Abalone polypeptide for improving balance ability and application

Abstract

The application belongs to the field of small molecule polypeptide preparation, and particularly relates to an abalone polypeptide for improving balance ability and application. The amino acid sequence of the abalone polypeptide for improving balance ability is NFLVWVNEEDHLR. The abalone polypeptide has small molecular weight and is easy to absorb. The abalone polypeptide can not only increase the survival time of nematodes, but also has excellent antioxidant capacity. Moreover, the abalone polypeptide can significantly improve the balance ability of Caenorhabditis elegans in multiple dimensions. The balance ability refers to the swing frequency, turning ability and paralysis ratio of Caenorhabditis elegans. The abalone polypeptide can be further used for the development of products such as medicines and health products, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of small molecule polypeptide preparation, and particularly relates to a haliotis discus hannai polypeptide for improving balance ability and application. BACKGROUND

[0002] In the process of human growth and development, various movement behaviors occur. Although the movement ability has certain correlation with the individual health condition, the level of movement ability cannot be completely equal to the good or bad of the health condition. Human gait refers to a walking manner, and refers to the movement realized by the continuous activities of the hip, knee, ankle and toes of the human body. It is a two-foot movement of moving the center of gravity in a certain direction. The walking ability has two basic components: balance and movement. The balance refers to the ability to take an upright posture and maintain balance; the movement refers to the ability to initiate and maintain a rhythmic pace. However, when the human body gradually ages or has diseases, these gait characteristics will change obviously. For example, the muscle mass and strength of the elderly decrease rapidly with age, which is manifested as weak lower limb muscle strength, and will lead to abnormal balance and gait; a considerable part of patients with diseases will also show abnormal conditions such as unstable gait, uneven foot pressure and walking disorder. Therefore, the difference in gait change can obviously reflect the change of the health condition of the organism, and is expected to be used as a prediction index of the pre-aging or pre-disease.

[0003] Similar to human aging, the decline of movement ability is also one of the obvious characteristics of aging of model animals Caenorhabditis elegans. Studies have shown that the decline of movement ability can be used as a health indicator of Caenorhabditis elegans. Caenorhabditis elegans can be cultured and measured in two different movement states of crawling and swimming in solid and liquid culture systems. In principle, the surface tension of agar, the force of embedding the groove of the nematode, the friction between the two and other factors also need to be considered in the behavioral study on the solid substrate. In the liquid substrate, Caenorhabditis elegans is less disturbed by external factors, and it is easier to process the characteristics of its movement gait. In addition, the amplitude of the swimming nematode movement is longer, and the time frequency is faster, and these differences also emphasize the physical adaptability of the movement gait to the external medium. The movement patterns in the two different states have their own characteristics, and researchers can choose the appropriate culture method according to the specific needs, so Caenorhabditis elegans is a very suitable model for studying balance ability.

[0004] Abalone is an animal of the genus Haliotis in the family Haliotidae of the order Archaeogastropoda of the phylum Mollusca, which is widely distributed in coastal waters of temperate and tropical zones. Abalone, known as the "crown of seafood", is popular in China. As a kind of delicious seafood with high economic value, the artificial culture of abalone has been rapidly developed in China in recent years. Abalone is characterized by high protein, low fat and complete amino acids, and is rich in vitamin E and trace elements, and its nutritional value is widely recognized in different countries and cultures. Recent studies have shown that abalone contains various bioactive molecules such as polysaccharides, proteins and fatty acids, and has potential activities of antioxidant, anti-thrombosis, anti-inflammatory, antibacterial and anti-cancer. Studies have shown that the protein hydrolysate derived from abalone has various biological activities. SUMMARY

[0005] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present application is to provide an abalone polypeptide for improving balance ability. The abalone polypeptide can not only increase the survival time of Caenorhabditis elegans, but also has excellent antioxidant capacity and can improve balance ability in multiple dimensions.

[0006] Another purpose of the present application is to provide a preparation method of the abalone polypeptide for improving balance ability.

[0007] Still another purpose of the present application is to provide the application of the abalone polypeptide for improving balance ability.

[0008] The purposes of the present application are achieved by the following technical solutions:

[0009] An abalone polypeptide for improving balance ability, wherein the amino acid sequence is NFLVWVNEEDHLR.

