Preparation method of soybean protein source polypeptide puree with anti-oxidation effect

Abstract

The invention discloses a preparation method of soybean protein source polypeptide primary pulp with an anti-oxidation effect, and belongs to the field of soybean protein food processing, and the preparation method comprises the following steps: selecting and soaking soybeans, and grinding to obtain soybean milk; the preparation method comprises the following steps: heating soybean milk, pretreating with a high-voltage pulsed electric field, and performing enzymolysis with protease to obtain soybean protein source polypeptide primary pulp with an antioxidant effect; according to the invention, pepsin, chymotrypsin and bromelain are added step by step for enzymolysis, so that soybean protein can be decomposed into micromolecule polypeptide with antioxidant activity; different enzyme reaction temperatures, pH values and time are adopted according to enzymatic characteristics of different proteases, the yield of polypeptide can be increased, and the prepared soybean protein source polypeptide puree is better in oxidation resistance.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptide puree preparation, in particular to a method for preparing soybean protein source polypeptide puree with antioxidant effect. Background Art

[0002] Soy milk made from soybeans contains high-quality protein and is rich in nutrients such as linoleic acid, vitamin E, and lecithin. Therefore, as a natural functional food, its consumption is increasing year by year. As a water extract, soy milk contains a water-in-oil emulsion system with complex protein components. Compared with cow's milk and human milk, soy milk contains more iron and potassium, and does not contain lactose and cholesterol. It can be used in the preparation of special foods. For example, infants or children who are allergic to cow's milk and those who lack lactase can use soy milk instead of cow's milk.

[0003] Chinese patent (publication number CN111066895A) discloses a micro-enzyme hydrolyzed soybean milk and its preparation method. The method comprises the following steps: (1) treating soybeans with NH 4 HCO 3 After soaking, washing, grinding and filtering are performed to obtain slurry; (2) the slurry is vacuum concentrated and homogenized to obtain a homogenized liquid; (3) the homogenized liquid is enzymatically hydrolyzed with neutral protease, pectinase and cellulase to obtain an enzymatic solution; (4) the enzymatic solution is subjected to high-pressure steam and sterilization to obtain sterile cooked soy milk; (5) the sterile cooked soy milk is vacuum heated, dehydrated and cooled to obtain micro-enzymatic hydrolysis soy milk. Although the enzymatic hydrolysis soy milk technology provided can hydrolyze soybean protein into polypeptides, the antioxidant property of the polypeptide soy milk prepared by the enzymatic hydrolysis soy milk is poor. In order to improve the antioxidant property of polypeptide soy milk, the present invention provides a method for preparing a soybean protein source polypeptide slurry with antioxidant effect. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a soybean protein source polypeptide puree with antioxidant effect, so as to solve the problems in the prior art.

[0005] The working principle of the present invention is: after removing impurities and washing, the soybean seeds are soaked in water to make the soybeans fully absorb water and swell, in preparation for subsequent grinding; the soaked soybeans are ground, filtered after grinding, and soybean milk is obtained; after heating the soybean milk, it is pretreated with a high-voltage pulse electric field: the pulse intensity of the high-voltage pulse electric field is 28-32kV / cm, the pulse time is 140-420μs, the pulse flow rate is 50-70mL / min, and the pulse frequency is 460-540Hz. The high-voltage pulse electric field used in the present invention can effectively kill harmful microorganisms such as bacteria and molds, and passivate enzymes such as lipase in soybean milk, thereby preventing soybean milk from deteriorating; high-voltage pulse electric field treatment changes the protein structure, which is conducive to the subsequent protease hydrolysis of protein in soybean milk, obtaining more soybean polypeptides with antioxidant effects, and making the soybean milk more delicate and smooth.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing soybean protein source polypeptide puree with antioxidant effect, comprising the following steps:

[0007] S1, soybean soaking;

[0008] S2, grinding to obtain soy milk;

[0009] S3, high-voltage pulse electric field pretreatment: after the soy milk is heated, it is pretreated with a high-voltage pulse electric field;

[0010] S4. Protease hydrolysis: Pepsin, chymotrypsin and bromelain are added step by step to hydrolyze the protein in the soy milk.

