A preparation method for extracting biologically active small molecule protein from soybean water

Abstract

The invention discloses a preparation method for extracting biologically active small molecule proteins from soybean water. The method comprises the following steps: firstly clarifying, microwave irradiating and concentrating the soybean water in sequence to obtain concentrated soybean water; then freezing the concentrated soybean water, thawing it with microwaves, and subjecting it to ultrasonic oscillation treatment. Under the action of a pulsed magnetic field, the concentrated soybean water is subjected to salting out by ammonium sulfate, and solids are filtered to obtain a salting out product; finally, the salting out product is ultrasonically dispersed in distilled water, subjected to high-pressure spraying treatment, a composite enzyme is added, enzymolysis is performed, enzyme inactivation is performed, and vacuum freeze drying is performed to obtain the product. The invention extracts biologically active small molecule proteins from soybean water, realizes the recycling of waste soybean water, is green and environmentally friendly, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to a preparation method for extracting biologically active small molecule protein from soybean water, belonging to the technical field of soybean processing. Background Art

[0002] At present, soy protein isolate is usually obtained by alkali dissolution and acid precipitation from soybean meal after oil removal. In the acid precipitation step (pH about 4.5), after the soy protein isolate is precipitated, it is separated by a horizontal screw centrifuge to obtain soy protein isolate and soy water. Soy water, also known as soybean whey wastewater, is usually discharged as waste liquid directly after being treated in a sewage treatment system. However, the amount of soy water produced is large, the BOD and COD are high, and the cost of sewage treatment is high.

[0003] In addition, bean curd water also contains the following recyclable substances, and direct discharge will undoubtedly cause a waste of resources:

[0004] 1. Protein;

[0005] 2. Oligosaccharides: sucrose, raffinose, stachyose and a small amount of verbascose, etc. Soybean oligosaccharides have good thermal stability and have multiple functions such as improving intestinal flora and treating constipation;

[0006] 3. Isoflavones: daidzein, daidzein, genistein, genistein, soybean glycoside, soybean aglycone, etc.; soy isoflavones are plant-based estrogen, which have special effects on relieving menopausal symptoms in women and have multiple biological functions such as preventing osteoporosis, anti-aging, and anti-tumor.

[0007] Among them, protein is one of the seven essential nutrients for the human body. It is very necessary to recycle and reuse the protein in soy water. Generally speaking, small molecule proteins are more likely to enter cells and between cells, thereby producing biological activity. The superiority and safety of small molecule proteins have been widely scientifically verified. Therefore, recovering protein from soy water and reducing the molecular weight of protein as much as possible has a broader application prospect.

[0008] Patent CN108308368B discloses a soy protein bean water treatment system and method. The bean water is first pumped into a steam direct contact heater through a centrifugal pump, the steam pipeline valve is opened, and the heating temperature is controlled by a back pressure valve; the heated bean water is directly pumped into a disc centrifugal separator, and the separation supernatant obtained after separation is directly entered into a separation liquid temporary storage tank; the centrifugal pump is turned on, and the separation supernatant in the separation liquid temporary storage tank is pumped into a three-effect tubular falling film evaporator for evaporation and concentration, and the concentrated liquid enters the concentrated liquid temporary storage tank; the concentrated liquid in the concentrated liquid temporary storage tank is sent to a high-pressure pump through a rotor pump, and the high-pressure pump is turned on to pressure-spray the concentrated liquid into a drying tower, and the obtained product is packaged after spray drying is completed. The protein product recovered by this patented technology has a high molecular weight and a limited scope of application. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for extracting biologically active small molecule proteins from soybean water.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] A preparation method for extracting biologically active small molecule proteins from soy milk, the specific steps are as follows:

[0012] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0013] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product;

[0014] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzymes, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain biologically active small molecule proteins.

[0015] Preferably, in step (1), the insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane.

[0016] Preferably, in step (1), the process conditions of microwave irradiation are: 300-500W microwave irradiation for 3-5 minutes.

