Micromolecular albumin peptide capable of promoting growth and development as well as preparation method and application of micromolecular albumin peptide

Abstract

The invention provides a micromolecular albumin peptide capable of promoting growth and development as well as a preparation method and application thereof, and belongs to the technical field of biology. According to the invention, bromelain, alkaline protease and flavourzyme are creatively adopted to carry out step-by-step mixed enzymolysis on egg white. Through scientific compounding and synergistic effects of various enzymes, the hydrolysis efficiency of the micromolecule albumin peptide is greatly improved, the taste of the micromolecule albumin peptide is improved, compared with similar products on the market, the micromolecule albumin peptide has the flavor advantage, and the problems of low yield, obvious bitter taste and the like in a traditional process are effectively solved. The average relative molecular weight of the small molecule peptide prepared by the process is stabilized in a range of 200-1000Da, the property is more stable, and the small molecule peptide has the characteristic of being extremely easily absorbed by a human body. Experimental results prove that the micromolecular protein peptide also has a remarkable effect of promoting growth and development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a small molecule albumin peptide capable of promoting growth and development, and a preparation method and application thereof. Background Art

[0002] Egg white protein is a complete protein with an extremely high protein content and an amino acid profile that closely matches human needs, containing all eight essential amino acids, making it an ideal protein source. Traditionally consumed, egg white requires gastrointestinal enzymatic breakdown into amino acids for absorption. However, digestion efficiency varies among individuals, and some people (such as those with chronic diseases and those with weak digestive systems) have difficulty fully breaking down large protein molecules, resulting in reduced nutrient utilization and even increased metabolic burden.

[0003] The preparation of small-molecule protein peptides is an important method for deep processing of egg white protein. This method involves hydrolyzing egg white protein into small-molecule protein peptides through targeted enzymatic hydrolysis. These small-molecule protein peptides are characterized by their small molecular weight and simple structure. Compared with large-molecule proteins, small-molecule protein peptides are absorbed faster and more efficiently by the human body, providing rapid nutritional supplementation. They are particularly suitable for people with weak digestive functions and can be used as nutritional reagents.

[0004] Calcium and Vitamin D are common nutritional supplements for growth and development. However, traditional nutritional supplements typically focus on providing a single nutrient and have relatively limited functionality. Some of these supplements also have limitations in absorption. For example, calcium absorption is easily affected by the gastrointestinal environment and other food components. When the human gastrointestinal tract is not functioning well, its absorption efficiency is significantly reduced. Furthermore, some supplements may contain numerous additives to ensure stability and shelf life. Long-term, high-dose use may potentially impact human health.

[0005] Therefore, small molecule protein peptides extracted from albumin have attracted widespread attention from more and more consumers due to their safety and effectiveness. However, the small molecule protein peptides currently extracted from albumin have problems such as low yield and severe bitterness. Therefore, it is crucial to develop small molecule protein peptide products with better taste and efficacy. Summary of the Invention

[0006] The purpose of the present invention is to provide a small molecule albumin peptide that can promote growth and development, as well as its preparation method and application. The small molecule albumin peptide has an average relative molecular weight of 200-1000Da, is stable in nature, is easily absorbed by the human body, and has the effect of promoting growth and development.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a small molecule albumin peptide capable of promoting growth and development, comprising the following steps:

[0009] (1) dissolving egg white powder and adjusting the pH to obtain an egg white powder extract;

[0010] (2) denaturing the egg white powder in the egg white powder extract of step (1), and then adding protease to perform a first hydrolysis;

[0011] (3) After the first hydrolysis, a composite protease is added for a second hydrolysis, and after the hydrolysis is completed, the protease is inactivated to obtain a hydrolyzate;

[0012] (4) Separating, concentrating, desalting, sterilizing, and drying the unhydrolyzed protein in the hydrolyzate to obtain small molecule albumin peptides.

[0013] Preferably, in step (1), the mass ratio of the egg white powder to water when the egg white powder is dissolved is 1:15-25; and the pH is 9-10.

[0014] Preferably, in step (2), the denaturation conditions are: pressure 0-0.5 MPa, temperature 110° C.-120° C., time 20 min-30 min;

[0015] Preferably, in step (2), the protease comprises bromelain and alkaline protease; the added amount of the bromelain is 0.5%-0.7% by weight of the dry matter of the egg white powder; and the added amount of the alkaline protease is 0.3%-0.5% by weight of the dry matter of the egg white powder.

