AU2021100134A4 - Active peptide with immunomodulatory effect, and preparation method and use thereof - Google Patents

Abstract

The present disclosure provides an active peptide with an immunomodulatory effect, and a preparation method and use thereof, and belongs to the technical field of polypeptide separation and identification. The active peptide with an immunomodulatory effect includes an albumin peptide B1 or a salt form of the albumin peptide B1; and the albumin peptide B1 has an amino acid sequence shown in SEQ ID No: 1. The active peptide is obtained by subjecting an ovalbumin solution to enzymolysis with papain and alkaline protease successively, separating and purifying, which is an octapeptide derived from egg white. Experiments have proved that the albumin peptide B1 has high immunomodulatory activity and shows an ideal improvement effect on immunodeficiencies in zebrafish. Therefore, the present disclosure provides use of the active peptide in a health product or a food with an immunomodulatory function. DRAWINGS I nte n sity(%VNJ15§ YJY§E 100 EV P- Y TY-----byax 2 bb I IIG I

Description

DRAWINGS I nte n sity(%VNJ15§ YJY§E

100 EV P- Y TY-----byax

2 bb

I IIG I I ACTIVE PEPTIDE WITH IMMUNOMODULATORY EFFECT, AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD The present disclosure belongs to the technical field of active peptide separation and identification, and particularly relates to an active peptide with an immunomodulatory effect, and a preparation method and use thereof.

BACKGROUND With the progression of the times and the increasingly fast-paced life, many people are in a sub-healthy state for a long time, and long-term development will lead to low autoimmunity. A body with low immunity is susceptible to infection of bacteria, viruses, and fungi. Therefore, the most direct manifestations in populations with low immunity are physical weakness, susceptibility to diseases, malnutrition, sleep disorders, and the like. If things go on like this, more serious health problems will occur. Immunodeficiency refers to low anti-infection ability of the human body due to developmental defects or immune response disorders of the human immune system, with clinical manifestations of repeated infections or severe infectious diseases. Immunodeficiency diseases can be divided into congenital hereditary immunodeficiency diseases and acquired secondary immunodeficiency diseases according to pathogeny, both of which can lead to poor or deficient immunological function and are prone to serious infection or tumor formation. Active peptides are biologically active fragments in proteins, which generally cannot exert their physiological activities because being hidden in the complex structure of a parent protein. The active peptides, when released from a parent protein by biochemical reactions such as in vitro proteolysis, gastrointestinal digestion, or fermentation, can exert a variety of physiological functions and affect the human health. So-called active peptides are composed of 2 to 20 amino acids in different arrangement and combination modes, with a molecular weight usually less than 6 KDa. These active peptides have different physiological activities, which has been proved by studies to be closely related to the types and sequences of composed amino acids. The active peptides can participate in or modulate the human immune system, endocrine system, cardiovascular system, and nervous system and affect the utilization of nutrients by the body. There have been many reports on active peptides with an immunomodulatory function in the prior art. The active peptides include pure polypeptides and alkyl modification-containing active peptides based on composition; include dipeptides and polypeptides based on the number of amino acid residues; include synthetic polypeptides and polypeptides isolated from different animal sources (such as bovine placenta and duck) based on isolation sources. There are few reports in the prior art that peptides isolated from egg white have the function of modulating immunodeficiency.

SUMMARY In view of this, the present disclosure is intended to provide a novel active peptide with an immunomodulatory effect, and a preparation method and use thereof. The present disclosure provides an active peptide with an immunomodulatory effect, including an albumin peptide B1 or a salt form of the albumin peptide B1, where, the albumin peptide B Ihas an amino acid sequence shown in SEQ ID No: 1. The present disclosure further provides a method for preparing the above active peptide with an immunomodulatory effect, including the following steps: 1) grinding and homogenizing an ovalbumin solution to obtain a stable homogenate; 2) keeping the homogenate at 50°C to 60°C for 1 h to 1.5 h to obtain a pretreated homogenate; 3) subjecting the pretreated homogenate to enzymolysis with papain and alkaline protease successively and to enzymatic inactivation to obtain an enzymatic hydrolysate; 4) filtering the enzymatic hydrolysate through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and collecting a resulting filtrate to obtain an albumin peptide mixture; 5) subjecting the albumin peptide mixture to high-performance liquid chromatography (HPLC) and collecting a component with peak time of 24 min to 27 min; and 6) subjecting the component with peak time of 24 min to 27 min to chromatographic analysis using a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain an active peptide, namely, an albumin peptide B1. Preferably, conditions of the chromatographic analysis in step 6) are as follows: mobile phase: phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100:0.1; phase B: a mixture of acetonitrile and formic acid, with an acetonitrile-formic acid volume ratio of 100:0.1; flow rate of the mobile phase: 300 nl/min; injection volume: 1IpL supernatant; gradient procedure of the mobile phase:

