An albumin peptide for promoting growth and development, its preparation method and application

Abstract

The present invention provides an albumin peptide for promoting growth and development, its preparation method and applications, belonging to the field of food science and technology. The albumin peptide provided by the present invention uses denatured egg white as a substrate. First, it is pre-hydrolyzed with chymotrypsin. After the pre-hydrolysis is completed, the temperature and pH are adjusted, and then alkaline protease and chymotrypsin are added for synergistic hydrolysis. The albumin peptide prepared by the present invention has a small molecular weight, is easy to digest and absorb, and has the effects of promoting growth and development, increasing the proliferation of macrophages, and enhancing immunity, etc.

Description

Technical Field

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

[0002] In eggs, ovalbumin accounts for about 10%-12% of the egg white mass, and is mainly composed of ovalbumin, ovotransferrin, ovomucoid, lysozyme, globulin, etc. Ovalbumin contains various amino acids essential for the human body, and the composition ratio is appropriate, which is extremely close to human needs, and it is an ideal protein resource. However, ovalbumin has disadvantages such as high viscosity, poor thermal stability, easy foaming, and allergenicity. Moreover, during the preparation of ovalbumin powder, some functional properties are also likely to decline, which limits its application in food processing. Research has found that enzymatic hydrolysis can not only reduce the relative molecular mass of egg white ovalbumin, reduce allergenicity, but also the generated albumin peptide is more easily digested and absorbed by the human body. In addition, the functional properties such as solubility, viscosity, and foaming property of the enzymatic hydrolysis product have been improved compared with the original protein, and it has physiological functions such as blood pressure lowering, antioxidant, anticoagulant, antibacterial, and immune regulation, which greatly expands the application scope of egg white ovalbumin in the food industry.

[0003] It has been confirmed by research that albumin peptide, as a functional active peptide, has good biological properties, and its biological activities are mainly manifested in the following aspects: 1. Easy to digest and absorb, and promote the functional recovery of immune tissues; 2. Adjust the intestinal flora and improve the food conversion rate; 3. Promote the absorption of substances such as calcium, iron, and nucleic acids; 4. Improve hypoproteinemia. Tian Gang, etc. [1] Research has found that 5 kinds of 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 and promote the proliferation of lymphocytes, and have the characteristics of enhancing immunity. Jin Lei, etc. [2] The antioxidant property of the egg white protease hydrolysis product was studied by chemiluminescence method, and the results showed that the egg white protease hydrolysis products with different concentrations and degrees of hydrolysis all have the ability to scavenge reactive oxygen species and resist lipid oxidation. As the concentration of the egg white protease hydrolysis product increases, the scavenging ability increases, and the antioxidant property also increases.

[0004] Currently, more and more research has been conducted on albumin, and people are constantly looking for more economical and practical methods to develop albumin series products. It has been confirmed by research that further hydrolyzing albumin to obtain polypeptide products is more conducive to absorption and will play a greater role in human health care.

[0005] [1] Tian Gang, Chen Daiwen, Yu Bing, etc. Effects of enzymatic hydrolysis of egg white small peptide mixture on immune function of mice [J]. Chinese Journal of Animal Science, 2005, (05): 14-17.

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

[0007] To solve the above problems, the present invention provides an albumin peptide for promoting growth and development, its preparation method and application.

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

[0009] Specifically, the albumin peptide is prepared by double-enzyme synergistic hydrolysis using egg white as a substrate, and the two enzymes are chymotrypsin and alkaline protease respectively.

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

[0011] More specifically, the molecular weight of the albumin peptide is not greater than 1 kDa.

[0012] On the other hand, the present invention provides a method for preparing the above albumin peptide.

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

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

[0015] (2) Hydrolysis with chymotrypsin;

[0016] (3) Double-enzyme synergistic hydrolysis with chymotrypsin 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: mixing egg white and sterilized distilled water according to a volume ratio of 1-3:3-9, heating and denaturing at 85°C - 100°C for 15 - 25 minutes to obtain the hydrolysis raw material.

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

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

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

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

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

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

[0025] More specifically, after step (3), it further includes a step of heating the product of the synergistic hydrolysis of the two enzymes to terminate the enzymatic hydrolysis reaction.