[0010] The preparation method of the abalone polypeptide for improving balance ability comprises the following steps:

[0011] The abalone polypeptide is directly prepared by solid-phase synthesis, or is prepared by combining trypsin and papain enzymolysis and purification using Haliotis diversicolor as raw material;

[0012] The specific operation of the combined trypsin and papain enzymolysis is preferably as follows:

[0013] (1) The pre-processed Haliotis diversicolor living body is cut into small pieces and homogenized, and the homogenate is freeze-dried to obtain abalone meat freeze-dried powder;

[0014] (2) The abalone meat freeze-dried powder and water are mixed, preheated in a water bath at 45 DEG C for 10-20 min, then the pH of the system is adjusted to 8.0, trypsin is added, and the reaction is carried out at 45 DEG C for 2-6 h; then the enzymolysis solution is preheated in a water bath at 60 DEG C for 10-20 min, the pH of the system is adjusted to 6.0, papain is added, and the reaction is carried out at 60 DEG C for 2-6 h; inactivation, to obtain abalone protein enzymolysis solution;

[0015] The purification comprises steps of alcohol extraction, membrane ultrafiltration separation and gel chromatography separation, etc.

[0016] The specific operation of the alcohol extraction is preferably as follows:

[0017] The abalone proteolysate and ethanol are mixed, and then centrifuged by suction filtration, the supernatant is collected, the ethanol in the supernatant is further removed, and freeze-drying is performed to obtain abalone crude polypeptide dry powder;

[0018] The specific operation of the ultrafiltration separation is as follows:

[0019] Ultracel-3 and Ultracel-10 filter membranes are used for separation, and components with a molecular weight less than 3 kDa are collected;

[0020] The specific operation of the gel chromatography separation is as follows:

[0021] After the components after ultrafiltration are desalted, Sephadex G-25 dextran gel chromatography column is used for separation;

[0022] The impurities are preferably removed by using a 0.45 μm filter membrane;

[0023] The abalone polypeptide for improving balance ability is applied to preparation of an antioxidant product;

[0024] The abalone polypeptide for improving balance ability is applied to preparation of a balance ability improving product;

[0025] The balance ability is at least one of swing frequency, turning ability and paralysis ratio;

[0026] The abalone polypeptide for improving balance ability is applied to the field of pharmaceutical products or health products;

[0027] An antioxidant product comprises at least one of the abalone polypeptide, abalone proteolysate containing the abalone polypeptide and hydrolysate containing the abalone polypeptide as an active ingredient;

[0028] A balance ability improving product comprises at least one of the abalone polypeptide, abalone proteolysate containing the abalone polypeptide and hydrolysate containing the abalone polypeptide as an active ingredient;

[0029] A pharmaceutical product comprises at least one of the abalone polypeptide, abalone proteolysate containing the abalone polypeptide and hydrolysate containing the abalone polypeptide as an active ingredient;

[0030] The present application has the following advantages and effects relative to the prior art:

[0031] (1) The polypeptide obtained from H. discus is brand new, and its amino acid sequence is NFLVWVNEEDHLR. The polypeptide can be obtained by separation and purification from H. discus or by chemical synthesis.

[0032] (2) The polypeptide has small molecular weight and is easy to absorb, can increase the survival time of nematodes, and has excellent antioxidant capacity.

[0033] (3) The polypeptide significantly improves the balance ability of C. elegans in multiple dimensions, wherein the balance ability refers to the swing frequency, turning ability and paralysis ratio of C. elegans.

[0034] (4) The polypeptide can be further used for the development of products such as medicines and health products, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a Sephadex G-25 dextran gel chromatography separation chromatogram.

[0036] Figure 2 is an antioxidant capacity screening result diagram of the polypeptide.

[0037] Figure 3 is an AbaPep#383 polypeptide swing frequency result analysis diagram of C. elegans.

[0038] Figure 4 is an AbaPep#383 polypeptide turning ability result analysis diagram of C. elegans.

[0039] Figure 5 is an AbaPep#383 polypeptide paralysis ratio result analysis diagram of C. elegans. DETAILED DESCRIPTION

[0040] The application will be further described in detail below in combination with the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0041] The concentrated NA22 bacterial solution in the embodiment is the food of C. elegans, and the preparation method is as follows: after the activated E. coli NA22 is inoculated into the LB liquid medium and oscillated and cultured, the bacterial body is collected by centrifugation to obtain the concentrated NA22 bacterial solution.

[0042] The wild type C. elegans N2 is from the Caenorhabditis Genetics Center of the University of Minnesota, USA.

[0043] Example 1: Isolation and identification of abalone polypeptide

[0044] 1. Enzymatic separation

[0045] (1) Take the live H. discus, remove the shell and internal organs, wash with clean water, cut into small pieces, homogenize, freeze-dry the homogenate, take 5 g of the freeze-dried powder of H. discus, add 100 mL of deionized water, stir with a glass rod to make it uniform, preheat in a 45°C constant temperature water bath, then adjust the pH of the solution to 8.0 with NaOH, add 1 mL of trypsin (15000 U / mL), react at 45°C for 4 h; then adjust the pH of the solution to 6.0 with HCl, add 1 mL of papain (15000 U / mL), react at 60°C for 4 h; boil to inactivate, to obtain the H. discus protein hydrolysate;

[0046] (2) After the H. discus protein hydrolysate prepared in step (1) is naturally cooled to room temperature, 170 mL of 95% ethanol by volume is slowly added under stirring with a glass rod, and the mixture is left to stand at room temperature overnight, then subjected to reduced pressure filtration and centrifugation (4°C, 4000 rpm, 5 min) to remove the precipitate, the supernatant is subjected to reduced pressure rotary evaporation to remove ethanol, then freeze-dried to obtain H. discus crude polypeptide dry powder.