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

Volume percentage of phase A 97% 97% 63% 10% 10% 97% 97%

Volume percentage of phase B 3% 3% 37% 90% 90% 3% 3%

Preferably, parameters of the HPLC in step 5) are as follows: a mixed solution of mobile phase A and mobile phase B used for the following gradient elution: from 0 min to 5 min, a volume percentage of mobile phase B gradiently increasing from % to 5%; from 5 min to 31 min, the volume percentage of mobile phase B gradiently increasing from 5% to 78%; from 31 min to 33 min, the volume percentage of mobile phase B remaining at 78%; and from 33 min to 36 min, the volume percentage of mobile phase B gradiently decreasing from 78% to 5%; where, the mobile phase A is a mixture of trifluoroacetic acid (TFA) and ultrapure water (UPW), with a TFA-UPW volume ratio of 0.1:99.9; and the mobile phase B is a mixture of TFA and acetonitrile, with a TFA-acetonitrile volume ratio of 0.1:99.9; detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21°C to 25°C. Preferably, in step 3), the papain has a mass concentration of 1% to 2% in the pretreated homogenate, and enzymolysis with the papain is conducted for 3 h to 4 h; and before the papain is added, pH of the pretreated homogenate is adjusted to neutral. Preferably, the alkaline protease has a mass concentration of 0.5% to 1% in the pretreated homogenate, and enzymolysis with the alkaline protease is conducted for 3 h to 4 h; and before the enzymolysis with the alkaline protease is conducted, pH of a resulting system is adjusted to 8.0 to 9.0. Preferably, in step 1), particles in the homogenate have a particle size of 50 m to 150 [m. The present disclosure further provides a health product or a food with an immunomodulatory effect, including the above active peptide. The present disclosure provides use of the above active peptide or an active peptide prepared by the above preparation method in the preparation of an accelerator for promoting the proliferation of macrophages. The present disclosure provides use of the above active peptide or an active peptide prepared by the above preparation method in a health product or a food with an immunomodulatory function.

The active peptide with an immunomodulatory effect provided in the present disclosure is obtained by subjecting an ovalbumin solution to enzymolysis with papain and alkaline protease successively, separating and purifying, which is an octapeptide derived from egg white. In the present disclosure, transgenic zebrafish with green fluorescence-labeled macrophages are used as a material to establish immunodeficiency zebrafish models, different concentrations of albumin peptide B Iare used for administration groups, berbamine hydrochloride is adopted as a positive control drug, and a normal control group (treating zebrafish with fish farming water) and a model control group are set. An improvement effect of the albumin peptide B1 on immunodeficiencies in zebrafish is evaluated by the statistical analysis of fluorescence intensity. Results show that, compared with the model control group (338,583 pixels), the albumin peptide B1 administration groups show extremely significant differences in the macrophage fluorescence intensity; and the fluorescence intensity is related to the concentration of albumin peptide B1, where, a low concentration of albumin peptide B1 results in a macrophage fluorescence intensity close to that of the positive control group, and medium and high concentrations of albumin peptide BI lead to macrophage fluorescence intensities significantly higher than that of the positive control drug (berbamine hydrochloride) group, which exhibit a significant improvement effect on immunodeficiencies in zebrafish, indicating that the albumin peptide B1 has high immunomodulatory activity. Therefore, the present disclosure has verified that the separated albumin peptide B1 has high activity and shows an ideal improvement effect on immunodeficiencies in zebrafish.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a mass spectrum of the albumin peptide BI; FIG. 2 is a phenotype diagram of the improvement of the albumin peptide BI on immunodeficiencies; FIG. 3 shows the macrophage fluorescence intensity after the albumin peptide B1 treatment, where, as compared with the model control group, ***p < 0.001; and FIG. 4 shows the improvement effect of the albumin peptide B1 on immunodeficiencies in zebrafish, where, as compared with the model control group, ***p < 0.001.

