Preparation method and application of high water-soluble small molecule peptide

Abstract

The application belongs to the technical field of protein deep processing, and specifically provides a preparation method and application of high-water-solubility small-molecule peptides. The preparation method of the high-water-solubility small-molecule peptides comprises the following steps: uniformly stirring protein powder with water, adjusting pH and heating for pretreatment; adding alkaline protease to the pretreated solution for primary enzymolysis; adding neutral protease and flavor protease to the primary enzymolysis solution for secondary enzymolysis; centrifuging the secondary enzymolysis solution to collect supernatant; fractionally purifying the supernatant through an ultrafiltration membrane to collect permeate, and concentrating and drying the permeate to obtain small-molecule peptides. The method is targeted at the characteristics of alcohol-soluble protein, such as compact spatial structure and strong hydrophobicity, and realizes step-by-step directional enzymolysis, protein structure unlocking and precise cutting, significantly improves hydrolysis efficiency and product uniformity, and avoids excessive degradation or insufficient degradation. The prepared protein peptides have high degree of hydrolysis, excellent water solubility, high proportion of small-molecule peptides, light color, no bitter taste, and clear and non-sedimented solution.

Description

Technical Field

[0001] This invention belongs to the field of protein deep processing technology, specifically relating to a method for preparing and applying highly water-soluble small molecule peptides. Background Technology

[0002] Corn gluten meal is a major byproduct of the corn starch industry. It has a high crude protein content, readily available raw materials, and low cost. However, its main component is corn gliadin, which has a highly dense molecular spatial structure and a large number of hydrophobic groups. This results in poor water solubility, low in vitro digestibility and absorption, dark product color, and a strong inherent bitter taste, which limits its application in high-end foods and functional feeds.

[0003] Currently, the industrial preparation of corn gluten peptides mainly falls into two categories: single-enzyme hydrolysis and stepwise enzymatic hydrolysis using two enzymes. CN101096696A discloses a process for stepwise enzymatic hydrolysis of corn gluten powder using a combination of high-temperature amylase, alkaline protease, and neutral protease. This process uses a high-temperature soaking treatment of raw materials at 95-98℃, which easily causes denaturation and aggregation of corn proteins, thus reducing the accessibility of enzymatic hydrolysis. Moreover, the process only uses a combination of two proteases, which can only achieve preliminary degradation of large-molecule proteins and cannot effectively decompose the bitter groups at the ends of peptide chains, resulting in a noticeable bitter taste in the finished product and a low proportion of small-molecule peptides. CN107177651A discloses the application of flavor proteases for debittering and improving the degree of hydrolysis of aquatic proteins. However, this scheme is designed for aquatic proteins such as ribbonfish, and the protein matrix and enzymatic hydrolysis adaptation system are very different from those of corn gliadin, making it unsuitable for direct application in corn gluten powder processing.

[0004] Conventional single-enzymatic hydrolysis processes suffer from problems such as limited enzyme cleavage sites and uneven degradation. Simple compound enzymatic hydrolysis does not have a dedicated pretreatment and graded enzymatic hydrolysis scheme designed for the dense structure of zein, and generally suffers from problems such as low hydrolysis efficiency, wide molecular weight distribution of products, poor water solubility, and residual bitterness. As a result, the product quality is unstable and it is difficult to meet the requirements of high-end industrial applications. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of insufficient enzymatic hydrolysis of corn protein peptides, poor water solubility of the products, strong bitterness, and low proportion of small molecule peptides in the prior art.

[0006] Therefore, the present invention provides a method for preparing highly water-soluble small molecule peptides, comprising the following steps:

[0007] The protein powder is mixed with water until smooth, the pH is adjusted, and the temperature is raised for pretreatment.

[0008] Alkaline protease was added to the pretreated solution for primary enzymatic hydrolysis.

[0009] Neutral protease and flavor protease were added to the primary enzymatic hydrolysate for secondary enzymatic hydrolysis.

[0010] Centrifuge the secondary enzymatic hydrolysate and collect the supernatant;

[0011] The supernatant was purified by ultrafiltration membrane fractionation, and the permeate was collected, concentrated, and dried to obtain small molecule peptides.