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

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

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

[0029] On the other hand, the present invention provides the use of the above albumin peptide in the preparation of a product for promoting growth and development.

[0030] Specifically, the product includes: drugs, foods or food additives.

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

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

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

[0034] The albumin peptide prepared in this application has the effects of promoting growth and development and enhancing immunity, and has a small molecular weight, making it easier to digest and absorb. Description of the Drawings

[0035] Figure 1 It is the detection result of the body length of zebrafish in Experimental Example 2. Detailed Embodiments

[0036] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions indicated in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0037] Table 1

[0038] Material Purchase Source Article Number Chymotrypsin Sigma C3142 Alkaline Protease Sigma P4860 Trypsin Sigma T1426

[0039] Example 1

[0040] 1. Preparation of hydrolysis raw material:

[0041] Wash fresh eggs, then separate the egg white. After mixing the egg white and sterilized distilled water at a volume ratio of 1:3, heat and denature it in a sterilizer at 95°C for 15 minutes. After cooling to room temperature, add phosphate buffer solution, and adjust the mass concentration of ovalbumin to 5% and the pH to 8.0 to obtain the hydrolysis raw material.

[0042] 2. Chymotrypsin hydrolysis:

[0043] Add chymotrypsin to the ovalbumin solution so that the mass ratio of the enzyme to the substrate is 1:50.

[0044] Perform pre-hydrolysis at 40°C for 2 hours, and shake it on a shaker at a speed of 150 rpm during this period.

[0045] 3. Double-enzyme synergistic hydrolysis:

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

[0047] Perform hydrolysis at 50°C for 4 hours, and shake it on a shaker at a speed of 150 rpm during this period. After the reaction is completed, heat it to 90°C and treat it 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 cut-off of 1 kDa to separate and purify the polypeptides.

[0050] 5. Freeze-drying:

[0051] Perform freeze-drying on the purified polypeptide solution to obtain polypeptide powder.

[0052] Example 2

[0053] The difference from Example 1 is that in step 2, the mass ratio of chymotrypsin to the substrate is 1:100, and in step 3, the mass ratio of the added alkaline protease to the chymotrypsin 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 chymotrypsin to the substrate is 1.5:100, and in step 3, the mass ratio of the added alkaline protease to the chymotrypsin added in step 2 is 1:3.

[0056] Comparative Example 1

[0057] The difference from Example 1 is that: in Step 2, chymotrypsin was replaced with an equal mass of alkaline protease.

[0058] Comparative Example 2

[0059] The difference from Example 1 is that: in Step 3, alkaline protease was replaced with an equal mass of chymotrypsin.

[0060] Comparative Example 3

[0061] The difference from Example 1 is that: in Step 2, chymotrypsin was replaced with an equal mass of trypsin. Comparative Example 4

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

[0063] Experimental Example 1

[0064] Wild-type AB strain zebrafish at 3 days after fertilization were randomly selected and placed in beakers, with 30 zebrafish in each beaker. They were divided into six groups in total, among which five groups were experimental groups, and they were respectively given the albumin peptide powder prepared in the examples or comparative examples of the present invention, and the concentrations are shown in Table 2; the control group was not treated with anything. They were treated at 28 °C until 3 days after fertilization and then fixed, stained with alizarin red. After staining, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing. The NIS-Elements D3.20 advanced image processing software was used to analyze and collect data, and the fluorescence intensity of the zebrafish skull was analyzed. The growth and development promoting efficacy of the albumin peptides prepared by each preparation method was evaluated based on the statistical analysis results of this index. The statistical processing results were expressed as mean ± SE, and SPSS 26.0 software was used for statistical analysis. 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] Explanation of the data in Table 2: The albumin peptide prepared by the present invention has the function of promoting the bone development of zebrafish.

[0069] Experimental Example 2

[0070] Randomly select 3-dpf wild-type AB strain zebrafish and place them in beakers, with 30 zebrafish in each beaker. Administer the albumin peptide powder prepared in the examples or comparative examples of the present invention by dissolving it in water. The control group is not treated with anything. The capacity of each beaker is 20 mL. Change the liquid every day. After treating at 28 °C for 3 days, randomly select 10 zebrafish from each experimental group and place them under a dissection microscope for photographing. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyze the body length of the zebrafish. Evaluate the growth-promoting effect of the sample based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis. p < 0.05 indicates that the difference is 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 in the present invention has the function of promoting the growth of zebrafish.