DETAILED DESCRIPTION The present disclosure provides an active peptide with an immunomodulatory effect, including an albumin peptide B1 with an amino acid sequence of VNPIPYYE (Val-Asn-Pro Ile-Pro-Tyr-Tyr-Glu), which includes 8 amino acid residues and has a molecular weight of 1320.6 Da. The active peptide with an immunomodulatory effect provided in the present disclosure may also include a salt form of the albumin peptide B1, such as an acetate form and a hydrochloride form. The present disclosure has no specific limitation on a source of the active peptide, which can be obtained by artificial synthesis or a separation and purification method. In the present disclosure, the albumin peptide B1 used in the animal experiment is entrusted to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis. The present disclosure provides a method for preparing the active peptide with an immunomodulatory effect, including the following steps: 1) grinding and homogenizing an ovalbumin solution to obtain a stable homogenate; 2) keeping the homogenate at 50°C to 60°C for 1 h to 1.5 h to obtain a pretreated homogenate; 3) subjecting the pretreated homogenate to enzymolysis with papain and alkaline protease successively and to enzymatic inactivation to obtain an enzymatic hydrolysate; 4) filtering the enzymatic hydrolysate through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and collecting a resulting filtrate to obtain an albumin peptide mixture; 5) subjecting the albumin peptide mixture to high-performance liquid chromatography (HPLC) and collecting a component with peak time of 24 min to 27 min; and 6) subjecting the component with peak time of 24 min to 27 min to chromatographic analysis using a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain an active peptide, namely, an albumin peptide B1. In the present disclosure, an ovalbumin solution is ground and homogenized to obtain a stable homogenate. In the present disclosure, the ovalbumin solution may have a concentration preferably of 3% to 10%, more preferably of 4% to 8%, and most preferably of 5%. The present disclosure has no specific limitation on a preparation method of the ovalbumin solution, and a preparation method of a protein solution well known in the art may be adopted. In the present disclosure, the grinding may preferably be conducted with a colloidal mill. The grinding with a colloidal mill may be conducted preferably for 1h to 2 h and more preferably for 1.5 h. The homogenization may preferably be conducted at a pressure of 25 MPa for 2 h. After the homogenization, particles in the homogenate may have a particle size preferably of 50 m to 150 m, more preferably of 80 m to 120 [m, and most preferably of 100 jm. The prepared homogenate has a fine texture and is not separated into layers, which is beneficial to the stability of the homogenate, thereby increasing the content of albumin peptide BI. In the present disclosure, after a homogenate is obtained, the homogenate is kept at 50°C to 60°C for 1 h to 1.5 h to obtain a pretreated homogenate. In the present disclosure, the temperature may preferably be kept at 55°C. In the present disclosure, the temperature may preferably be kept for 1.2 h. A too-high temperature may cause denaturation of protein particles in the homogenate, and a too-low temperature is not conducive to the full dissolution of materials. The temperature-keeping treatment of the homogenate is beneficial to improve the solubility of materials and facilitate the enzymolysis process. In the present disclosure, after a pretreated homogenate is obtained, the pretreated homogenate is subjected to enzymolysis with papain and alkaline protease successively and then to enzymatic inactivation to obtain an enzymatic hydrolysate. In the present disclosure, the papain in the pretreated homogenate may have a mass concentration preferably of 1% to 2% and more preferably of 1.5%.The enzymolysis with the papain may be conducted preferably for 3 h to 4 h and more preferably for 3.5 h. With strong enzyme cleavage characteristics, the papain can enzymatically destroy the spatial structure of ovalbumin to expose peptide chains and thus enzymatically cleave the chains into large fragment polypeptides at a specific site. Before the papain is added, pH of the pretreated homogenate may be adjusted preferably to neutral and more preferably to 7.2.A solution for adjusting the pH of the pretreated homogenate is not specifically limited, and a reagent for adjusting pH well known in the art may be used, such as a sodium hydroxide solution and a hydrochloric acid solution. After the enzymolysis with papain is conducted, enzymolysis with alkaline protease is conducted. Before the enzymolysis with alkaline protease is conducted, pH of a resulting system may be adjusted preferably to 8.0 to 9.0 and more preferably to 8.5. The alkaline protease in the pretreated homogenate may have a mass concentration preferably of 0.5% to 1% and more preferably of 0.8%; and the enzymolysis with alkaline protease may be conducted preferably for 3 h to 4 h and more preferably for 3.5 h. On the basis of the enzymolysis with papain, the enzymolysis with alkaline protease is conducted to enzymatically cleave obtained large fragment polypeptides at a specific site, where, an amino acid sequence of the active peptide will not be cut during this enzymatic cleavage process, and the active peptide is obtained from small fragments resulting from the enzymatic cleavage with alkaline protease. During the enzymolysis process, the present disclosure tries to use other kinds of proteases alone or in combination to enzymatically cleave ovalbumin particles, but an albumin peptide B1 cannot be obtained or an obtained albumin peptide B1 shows a poor effect. Moreover, the use of alkaline protease can better control the taste of a product and allows the molecular weight of an obtained product to meet the quality requirements.

The present disclosure has no specific limitation on a method for the enzymatic inactivation, and a scheme for enzymatic inactivation well known in the art may be adopted, such as high temperature enzymatic inactivation.

In the present disclosure, after an enzymatic hydrolysate is obtained, the enzymatic hydrolysate is filtered through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and a resulting filtrate is collected to obtain an albumin peptide mixture. In the present disclosure, after the enzymatic hydrolysate is filtered through the 30,000 Da ceramic membrane, a resulting filtrate is collected. The filtrate is filtered through the1 KDa organic membrane. The present disclosure has no specific limitation on sources of the ceramic membrane and the organic membrane, and sources of the ceramic membrane and the organic membrane well known in the art may be adopted. In an example of the present disclosure, the ceramic membrane and the organic membrane are purchased from Jiangsu Jiuwu Hi-Tech Co., Ltd. The filtration with the ceramic membrane and organic membrane is conducted to remove large fragment polypeptides in the enzymatic hydrolysate and collect polypeptides with a small molecular weight below 1 KDa. In the present disclosure, after an albumin peptide mixture is obtained, the albumin peptide mixture is subjected to HPLC, and a component with peak time of24 min to 27 min is collected. In the present disclosure, parameters for the HPLC are as follows: a mixed solution of mobile phase A and mobile phase B used for the following gradient elution: from 0 min to 5 min, a volume percentage of mobile phase B gradiently increasing from % to 5%; from 5 min to 31 min, the volume percentage of mobile phase B gradiently increasing from 5% to 78%; from 31 min to 33 min, the volume percentage of mobile phase B remaining at 78%; and from 33 min to 36 min, the volume percentage of mobile phase B gradiently decreasing from 78% to 5%; where, the mobile phase A is a mixture of trifluoroacetic acid (TFA) and ultrapure water (UPW), with a TFA-UPW volume ratio of 0.1:99.9; and the mobile phase B is a mixture of TFA and acetonitrile, with a TFA-acetonitrile volume ratio of 0.1:99.9; detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21C to °C. The HPLC can be used to exclude polypeptides of less than 1 KDa collected in the present disclosure in the order of time, which is beneficial to the fine separation of polypeptides of various molecular weights. In the separation of albumin peptide components by HPLC, a collection tube is changed every 3 min to obtain 12 components. The in vitro immune activity assay of the 12 components shows that a component 9 (the component with peak time of 24 min to 27 min) has an excellent proliferation-promoting effect. In the present disclosure, after a component with peak time of24 min to 27 min is collected, the component with peak time of 24 min to 27 min is subjected to chromatographic analysis using a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain an active peptide, namely, an albumin peptide B1. In the present disclosure, the liquid chromatography-Q EXACTIVE mass spectrometry system may preferably be a nano-liquid chromatography-Q EXACTIVE mass spectrometry system. Conditions of the chromatographic analysis may preferably be as follows: mobile phase: phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100:0.1; phase B: a mixture of acetonitrile and formic acid, with an acetonitrile-formic acid volume ratio of 100:0.1; flow rate of the mobile phase: 300 nl/min; injection volume: 1IpL supernatant; gradient procedure of the mobile phase:

