Albumin peptide for promoting growth and development as well as preparation method and application thereof

Abstract

The invention provides an albumin peptide for promoting growth and development as well as a preparation method and application thereof, and belongs to the technical field of food science. According to the albumin peptide provided by the invention, denatured egg white is taken as a substrate, firstly, chymotrypsin is used for prehydrolysis, after prehydrolysis is completed, the temperature and pH are adjusted, and then alkaline protease and chymotrypsin are added for synergistic hydrolysis. The prepared albumin peptide is small in molecular weight and easy to digest and absorb, and has the effects of promoting growth and development, improving macrophage proliferation capacity, improving immunity and the like.

Description

Technical Field

[0001] The invention belongs to the field of food science, and specifically relates to an albumin peptide for promoting growth and development, and a preparation method and application thereof. Background Art

[0002] Egg white protein accounts for about 10%-12% of the egg white mass, and is mainly composed of ovalbumin, ovotransferrin, ovomucoid, lysozyme, globulin, etc. Egg white protein contains a variety of amino acids necessary for the human body, and the composition ratio is appropriate, which is very close to the needs of the human body. It is an ideal protein resource. However, egg white protein has disadvantages such as high viscosity, poor thermal stability, easy foaming and allergenicity. In addition, the preparation process of egg white protein powder is also prone to cause some functional properties to decline, which limits its application in food processing. Studies have found that enzymatic hydrolysis can not only reduce the relative molecular mass of egg white protein and reduce allergenicity, but also the albumin peptides generated by enzymatic hydrolysis are easier for human digestion and absorption. In addition, the functional properties of the enzymatic hydrolysis products such as solubility, viscosity and foaming have been improved compared with the original protein, and they have physiological functions such as lowering blood pressure, anti-oxidation, anti-coagulation, antibacterial and immunomodulation, which greatly improves the application range of egg white protein in the food industry.

[0003] Studies have confirmed that albumin peptides, as functional active peptides, have good biological properties. Their biological activities are mainly manifested in the following aspects: 1. Easy to digest and absorb, promoting the functional recovery of immune tissues; 2. Adjusting intestinal flora and improving food conversion rate; 3. Promoting the absorption of calcium, iron, nucleic acid and other substances; 4. Improving hypoproteinemia. Tian Gang et al. [1] The study found that the five oligopeptide mixtures obtained by enzymatic hydrolysis of egg white with flavor protease, alkaline protease, pepsin, trypsin and papain can significantly or extremely significantly improve the phagocytic ability of macrophages, promote the proliferation of lymphocytes, and have the characteristics of enhancing immunity. Jin Lei et al. [2] The antioxidant activity of egg white protein hydrolysate was studied by chemiluminescence. The results showed that egg white protein hydrolysate with different concentrations and hydrolysis degrees had the ability to scavenge reactive oxygen species and resist lipid oxidation. As the concentration of egg white protein hydrolysate increased, the scavenging ability increased and the antioxidant activity also increased.

[0004] At present, more and more research is being done on albumin, and people are constantly looking for more economical and practical methods to develop albumin products. Studies have shown that further hydrolysis of albumin to obtain peptide products is more conducive to absorption and will play a greater role in human health.

[0005] [1] Tian Gang, Chen Daiwen, Yu Bing, et al. Effects of enzymatic hydrolyzed egg white peptide mixture on immune function of mice [J]. Chinese Animal Husbandry Magazine, 2005, (05): 14-17.

[0006] [2] Jin Lei, Wang Jing, Li Xinhua. Preparation of antioxidant peptides from egg white by enzymatic hydrolysis with Alcalase alkaline protease [J]. Food Research and Development, 2009, 30(06): 59-62. Summary of the invention

[0007] In order to solve the above problems, the present invention provides an albumin peptide for promoting growth and development, and a preparation method and application thereof.

[0008] In one aspect, the present invention provides an albumin peptide for promoting growth and development.

[0009] Specifically, the albumin peptide is prepared by using egg white as a substrate through the coordinated hydrolysis of two enzymes, wherein the two enzymes are chymosin and alkaline protease.

[0010] More specifically, the egg white is denatured egg white.

[0011] More specifically, the molecular weight of the albumin peptide is no greater than 1 kDa.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned albumin peptide.

[0013] Specifically, the preparation method comprises the following steps:

[0014] (1) Preparation of hydrolysis raw materials;

[0015] (2) chymotrypsin hydrolysis;

[0016] (3) synergistic hydrolysis by chymosin and alkaline protease;

[0017] (4) Separation and purification of polypeptides.