[0016] Preferably, in step (2), the conditions for the first hydrolysis are: temperature 50° C. to 54° C., time 1.5 to 2.5 h.

[0017] Preferably, in step (3), the compound protease comprises Flavourzyme, and the added amount of the compound protease is 0.1%-0.15% of the dry matter weight of the egg white powder.

[0018] Preferably, in step (3), the conditions for the second hydrolysis are: temperature 50° C. to 54° C., time 1.5 to 2.5 h.

[0019] The present invention also provides a small molecule albumin peptide capable of promoting growth and development, which is prepared by the method for preparing the small molecule albumin peptide capable of promoting growth and development.

[0020] The present invention also provides a use of the small molecule albumin peptide capable of promoting growth and development in the preparation of a product promoting growth and development.

[0021] The beneficial effects of the present invention compared with the prior art are:

[0022] The present invention creatively uses bromelain, alkaline protease and flavor protease to carry out step-by-step mixed enzymolysis on egg white. Through the scientific compounding and synergistic effect of multiple enzymes, not only the hydrolysis efficiency of small molecule albumin peptides is greatly improved, but also the taste of small molecule albumin peptides is improved. Compared with similar products on the market, it has more flavor advantages and effectively solves the problems of low yield and obvious bitterness in traditional processes. The small molecule peptides obtained by the process of the present invention have an average relative molecular weight that is stable in the range of 200-1000Da, are more stable in nature, and have the characteristics of being easily absorbed by the human body. Experimental results confirm that the small molecule protein peptide also has a significant effect of promoting growth and development. Moreover, after scaled-up production, the product performance is stable, the yield is increased to 60%, and the production cost is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is a typical graph of the fluorescence intensity of zebrafish skulls in each group in Experimental Example 1 of the present invention. The yellow dotted box is the analysis area. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The present invention provides a method for preparing a small molecule albumin peptide capable of promoting growth and development, comprising the following steps:

[0031] (1) dissolving egg white powder and adjusting the pH to obtain an egg white powder extract;

[0032] (2) denaturing the egg white powder in the egg white powder extract of step (1), and then adding protease to perform a first hydrolysis;

[0033] (3) After the first hydrolysis, a composite protease is added for a second hydrolysis, and after the hydrolysis is completed, the protease is inactivated to obtain a hydrolyzate;

[0034] (4) Separating, concentrating, desalting, sterilizing, and drying the unhydrolyzed protein in the hydrolyzate to obtain small molecule albumin peptides.

[0035] Preferably, in step (1), the mass ratio of the egg white powder to water when the egg white powder is dissolved is 1:15-25; the pH is 9-10, more preferably 1:20.

[0036] Preferably, in step (2), the denaturation conditions are: pressure 0-0.5 MPa, more preferably 0-0.2 MPa; temperature 110° C.-120° C., time 20 min-30 min;

[0037] Preferably, in step (2), the protease comprises bromelain and alkaline protease; the added amount of the bromelain is 0.5%-0.7% by weight of the dry matter of the egg white powder; and the added amount of the alkaline protease is 0.3%-0.5% by weight of the dry matter of the egg white powder.

[0038] Preferably, in step (2), the conditions for the first hydrolysis are: temperature 50°C to 54°C, time 1.5 to 2.5 hours, more preferably 2 hours.

[0039] Preferably, in step (3), the compound protease comprises Flavourzyme, and the added amount of the compound protease is 0.1%-0.15% of the dry matter weight of the egg white powder.

[0040] Preferably, in step (3), the conditions for the second hydrolysis are: temperature 50°C to 54°C, time 1.5 to 2.5 hours, more preferably 2 hours.

[0041] Preferably, in step (4), the step of separating the unhydrolyzed protein in the hydrolyzate comprises: adjusting the pH of the hydrolyzate obtained in step (3) to 4-4.5 to precipitate the unhydrolyzed protein, adding biochar to adsorb the unhydrolyzed protein, and separating the biochar and the fines in the hydrolyzate.

[0042] Preferably, in step (4), the sterilization step comprises: filtering and sterilizing with a 0.1 micron filter membrane at a temperature of 40°C to 45°C.