[0012] Specifically, the above pretreatment is as follows: add water to the protein powder and stir evenly, adjust the pH to 7.5~8.5, heat to 55~65℃, keep warm and stir continuously for 20~40 minutes.

[0013] Specifically, the enzyme activity of the alkaline protease is ≥200,000 U / g; the amount of alkaline protease added is 0.2%~0.6% based on the weight of protein powder.

[0014] Specifically, the enzymatic hydrolysis temperature for the first-stage enzymatic hydrolysis is 55~60℃, the pH is 8.0~9.0, and the hydrolysis time is 2~3h.

[0015] Specifically, the activity of the neutral protease is ≥100,000 U / g, and the activity of the flavor protease is ≥80,000 U / g; based on the weight of protein powder, the amount of neutral protease added is 0.15%~0.4%, and the amount of flavor protease added is 0.1%~0.3%.

[0016] Specifically, the enzymatic hydrolysis temperature for the above-mentioned secondary enzymatic hydrolysis is 45~50℃, the pH is 6.5~7.5, and the hydrolysis time is 3~5h.

[0017] Specifically, the supernatant was purified by fractionation using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da.

[0018] Specifically, the permeate is concentrated under vacuum to a solid content of 25% to 35%, and then spray-dried with an inlet air temperature of 160 to 170°C and an outlet air temperature of 75 to 85°C.

[0019] The present invention also provides a highly water-soluble small molecule peptide, prepared by any of the preparation methods described above.

[0020] The present invention also provides the application of the above preparation method or highly water-soluble small molecule peptides in food and functional feed.

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

[0022] This invention provides a method for preparing highly water-soluble small molecule peptides that addresses the characteristics of alcohol-soluble proteins, such as their tightly packed spatial structure and strong hydrophobicity. It employs stepwise, directional enzymatic hydrolysis to achieve progressive unlocking and precise cleavage of the protein structure, significantly improving hydrolysis efficiency and product uniformity while avoiding over- or under-degradation. The process conditions are mild, precisely controlled, and highly stable, making it suitable for large-scale continuous production. The prepared protein peptides exhibit high degree of hydrolysis, excellent water solubility, a high proportion of small molecule peptides, light color, no bitter taste, and clear dissolution without precipitation, greatly expanding their application range. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0024] This invention provides a method for preparing highly water-soluble small molecule peptides, comprising the following steps:

[0025] (1) Pretreatment of slurry preparation and structural stretching

[0026] Mix protein powder and water at a ratio of 1:8 to 1:12 and stir until a uniform suspension without lumps or stratification is formed. Adjust the pH to 7.5 to 8.5 and heat to 55 to 65°C. Keep warm and stir continuously for 20 to 40 minutes for pretreatment to fully expand the spatial structure of zein and improve the accessibility of enzymatic hydrolysis.

[0027] (2) Primary enzymatic hydrolysis

[0028] Add alkaline protease with an enzyme activity ≥200,000 U / g to the pretreated system for primary enzymatic hydrolysis; the amount of alkaline protease added is 0.2%~0.6% based on the weight of protein powder; control the temperature of the enzymatic hydrolysis system to be stable at 55~60℃, continuously stir and maintain pH 8.0~9.0, and enzymatic hydrolysis for 2~3 hours; in this stage, large protein molecules are gradually broken down into polypeptide fragments, reducing the viscosity of the system and providing a basis for subsequent directional shearing.

[0029] (3) Secondary enzymatic hydrolysis

[0030] The temperature of the primary enzymatic hydrolysate was lowered to 45-50℃, the pH was adjusted to 6.5-7.5, and a neutral protease with an enzyme activity ≥100,000 U / g and a flavor protease with an enzyme activity ≥80,000 U / g were added for secondary enzymatic hydrolysis. The amount of neutral protease added was 0.15%-0.4% and the amount of flavor protease added was 0.1%-0.3% by weight of protein powder. The enzymatic hydrolysis was carried out at a constant temperature for 3-5 hours to achieve precise cleavage of peptide chains and significantly increase the proportion of small molecule peptides and water-soluble proteins.

