A lactoglobulin peptide with desensitization effect and its application

Abstract

The present invention provides a desensitized lactoglobulin peptide, which is prepared by placing lactoglobulin in an extremely strong magnetic field. The polypeptide provided by the present invention removes the allergenicity of lactoglobulin and has the ability to improve immunity. It has been verified by a mouse model that it can improve the cellular immunity and humoral immunity of mice, and can be used to prepare functional foods or medicines for improving immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polypeptide preparation, and particularly relates to a lactoglobulin peptide with desensitization effect and application thereof. Background Art

[0002] Protein is one of the three major nutrients in living organisms. It plays an important physiological function in many aspects, such as the composition of living organisms, maintaining normal metabolism, repairing and renewing body tissues, maintaining the activity of various enzymes in the body, electrolyte balance and providing energy.

[0003] With the growing global demand for high-quality protein raw materials, the shortage of high-quality protein resources in my country is becoming increasingly apparent. As the concepts of nutrition, health and sustainable development are gradually recognized, and along with the development of modern nutrition science and feed science, more proteins with excellent characteristics can be developed through scientific processing and technological innovation.

[0004] my country is currently one of the countries with the fastest growth in the global protein market, and has developed a variety of protein products such as whey protein, soy protein, wheat gluten protein, peanut protein, cottonseed protein, etc. In the process of protein resource development, the quality of protein varieties and their nutritional and functional characteristics are the focus of attention. In addition to in-depth research on protein content, amino acid balance, low molecular weight protein and small peptide content, the allergens contained in the protein and the allergic reactions they cause, as well as how to reduce allergenicity, are also receiving more and more attention. Summary of the invention

[0005] The present invention provides a lactoglobulin peptide with desensitization effect and application thereof, thereby making up for the deficiencies of the prior art.

[0006] The present invention first provides a desensitized lactoglobulin peptide, which is prepared by placing lactoglobulin in an extremely strong magnetic field;

[0007] As a specific record of the embodiment, the extremely strong magnetic field has a magnetic field strength of 15T;

[0008] As a record of the embodiment, the processing time is 15s;

[0009] The lactoglobulin peptide has an amino acid sequence of LLDAQSAPLR (SEQ ID NO: 1) EGDLEILLQK (SEQ ID NO: 2).

[0010] The polypeptide provided by the present invention also has the ability to improve immunity, and has been verified by a mouse model to improve the cellular immunity and humoral immunity of mice;

[0011] In yet another aspect, the present invention provides a medicine containing the above-mentioned desensitized lactoglobulin peptide.

[0012] The lactoglobulin peptide prepared by the invention effectively removes the allergenicity of lactoglobulin, can improve the immunity of the body, and can be used for preparing medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the relationship diagram between magnetic field intensity and protein decomposition degree;

[0014] Figure 2 is the relationship between processing time and protein degradation degree;

[0015] Figure 3 Figure 2 is the weight change of mice in each group during the experiment;

[0016] Figure 4 Figure 1 is the effect of lactoglobulin peptide on the intestinal tissue of mice, wherein Figure A is the blank control group, Figure B is the low-dose lactoglobulin peptide group, Figure C is the medium-dose lactoglobulin peptide group, Figure D is the high-dose lactoglobulin peptide group, and Figure E is the original lactoglobulin group;

[0017] Figure 5 This is the RP-HPLC separation elution diagram of the middle desensitized component of lactoglobulin peptide;

[0018] Figure 6 Figure 1 is a mass spectrometry identification diagram of the polypeptide, wherein Figure A is a mass spectrometry identification diagram of sequence 1, and Figure B is a mass spectrometry identification diagram of sequence 2. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with embodiments and drawings.

[0020] Example 1: Preparation of lactoglobulin peptide

[0021] The production process of lactoglobulin powder is as follows:

[0022] 1) Place a certain amount of DEAE52 (weak anion exchange resin of diethylaminoethyl cellulose) into a beaker, add 0.01Mol / L pH 8.0 phosphate buffer, let stand for 30 minutes, remove the supernatant particles, and repeat once.

[0023] 2) Filter using a Buchner funnel (with two layers of filter paper inside).

