A method for preparing polypeptides from high isoflavone soybeans

Abstract

The application relates to the food processing technical field and provides a method for preparing polypeptides from high-isoflavone soybeans, which comprises the following steps: step one, selecting high-isoflavone soybeans as raw materials, grinding and squeezing the high-isoflavone soybeans to obtain defatted soybean meal, and purifying the defatted soybean meal to obtain a soybean protein solution; step two, adjusting the pH of the soybean protein solution to 7.0-10.0, adding alkaline protease, and performing an enzymolysis reaction in a microwave reactor, wherein the microwave power is 100-1000 W; and step three, inactivating the enzyme of the solution after the enzymolysis, adjusting the pH to neutral, and obtaining a soybean polypeptide stock solution after centrifugation. Through synchronous regulation of pH, temperature, power and time, the soybean protein is rapidly unfolded and completes site cutting, the enzymolysis period is significantly shortened, the generation of bitter peptides is reduced, the soluble nitrogen yield is improved, the isoflavone conversion caused by high-temperature concentration can be avoided, the decolorization, debittering and activity reservation are cooperated, the final peptide powder can keep the high-isoflavone characteristics, and the quality of the finished product is ensured.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for preparing polypeptides from soybeans high in isoflavones. Background Technology

[0002] Soybean peptides, as hydrolysates of soybean protein, possess advantages such as small molecular weight, easy absorption, low antigenicity, and diverse physiological functions, and are widely used in functional foods, feed, and pharmaceuticals. Traditional preparation methods typically use soybean residue or soy protein isolate as raw materials, obtaining peptides through enzymatic hydrolysis, centrifugation, and drying. However, existing technologies generally suffer from low enzymatic hydrolysis efficiency, bitter taste, dark color, and complex processes. This is especially true for special raw materials like soybeans high in isoflavones, where isoflavone components are easily lost during processing, making it difficult to balance peptide yield with the retention of active ingredients.

[0003] High-isoflavone soybeans are a type of unique germplasm obtained through systematic breeding or physical mutagenesis. Isoflavones in the seeds are enriched in the form of malonyl glycosides, making them an ideal raw material for developing women's nutrition, sports recovery, and special medical foods. However, isoflavones are extremely sensitive to heat, acids, alkalis, and prolonged enzymatic hydrolysis. In the conventional "alkali dissolution, acid precipitation, enzymatic hydrolysis, concentration, and drying" route, they are prone to deglycosylation or degradation, resulting in a significant reduction in activity. Simultaneously, high-isoflavone soybeans have a denser protein conformation with deeper hydrophobic regions, leading to low extraction rates, slow enzymatic hydrolysis, and increased formation of bitter peptides and pigments. Furthermore, isoflavones are lost simultaneously during the desalination process.

[0004] Current technologies have not yet achieved a balance between "efficient peptide production" and "activity retention," severely hindering the industrial application of high-isoflavone soybeans in high-end peptide-based functional foods. Therefore, a method for preparing peptides from high-isoflavone soybeans is needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing peptides from soybeans high in isoflavones, solving the problem that existing technologies have not yet achieved both "efficient peptide preparation" and "activity retention".

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing polypeptides from soybeans high in isoflavones, comprising: Step 1: Select high-isoflavone soybeans as raw materials, grind and press them to obtain defatted soybean meal, purify the defatted soybean meal to obtain soybean protein solution; Step 2: Adjust the pH of the separated soybean protein solution to 7.0-10.0, add alkaline protease, and carry out the enzymatic hydrolysis reaction in a microwave reactor with a microwave power of 100-1000W; Step 3: Inactivate the enzymes in the enzymatically hydrolyzed solution, adjust the pH to neutral, and centrifuge to obtain soybean polypeptide stock solution; Step 4: Mix soybean polypeptide stock solution with activated carbon powder at a solid-liquid ratio of 1:10, stir at 40-50℃ for 25-45 minutes for decolorization and debittering, and then pass the soybean polypeptide stock solution sequentially through H-type cation exchange resin and OH-type anion exchange resin at a flow rate of 10 column volumes / h to remove Na+. + and Cl - Desalination is carried out; Step 5: Filter the decolorized and debittered solution through an ultrafiltration membrane with a molecular weight of 3000-5000 Da to obtain the filtrate, and then concentrate it under reduced pressure to obtain a soybean polypeptide concentrate with a solid content of 25-40%. Step 6: Dry the soybean polypeptide concentrate to obtain soybean polypeptide powder.