[0018] More specifically, in step (1), the preparation step of the hydrolysis raw material is: egg white and sterile distilled water are mixed in a volume ratio of 1-3:3-9, and then heated and denatured at 85°C-100°C for 15-25 minutes to obtain the hydrolysis raw material.

[0019] In certain specific embodiments of the present invention, the egg white is chicken egg white.

[0020] Preferably, the pH range of the hydrolyzed raw material is 7.5-8.5.

[0021] More specifically, in step (2), the amount of chymosin added is 0.8-2.5 wt %.

[0022] More specifically, in step (2), the conditions for chymosin hydrolysis are: hydrolysis at 35-45° C. for 1.5-2.5 h.

[0023] More specifically, in step (3), the amount of alkaline protease added is 0.8-2.5 wt %.

[0024] More specifically, in step (3), the conditions for the dual enzyme synergistic hydrolysis are: 45-55°C, and hydrolysis for 3-5h.

[0025] More specifically, after step (3), the method further includes heating the product of the double-enzyme synergistic hydrolysis to terminate the enzymatic hydrolysis reaction.

[0026] More specifically, in step (4), the polypeptide separation and purification step is: taking the supernatant after centrifugation and passing it through an ultrafiltration membrane.

[0027] Preferably, the centrifugal conditions are: 3000-5000 rpm, centrifugation for 15-30 minutes.

[0028] Preferably, the molecular weight cut-off of the ultrafiltration membrane is 1 kDa.

[0029] In yet another aspect, the present invention provides the use of the above-mentioned albumin peptide in the preparation of a product for promoting growth and development.

[0030] Specifically, the products include: medicines, foods or food additives.

[0031] More specifically, when the product is a medicine, it also includes a pharmaceutically acceptable excipient.

[0032] More specifically, the food includes but is not limited to: health products or drinks.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] The albumin peptide prepared in the present application has the effects of promoting growth and development and increasing immunity, and has a small molecular weight and is easier to digest and absorb. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the zebrafish body length detection result in Experimental Example 2. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0037] Table 1

[0038] Material Where to buy Part Number Chymotrypsin Sigma C3142 Alkaline protease Sigma P4860 Trypsin Sigma T1426

[0039] Example 1

[0040] 1. Preparation of hydrolysis raw materials:

[0041] Wash fresh eggs, separate the egg white, mix the egg white with sterile distilled water in a volume ratio of 1:3, heat and denature at 95°C for 15 minutes in an autoclave, cool to room temperature, add phosphate buffer, adjust the egg white protein concentration to 5%, and the pH to 8.0 to obtain a hydrolyzed raw material.

[0042] 2. Chymotrypsin hydrolysis:

[0043] Chymotrypsin was added to the ovalbumin solution so that the mass ratio of enzyme to substrate was 1:50.

[0044] Pre-hydrolysis was performed at 40°C for 2 hours with shaking at 150 rpm on a shaker.

[0045] 3. Double enzyme synergistic hydrolysis:

[0046] After the hydrolysis in step 2 is completed, a 1% NaOH solution is added to adjust the pH to 8.5, and alkaline protease is added. The mass ratio of the added alkaline protease to the chymosin added in step 2 is 1:1.

[0047] The hydrolysis was carried out at 50°C for 4 hours, with shaking at 150 rpm. After the reaction, the mixture was heated to 90°C for 10 minutes to terminate the enzymatic reaction.

[0048] 4. Separation and purification of polypeptides:

[0049] Centrifuge at 4000 r / min for 15 minutes, take the supernatant, and use an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to separate and purify the polypeptide.

[0050] 5. Freeze drying:

[0051] The purified polypeptide solution is freeze-dried to obtain polypeptide powder.

[0052] Example 2

[0053] The difference from Example 1 is that in step 2, the mass ratio of chymosin to substrate is 1:100, and in step 3, the mass ratio of the added alkaline protease to the chymosin added in step 2 is 3:1.

[0054] Example 3

[0055] The difference from Example 1 is that in step 2, the mass ratio of chymosin to substrate is 1.5:100, and in step 3, the mass ratio of the added alkaline protease to the chymosin added in step 2 is 1:3.

[0056] Comparative Example 1

[0057] The difference from Example 1 is that in step 2, chymosin is replaced by alkaline protease of equal mass.