[0043] Example 1

[0044] Example 1 of the present invention provides a method for preparing a small molecule albumin peptide that promotes growth and development, and the specific steps are as follows:

[0045] (1) Take egg white powder and completely dissolve it with pure water according to the material-liquid mass ratio of egg white powder to water = 1:20. After complete dissolution, use food-grade sodium carbonate to adjust the pH to 9.5 to obtain egg white powder extract.

[0046] (2) denaturing the egg white powder extract obtained in step (1) using a high-temperature and high-pressure extraction device at a pressure of 0.2 MPa, a temperature of 110° C., and a time of 30 min;

[0047] (3) adding 0.5% bromelain (enzyme activity 1.2 million U / g) based on the dry matter weight of the egg white powder and 0.3% alkaline protease (enzyme activity 200,000 U / g) based on the dry matter weight of the egg white powder to the egg white powder extract after denaturation treatment in step (2), hydrolyzing at 54°C for 2 hours, and then adding 0.1% flavorzyme (enzyme activity 100,000 U / g) based on the dry matter weight of the egg white powder to the obtained enzymatic solution, hydrolyzing at 50°C for 2 hours, and then inactivating the enzyme at 85°C for 15 minutes;

[0048] (4) Protein precipitation treatment: The hydrolyzate obtained in step (3) is adjusted to pH 4.5 at room temperature using food-grade glacial acetic acid, and the unhydrolyzed protein is precipitated by adjusting to the isoelectric point of the material. Then, food-grade wood activated carbon with a solid content of 3%-5% is added, and the temperature is 60°C for adsorption for 1 hour. After the adsorption is completed, the material liquid is deslagging through a disc separator, and the suspended fragments, part of the activated carbon and the liquid in the enzymatic hydrolyzate are completely separated to obtain a deslagging liquid; the deslagging liquid is ultrafiltered by a multifunctional inorganic ceramic membrane separation device to efficiently remove the activated carbon, and the filtrate is filtered into a clear liquid storage tank.

[0049] (5) Nanofiltration concentration and desalination: After the filtrate is concentrated by multiple nanofiltration membranes, the concentrate is diluted with water multiple times for desalination;

[0050] (6) Filtration and sterilization: To ensure the activity of small molecule peptides in the liquid, filtration and sterilization were performed at 45°C using a 0.1 μm filter membrane. The sterile liquid was stored in a sterile tank, and the pipes were steam sterilized before filtration.

[0051] (7) High-speed centrifugal spray drying: The sterile liquid is powdered and dried by a high-speed centrifugal spray drying granulator to obtain small molecule albumin peptides that promote growth and development.

[0052] The small molecule protein peptide obtained by the present invention has an average relative molecular weight of 200 to 1000 Da, is stable in nature, is easily absorbed by the human body, and has the effect of promoting growth and development.

[0053] Test Example 1

[0054] In this aspect, Experimental Example 1 tested the effect of the small molecule albumin peptide prepared in Example 1 on promoting growth and development. The specific steps are as follows:

[0055] (1) Group processing:

[0056] The zebrafish were divided into three groups: normal control group (no treatment), small molecule albumin peptide group, and positive control group, with 30 zebrafish in each group.

[0057] (2) MTC determination

[0058] Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in beakers, with 30 zebrafish per beaker. Water-soluble albumin peptide powder (concentrations shown in Table 1) was administered, along with a normal control group. Each beaker held 20 mL of water. The medium was changed daily. After treatment at 28°C until 6 dpf, the MTC of the samples in normal zebrafish was measured. The results are shown in Table 1.

[0059] Table 1 Results of small molecule albumin peptides promoting growth and development

[0060]

[0061] Zebrafish in the 500 and 1000 μg / mL groups of small molecule protein peptide showed intolerance, proving that the tolerance concentration of zebrafish to this product is 250 μg / mL, but it is not directly related to the safety of human consumption.