[0031] (3) Enzyme inactivation and solid-liquid separation

[0032] After enzymatic hydrolysis, rapidly heat to 90-95℃ and incubate for 10 minutes to completely inactivate the enzyme. After cooling, centrifuge at 4000-6000 r / min for 10-15 minutes to remove insoluble impurities and residues, and collect the supernatant.

[0033] (4) Membrane separation purification and drying

[0034] The supernatant was fractionated and purified by ultrafiltration membrane with a molecular weight cutoff of 3000 Da; the permeate was collected and concentrated under vacuum to a solid content of 25%~35%; spray drying was performed with an inlet air temperature of 160~170℃ and an outlet air temperature of 75~85℃ to obtain the protein peptide product.

[0035] The preparation method and application effects of the highly water-soluble small molecule peptides of the present invention are studied through specific embodiments below. The alkaline protease used in the embodiments of the present invention has an enzyme activity of 200,000 U / g, the neutral protease has an enzyme activity of 100,000 U / g, and the flavor protease has an enzyme activity of 80,000 U / g; the spray drying inlet air temperature is 160~170℃, and the outlet air temperature is 75~85℃.

[0036] Example 1:

[0037] This embodiment provides a method for preparing highly water-soluble small molecule peptides, including the following steps:

[0038] Corn gluten powder and deionized water were mixed at a ratio of 1:8 and added to an enzymatic hydrolysis tank. The mixture was stirred thoroughly to form a uniform suspension. The pH of the system was adjusted to 7.5 using sodium hydroxide, and the temperature was raised to 55°C. The mixture was then stirred at this temperature for 20 minutes for pretreatment. 0.2% alkaline protease (by weight of gluten powder) was added to the system. The reaction temperature was controlled at 55°C and the pH at 8.0. The mixture was then hydrolyzed at this temperature for 2 hours. After hydrolysis, cooling water was introduced to lower the temperature to 45°C. The pH was adjusted to 6.5 using hydrochloric acid. 0.15% neutral protease and 0.1% flavor protease (by weight of gluten powder) were added. The mixture was then hydrolyzed at this temperature for 3 hours. After the reaction, the temperature was raised to 90°C and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 4000 rpm for 10 minutes to obtain the supernatant. The supernatant was purified by 3000 Da ultrafiltration membrane fractionation, concentrated under vacuum, and then spray-dried to obtain the final corn gluten peptide.

[0039] Example 2:

[0040] This embodiment provides a method for preparing highly water-soluble small molecule peptides, including the following steps:

[0041] Corn gluten powder and deionized water were mixed at a ratio of 1:10 and added to an enzymatic hydrolysis tank. The mixture was stirred thoroughly to form a homogeneous suspension. The pH of the system was adjusted to 8.0 using sodium hydroxide, and the temperature was raised to 60°C. The mixture was then stirred at this temperature for 30 minutes for pretreatment. 0.4% alkaline protease (by weight of gluten powder) was added to the system. The reaction temperature was controlled at 58°C and the pH at 8.5. The mixture was then hydrolyzed at this temperature for 2.5 hours. After hydrolysis, cooling water was introduced to lower the temperature to 48°C. The pH was adjusted to 7.0 using hydrochloric acid. 0.25% neutral protease and 0.2% flavor protease (by weight of gluten powder) were added. The mixture was then hydrolyzed at this temperature for 4 hours. After the reaction, the temperature was raised to 93°C and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 5000 rpm for 12 minutes to obtain the supernatant. The supernatant was purified by 3000 Da ultrafiltration membrane fractionation, concentrated under vacuum, and then spray-dried to obtain the final corn gluten peptide.