[0024] 3) Add all ingredients in a ratio of 500 g wet weight of DEAE-cellulose to 100 ml of milk and 300 ml of distilled water, and stir thoroughly.

[0025] 4) Place at 4°C for 1 hour, stirring several times.

[0026] 5) Filter with a Buchner funnel, then rinse the cellulose with 0.01Mol / L pH 8.0 PB buffer and filter with suction. The filtrate is the extracted whey protein solution.

[0027] 6) The lactoglobulin powder is obtained by freeze drying.

[0028] 5 g of lactoglobulin powder was soaked with 30% water and placed in an ultra-high magnetic field (UMF) device. The magnetic field strength was set to 5 T, 10 T, 15 T and 20 T respectively through charging voltage control and discharge current monitoring.

[0029] Depend on Figure 1 It can be seen that with the increase of magnetic field intensity, the decomposition rate of protein gradually increases. When the magnetic field intensity is 15T, the decomposition rate reaches the maximum. When it exceeds 15T, the hydrolysis rate gradually decreases.

[0030] The magnetic field intensity was set to a fixed value of 15 T, and the effect of different treatment times (5s, 10s, 15s, 20s, 25s) on the decomposition rate of protein peptides was studied.

[0031] Figure 2 It can be seen that under the condition of a magnetic field intensity of 15 T, with the increase of treatment time, the decomposition rate of protein peptides showed an upward trend and gradually stabilized after 15 s, so a treatment time of 15 s was selected as the optimal condition.

[0032] Therefore, the best effect of preparing lactoglobulin peptide was achieved under the conditions of magnetic field intensity of 15T and treatment time of 15s.

[0033] Example 2: Desensitization effect of lactoglobulin peptide

[0034] Select several SPF-grade 4-week-old female BALB / c mice and adapt them to the new environment for one week before the experiment. The feeding conditions are 23℃±2℃ and 60%±20% relative humidity. The mice change drinking water and feed every day, and they can eat and drink freely. There are 12 hours of light and 12 hours of darkness every day. On the first, third and seventh days of the experiment, 0.2mL of sterile saline containing 50μg lactoglobulin and 0.1mL of Freund's adjuvant was used for intraperitoneal injection for sensitization. The modeled mice showed allergic symptoms such as scratching, messy hair, weight loss, reduced autonomous activity, diarrhea, and purple lips, which proved that the food allergy modeling of mice was successful.

[0035] The mice after modeling were randomly divided into 4 groups, with 6 mice in each group, including the low, medium and high dose groups of lactoglobulin peptide feeding [50, 100, 200 mg / (kg·d)], the original lactoglobulin group [100 mg / (kg·d)], and the same-age mice without modeling as the blank control group (normal saline), which were gavaged once a day, and the mice were weighed and recorded before gavage for 28 consecutive days. During this period, the mice were free to eat and drink water.

[0036] 1. Effect of lactoglobulin peptide on body weight of allergic model mice

[0037] Depend on Figure 3 It can be seen that the body weight of mice in the blank group, high-, medium- and low-dose lactoglobulin peptide groups gradually increased with the extension of feeding time, while the body weight of mice in the original lactoglobulin group continued to decrease with the extension of feeding time, and even died, indicating that lactoglobulin caused and continued the allergic reaction of mice, and the lactoglobulin peptide groups were separated from the allergens after the start of the experiment, and the allergic reaction of the modeling mice stopped.

[0038] 2. Effects of lactoglobulin peptide on the intestines of allergic model mice

[0039] Jejunal tissue pathological sections can reflect the intestinal damage status of mice caused by food allergy ( Figure 4 ). In order to explore the intestinal damage of mice caused by food allergy in each group, the jejunal damage of mice was compared through HE pathological sections of jejunum tissue. The results showed that compared with the blank control group, the small intestinal villi in the lactoglobulin original group had a certain degree of shedding, and the arrangement was loose, there was inflammatory cell infiltration, and the lamina propria was loosely arranged, indicating that lactoglobulin caused allergic reactions in mice, while the allergic reactions of mice in the lactoglobulin peptide groups were alleviated, and this result was consistent with the changes in the body weight of mice.