[0062] (3) Evaluation of growth and development promotion efficacy (bone development)

[0063] 3dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish in each beaker. The small molecule albumin peptide group was given water-soluble albumin peptide powder (concentrations are shown in Table 2), and the positive control was Swisse "Calcium & Vitamin D" at a concentration of 250μg / mL. A normal control group was also set up, and the capacity of each beaker was 20mL. The medium was changed every day. After treatment at 28℃ until 6dpf, it was fixed and stained with phycocyanin. After staining, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. NIS-ElementsD3.20 advanced image processing software was used to analyze and collect data, and the fluorescence intensity of the zebrafish skull was analyzed. The statistical analysis results of this indicator were used to evaluate the effectiveness of the sample in promoting growth and development. The results are shown in the figure. Figure 1 The results are shown in Table 2. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated a statistically significant difference.

[0064] Table 2 Results of the experimental evaluation of the efficacy of small molecule albumin peptides in promoting growth and development (bone development) (n=10)

[0065]

[0066] Note: Compared with the normal group, **p<0.01, ***p<0.001.

[0067] Depend on Figure 1 As shown in Table 2, the small molecule albumin peptide prepared in Example 1 of the present invention has the function of promoting bone development.

[0068] (4) Evaluation of growth and development promotion efficacy (body length)

[0069] Wild-type AB zebrafish (3 dp1) were randomly selected and placed in beakers, with 30 zebrafish per beaker. Water-soluble albumin peptide powder (concentrations shown in Table 3) was administered, along with a positive control of Swisse "Calcium & Vitamin D" at a concentration of 250 μg / mL. A normal control group was also established, with each beaker containing 20 ml of water. The medium was changed daily. After three days of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. Zebrafish body length was analyzed, and the growth-promoting efficacy of the samples was evaluated using statistical analysis of this indicator. The results are shown in Table 3. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated statistical significance.

[0070] Table 3 Results of the experimental evaluation of the growth-promoting effect of small molecule albumin peptides (body length) (n=10)

[0071]

[0072]

[0073] Note: Compared with the normal group, *p<0.05, **p<0.01.

[0074] As shown in Table 3, the small molecule albumin peptide prepared in Example 1 of the present invention has the function of promoting the growth of zebrafish.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a small molecule albumin peptide that can promote growth and development, characterized in that: The steps include: (1) Dissolve the egg white powder and adjust the pH to obtain an egg white powder extract; (2) denaturing the egg white powder in the egg white powder extract of step (1), and then adding protease to perform a first hydrolysis; (3) After the first hydrolysis, a composite protease is added for a second hydrolysis, and the protease is inactivated after the hydrolysis is completed to obtain a hydrolyzate; (4) The unhydrolyzed protein in the hydrolyzate is separated, concentrated, desalted, sterilized, and dried to obtain small molecule albumin peptides.

2. The method for preparing the small molecule albumin peptide capable of promoting growth and development according to claim 1, characterized in that: In step (1), when the egg white powder is dissolved, the mass ratio of the egg white powder to water is 1:15-25; and the pH is 9-10.

3. The method for preparing the small molecule albumin peptide capable of promoting growth and development according to claim 1, characterized in that: In step (2), the denaturation conditions are: pressure 0-0.5 MPa, temperature 110°C-120°C, and time 20 min-30 min.

4. The method for preparing the small molecule albumin peptide capable of promoting growth and development according to claim 1, characterized in that: In step (2), the protease includes bromelain and alkaline protease; the added amount of bromelain is 0.5%-0.7% of the weight of the dry matter of the egg white powder; the added amount of alkaline protease is 0.3%-0.5% of the weight of the dry matter of the egg white powder.

5. The method for preparing the small molecule albumin peptide capable of promoting growth and development according to claim 1, characterized in that: In step (2), the conditions for the first hydrolysis are: temperature 50°C to 54°C, time 1.5 to 2.5 hours.

6. The method for preparing the small molecule albumin peptide capable of promoting growth and development according to claim 1, characterized in that: In step (3), the compound protease includes Flavourzyme, and the added amount of the compound protease is 0.1%-0.15% of the dry matter weight of the egg white powder.

7. The method for preparing the small molecule albumin peptide capable of promoting growth and development according to claim 1, characterized in that: In step (3), the conditions for the second hydrolysis are: temperature 50°C~54°C, time 1.5~2.5h.

8. A small molecule albumin peptide capable of promoting growth and development, prepared by the method for preparing a small molecule albumin peptide capable of promoting growth and development according to any one of claims 1 to 7.

9. Use of the small molecule albumin peptide capable of promoting growth and development according to claim 8 in the preparation of a product promoting growth and development.

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