[0011] Soaking soybeans in S1: Soybeans are placed in a container and soaked in water at a temperature of 20-25° C. for 8-12 hours, during which the water is changed 1-2 times.

[0012] The soybean grinding in S2 is as follows: the soaked soybeans are added with 85-95° C. pure water in a ratio of 1:4-6 (w / v), the soybeans are ground with a grinding wheel, the slurry is sieved through an 80-mesh filter cloth, the slurry is sent to a slurry storage tank through a pipeline, the slurry is boiled at 95° C. to make the slurry temperature reach 75° C., and then filtered with a 250-mesh sieve to obtain soybean milk.

[0013] The pulse intensity of the high-voltage pulse electric field in S3 is 20-50 kV / cm, the pulse time is 70-550 μs, the pulse flow rate is 50-70 mL / min, and the pulse frequency is 400-600 Hz.

[0014] The protease hydrolysis in S4 is as follows: adjusting the pH of the soy milk after the pulse electric field treatment, adding pepsin, chymotrypsin and bromelain step by step for hydrolysis, heating in a boiling water bath for 10 minutes to terminate the hydrolysis, and obtaining soybean polypeptide puree of components with a molecular weight less than 3KDa.

[0015] Preferably, pepsin is added first; the conditions are: pH 1.5-3.0, temperature 20-50°C, enzyme-substrate ratio 1000-3000U / ml, and enzymolysis time 1-3h.

[0016] Further, pepsin was added under the following conditions: pH 2.0, temperature 37°C, enzyme-substrate ratio 1000U / ml, and enzymolysis time 2h.

[0017] Preferably, chymotrypsin or bromelain is then added under the following conditions: pH 6-8, temperature 40-60° C., ratio of both enzymes to substrate 1000-3000 U / ml, and enzymolysis time 2-6 h.

[0018] Further, chymotrypsin or bromelain was added under the following conditions: pH 7.5, temperature 50°C, the ratio of both enzymes to substrate was 2000U / ml, and the enzymolysis time was 4h.

[0019] The prepared puree has an antioxidant soybean protein source polypeptide content of more than 10%.

[0020] Beneficial Effects

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention performs high-voltage pulse electric field pretreatment before enzymolysis, which can effectively kill harmful microorganisms such as bacteria and molds, and passivate enzymes such as lipase in soy milk, thereby preventing soy milk from deteriorating; high-voltage pulse electric field treatment changes the protein structure, which is beneficial to the subsequent protease hydrolysis of protein in soy milk, obtains more soybean polypeptides with antioxidant effects, and makes the soy milk more delicate and smooth.

[0023] 2. The present invention adopts the method of adding pepsin, chymotrypsin and bromelain step by step for enzymatic hydrolysis, so as to hydrolyze soybean protein into small molecular polypeptides with antioxidant activity, thereby improving the antioxidant property of the prepared soybean puree.

[0024] 3. The present invention adopts different temperatures, pH values ​​and times for different proteases, thereby ensuring the activity of the proteases, which is beneficial to increasing the yield of the polypeptides and making their antioxidant properties higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 Technical flow chart of the present invention (a method for preparing soybean protein source polypeptide puree with antioxidant effect). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0028] Embodiment 1

[0029] See also Figure 1 In an embodiment of the present invention, a method for preparing a soybean protein source polypeptide stock paste having an antioxidant effect comprises the following steps:

[0030] S1. Soaking soybeans: Soak soybeans in a container with water at a temperature of 20-25°C for 8-12 hours, changing the water 1-2 times during the soaking period;

[0031] S2, soybean grinding: add 85-95°C pure water to the soaked soybeans in a ratio of 1:4-6 (w / v), grind with a grinding wheel, sieve through an 80-mesh filter cloth, send to a pulp storage tank through a pipeline, boil at 95°C until the pulp temperature reaches 75°C, and then filter with a 250-mesh screen to obtain soybean milk;

[0032] S3, high-voltage pulse electric field pretreatment: after the soy milk is heated, it is pretreated with a high-voltage pulse electric field, with a pulse intensity of 20 kV / cm, a pulse time of 70 μs, a pulse flow rate of 50 mL / min, and a pulse frequency of 400 Hz;

[0033] S4. Protease hydrolysis: adjust the pH of the soy milk after the pulse electric field treatment, add pepsin, chymotrypsin and bromelain step by step for hydrolysis, heat in a boiling water bath for 10 minutes to terminate the hydrolysis, and obtain soybean polypeptide puree with a molecular weight less than 3KDa.