[0040] The above results indicate that the lactoglobulin peptide decomposed after being subjected to extreme magnetic field strength has an alleviating effect on weight loss and pathological symptoms caused by food allergy in mice. The results indicate that the prepared lactoglobulin peptide removes the allergenic substances carried by lactoglobulin and reduces the allergenicity of lactoglobulin.

[0041] Example 3: Purification and sequence identification of lactoglobulin peptides

[0042] As can be seen from Example 2, lactoglobulin peptide showed a good beneficial effect in alleviating the body weight of allergic mice, and during the 4-week experiment, the growth curve of body weight of mice in each lactoglobulin peptide group was higher than that of the blank control group.

[0043] The lactoglobulin peptide prepared in Example 1 was separated and purified by RP-HPLC to obtain three elution components with different molecular weights ( Figure 5 ).

[0044] 40 SPF BALB / c male mice (weight 18-22g) were randomly divided into 4 groups. The lactoglobulin peptide was fed to the A1, A2, A3 and white control groups (normal saline). The mice were gavaged with a volume of 0.2mL / 10gbw once a day. The gavage was continued for 30 days, and the body weight was measured before and after the experiment. After the mice were weighed, they were killed by cervical dislocation, dissected, and the spleen and thymus were removed. The blood was dried with filter paper and weighed. The organ / body weight ratio was determined.

[0045] Table 1: Effects of the test substances in each dose group on the body weight of the experimental group mice

[0046]

[0047] As shown in Table 1, the mice in each group grew and developed well, and their weight increased steadily. Compared with the negative control group, there was no significant change in the weight of the mice in each dose group.

[0048] Table 2: Effects of the test substances in each dose group on the ratio of lymphoid organs to body weight in mice

[0049]

[0050] After analysis (Table 2), there were extremely significant differences in the thymus / body weight ratio between the A2 group and the control group, P<0.01. There were no significant differences in the spleen weight ratio between the various dose groups and the control group, P>0.05.

[0051] The spleen is the largest lymphoid organ in the human body and is involved in a variety of immune responses. T lymphocytes are indispensable to the development of the entire lymphoid organ and the body's immunity, and the thymus produces T lymphocytes, so the enlargement of the thymus will lead to an increase in T lymphocytes and improve the body's immunity. As shown in Table 2, the A2 component has a good effect on improving the body's immunity.

[0052] The peptide sequence of component A2 was analyzed by LC-MS / MS, and the peptide sequences with confidence interval>96% were selected. Two highly active peptides were identified by mass spectrometry, and their sequences were LLDAQSAPLR (SEQ ID NO: 1) EGDLEILLQK (SEQ ID NO: 2). The mass spectrometry identification figure is shown in Figure 6 shown.

[0053] The polypeptides with the sequences of LLDAQSAPLR and EGDLEILLQK were synthesized, and after modeling experiments, 0.2 mL of 2 mg / mL allergen (1 mL of Freund's adjuvant + 1 mL of 2 mg / mL lactoglobulin) was intraperitoneally injected into the model mice, and 0.2 mL of 2 mg / mL polypeptide solution was injected into the experimental group, and the IgE content in the serum of each group of mice was measured. The experimental results showed that the desensitization of mice injected with polypeptides LLDAQSAPLR and EGDLEILLQK was significantly improved compared with the model group of the polypeptide prepared in Example 2, and the IgE level in the serum was significantly reduced, which verified that the two polypeptides effectively avoided the allergy of mice induced by lactoglobulin.

[0054] Example 4: Immunity-enhancing effect of lactoglobulin peptide

[0055] Thirty SPF BALB / c male mice (body weight 18-22 g) were randomly divided into three groups, group 1 (polypeptide sequence: LLDAQSAPLR), group 2 (polypeptide sequence: EGDLEILLQK) fed with lactoglobulin peptide at a feeding rate of 100 mg / (kg·d) and a blank control group (normal saline), the gavage volume of mice was 0.2 mL / 10 gbw, and gavage was performed once a day.

[0056] 1. Effect of lactoglobulin peptide on immune function of mice

[0057] After continuous gavage for 30 days, the abdominal skin of each mouse was depilated with a depilatory agent, covering an area of ​​about 3cm×3cm, and then 50μL of DNFB solution was evenly applied to induce sensitization. After 5 days, 10μL of DNFB solution was evenly applied to both sides of the right ear of the mouse for attack. After 24 hours, the mice were killed by cervical dislocation, the left and right ears were cut off, and the ear pieces with a diameter of 5mm were removed with a puncher and weighed.