[0007] Preferably, the purification process in step one includes the following steps: S1. Mix defatted soybean meal with water at a solid-liquid ratio of 1:8-1:12 and heat to 30-60℃. S2. Add concentrated hydrochloric acid to the mixed liquid to adjust the pH value of the mixed liquid to 3.0-6.0, precipitating soybean protein; S3. Centrifuge the mixed liquid, then wash the precipitate with water, repeat the washing 2-3 times, collect the precipitate and add water to prepare a soy protein separation solution.

[0008] Preferably, the alkaline protease in step two is Bacillus subtilis serine alkaline protease, and the enzyme dosage is 2% to 4% in terms of E / S, where E is the enzyme mass and S is the substrate protein mass.

[0009] Preferably, in step two, the enzymatic hydrolysis reaction environment is maintained at 35–55°C, and the reaction time is 5–30 min.

[0010] Preferably, the enzyme inactivation conditions in step three are a temperature environment of 90-95°C for 10-20 minutes.

[0011] Preferably, the centrifugation conditions in step three are: the rotation speed is maintained at 12,000 to 15,000 rpm, and the time lasts for 15 to 30 minutes.

[0012] Preferably, in step five, the ultrafiltration membrane is a ceramic membrane or an organic membrane, the working pressure is 2 to 2.5 bar, and the transmembrane pressure difference is 1.5 to 2 bar.

[0013] Preferably, the vacuum concentration conditions in step five are: vacuum degree 600-700 mmHg, pressure 85-95 kPa, and time 25-40 min.

[0014] Preferably, the drying method in step six is ​​either spray drying or freeze drying.

[0015] Preferably, the alkaline protease in step two is a protease secreted by Basillus sp. 8A6, with an enzyme activity of 5000-30000 U / mL.

[0016] This invention provides a method for preparing polypeptides from soybeans high in isoflavones. It has the following beneficial effects: 1. This invention enables soybean protein to rapidly expand and complete targeted shearing by synchronously controlling pH, temperature, power and time, significantly shortening the enzymatic hydrolysis cycle, reducing the formation of bitter peptides, increasing the yield of soluble nitrogen, and avoiding isoflavone conversion caused by high-temperature concentration. It achieves synergistic decolorization and debittering with activity retention, ensuring that the final peptide powder maintains high isoflavone characteristics and guaranteeing the quality of the finished product.

[0017] 2. This invention uses an ultrafiltration membrane with a suitable molecular weight cutoff to perform one-step fractionation of the decolorizing solution in a low-pressure range, simultaneously completing the retention of bitter peptides and pigments, and the enrichment of active oligopeptides and isoflavones. The membrane flux is stable and the cleaning cycle is long. Furthermore, by combining vacuum concentration and gentle drying, energy consumption and heat loss are significantly reduced. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example: Please see the appendix Figure 1 This invention provides a method for preparing polypeptides from soybeans high in isoflavones, comprising: Step 1: Select high-isoflavone soybeans as raw material, grind and press them to obtain defatted soybean meal, and purify the defatted soybean meal to obtain a soybean protein solution. The specific steps of the purification process include: S1. Mix defatted soybean meal with water at a solid-liquid ratio of 1:8-1:12 and heat to 30-60℃. S2. Add concentrated hydrochloric acid to the mixed liquid to adjust the pH value of the mixed liquid to 3.0-6.0, precipitating soybean protein; S3. Centrifuge the mixed liquid, then wash the precipitate with water, repeat the washing 2-3 times, collect the precipitate and add water to prepare a soy protein separation solution. Step 2: Adjust the pH of the separated soybean protein solution to 7.0-10.0, add alkaline protease, and carry out enzymatic hydrolysis in a microwave reactor with a microwave power of 100-1000W. The alkaline protease is a protease secreted by Basillus sp. 8A6, with an enzyme activity of 5000-30000 U / mL. The preparation process involves inoculating the bacterial strain into an enzyme-producing medium containing chicken feathers, collecting the supernatant, and then culturing it to prepare the solution. The alkaline protease is Bacillus subtilis serine alkaline protease, and the enzyme dosage is 2%-4% (E / S), where E is the enzyme mass and S is the substrate protein mass. The enzymatic hydrolysis reaction environment is maintained at 35-55℃, and the reaction time is 5-30 min. Step 3: Inactivate the enzyme in the solution after enzymatic hydrolysis. The enzyme inactivation conditions are 90-95℃ for 10-20 minutes. Adjust the pH to neutral. After centrifugation, obtain soybean polypeptide stock solution. The centrifugation conditions are 12000-15000 rpm for 15-30 minutes. Step 4: Mix soybean polypeptide stock solution with activated carbon powder at a solid-liquid ratio of 1:10, stir at 40-50℃ for 25-45 minutes for decolorization and debittering, and then pass the soybean polypeptide stock solution sequentially through H-type cation exchange resin and OH-type anion exchange resin at a flow rate of 10 column volumes / h to remove Na+. + and Cl - Desalination is carried out; Step 5: Filter the decolorized and debittered solution through an ultrafiltration membrane with a molecular weight of 3000-5000 Da to obtain the filtrate. Then, concentrate the filtrate under reduced pressure to obtain a soybean polypeptide concentrate with a solid content of 25-40%. The reduced pressure concentration conditions are: vacuum degree 600-700 mmHg, pressure 85-95 kPa, time 25-40 min, ultrafiltration membrane is a ceramic membrane or organic membrane, working pressure is 2-2.5 bar, and transmembrane pressure difference is 1.5-2 bar. Step 6: Dry the soybean polypeptide concentrate to obtain soybean polypeptide powder. The drying method can be either spray drying or freeze drying.