[0042] Example 3:

[0043] This embodiment provides a method for preparing highly water-soluble small molecule peptides, including the following steps:

[0044] Corn gluten powder and deionized water were mixed at a ratio of 1:12 and added to an enzymatic hydrolysis tank. The mixture was stirred thoroughly to form a homogeneous suspension. The pH of the system was adjusted to 8.5 using sodium hydroxide, and the temperature was raised to 65°C. The mixture was then stirred at this temperature for 40 minutes. Alkaline protease (0.6% of the protein powder mass) was added to the system. The reaction temperature was controlled at 60°C and the pH at 9.0. The mixture was then hydrolyzed at this temperature for 3 hours. After hydrolysis, cooling water was introduced to lower the temperature to 50°C. The pH was adjusted to 7.5 using hydrochloric acid. Neutral protease (0.4% of the protein powder mass) and flavor protease (0.3% of the protein powder mass) were added. The mixture was then hydrolyzed at this temperature for 5 hours. After the reaction, the temperature was raised to 95°C and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 6000 rpm for 15 minutes to obtain the supernatant. The supernatant was purified by 3000 Da ultrafiltration membrane fractionation, concentrated under vacuum, and then spray-dried to obtain the final corn gluten peptide.

[0045] Comparative Example 1:

[0046] This comparative example provides a method for preparing corn protein peptides, including the following steps:

[0047] Corn gluten powder and deionized water were mixed at a ratio of 1:10 and added to an enzymatic hydrolysis tank. The mixture was stirred thoroughly to form a homogeneous suspension. The pH of the system was adjusted to 8.0 using sodium hydroxide, and the temperature was raised to 60°C and pretreated with stirring at this temperature for 30 minutes. Alkaline protease (0.85% of the protein powder mass) was added to the system, and the reaction temperature was controlled at 58°C and the pH at 8.5. Enzymatic hydrolysis was carried out at this temperature for 6 hours. After the reaction, the temperature was raised to 93°C and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 5000 rpm for 12 minutes to obtain the supernatant. The supernatant was purified by 3000 Da ultrafiltration, concentrated under vacuum, and then spray-dried to obtain the final corn gluten peptide.

[0048] Comparative Example 2:

[0049] This comparative example provides a method for preparing corn protein peptides, including the following steps:

[0050] Corn gluten powder and deionized water were mixed at a ratio of 1:10 and added to an enzymatic hydrolysis tank. The mixture was stirred thoroughly to form a homogeneous suspension. 0.4% alkaline protease (by weight of gluten powder) was added to the system. The reaction temperature was controlled at 58℃ and the pH at 8.5. The hydrolysis was carried out at this constant temperature for 2.5 hours. After hydrolysis, cooling water was introduced to lower the temperature to 48℃. The pH was adjusted to 7.0 with hydrochloric acid. 0.25% neutral protease and 0.2% flavor protease (by weight of gluten powder) were added. The hydrolysis was continued at this constant temperature for 4 hours. After the reaction, the temperature was raised to 93℃ and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 5000 rpm for 12 minutes to obtain the supernatant. The supernatant was purified by 3000 Da ultrafiltration membrane fractionation, concentrated under vacuum, and then spray-dried to obtain the finished corn gluten peptide.

[0051] Comparative Example 3:

[0052] This comparative example provides a method for preparing corn protein peptides, including the following steps:

[0053] Corn gluten powder and deionized water were mixed at a ratio of 1:10 and added to an enzymatic hydrolysis tank. The mixture was stirred thoroughly to form a homogeneous suspension. The pH of the system was adjusted to 8.0 using sodium hydroxide, and the temperature was raised to 60°C. The mixture was then stirred at this temperature for 30 minutes. Alkaline protease (0.4% of the protein powder mass) was added to the system, and the reaction temperature was controlled at 58°C and the pH at 8.5. The mixture was then hydrolyzed at this temperature for 2.5 hours. After hydrolysis, cooling water was introduced to lower the temperature to 48°C. The pH was adjusted to 7.0 using hydrochloric acid, and neutral protease (0.45% of the protein powder mass) was added. The mixture was then hydrolyzed at this temperature for 4 hours. After the reaction, the temperature was raised to 93°C and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 5000 rpm for 12 minutes to obtain the supernatant. The supernatant was purified by 3000 Da ultrafiltration membrane fractionation, concentrated under vacuum, and then spray-dried to obtain the final corn gluten peptide.