[0017] Preferably, in step (1), concentration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentration ratio is 5 to 7 times.

[0018] Preferably, in step (2), the process conditions for the freezing treatment are: freezing at -10 to -15°C for 24 to 30 hours.

[0019] Preferably, in step (2), the process conditions for microwave thawing are: microwave treatment at 300-450 W for 2-3 minutes, stop for 1-2 minutes, microwave treatment at 300-450 W for 2-3 minutes, stop for 1-2 minutes, and microwave treatment at 300-450 W for 2-3 minutes.

[0020] Preferably, in step (2), the process conditions of ultrasonic oscillation treatment are: 300-500W ultrasonic oscillation for 3-5 minutes, stop for 1-2 minutes, and 700-900W ultrasonic oscillation for 3-5 minutes.

[0021] Preferably, in step (2), the pulse magnetic field conditions are: pulse frequency 500-700 Hz, pulse width 200 ms, and magnetic field strength 5-7T.

[0022] Preferably, in step (2), the specific method of salting out is: adding solid ammonium sulfate to 32-35% saturation, stirring for 5-6 hours, standing for 2-3 hours, and centrifuging to obtain a precipitate.

[0023] Preferably, in step (3), the mass ratio of the salting-out product, distilled water and complex enzyme is 1:5-6:0.02-0.03, and the complex enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0024] Preferably, in step (3), the process conditions of high pressure spraying treatment are: pressure 100-200 MPa, flow rate 0.5-0.7 m 3 / h.

[0025] Preferably, in step (3), the process conditions of enzymatic hydrolysis are: 45-48° C., and enzymatic hydrolysis for 2-3 hours.

[0026] Preferably, in step (3), the process conditions for vacuum freeze drying are: freezing at -40 to -50°C for 6 to 8 hours, evacuating to 5 to 7 Pa, heating to 25 to 30°C, and maintaining temperature and pressure for 50 to 60 hours.

[0027] Beneficial effects of the present invention:

[0028] The present invention first clarifies, microwaves and concentrates soybean water in sequence to obtain concentrated soybean water; then freezes the concentrated soybean water, thaws it with microwaves, and performs ultrasonic oscillation treatment, and salts out the concentrated soybean water through ammonium sulfate under the action of a pulsed magnetic field, and filters out solids to obtain a salting-out product; finally, the salting-out product is ultrasonically dispersed in distilled water, subjected to high-pressure spray treatment, and a composite enzyme is added, enzymolyzed, enzymes are inactivated, and vacuum freeze-dried to obtain a bioactive small molecule protein. The present invention extracts bioactive small molecule proteins from soybean water, realizes the recycling of waste soybean water, is green and environmentally friendly, and has broad application prospects.

[0029] Clarification, microwave irradiation and concentration of soy water can remove residual insoluble matter in the soy water, kill harmful microorganisms, and facilitate subsequent processing and product quality.

[0030] Freezing, microwave and ultrasonic oscillation treatment of concentrated soybean water are helpful to break the macromolecular protein structure and reduce the molecular weight of the product. The present invention uses ammonium sulfate for salting out, and the salting out is carried out under the action of a pulsed magnetic field. The salting out effect is good, which promotes the full separation of proteins and further reduces the molecular weight.

[0031] After the salting-out product is ultrasonically dispersed in distilled water, it is subjected to high-pressure spraying treatment to promote subsequent enzymolysis. The present invention utilizes a composite enzyme obtained by mixing ficin, trypsin and subtilisin to perform enzymolysis to reduce the molecular weight of the product, and the obtained product is a small molecule protein with biological activity. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0033] Embodiment 1:

[0034] A preparation method for extracting biologically active small molecule proteins from soy milk, the specific steps are as follows:

[0035] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0036] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0037] Microwave irradiation: 300W microwave irradiation for 3 minutes;

[0038] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 5 times;

[0039] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product;