[0058] Comparative Example 2

[0059] The difference from Example 1 is that in step 3, the alkaline protease is replaced by an equal mass of chymosin.

[0060] Comparative Example 3

[0061] The difference from Example 1 is that in step 2, chymosin is replaced by an equal amount of trypsin.

[0062] The difference from Example 1 is that after step 2, the mixture is heated to 90° C. for 10 minutes to terminate the enzymatic reaction, and alkaline protease is not added in step 3.

[0063] Experimental Example 1

[0064] Wild-type AB strain zebrafish 3 days after fertilization were randomly selected in beakers, with 30 zebrafish in each beaker, and divided into six groups, of which five groups were experimental groups, and the albumin peptide powder prepared in the embodiments of the present invention or the comparative examples was given respectively, and the concentrations were shown in Table 2; the control group did not receive any treatment. After treatment at 28°C for 3 days after fertilization, it was fixed and stained with alizarin red. After staining, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photography. The data were analyzed and collected using NIS-ElementsD3.20 advanced image processing software, and the fluorescence intensity of the zebrafish skull was analyzed. The statistical analysis results of this indicator were used to evaluate the growth and development promoting effect of the albumin peptides prepared by each preparation method. The statistical processing results were expressed as mean±SE, and statistical analysis was performed using SPSS26.0 software. p<0.05 indicated that the difference was statistically significant. The experimental results are shown in Table 2.

[0065] Table 2

[0066] Group Concentration (μg / mL) Skull fluorescence intensity (pixels, mean ± SE) Control group - 1142623±21125 Example 1 250 1342568±51332** Example 2 250 1326456±42156** Example 3 250 1334562±43214** Comparative Example 1 250 1192349±23512 Comparative Example 2 250 1183544±32154 Comparative Example 3 250 1161358±42368 Comparative Example 4 250 1148953±38749

[0067] Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0068] The data in Table 2 illustrate that the albumin peptide prepared by the present invention has the function of promoting the skeletal development of zebrafish.

[0069] Experimental Example 2

[0070] 3dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish in each beaker. The albumin peptide powder prepared in the embodiments of the present invention or the comparative examples was administered in water, and the control group did not receive any treatment. The capacity of each beaker was 20mL. The liquid was changed every day. After treatment at 28°C for 3 days, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. NIS-ElementsD3.20 advanced image processing software was used to analyze and collect data, and the body length of the zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the effectiveness of the samples in promoting growth and development. The statistical processing results are expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant. The experimental results are shown in Figure 1 and Table 3.

[0071] Table 3

[0072]

[0073] Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0074] The data in Table 3 illustrate that the albumin peptide prepared by the present invention has the function of promoting the growth of zebrafish.

[0075] Experimental Example 3

[0076] Experimental animals: 4-week-old weaned SD rats were used.

[0077] The experimental groups are shown in Table 4, with 8 rats in each group. The rats in the experimental group were gavaged with the test sample once a day, and the rats in the control group were gavaged with equal-mass saline. The test sample was administered for 42 days.

[0078] Table 4

[0079]

[0080]

[0081] During the administration period, the rats' food intake was recorded every day, their body weight was tested twice a week, and their body weight changes were observed and recorded. The statistical analysis results were expressed as mean and standard deviation (Mean±SD). SPSS25.0 was used for statistical analysis. There was a significant difference between the groups when P<0.05, and there was no significant statistical difference when P>0.05.

[0082] The food intake of rats is shown in Table 5:

[0083] Table 5

[0084]

[0085] Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0086] The changes in rat body weight are shown in Table 6:

[0087] Table 6

[0088] Group Body weight change (g, mean ± SE) Control group 282.19±21.23 Experimental Group 1 312.73±22.15* Experimental Group 2 309.92±26.46* Experimental Group 3 310.32±25.71* Experimental Group 4 293.31±15.28 Experimental Group 5 286.48±25.78 Experimental Group 6 294.34±29.62 Experimental Group 7 279.91±20.98

[0089] Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0090] From the above experimental results, it can be seen that the height of experimental group 1-3 is higher than that of normal control group and experimental group 4-5, and the difference is significant. At the same time, the food utilization rate is not significantly lower than that of normal control group. It can be judged that the test sample has positive results in improving growth and development animal experiments.