[0034] Among them, pepsin is added first; the conditions are: pH 1.5-3.0, temperature 20-50°C, enzyme-substrate ratio 1000-3000U / ml, and enzymolysis time 1-3h; the optimal conditions are: pH 2.0, temperature 37°C, enzyme-substrate ratio 1000U / ml, and enzymolysis time 2h.

[0035] Then, chymotrypsin or bromelain is added under the following conditions: pH 6-8, temperature 40-60°C, ratio of both enzymes to substrates 1000-3000U / ml, and enzymolysis time 2-6h; optimal conditions: pH 7.5, temperature 50°C, ratio of both enzymes to substrates 2000U / ml, and enzymolysis time 4h, and the prepared puree contains antioxidant soybean protein source polypeptides.

[0036] Embodiment 2

[0037] See also Figure 1 In an embodiment of the present invention, a method for preparing a soybean protein source polypeptide stock paste having an antioxidant effect comprises the following steps:

[0038] S1, same as in Example 1;

[0039] S2, same as in Example 1;

[0040] S3. High-voltage pulse electric field pretreatment: After the soy milk is heated, it is pretreated with a high-voltage pulse electric field; the pulse intensity of the high-voltage pulse electric field is 50 kV / cm, the pulse time is 550 μs, the pulse flow rate is 70 mL / min, and the pulse frequency is 600 Hz.

[0041] S4. Protease hydrolysis: adjust the pH of the soy milk after the pulse electric field treatment, add pepsin, chymotrypsin and bromelain step by step for hydrolysis, heat in a boiling water bath for 10 minutes to terminate the hydrolysis, and obtain soybean polypeptide puree with a molecular weight less than 3KDa.

[0042] Among them, pepsin is added first, and the conditions are: pH 1.5-3.5, temperature 20-50°C, enzyme-substrate ratio 1000-3000U / g, and enzymolysis time 1-3h; the optimal conditions are: pH 2.0, temperature 37°C, enzyme-substrate ratio 1000U / g, and enzymolysis time 2h.

[0043] Then, chymotrypsin or bromelain is added under the following conditions: pH 6-8, temperature 40-60°C, ratio of both enzymes to substrates 1000-3000U / g, and enzymolysis time 2-6h; optimal conditions: pH 7.5, temperature 50°C, ratio of both enzymes to substrates 2000U / g, and enzymolysis time 4h, and the prepared raw pulp has antioxidant soybean protein source polypeptides.

[0044] Embodiment 3

[0045] See also Figure 1 In an embodiment of the present invention, a method for preparing a soybean protein source polypeptide stock paste having an antioxidant effect comprises the following steps:

[0046] S1, same as in Example 1;

[0047] S2, same as in Example 1;

[0048] S3. High-voltage pulse electric field pretreatment: After the soy milk is heated, it is pretreated with a high-voltage pulse electric field; the pulse intensity of the high-voltage pulse electric field is 30 kV / cm, the pulse time is 400 μs, the pulse flow rate is 60 mL / min, and the pulse frequency is 400 Hz.

[0049] S4, protease hydrolysis; adjusting the pH of the soy milk after the pulse electric field treatment, adding pepsin, chymotrypsin and bromelain step by step for hydrolysis, heating in a boiling water bath for 10 minutes to terminate the hydrolysis, and obtaining soybean polypeptide puree of components with a molecular weight less than 3KDa.

[0050] Among them, pepsin is added first, and the conditions are: pH 1.5-3.5, temperature 20-50°C, enzyme-substrate ratio 1000-3000U / g, and enzymolysis time 1-3h; the optimal conditions are: pH 2.0, temperature 37°C, enzyme-substrate ratio 1000U / g, and enzymolysis time 2h.