[0040] The process conditions of freezing treatment are: freezing at -10℃ for 24 hours;

[0041] The process conditions of microwave thawing were as follows: 300 W microwave treatment for 2 minutes, 1 minute rest, 300 W microwave treatment for 2 minutes, 1 minute rest, 300 W microwave treatment for 2 minutes;

[0042] The process conditions of ultrasonic oscillation treatment were: 300W ultrasonic oscillation for 3 minutes, 1 minute of rest, and 700W ultrasonic oscillation for 3 minutes;

[0043] The pulse magnetic field conditions were: pulse frequency 500 Hz, pulse width 200 ms, and magnetic field intensity 5 T;

[0044] The specific method of salting out is: adding solid ammonium sulfate to 32% saturation, stirring for 5 hours, standing for 2 hours, and centrifuging to obtain a precipitate;

[0045] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a biologically active small molecule protein;

[0046] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:5:0.02, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0047] The process conditions of high pressure jet treatment are: pressure 100MPa, flow rate 0.5m 3 / h.

[0048] The process conditions of enzymatic hydrolysis are: 45°C, enzymatic hydrolysis for 2 hours.

[0049] The process conditions of vacuum freeze drying are: freezing at -40°C for 6 hours, evacuating to 5 Pa, heating to 25°C, and keeping warm and at pressure for 50 hours.

[0050] Embodiment 2:

[0051] A preparation method for extracting biologically active small molecule proteins from soy milk, the specific steps are as follows:

[0052] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0053] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0054] Microwave irradiation: 500W microwave irradiation for 5 minutes;

[0055] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 7 times;

[0056] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product;

[0057] The process conditions of freezing treatment are: freezing at -15°C for 30 hours;

[0058] The process conditions of microwave thawing were as follows: 450 W microwave treatment for 3 minutes, stop for 2 minutes, 450 W microwave treatment for 3 minutes, stop for 2 minutes, and 450 W microwave treatment for 3 minutes;

[0059] The process conditions of ultrasonic oscillation treatment were: 500W ultrasonic oscillation for 5 minutes, stop for 2 minutes, and 900W ultrasonic oscillation for 5 minutes;

[0060] The pulse magnetic field conditions were: pulse frequency 700 Hz, pulse width 200 ms, and magnetic field intensity 7 T;

[0061] The specific method of salting out is: adding solid ammonium sulfate to 35% saturation, stirring for 6 hours, standing for 3 hours, and centrifuging to obtain a precipitate;

[0062] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a biologically active small molecule protein;

[0063] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:6:0.03, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0064] The process conditions of high pressure jet treatment are: pressure 200MPa, flow rate 0.7m 3 / h.

[0065] The process conditions of enzymatic hydrolysis are: 48°C and 3 hours.

[0066] The process conditions of vacuum freeze drying are: freezing at -50°C for 8 hours, evacuating to 7 Pa, heating to 30°C, and keeping the temperature and pressure for 60 hours.

[0067] Embodiment 3:

[0068] A preparation method for extracting biologically active small molecule proteins from soy milk, the specific steps are as follows:

[0069] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0070] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0071] Microwave irradiation: 300W microwave irradiation for 5 minutes;

[0072] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 5 times;

[0073] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product;

[0074] The process conditions of freezing treatment are: freezing at -15℃ for 24 hours;

[0075] The process conditions of microwave thawing were as follows: 450 W microwave treatment for 2 minutes, 2 minutes of rest, 300 W microwave treatment for 3 minutes, 1 minute of rest, and 450 W microwave treatment for 2 minutes;

[0076] The process conditions of ultrasonic oscillation treatment were: 500W ultrasonic oscillation for 3 minutes, stop for 2 minutes, and 700W ultrasonic oscillation for 5 minutes;

[0077] The pulse magnetic field conditions were: pulse frequency 500 Hz, pulse width 200 ms, and magnetic field intensity 7 T;

[0078] The specific method of salting out is: adding solid ammonium sulfate to 32% saturation, stirring for 6 hours, standing for 2 hours, and centrifuging to obtain a precipitate;

[0079] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a biologically active small molecule protein;

[0080] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:6:0.02, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0081] The process conditions of high pressure jet treatment are: pressure 200MPa, flow rate 0.5m 3 / h.