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

A (%) 97 97 63 10 10 97 97

B 3 3 37 90 90 3 3

It is determined by the liquid chromatography-mass spectrometry technique that the albumin peptide B1 accounts for 39% of the total mass of component 9, and it is determined by the liquid chromatography-mass spectrometry technique that a mass percentage of the single-chain polypeptide in the albumin peptide mixture is 1.1%. The present disclosure uses different concentrations of albumin peptide B1 as drugs for treating a material of immunodeficiency zebrafish models, and results show that the albumin peptide B1 can significantly increase the fluorescence signal intensity of macrophages in zebrafish, that is, the number of macrophages is increased. Therefore, the present disclosure provides use of the active peptide or an active peptide prepared by the preparation method in the preparation of an accelerator for promoting the proliferation of macrophages. The present disclosure adopts administration groups where immunodeficiency zebrafish models are treated with different concentrations of albumin peptide B1. Compared with the model control group, the albumin peptide B1 can significantly increase the number of macrophages in zebrafish, and the increase in the number of macrophages can enhance the immunomodulatory ability of the body. Therefore, the albumin peptide B Ishows a significant improvement effect on immunodeficiencies in zebrafish. Based on this, the present disclosure provides a health product or a food with an immunomodulatory effect, including the active peptide. The present disclosure has no specific limitation on a type of the health product or food, and a type and form of health products and foods well known in the art may be adopted, such as granules, oral liquids, and solid beverages. In the health product or food, the albumin peptide B1 may have a concentration of 1.1%. The present disclosure has no specific limitation on a preparation method of the health product or food, and a preparation method of health products or foods well known in the art may be adopted. The present disclosure provides use of the active peptide or an active peptide prepared by the preparation method in a health product or a food with an immunomodulatory function. Moreover, the present disclosure also provides use of the active peptide or an active peptide prepared by the preparation method in the preparation of a drug for treating immunodeficiency diseases. Those skilled in the art do not make specific limitations on the immunodeficiency diseases, and all diseases that occur due to a low autoimmunity are in the claimed scope of the present disclosure. The active peptide provided in the present disclosure can be used to treat immunodeficiency diseases. The active peptide with an immunomodulatory effect, and a preparation method and use thereof provided in the present disclosure will be described in detail below with reference to examples, but these examples cannot be understood as limiting the claimed scope of the present disclosure.

Example 1 Preparation method of an albumin peptide Step 1. 1,000 L of deionized water was prepared and heated to 50°C, and 50 kg of ovalbumin was added to obtain an ovalbumin solution with a concentration of 5%. Step 2. The ovalbumin solution was thoroughly stirred, treated with a colloidal mill for 1.5 h, and then treated with a high-pressure homogenizer for 2 h at a pressure of 25 MPa, where, protein particles in a resulting homogenate had a particle size of 50 m to 150 [m. Step 3. The homogenate obtained from high-pressure homogenization was heated to 55°C and stirred at the temperature for 1 h. Step 4. pH of an incubated ovalbumin solution was adjusted to 7.2 with a 0.5 M NaOH solution, and the temperature was kept constant. Step 5. 1% papain (0.5 Kg) was added to the pH-adjusted ovalbumin solution, and enzymolysis was conducted for 3 h; the pH was adjusted to 8.5 with a 0.5 M NaOH solution; and 0.6% alkaline protease (0.3 kg) was then added, and enzymolysis was further conducted for 4 h. Step 6. An obtained enzymatic hydrolysate was heated to 110C and kept at the temperature for 5 min to inactivate the proteases. Step 7. An enzyme-inactivated enzymatic hydrolysate was cooled to room temperature and filtered through a 30,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was collected and spray dried to obtain an albumin peptide mixture. Determination indexes of the basic physical and chemical properties of the albumin peptide mixture: protein content determination (GB5009.9), moisture content determination (GB5009.3), and ash content determination (GB5009.4). Results are shown in Table 1.