[0075] Experimental Example 3

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

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

[0078] Table 4

[0079]

[0080]

[0081] During the administration period, record the food intake of the rats every day, measure the body weight 2 times a week, observe and record the changes in their body weight. The statistical analysis results are expressed as the mean and standard deviation (Mean ± SD). Use SPSS 25.0 for statistical analysis. A significant difference between groups is indicated by P < 0.05, and no significant statistical difference is indicated by P > 0.05.

[0082] The food intake of the 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 body weight changes of the rats are shown in Table 6 as follows:

[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 body lengths of experimental groups 1 - 3 are higher than those of the normal control group and experimental groups 4 - 5, and the differences are significant. At the same time, the food utilization rate is not significantly lower than that of the normal control group. It can be determined that the test sample has a positive result in improving the growth and development of animals.

[0091] Experimental Example 4

[0092] SD rats were intraperitoneally injected with 2 mL of 2% (w / w) sterilized starch solution for three consecutive days. They were decapitated 24 hours after the last injection. The abdominal skin was removed, and 4℃ phosphate - buffered saline (PBS) was aspirated with a syringe to repeatedly rinse the abdominal cavity. After collecting the rinsing fluid in a centrifuge tube, it was centrifuged (1000 rpm, 4℃) for 10 minutes, and then the supernatant was discarded. It was washed twice with 4℃ RPMI 1640 complete culture medium (containing 10% FBS), stained with 0.2% trypan blue solution for cell viability detection, and it was confirmed that more than 95% of the collected viable macrophages were obtained. After reading with a cell counting chamber, the cell concentration was adjusted to 2×10 5 cells / mL.

[0093] 100 μL of cell suspension was added to the wells. After adherent 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 continuously cultured for 48 hours. The inflammatory group was added with LPS to a final concentration of 100 ng / mL after culturing for 24 hours. At 44 hours of culture, 20 μL / well of 5% MTT was added. At 48 hours of culture, 100 μL / well of triple dissolution solution was added to terminate the culture. After overnight dissolution, the absorbance value (OD 570) of each well was measured with an enzyme - linked immunosorbent assay reader at a wavelength of 570 nm. The calculation formula for the growth index (Growth Indices) is as follows:

[0095]

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

[0097] Table 7

[0098] Group Normal Group GI Inflammatory 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 non - specific immune ability of the body.

[0101] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Simple modifications or equivalent replacements made by those of ordinary skill in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A preparation method of albumin peptide for promoting growth and development, characterized in that, It includes the following steps: (1) Prepare the hydrolysis raw material. The steps for preparing the hydrolysis raw material are as follows: Mix egg white and sterilized distilled water in a volume ratio of 1 - 3:3 - 9, heat and denature at 85°C - 100°C for 15 - 25 minutes to obtain the hydrolysis raw material, and the pH of the hydrolysis raw material is 8.0; (2) Hydrolyze with chymotrypsin. The addition amount of chymotrypsin is 0.8 - 2.5 wt%, and the hydrolysis conditions of chymotrypsin are: hydrolyze at 35 - 45°C for 1.5 - 2.5 h; (3) Double - enzyme synergistic hydrolysis with chymotrypsin and alkaline protease. Adjust the pH to 8.5, add alkaline protease, the addition amount of the alkaline protease is 0.8 - 2.5 wt%, and the conditions for the double - enzyme synergistic hydrolysis are: 45 - 55°C, hydrolyze for 3 - 5 h; (4) Separation and purification of polypeptides. The steps for the separation and purification of polypeptides are as follows: After centrifugation, take the supernatant and pass it through an ultrafiltration membrane; the conditions for centrifugation are: 3000 - 5000 rpm, centrifuge for 15 - 30 minutes; The cut - off molecular weight of the ultrafiltration membrane is 1 KDa.

2. Use of the albumin peptide prepared by the preparation method according to claim 1 in the preparation of a product for promoting growth and development, and the product is a food.

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