[0082] The process conditions of enzymatic hydrolysis are: 48°C, enzymatic hydrolysis for 2 hours.

[0083] The process conditions of vacuum freeze drying are: freezing at -50°C for 6 hours, evacuating to 7 Pa, heating to 25°C, and keeping the temperature and pressure for 60 hours.

[0084] Embodiment 4:

[0085] A preparation method for extracting biologically active small molecule proteins from soy milk, the specific steps are as follows:

[0086] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0087] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0088] Microwave irradiation: 450W microwave irradiation for 4 minutes;

[0089] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 6 times;

[0090] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product;

[0091] The process conditions of freezing treatment are: freezing at -12℃ for 28 hours;

[0092] The process conditions of microwave thawing were as follows: 450 W microwave treatment for 2 minutes, 1 minute off, 300 W microwave treatment for 2 minutes, 2 minutes off, and 450 W microwave treatment for 3 minutes;

[0093] The process conditions of ultrasonic oscillation treatment were: 400W ultrasonic oscillation for 4 minutes, 2 minutes of rest, and 800W ultrasonic oscillation for 4 minutes;

[0094] The pulse magnetic field conditions were: pulse frequency 600 Hz, pulse width 200 ms, and magnetic field intensity 6 T;

[0095] The specific method of salting out is: adding solid ammonium sulfate to 33% saturation, stirring for 5 hours, standing for 2 hours, and centrifuging to obtain a precipitate;

[0096] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a biologically active small molecule protein;

[0097] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:5.5:0.02, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0098] The process conditions of high pressure jet treatment are: pressure 150MPa, flow rate 0.6m 3 / h.

[0099] The process conditions of enzymatic hydrolysis are: 46°C, 2 hours.

[0100] The process conditions of vacuum freeze drying are: freezing at -45°C for 7 hours, evacuating to 6 Pa, heating to 28°C, and keeping warm and at pressure for 55 hours.

[0101] Comparative Example 1

[0102] A preparation method for extracting protein from soy milk, the specific steps are as follows:

[0103] (1) first clarifying and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0104] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0105] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 5 times;

[0106] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product;

[0107] The process conditions of freezing treatment are: freezing at -10℃ for 24 hours;

[0108] The process conditions of microwave thawing were as follows: 300 W microwave treatment for 2 minutes, 1 minute rest, 300 W microwave treatment for 2 minutes, 1 minute rest, 300 W microwave treatment for 2 minutes;

[0109] The process conditions of ultrasonic oscillation treatment were: 300W ultrasonic oscillation for 3 minutes, 1 minute of rest, and 700W ultrasonic oscillation for 3 minutes;

[0110] The pulse magnetic field conditions were: pulse frequency 500 Hz, pulse width 200 ms, and magnetic field intensity 5 T;

[0111] The specific method of salting out is: adding solid ammonium sulfate to 32% saturation, stirring for 5 hours, standing for 2 hours, and centrifuging to obtain a precipitate;

[0112] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a protein product;

[0113] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:5:0.02, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0114] The process conditions of high pressure jet treatment are: pressure 100MPa, flow rate 0.5m 3 / h.

[0115] The process conditions of enzymatic hydrolysis are: 45°C, enzymatic hydrolysis for 2 hours.

[0116] The process conditions of vacuum freeze drying are: freezing at -40°C for 6 hours, evacuating to 5 Pa, heating to 25°C, and keeping warm and at pressure for 50 hours.