Table 1 Basic physical and chemical properties of the albumin peptide mixture

Item Protein content ()Moisture content ()Ash content (%)

Casein peptide mixture 92 3.6 4.4

Example 2 The preparative HPLC was used to separate and purify an albumin peptide from the albumin peptide mixture. (1) Mobile phase A: 0.1% TFA + 99.9% UPW; mobile phase B: 0.1% TFA + 99.9% acetonitrile. (2) Detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21°C to 25°C. (3) Equilibration was conducted using 6 column volumes of mobile phase A at a speed of mL/min until a baseline was stable. (4) The obtained albumin peptide mixture was dissolved with mobile phase A to obtain an albumin peptide mixture solution with a concentration of 10 mg/mL; the albumin peptide mixture solution was centrifuged at 10,000 r/min for 5 min; and a resulting supernatant was filtered with a 0.22 m filter membrane and injected at a volume of 15 mL. (5) A mixed solution of mobile phase A and mobile phase B was used for the following gradient elution: from 0 min to 5 min, a concentration of mobile phase B gradiently increasing from 0% to %; from 5 min to 31 min, the concentration of mobile phase B gradiently increasing from 5% to 78%; from 31 min to 33 min, the concentration of mobile phase B remaining at 78%; and from 33 min to 36 min, the concentration of mobile phase B gradiently decreasing from 78% to 5%. (6) Different components were collected for activity assay. 12 components were collected. The peak time of the 12 components is shown in Table 2.

Table 2 Peak time of 12 components

Component Peak time Component Peak time Component Peak time

1 0 min to 3 min 2 3 min to 6 min 3 6 min to 9 min

4 9 min to 12 min 5 12 min to 15 min 6 15 min to 18 min

7 18 min to 21 min 8 21 min to 24 min 9 24 min to 27 min

10 27 min to 30 min 11 30 min to 33 min 12 33 min to 36 min

Example 3 In vitro immune activity assay Macrophage proliferation test: Macrophages RAW264.7 were inoculated in high glucose DMEM complete medium (with 10% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin), and cultivated in a 37°C incubator with 5% C02. Macrophages RAW264.7 at logarithmic growth phase were collected and inoculated in high glucose DMEM complete medium (with % FBS, 100 U/mL streptomycin, and 100 U/mL penicillin) in a 96-well plate, with 1 x 105

RAW264.7 cells per well. The macrophages were cultivated in a 37°C incubator with 5% C02. After the cells grew adherently, the supernatant in each well was removed, and the wells were grouped as follows: group A: blank group (with medium only); groups B: experimental groups (with the 12 components obtained from albumin peptide separation and purification, respectively), with 6 replicates for each group. The plate was incubated in a 37°C incubator with 5% C02 for 44 h, then 20 L of 5 mg/mL MTT was added to each well, and the plate was further incubated for 4 h. A supernatant was removed, 150 pL of DMSO was added to each well, and the plate was shaken at 37°C for 10 min. The OD value was determined at 492 nm. A cell proliferation rate was calculated according to formula I. Cell proliferation rate(% = OD value of an experimental group - OD value of a blank group x 100% OD value of a control group - OD value of a blank group formula I.

Table 3 OD values of RAW264.7 macrophages treated with the 12 components

Component Blank control 1 2 3 4 5

OD value 0.89±0.16 0.90±0.12 0.96 ±0.21 1.11±0.32 0.92±0.19 0.98±0.65

Cell proliferation rate - 1.12% 7.87% 24.71% 3.37% 10.11%

Component 7 8 9 10 11 12

OD value 0.91 ±0.33 1.43±0.36 1.52 ±0.72 1.24±0.20 1.31 ±0.29 0.95±0.31

Cell proliferation rate 2.25% 60.67% 70.79% 39.33% 47.19% 6.74%

Example 4 Identification of immunologically-active peptide sequences From the macrophage proliferation test in Example 3, it was found that the component 9 had an excellent proliferation-promoting effect. Therefore, the purity and structure of component 9 were identified. The purity and structure of the component 9 in Example 3 were identified by the nano liquid chromatography-Q EXACTIVE mass spectrometry system. 1. Chromatographic conditions: (1) mobile phase: phase A: 100% purified water + 0.1% formic acid; phase B: 100% acetonitrile + 0.1% formic acid; (2) flow rate of the mobile phase: 300 nl/min (3) injection volume: 1IpL supernatant (4) gradient procedure of the mobile phase as shown in Table 4. Table 4 Gradient procedure of the mobile phase

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

A(%) 97 97 63 10 10 97 97

B(%) 3 3 37 90 90 3 3

The nano-liquid chromatography-Q EXACTIVE mass spectrometry system was used to purify an active polypeptide from the component 9, whose structure was identified as VNPIPYYE (Val-Asn-Pro-Ile-Pro-Tyr-TyrGlu), with a molecular weight of 1320.6 Da, as shown in FIG. 1. It was determined by the liquid chromatography-mass spectrometry technique that the single-chain polypeptide accounted for 39% of the total mass of component 9, and it was determined by the liquid chromatography-mass spectrometry technique that a mass percentage of the single-chain polypeptide in the albumin peptide mixture was 1.1%. Therefore, the VNPIPYYE was named albumin peptide B.