[0054] Example 4:

[0055] This embodiment tests the indicators of the corn protein peptides prepared in Examples 1-3 and Comparative Examples 1-3. The water-soluble protein content was determined according to the method specified in NY / T 1205-2006; the degree of hydrolysis, peptide content, and molecular weight were determined according to the method specified in GB / T 22492-2008, with the degree of hydrolysis (%) = (free amino nitrogen after hydrolysis - free amino nitrogen from raw material) / total nitrogen × 100%; the moisture content was determined according to the method specified in GB 5009.3-2016. The results are shown in Table 1.

[0056] Table 1 Test Results

[0057]

[0058] Table 1 shows that Comparative Example 2, lacking a heating and relaxation pretreatment step, had a compact protein structure and insufficient exposure of enzymatic hydrolysis sites, resulting in significantly lower degrees of hydrolysis, small peptide content, and solubility compared to other examples. This indicates that pretreatment can effectively relax the dense structure of zein, significantly improving the cleavage efficiency of subsequent proteases. Comparative Example 1 used a traditional single alkaline protease for one-step enzymatic hydrolysis, resulting in a single cleavage site and insufficient degradation. The products were mostly long-chain peptides with the lowest yield of small peptides, and also had a strong bitter taste and dark color, proving that single enzymatic hydrolysis cannot achieve deep modification and flavor optimization. Comparative Example 3 lacked flavor protease participation in secondary enzymatic hydrolysis, relying solely on neutral protease to cleave peptide chains. The proportion of small peptides was low, and the product still had a slight bitter taste. This indicates that flavor protease can further finely cleave peptide ends and decompose bitter groups, which is key to achieving a bitter-free, high-purity small peptide product.

[0059] Compared to traditional single enzymatic hydrolysis, no pretreatment, and enzyme-deficient modification processes, the method provided by this invention adopts a step-by-step mode that combines structural expansion with primary coarse hydrolysis and secondary fine enzymatic hydrolysis. This method can significantly improve protein water solubility, degree of hydrolysis, and small molecule peptide yield while retaining the original crude protein nutrition, and at the same time improve the product color and flavor.

[0060] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing highly water-soluble small molecule peptides, characterized in that, Includes the following steps: The protein powder is mixed with water until smooth, the pH is adjusted, and the temperature is raised for pretreatment. Alkaline protease was added to the pretreated solution for primary enzymatic hydrolysis. Neutral protease and flavor protease were added to the primary enzymatic hydrolysate for secondary enzymatic hydrolysis. Centrifuge the secondary enzymatic hydrolysate and collect the supernatant; The supernatant was purified by ultrafiltration membrane fractionation, the permeate was collected, concentrated and dried to obtain small molecule peptides.

2. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that, The pretreatment specifically involves: adding water to the protein powder and stirring until homogeneous, adjusting the pH to 7.5-8.5, heating to 55-65°C, maintaining the temperature and stirring continuously for 20-40 minutes.

3. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that: The alkaline protease has an enzyme activity ≥200,000 U / g; the amount of alkaline protease added is 0.2%~0.6% based on the weight of the protein powder.

4. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that: The first-stage enzymatic hydrolysis is performed at a temperature of 55-60℃, a pH of 8.0-9.0, and a hydrolysis time of 2-3 hours.

5. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that: The neutral protease has an enzyme activity of ≥100,000 U / g, and the flavor protease has an enzyme activity of ≥80,000 U / g; based on the weight of protein powder, the amount of neutral protease added is 0.15%~0.4%, and the amount of flavor protease added is 0.1%~0.3%.

6. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that: The secondary enzymatic hydrolysis is performed at a temperature of 45-50℃, a pH of 6.5-7.5, and a hydrolysis time of 3-5 hours.

7. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that: The supernatant was purified by fractionation using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da.

8. The method for preparing highly water-soluble small molecule peptides as described in claim 1, characterized in that: The permeate is concentrated under vacuum to a solid content of 25% to 35%, and then spray-dried with an inlet air temperature of 160 to 170°C and an outlet air temperature of 75 to 85°C.

9. A highly water-soluble small molecule peptide, characterized in that, Prepared using the preparation method described in any one of claims 1-8.

10. The preparation method according to any one of claims 1-8 or the application of the highly water-soluble small molecule peptide according to claim 9 in food and functional feed.

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