[0117] Comparative Example 2

[0118] A preparation method for extracting protein from soy milk, the specific steps are as follows:

[0119] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0120] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0121] Microwave irradiation: 300W microwave irradiation for 3 minutes;

[0122] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 5 times;

[0123] (2) Salting out: The concentrated soybean water is subjected to ultrasonic oscillation treatment, and salting out is carried out by ammonium sulfate under the action of a pulsed magnetic field, and the solid is filtered to obtain a salting out product;

[0124] The process conditions of ultrasonic oscillation treatment were: 300W ultrasonic oscillation for 3 minutes, 1 minute of rest, and 700W ultrasonic oscillation for 3 minutes;

[0125] The pulse magnetic field conditions were: pulse frequency 500 Hz, pulse width 200 ms, and magnetic field intensity 5 T;

[0126] The specific method of salting out is: adding solid ammonium sulfate to 32% saturation, stirring for 5 hours, standing for 2 hours, and centrifuging to obtain a precipitate;

[0127] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a protein product;

[0128] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:5:0.02, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0129] The process conditions of high pressure jet treatment are: pressure 100MPa, flow rate 0.5m 3 / h.

[0130] The process conditions of enzymatic hydrolysis are: 45°C, enzymatic hydrolysis for 2 hours.

[0131] The process conditions of vacuum freeze drying are: freezing at -40°C for 6 hours, evacuating to 5 Pa, heating to 25°C, and keeping warm and at pressure for 50 hours.

[0132] Comparative Example 3

[0133] A preparation method for extracting protein from soy milk, the specific steps are as follows:

[0134] (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk;

[0135] Clarification: The insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane;

[0136] Microwave irradiation: 300W microwave irradiation for 3 minutes;

[0137] Concentration: Concentrate by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, with a concentration ratio of 5 times;

[0138] (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic vibration, salting it out with ammonium sulfate, filtering out the solid, and obtaining a salting out product;

[0139] The process conditions of freezing treatment are: freezing at -10℃ for 24 hours;

[0140] The process conditions of microwave thawing were as follows: 300 W microwave treatment for 2 minutes, 1 minute rest, 300 W microwave treatment for 2 minutes, 1 minute rest, 300 W microwave treatment for 2 minutes;

[0141] The process conditions of ultrasonic oscillation treatment were: 300W ultrasonic oscillation for 3 minutes, 1 minute of rest, and 700W ultrasonic oscillation for 3 minutes;

[0142] The specific method of salting out is: adding solid ammonium sulfate to 32% saturation, stirring for 5 hours, standing for 2 hours, and centrifuging to obtain a precipitate;

[0143] (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzyme, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain a protein product;

[0144] The mass ratio of the salting-out product, distilled water and compound enzyme is 1:5:0.02, and the compound enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

[0145] The process conditions of high pressure jet treatment are: pressure 100MPa, flow rate 0.5m 3 / h.

[0146] The process conditions of enzymatic hydrolysis are: 45°C, enzymatic hydrolysis for 2 hours.

[0147] The process conditions of vacuum freeze drying are: freezing at -40°C for 6 hours, evacuating to 5 Pa, heating to 25°C, and keeping warm and at pressure for 50 hours.

[0148] Test example

[0149] The molecular weights of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 3 were measured respectively, and antibacterial tests were performed. The specific methods are as follows:

[0150] Enterococcus faecalis and Candida albicans were cultured in LB medium at a concentration of 2×10 5CFU / mL, and two bacterial solutions were obtained. The products obtained in Examples 1 to 4 or Comparative Examples 1 to 3 were dissolved and diluted with 7 times the volume of PBS solution, and then sterilized by filtering with a 0.22 μm filter to obtain a protein solution. The protein solution was added to the two bacterial solutions respectively, so that the final protein concentrations were: 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL, respectively. The culture was shaken at 37°C for 12 hours, and the OD620 was detected to calculate the number of Enterococcus faecalis and Candida albicans after culture. The minimum inhibitory concentration (MIC) is expressed as the minimum concentration that causes 100% inhibition of growth.