[0021] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing polypeptides from soybeans high in isoflavones, characterized in that, include: Step 1: Select high-isoflavone soybeans as raw materials, grind and press them to obtain defatted soybean meal, purify the defatted soybean meal to obtain soybean protein solution; Step 2: Adjust the pH of the separated soybean protein solution to 7.0-10.0, add alkaline protease, and carry out the enzymatic hydrolysis reaction in a microwave reactor with a microwave power of 100-1000W; Step 3: Inactivate the enzymes in the enzymatically hydrolyzed solution, adjust the pH to neutral, and centrifuge to obtain soybean polypeptide stock solution; Step 4: Mix soybean polypeptide stock solution with activated carbon powder at a solid-liquid ratio of 1:10, stir at 40-50℃ for 25-45 minutes for decolorization and debittering, and then pass the soybean polypeptide stock solution sequentially through H-type cation exchange resin and OH-type anion exchange resin at a flow rate of 10 column volumes / h to remove Na+. + and Cl - Desalination is carried out; Step 5: Filter the decolorized and debittered solution through an ultrafiltration membrane with a molecular weight of 3000-5000 Da to obtain the filtrate, and then concentrate it under reduced pressure to obtain a soybean polypeptide concentrate with a solid content of 25-40%. Step 6: Dry the soybean polypeptide concentrate to obtain soybean polypeptide powder.

2. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, The specific steps of the purification process in step one include: S1. Mix defatted soybean meal with water at a solid-liquid ratio of 1:8-1:12 and heat to 30-60℃. S2. Add concentrated hydrochloric acid to the mixed liquid to adjust the pH value of the mixed liquid to 3.0-6.0, precipitating soybean protein; S3. Centrifuge the mixed liquid, then wash the precipitate with water, repeat the washing 2-3 times, collect the precipitate and add water to prepare a soy protein separation solution.

3. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, In step two, the alkaline protease is Bacillus subtilis serine alkaline protease, and the enzyme dosage is 2% to 4% in terms of E / S, where E is the enzyme mass and S is the substrate protein mass.

4. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, In step two, the enzymatic hydrolysis reaction environment is maintained at 35–55°C, and the reaction time is 5–30 minutes.

5. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, In step three, the enzyme inactivation conditions are a temperature environment of 90-95°C for 10-20 minutes.

6. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, In step three, the centrifugation conditions are: the rotation speed is maintained at 12,000 to 15,000 rpm, and the time lasts for 15 to 30 minutes.

7. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, In step five, the ultrafiltration membrane is a ceramic membrane or an organic membrane, with an operating pressure of 2 to 2.5 bar and a transmembrane pressure difference of 1.5 to 2 bar.

8. The method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, The vacuum concentration conditions in step five are: vacuum degree 600-700 mmHg, pressure 85-95 kPa, and time 25-40 min.

9. A method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, In step six, the drying method can be either spray drying or freeze drying.

10. A method for preparing polypeptides from high-isoflavone soybeans according to claim 1, characterized in that, The alkaline protease in step two is a protease secreted by Basillus sp. 8A6, with an enzyme activity of 5000-30000 U / mL.

Images