Example 5 Evaluation of the improvement effect of the albumin peptide on immunodeficiencies Experimental purpose: Evaluating the improvement effect of the albumin peptide BI on immunodeficiencies. Experimental animals: Transgenic zebrafish with green fluorescence-labeled macrophages (constructed by Hunter Biotechnology Co., Ltd.) aged 2 dpf, 390 fish in total, and 30 fish for each experimental group. The zebrafish were used to determine the maximum tolerated concentration (MTC) of the albumin peptide B1 and the improvement effect of the albumin peptide on immunodeficiencies. The zebrafish were fed in fish farming water (water quality: 200 mg of instant sea salt was added per IL reverse osmosis water, with a conductivity of 480 S/cm to 510 S/cm; a pH of 6.9 to 7.2; and a hardness of 53.7 to 71.6 mg/L CaCO3) at 28°C, and use license of experimental animals was: SYXK (Zhejiang) 2012-0171. The feeding management met the requirements of international AAALAC certification. Experimental drug: Albumin peptide B1, which was entrusted to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis, stored in a cool cabinet at -20°C, and prepared into a 20 mg/mL solution with the fish farming water before use; vinorelbine tartrate injection, transparent and clear, batch No. 170513C, from Hangzhou Minsheng Pharmaceutical Co., Ltd, which was prepared into a 0.1 mg/mL solution using normal saline (NS) and stored at 4°C before use; and berbamine hydrochloride, white powder, batch No. R29J6F1, from Shanghai Yuanye Biotechnology Co., Ltd., which was prepared into a 6.8 mg/mL stock solution using DMSO and stored at -20°C before use. Instruments and reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertAl, Shanghai Tusen Vision Technology Co., Ltd.); microinjector (IM300, NARISHIGE, Japan); motorized focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, America); 6-well plate (Nest Biotech, Shanghai, China). Concentration groups Experimental group 1: normal control group; experimental group 2: model control group; experimental group 3: positive control drug of berbamine hydrochloride at 27.3 [g/mL; experimental group 4: albumin peptide B Iat11 g/mL; experimental group 5: albumin peptide B Iat 33 g /mL; and experimental group 6: albumin peptide B Iat 100 [g/mL. Basis for concentration determination According to the concentration exploration experiment, the MTC for evaluating the improvement effect of the albumin peptide BI on immunodeficiencies was 100 pg/mL. Experimental concentrations of the albumin peptide B1were set to 11 g/mL, 33 pg/mL, and 100 pg/mL. Establishment of models The vinorelbine tartrate injection was injected into the 2 dpf transgenic zebrafish with green fluorescence-labeled macrophages at a dosage of 1 ng/fish to establish immunodeficiency zebrafish models.

Experimental method 1. Determination of MTC 210 transgenic zebrafish (with green fluorescence-labeled macrophages, 2 dpf) were randomly selected and added to a 6-well plate, with 30 fish per well. The albumin peptide BI was administered by water-soluble administration at concentrations of 50 pg/mL, 100 pg/mL, 250 pg/mL, 500 pg/mL, 1,000 pg/mL, and 2,000 pg/mL, and a normal control group was set. Each well (experimental group) had a volume of 3 mL. The plate was incubated in an incubator at 28°C for 24 h. The death of zebrafish was counted, and the MTC of the polypeptide on zebrafish was determined based on the toxicity and death of zebrafish. 2. Evaluation of the improvement effect on immunodeficiencies 180 transgenic zebrafish (with green fluorescence-labeled macrophages, 2 dpf) were randomly selected and added to a 6-well plate, with 30 fish per well. The albumin peptide BI was administered by water-soluble administration at concentrations of 11 g/mL, 33 pg/mL, and 100 pg/mL, the positive control drug of berbamine hydrochloride was administered at a concentration of 27.3 [g/mL, and a normal control group (treating zebrafish with fish farming water) and a model control group were set. Each well (experimental group) had a volume of 3 mL. After the plate was incubated for 24 h in an incubator at 28°C, 10 zebrafish were randomly selected from each group to acquire the macrophage fluorescence intensity of zebrafish under a fluorescence microscope. The statistical analysis of fluorescence intensity was used to evaluate the improvement effect of the albumin peptide B1 on immunodeficiencies in zebrafish. The fluorescence intensity was expressed by fluorescent pixels, and the fluorescent pixels were counted by the motorized focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan) software. The improvement effect of the albumin peptide B1 on immunodeficiencies in zebrafish was calculated according to the following formula II:

Immunodeficiency-improving effect (%) I= S (test sample) - S (model control group) oecotogru)x 1000% S (model control group) formula II. Statistical analysis was conducted using one-way analysis of variance (ANOVA) and Dunnett's T test. p < 0.05 indicates a significant difference. A representative experimental map was provided. Experimental results 1. MTC The albumin peptide B1 induced a pericardial edema incidence of 30% and a mortality of 6.7% at a concentration of 250 pg/mL, and did not induce zebrafish death and exhibited no obvious toxic phenotype at a concentration of 100 pg/mL. Therefore, the MTC for determining the improvement effect of the albumin peptide B Ion immunodeficiencies in zebrafish was 100 pg/mL. Details can be seen in Table 5.