[0151] The results are shown in Table 1.

[0152] Table 1. Comparison of product molecular weight and antibacterial performance

[0153]

[0154] As shown in Table 1, the products obtained in Examples 1 to 4 have a lower average molecular weight, have a good antibacterial effect on Enterococcus faecalis and Candida albicans, and have good biological activity.

[0155] In comparative example 1, the microwave irradiation step of step (1) is omitted, in comparative example 2, the freezing treatment and microwave thawing steps of step (2) are omitted, and in comparative example 3, the pulsed magnetic field effect is omitted. The average molecular weight of the obtained product is higher, and the antibacterial effect on Enterobacter faecalis and Candida albicans is significantly deteriorated. This indicates that the present invention effectively reduces the molecular weight of the product and improves the biological activity of the product through the synergistic effects of microwave irradiation, freezing and thawing, pulsed magnetic field treatment, etc., combined with subsequent enzymatic hydrolysis.

[0156] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for extracting biologically active small molecule proteins from soy milk, characterized in that: The specific steps are as follows: (1) firstly clarifying, microwave irradiating and concentrating the soy milk in sequence to obtain concentrated soy milk; (2) Salting out: freezing the concentrated soybean water, thawing it with microwave, treating it with ultrasonic oscillation, salting it out with ammonium sulfate under the action of a pulsed magnetic field, filtering out the solid, and obtaining a salting out product; (3) ultrasonically dispersing the salting-out product in distilled water, high-pressure spraying treatment, adding complex enzymes, enzymolysis, enzyme inactivation, and vacuum freeze-drying to obtain biologically active small molecule proteins.

2. The preparation method according to claim 1, characterized in that: In step (1), the insoluble matter in the soy milk is clarified by a 250 kDa hollow fiber membrane; The process conditions of microwave irradiation are: 300-500W microwave irradiation for 3-5 minutes; The product was concentrated by ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentration ratio was 5 to 7 times.

3. The preparation method according to claim 1, characterized in that: In step (2), the process conditions of the freezing treatment are: freezing at -10 to -15°C for 24 to 30 hours.

4. The preparation method according to claim 1, characterized in that: In step (2), the process conditions of microwave thawing are: microwave treatment at 300-450 W for 2-3 minutes, stop for 1-2 minutes, microwave treatment at 300-450 W for 2-3 minutes, stop for 1-2 minutes, and microwave treatment at 300-450 W for 2-3 minutes.

5. The preparation method according to claim 1, characterized in that: In step (2), the process conditions of ultrasonic oscillation treatment are: 300-500W ultrasonic oscillation for 3-5 minutes, stop for 1-2 minutes, and 700-900W ultrasonic oscillation for 3-5 minutes.

6. The preparation method according to claim 1, characterized in that: In step (2), the pulse magnetic field conditions are: pulse frequency 500-700 Hz, pulse width 200 ms, and magnetic field strength 5-7 T.

7. The preparation method according to claim 1, characterized in that: In step (2), the specific method of salting out is: adding solid ammonium sulfate to 32-35% saturation, stirring for 5-6 hours, standing for 2-3 hours, and centrifuging to obtain a precipitate.

8. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the salting-out product, distilled water and composite enzyme is 1:5-6:0.02-0.03, and the composite enzyme is obtained by mixing the mass of ficin, trypsin, subtilisin and the like.

9. The preparation method according to claim 1, characterized in that: In step (3), the process conditions of high pressure spraying treatment are: pressure 100-200 MPa, flow rate 0.5-0.7 m 3 / h.

10. The preparation method according to claim 1, characterized in that: In step (3), the process conditions of enzymatic hydrolysis are: 45-48° C., enzymatic hydrolysis for 2-3 hours; The process conditions of vacuum freeze drying are: freezing at -40 to -50°C for 6 to 8 hours, evacuating to 5 to 7 Pa, heating to 25 to 30°C, and keeping warm and at pressure for 50 to 60 hours.

Images