[0091] Experimental Example 4

[0092] SD rats were intraperitoneally injected with 2 mL of 2% (w / w) sterilized starch solution for three consecutive days. The rats were killed by cervical dislocation 24 hours after the last injection. The abdominal skin was peeled off, and the abdominal cavity was repeatedly rinsed with 4°C phosphate buffered saline (PBS) using a syringe. The rinsing fluid was collected in a centrifuge tube, centrifuged (1000 rpm, 4°C) for 10 minutes, and the supernatant was discarded. The rats were washed twice with 4°C RPMI 1640 complete culture medium (containing 10% FBS), and stained with 0.2% trypan blue solution for cell viability detection to confirm that the collected active macrophages accounted for more than 95%. After the cell counting plate was read, the cell concentration was adjusted to 2×10 5 Pieces / mL.

[0093] 100 μL of cell suspension was added to the wells. After adhesion purification, 200 μL / well of RPMI 1640 complete culture medium (10% FBS) containing the polypeptide sample was added. A negative control group was set up, which was RPMI 1640 complete culture medium (10% FBS) with 500 μg / mL BSA.

[0094] The blank group was cultured for 48 hours, and LPS was added to the inflammation group after 24 hours of culture to a final concentration of 100 ng / mL. After 44 hours of culture, 20 μL / well of 5% MTT was added. After 48 hours of culture, 100 μL / well of the triple dissolution solution was added to terminate the culture. After overnight dissolution, the absorbance value (OD 570) of each well was measured at a wavelength of 570 nm using an ELISA reader. The growth index (GrowthIndices) was calculated as follows:

[0095]

[0096] The experimental results are shown in Table 7.

[0097] Table 7

[0098] Group Normal group GI Inflammation Group GI Blank group 1.024±0.012 1.058±0.032 Experimental Group 1 1.187±0.350** 1.070±0.342 Experimental Group 2 1.179±0.211** 1.065±0.423 Experimental Group 3 1.181±0.382** 1.068±0.356 Experimental Group 4 1.025±0.025 1.059±0.032 Experimental Group 5 1.020±0.045 1.055±0.028 Experimental Group 6 1.027±0.063 1.058±0.048 Experimental Group 7 1.025±0.035 1.060±0.051

[0099] Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0100] It can be seen from the experimental results in Table 7 that experimental groups 1-3 have the ability to promote macrophage proliferation and can effectively improve the body's nonspecific immune ability.

[0101] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An albumin peptide for promoting growth and development, characterized in that: The albumin peptide is prepared by using egg white as substrate through double enzyme synergistic hydrolysis, wherein the double enzymes are chymosin and alkaline protease.

2. The albumin peptide according to claim 1, characterized in that The molecular weight of the albumin peptide is no greater than 1 kDa.

3. The method for preparing the albumin peptide according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) preparing a hydrolysis raw material; (2) chymotrypsin hydrolysis; (3) synergistic hydrolysis by chymosin and alkaline protease; (4) Separation and purification of polypeptides.

4. The preparation method according to claim 3, characterized in that: In step (1), the step of preparing the hydrolysis raw material is: egg white and sterilized distilled water are mixed in a volume ratio of 1-3:3-9, and then heated and denatured at 85°C-100°C for 15-25 minutes to obtain the hydrolysis raw material, and the pH range of the hydrolysis raw material is 7.5-8.

5.

5. The preparation method according to claim 3, characterized in that: In step (2), the amount of chymosin added is 0.8-2.5 wt %, and the conditions for chymosin hydrolysis are: hydrolysis at 35-45° C. for 1.5-2.5 h.

6. The preparation method according to claim 3, characterized in that: In step (3), the amount of alkaline protease added is 0.8-2.5 wt %.

7. The preparation method according to claim 3, characterized in that: In step (3), the conditions for the dual enzyme synergistic hydrolysis are: 45-55°C, hydrolysis for 3-5h.

8. The preparation method according to claim 3, characterized in that: In step (4), the polypeptide separation and purification step is as follows: after centrifugation, the supernatant is taken and passed through an ultrafiltration membrane; the centrifugation conditions are: 3000-5000rpm, centrifugation for 15-30 minutes.

9. The preparation method according to claim 8, characterized in that: The molecular weight cut-off of the ultrafiltration membrane is 1 kDa.

10. Use of the albumin peptide according to any one of claims 1 to 2 and / or the albumin peptide prepared by the preparation method according to any one of claims 3 to 9 in the preparation of a product that promotes growth and development, the product comprising: Medicine or food; preferably, the food includes: ordinary food, functional food or health care product.

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