Table 5 "concentration-mortality" results after the albumin peptide B Itreatment (n = 30)

Group Concentration (pg/mL) Death count Mortality(%) Toxic phenotype

Normal control group - 0 0 no significant abnormalities

0 0 no significant 50 abnormalities

100 0 0 no significant abnormalities

Albumin peptide B1 250 2 6.7 30% pericardial edema

500 30 100

1000 30 100

2000 30 100

Evaluation of the improvement effect on immunodeficiencies The macrophage fluorescence intensity of the model control group was 338,583 pixels, withp < 0.001 as compared with the normal control group (479,591 pixels), indicating that the model was successful established. The macrophage fluorescence intensity of the group administered with berbamine hydrochloride at 27.3 [tg/mL was 420,243 pixels, with p < 0.001 as compared with the model control group (338,583 pixels) and an immunodeficiency improving effect of 24%, indicating that the positive control drug of berbamine hydrochloride exhibited a significant improvement effect on immunodeficiencies in zebrafish. The macrophage fluorescence intensities of groups administered with albumin peptide B1 at concentrations of 11 g/mL, 33 [g/mL, and 100 [tg/mL were 417,396 pixels, 432,597 pixels, and 432,227 pixels, respectively, with p < 0.001 as compared with the model control group (338,583 pixels) and immunodeficiency-improving effects of 23%, 28%, and 28%, respectively, indicating that the albumin peptide B1 exhibited a significant improvement effect on immunodeficiencies in zebrafish. Details can be seen in Table 6, FIG. 2, FIG. 3, and FIG. 4.

Table 6 Improvement effect of the albumin peptide BIon immunodeficiencies in zebrafish (n=10)

Concentration Macrophage fluorescence intensity Improvement effect on Group (pg/mL) (pixel, mean + SE) immunodeficiencies

Normal control group - 479591 ±6305

Model control group - 338583 +8581

Berbamine hydrochloride 27.3 420243 15297*** 24***

11 417396±14363*** 23***

Albumin peptide B1 33 432597 +11361*** 28***

100 432227+ 10133*** 28***

As compared with the model control group, ***P < 0.001.

Comparative Example 1 1. Optimization of single-enzyme hydrolysis conditions A 5% ovalbumin solution was prepared and heated to the optimal enzymolysis temperature of a protease using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to the optimal pH of a corresponding protease; a resulting solution was incubated for 30 min; then a weighed protease (see Table 7) was added, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis, see Table 7); pH was adjusted to neutral, and a resulting solution was placed in a 100°C water bath for 5 min to inactivate the protease; an enzymatic hydrolysate was filtered through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively; and a resulting filtrate was collected and lyophilized to obtain an albumin peptide mixture. The effect of the albumin peptide mixture on the proliferation rate of macrophages was tested according to the in vitro immune activity assay method described in Example 3, and a mass percentage of an albumin peptide B1 in the albumin peptide mixture was determined according to the chromatographic analysis method described in Example 4.

Table 7 List of enzymolysis conditions for different types of proteases

Protein Optimal enzymolysis Optimal enzymolysis Added amount Protease type concentration pH temperature time (0%)

(%) (C) (h)