[0051] Then, chymotrypsin or bromelain is added under the following conditions: pH 6-8, temperature 40-60°C, ratio of both enzymes to substrates 1000-3000U / g, and enzymolysis time 2-6h; optimal conditions: pH 7.5, temperature 50°C, ratio of both enzymes to substrates 2000U / g, and enzymolysis time 4h, and the prepared raw pulp has antioxidant soybean protein source polypeptides.

[0052] Comparative Example 1

[0053] The only difference from Example 1 is that there is no S3, that is, no pulse electric field treatment is performed;

[0054] The remaining operations are the same as those in Example 1.

[0055] The prepared puree contains antioxidant soybean protein source polypeptides.

[0056] Comparative Example 2

[0057] The difference from Example 1 is that during the enzymatic hydrolysis with S4 protease, only pepsin is added, and the remaining operations are the same as those in Example 1. The specific operating steps are as follows:

[0058] S4, protease hydrolysis: adding pepsin for hydrolysis, heating in a boiling water bath for 10 minutes to terminate the hydrolysis, and obtaining soybean polypeptide puree of components with a molecular weight less than 3 KDa.

[0059] Pepsin is added; the conditions are: pH 1.5-3.5, temperature 20-50°C, enzyme-substrate ratio 1000-3000U / ml, and enzymolysis time 1-3h; the optimal conditions are: pH 2.0, temperature 37°C, enzyme-substrate ratio 1000U / ml, and enzymolysis time 2h. The prepared puree has antioxidant soybean protein source polypeptides.

[0060] Test Example 1: Peptide Content Determination

[0061] The high performance liquid chromatography (HPLC) method is used to determine the polypeptide content in soybean milk, comprising the following steps:

[0062] 1. Sample preparation: Take an appropriate amount of soy milk sample, dilute and filter it appropriately, and then add an appropriate buffer solution to make its pH value moderate, which is conducive to the dissolution of protein.

[0063] 2. Protease digestion: Add specific proteases (such as trypsin, pepsin, etc.) to enzymatically hydrolyze proteins to generate small molecule peptides.

[0064] 3. Extraction and separation: Use solid phase extraction (SPE) columns to purify and concentrate the enzymatic hydrolysis products.

[0065] 4. HPLC analysis: The purified sample is injected into the HPLC system, the peptides are separated by a reverse phase column, and detected using an ultraviolet detector (UV) or a fluorescence detector.

[0066] 5. Data processing: record the peak area or height and calculate the peptide content by comparing with the standard curve.

[0067] Test Example 2: Antioxidant Activity Determination

[0068] In the above examples, the antioxidant activity of soy milk peptides is expressed by oxygen radical absorbance capacity (ORAC), and the determination method is as follows:

[0069] 1. Sample Preparation

[0070] Dilute the soy protein puree: Dilute the soy protein puree appropriately according to the expected antioxidant activity level;

[0071] Positive control: Choose a known standard antioxidant as a positive control;

[0072] 2. Buffer and Reagent Preparation

[0073] PBS buffer: Phosphate buffered saline (pH 7.4), used to maintain a stable pH value in the system.

[0074] AAPH (2,2'-azobisisobutyronitrile): As a source of peroxide free radicals, it must be freshly prepared.

[0075] Fluorescein: As a fluorescent probe, it is usually used at a final concentration of 70 nM.

[0076] 3. Add appropriate amount of soybean protein concentrate dilution, fluorescein and AAPH, and repeat the group three times to ensure the reliability of the data;

[0077] 4. Preheating: Place the microplate in a fluorescent microplate reader at 37°C for about 15 minutes to allow all components to reach temperature equilibrium;

[0078] 5. Initial reading: Set the excitation wavelength to 485 nm and the emission wavelength to 528 nm, and record the initial fluorescence intensity once before adding AAPH;

[0079] 6. Free radical generation: quickly add an equal amount of freshly prepared AAPH solution to each well to initiate the free radical reaction;

[0080] 7. Continuous monitoring: Read the fluorescence intensity at regular intervals (e.g., every 1 minute) for 60-90 minutes until the fluorescence intensity stabilizes;

[0081] 8. Calculate the ORAC value.

[0082] y=mx+b

[0083] Where, y - measured response value (absorbance);

[0084] x – concentration of analyte;

[0085] m - slope, which indicates the change in response value per unit change in concentration;

[0086] b-intercept, that is, the value of y when (x=0).