[0058] Table 3: Effect of lactoglobulin peptide on delayed-type hypersensitivity (DTH) in mice

[0059]

[0060] As shown in Table 3, Group 1 has a better immune effect.

[0061] 2. Effects of lactoglobulin peptide on humoral immunity in mice

[0062] After continuous gavage for 30 days, mice were immunized by intraperitoneal injection of 0.2 mL of cell suspension containing 2% (v / v) SRBC. After 5 days, the eyeballs of mice were removed and blood was collected in a centrifuge tube. The tube was placed for about 1 hour to allow the serum to precipitate. The serum was collected by centrifugation at 2000 r / min for 10 minutes. The serum was diluted with saline (300 times), 1 mL of the diluted serum was placed in a test tube, and 0.5 mL of 10% (v / v) SRBC and 1 mL of complement (diluted with saline at 1:10) were added in sequence. A control tube without serum was set up (replaced with saline), and the tube was placed in a 37°C constant temperature water bath for 15 to 30 minutes, and then the reaction was terminated by ice bath. Centrifuge at 2000r / min for 10min, take 1mL of supernatant and 3mL of Dubonnet's reagent into a test tube, and at the same time take 0.25mL of 10% (v / v) SRBC and add Dubonnet's reagent to 4mL, put into another test tube, mix thoroughly, let stand for 10min, and measure the optical density of each tube at 540nm using the control tube as blank.

[0063] Table 4: Half hemolytic value (HC) of lactoglobulin peptides on mice 50 )

[0064]

[0065] As shown in Table 4, Group 1 has a more obvious immune effect, while Group 2 is not much different from the control group.

[0066] 3. Effects of lactoglobulin peptide on cellular immunity in mice

[0067] After continuous gavage for 30 days, the mice were killed by cervical dislocation, and the spleen was removed aseptically and placed in a small dish containing an appropriate amount of sterile Hanks solution. The spleen was gently torn into pieces with tweezers and filtered through a 200-mesh sieve to make a single-cell suspension. The cells were washed three times with Hanks solution, each time centrifuged for 10 min (1000 r / min), and then the cells were suspended in 2 ml of complete culture medium. The number of live cells was counted by trypan blue staining (should be above 95%), and the cell concentration was adjusted to 5×10 with RPMI1640 complete culture medium. 6 cfu / mL. Target cells were subcultured 24 h before the test, washed 3 times with Hanks solution before use, and the cell concentration was adjusted to 1x10 with RPMI1640 complete culture medium. 5cfu / mL; 100mL of target cells and effector cells (effector-target ratio 50:1) were added to a 96-well culture plate: 100μL of target cells and culture medium were added to the target cell natural release well, and 100wL of target cells and 1% NP40 were added to the target cell maximum release well. Each of the above items was set up with 3 replicates. The cells were cultured in an incubator at 37°C and 5% CO for 4h, and then the culture plate was centrifuged (1500r / min) for 5min. 100μL of supernatant was aspirated from each well and placed in a 96-well culture plate. 100mL of LDH matrix solution was added at the same time. The reaction was continued for 3min. 30μL of 1mol / L HCl was added to each well, and the density value (OD) was measured at 490nm on an enzyme reader.

[0068] Table 5: Effect of lactoglobulin peptide on mouse NK cell activity

[0069]

[0070] From the results in Table 5, we can see that there is a significant difference between group 1 and the control group, P<0.05.

[0071] The above results indicate that the lactoglobulin peptide LLDAQSAPLR (SEQ ID NO: 1) has effects on both cellular and humoral immune functions and has the efficacy of enhancing immunity, while the polypeptide with the sequence EGDLEILLQK does not significantly improve the immune effect.

[0072] In summary, the lactoglobulin peptide prepared by the present invention effectively eliminates the allergenicity of lactoglobulin and can improve the body's immunity, providing a new idea for the research and development of anti-allergic functional products.

Claims

1. Use of a lactoglobulin peptide having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of a medicine for improving immunity.

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