Alkaline 5 1 8.5 55 4 protease

Papain 5 1 7.2 55 4

Neutral 5 1 7.0 55 4 protease

Trypsin 5 1 6.9 37 4

1.1 Enzymolysis results of alkaline protease A 5% ovalbumin solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 8.5 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then an alkaline protease was added, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to neutral, and a resulting solution was placed in a 100°C water bath for 5 min to inactivate the protease; an enzymatic hydrolysate was filtered through membranes; and a resulting filtrate was lyophilized to obtain an albumin peptide mixture. Activity assay was conducted for the albumin peptide mixture. A macrophage proliferation rate was 12.56% and a mass percentage of the albumin peptide B1 in the albumin peptide mixture was 0.59%. 1.2 Enzymolysis results of papain A 5% ovalbumin solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.2 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then papain was added, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to neutral, and a resulting solution was placed in a 100°C water bath for min to inactivate the protease; an enzymatic hydrolysate was filtered through membranes; and a resulting filtrate was lyophilized to obtain an albumin peptide mixture. Activity assay was conducted for the albumin peptide mixture. A macrophage proliferation rate was 9.89% and a mass percentage of the albumin peptide B1 in the albumin peptide mixture was 0.39%. 1.3 Enzymolysis results of neutral protease A 5% ovalbumin solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.0 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then a neutral protease was added, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to neutral, and a resulting solution was placed in a 100°C water bath for 5 min to inactivate the protease; an enzymatic hydrolysate was filtered through membranes; and a resulting filtrate was lyophilized to obtain an albumin peptide mixture. Activity assay was conducted for the albumin peptide mixture. A macrophage proliferation rate was 5.16% and a mass percentage of the albumin peptide B1 in the albumin peptide mixture was 0.19%. 1.4 Enzymolysis results of trypsin A 5% ovalbumin solution was prepared and heated to 37°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.0 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then 0.1 g of trypsin was added, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); a resulting solution was placed in a 100°C water bath for 5 min to inactivate the protease; an enzymatic hydrolysate was filtered through membranes; and a resulting filtrate was lyophilized to obtain an albumin peptide mixture. Activity assay was conducted for the albumin peptide mixture. A macrophage proliferation rate was 1.99% and a mass percentage of the albumin peptide B1 in the albumin peptide mixture was 0.09%. 2. Stepwise enzymolysis of ovalbumin using a combination of two enzymes A single one of alkaline protease, papain, neutral protease, and trypsin was first used to conduct hydrolysis under the optimal enzymolysis conditions of the single enzyme. According to the detection results of the proliferation activity of macrophages and the content of albumin peptide B1, it was intended to use the papain and alkaline protease for enzymolysis. 2.1 Stepwise enzymolysis results of a combination of alkaline protease and papain A 5% ovalbumin solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 8.5 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then an alkaline protease was added with a final concentration of 0.6%, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to 7.2, papain was added with a final concentration of 1%, and enzymolysis was further conducted for 3 h; a resulting solution was placed in a 100°C water bath for 5 min to inactivate the protease; an enzymatic hydrolysate was filtered through membranes; and a resulting filtrate was lyophilized to obtain an albumin peptide mixture. Activity assay was conducted for the albumin peptide mixture. A macrophage proliferation rate was 16.23% and a mass percentage of the albumin peptide B1 in the albumin peptide mixture was 0.72%. 2.2 Stepwise enzymolysis results of a combination of papain and alkaline protease A 5% ovalbumin solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.2 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then papain was added with a final concentration of 1%, and enzymolysis was conducted for 3 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to 8.5, an alkaline protease was added with a final concentration of 0.6%, and enzymolysis was further conducted for 4 h; a resulting solution was placed in a 100°C water bath for 5 min to inactivate the protease; an enzymatic hydrolysate was filtered through membranes; and a resulting filtrate was lyophilized to obtain an albumin peptide mixture. Activity assay was conducted for the albumin peptide mixture. A macrophage proliferation rate was 38.16% and a mass percentage of the albumin peptide B1 in the albumin peptide mixture was 1.09%.

The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims (5)

What is claimed is:

1. An active peptide with an immunomodulatory effect, comprising an albumin peptide B1 or a salt form of the albumin peptide B1, wherein, the albumin peptide B1 has an amino acid sequence shown in SEQ ID No: 1.

2. A method for preparing the active peptide with an immunomodulatory effect according to claim 1, comprising the following steps: 1) grinding and homogenizing an ovalbumin solution to obtain a stable homogenate; 2) keeping the homogenate at 50°C to 60°C for 1 h to 1.5 h to obtain a pretreated homogenate; 3) subjecting the pretreated homogenate to enzymolysis with papain and alkaline protease successively and to enzymatic inactivation to obtain an enzymatic hydrolysate; 4) filtering the enzymatic hydrolysate through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and collecting a resulting filtrate to obtain an albumin peptide mixture; 5) subjecting the albumin peptide mixture to high-performance liquid chromatography (HPLC) and collecting a component with peak time of 24 min to 27 min; and 6) subjecting the component with peak time of 24 min to 27 min to chromatographic analysis using a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain an active peptide, namely, an albumin peptide B1.

3. The preparation method according to claim 2, wherein, conditions of the chromatographic analysis in step 6) are as follows: mobile phase: phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100:0.1; phase B: a mixture of acetonitrile and formic acid, with an acetonitrile-formic acid volume ratio of 100:0.1; flow rate of the mobile phase: 300 nl/min; injection volume: 1IpL supernatant; gradient procedure of the mobile phase:

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

Volume percentage of phase A 97% 97% 63% 10% 10% 97% 97%

Volume percentage of phase B 3% 3% 37% 90% 90% 3% 3% wherein, parameters of the HPLC in step 5) are as follows: a mixed solution of mobile phase A and mobile phase B used for the following gradient elution: from 0 min to 5 min, a volume percentage of mobile phase B gradiently increasing from % to 5%; from 5 min to 31 min, the volume percentage of mobile phase B gradiently increasing from 5% to 7 8 %; from 31 min to 33 min, the volume percentage of mobile phase B remaining at 78%; and from 33 min to 36 min, the volume percentage of mobile phase B gradiently decreasing from 78% to 5%; wherein, the mobile phase A is a mixture of trifluoroacetic acid (TFA) and ultrapure water (UPW), with a TFA-UPW volume ratio of 0.1:99.9; and the mobile phase B is a mixture of TFA and acetonitrile, with a TFA-acetonitrile volume ratio of 0.1:99.9; detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21C to °C; wherein, in step 3), the papain has a mass concentration of 1% to 2% in the pretreated homogenate, and enzymolysis with the papain is conducted for 3 h to 4 h; and before the papain is added, pH of the pretreated homogenate is adjusted to neutral; wherein, the alkaline protease has a mass concentration of 0.5% to 1% in the pretreated homogenate, and enzymolysis with the alkaline protease is conducted for 3 h to 4 h; and before the enzymolysis with the alkaline protease is conducted, pH of a resulting system is adjusted to 8.0 to 9.0.

4. The preparation method according to any one of claims 2 to 3, wherein, in step 1), particles in the homogenate have a particle size of 50 m to 150 [m.

5. A health product or a food with an immunomodulatory effect, comprising the active peptide according to claim 1.