walnut; The centrifugal conditions are: 4000-5000 rpm, 10-15 min; The mass ratio of bromelain, bacillus subtilis neutral protease, papain and compound flavor protease is 1-2:1-2:1-2:1-2.

6. The extraction method according to claim 5, characterized in that In step (3), the volume ratio of water to bottom sediment 1 is 2 to 3:1; The stirring speed is 200-300 rpm and the time is 30-40 min; The centrifugal conditions are: 3000-3500 rpm, 10-15 min.

7. The extraction method according to claim 6, characterized in that The initial pH value of the second enzymatic hydrolysis in step (4) is 4.3-4.5, and the enzymatic hydrolysis conditions are: 40-55° C., 1-2 h; The saccharifying enzyme is α-amylase, and the added amount is 300-400 U / mL solution 3.

8. The extraction method according to claim 7, characterized in that The conditions for inactivating the enzyme in step (4) are: 90-95° C., water bath for 15-20 min; after inactivating the enzyme, the pH is adjusted to 3.8-4.0; The centrifugal conditions are 5000-6000 rpm, 5-10 min.

9. The extraction method according to claim 8, characterized in that In step (5), the ratio of water to bottom precipitate 2 is: 2-3 mL: 1 g; The volume fraction of the hydrogen peroxide is 2.8-3.4%, the volume ratio of the hydrogen peroxide to the solution 4 is 0.6-0.8:1, and the reaction time is 1.0-2.0h.

10. Walnut protein prepared by the preparation method according to any one of claims 1 to 9.