[0087] Table 1 Comparison of polypeptide content and antioxidant activity in soybean protein hydrolysates

[0088] Sample Type Peptide content (%) ORAC value (μmolTE / g) Example 1 12-26 30-40 Example 2 15-26 31-40 Example 3 12-20 30-38 Comparative Example 1 7-9 26-35 Comparative Example 2 5-7 15-28

[0089] As can be seen from the results in Table 1, the polypeptide content of Example 1, Example 2 and Example 3 is higher than that of Comparative Example 1 and Comparative Example 2. Among the three examples, the polypeptide content of Example 1 and Example 2 is higher than that of Example 3. This is because when pepsin is used for enzymatic hydrolysis, the amount of enzyme added and the enzymatic hydrolysis time are lower than those of Example 1 and Example 2. From the perspective of antioxidant activity, the ORAC values ​​of Example 1, Example 2 and Example 3 are higher than those of Comparative Example 1 and Comparative Example 2. Among them, the ORAC values ​​of Example 1 and Example 2 are both higher than those of Example 3. It can be seen that there is a dose-effect relationship between the peptide content and the antioxidant activity in the soy protein hydrolyzate, which also shows that the method for preparing a soy protein source polypeptide slurry with antioxidant effect of the present invention is excellent.

[0090] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a soybean protein-derived polypeptide puree having an antioxidant effect, characterized in that: The following steps are involved: S1, soybean soaking; S2, grinding to obtain soy milk; S3, high-voltage pulse electric field pretreatment: after the soy milk is heated, it is pretreated with a high-voltage pulse electric field; S4. Protease hydrolysis: add pepsin, chymotrypsin and bromelain step by step to hydrolyze the soy milk protein.

2. The preparation method according to claim 1, characterized in that: Soaking soybeans as described in S1: Soaking soybeans in a container with water at a temperature of 20-25° C. for 8-12 hours, during which the water is changed 1-2 times.

3. The preparation method according to claim 1, characterized in that: The soybean grinding described in S2: the soaked soybeans are added with 85-95°C pure water in a ratio of 1:4-6 (w / v), the slurry is ground with a grinding wheel, the slurry is sieved through an 80-mesh filter cloth, and the slurry is sent to a slurry storage tank through a pipeline, boiled at 95°C to make the slurry temperature reach 75°C, and then filtered with a 250-mesh sieve to obtain soy milk.

4. The preparation method according to claim 1, characterized in that: The pulse intensity of the high-voltage pulse electric field described in S3 is 20-50 kV / cm, the pulse time is 70-550 μs, the pulse flow rate is 50-70 mL / min, and the pulse frequency is 400-600 Hz.

5. The preparation method according to claim 1, characterized in that: When pepsin is added in S4, the pH is 1.5-3.0, the temperature is 20-50° C., the enzyme-substrate ratio is 1000-3000 U / ml, and the enzymolysis time is 1-3 h.

6. The preparation method according to claim 1, characterized in that: When pepsin is added as described in S4, the pH is 2.0, the temperature is 37° C., the enzyme-substrate ratio is 1000 U / ml, and the enzymatic hydrolysis time is 2 h.

7. The preparation method according to claim 1, characterized in that: When chymotrypsin or bromelain is added in S4, the pH is 6-8, the temperature is 40-60° C., the ratio of the two enzymes to the substrate is 1000-3000 U / ml, and the enzymolysis time is 2-6 hours.

8. The preparation method according to claim 1, characterized in that: When chymotrypsin or bromelain is added as described in S4, the pH is 7.5, the temperature is 50° C., the ratio of the two enzymes to the substrate is 2000 U / ml, and the enzymolysis time is 4 h.

9. The preparation method according to claim 1, characterized in that: The protease hydrolysis described in S4: after the soy protein is hydrolyzed by pepsin, chymotrypsin and bromelain, the soy milk is heated in a boiling water bath for 10 minutes to terminate the hydrolysis, and a soy polypeptide puree with a molecular weight less than 3KDa is obtained.

10. The soybean protein source polypeptide puree with antioxidant effect obtained by the preparation method according to any one of claims 1 to 9.

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