[0047] 2. Ultrafiltration purification

[0048] (1) The H. discus crude polypeptide dry powder prepared in step 1 is prepared into a 20 mg / mL solution, which is subjected to ultrafiltration using an Amicon Ultra-2 centrifugal filter equipped with an Ultracel-3, Ultracel-10 filter membrane of Millipore, and the component with a molecular weight less than 3 kDa is collected; then the impurities are removed through a 0.45 μm filter membrane, and the salts in the polypeptide sample are removed using a desalting column;

[0049] (2) The polypeptide component after desalting in step (1) is further separated using a Sephadex G-25 dextran gel chromatography column, eluted with deionized water at a flow rate of 1 mL / min, and monitored at 280 nm using a 785 UV / VIS detector while collecting the fractions of each absorption peak.

[0050] Figure 1 is a Sephadex G-25 dextran gel chromatography separation chromatogram, from which it can be seen that the polypeptide component after desalting in step (1) is further separated using Sephadex G-25 dextran gel, and four peaks are obtained, which are named F1, F2, F3 and F4 components, respectively, and the F2 component is freeze-dried to obtain polypeptide dry powder.

[0051] 3. LC-MS / MS identification

[0052] The polypeptide composition and amino acid sequence of the F2 component are identified by LC-MS / MS.

[0053] 4. Database comparison screening

[0054] According to the polypeptide sequence information identified in the BIOPEP-UWM database provided with antioxidant activity, the sequenced H. discus multiple polypeptide sequences were compared with the database for virtual activity screening. The specific operation steps are as follows: log in to the BIOPEP-UWM website, select the Bioactive peptides database, select search "antioxidative", by "Activity". The polypeptide sequences with antioxidant activity in the database were compared with the abovementioned polypeptide sequences of abalone identified by liquid chromatography-mass spectrometry (LC-MS) in step 3. The abalone polypeptide sequences containing 2 or more antioxidant amino acid fragments were selected as the polypeptide sequences of H. discus multiple with potential antioxidant activity for the following tests.

[0055] Example 2 Solid-phase synthesis of abalone polypeptide

[0056] The candidate peptide segments screened in Example 1 were synthesized by solid-phase synthesis by Shanghai Taopu Biological Technology Co., Ltd. The specific method is as follows:

[0057] The carboxyl group of the first amino acid is covalently linked to the carrier resin, and then the amino group of this amino acid is used as the reaction starting point to undergo acylation reaction with the carboxyl group of the adjacent amino acid to form a peptide bond. This process is repeated until the target polypeptide is synthesized. The Fmoc protecting group is removed and the resin is dried. Then, a cutting solution (97.50% TFA + 2.50% H2O, volume percentage) of 6 times the volume of the resin is used for cutting. The precipitate is washed with anhydrous ether for 3 times to obtain the crude polypeptide. The polypeptide with a purity of more than 95% is obtained by high-performance liquid chromatography separation and purification.

[0058] Example 3 Screening of antioxidant capacity of H. discus multiple polypeptide

[0059] In order to verify whether the candidate peptide segments prepared by solid-phase synthesis in Example 2 have antioxidant activity, this embodiment uses the Caenorhabditis elegans model to detect their antioxidant activity. The specific method is as follows:

[0060] (1) Add 80 μL of a mixture of 5-fluorouracil (5-FUdR), ampicillin (AMP), concentrated NA22 bacterial liquid and S. Medium culture solution to each well of a 96-well plate, and then add 10 μL of a polypeptide sample solution. The control group adds 10 μL of S. Medium, and then adds 10 μL of synchronized L4 stage wild-type Caenorhabditis elegans N2 (density about 20 worms / 10 μL, more than 100 worms per group) to each well. In the above 100 μL system, the final concentration of 5-FUdR is 75 μg / mL, the final concentration of AMP is 100 μg / mL, the OD 570 of the system is controlled to 0.5 after adding the concentrated NA22 bacterial liquid, and the final concentration of the polypeptide sample is 2.0 mM.

[0061] (2) After incubation at 20℃, 120 rpm for 24 h, paraquat was added to each well at a final concentration of 50 mmol / L, and then the survival of C. elegans in each well was counted every 12 h. The difference in the area under the survival curve between the polypeptide group and the control group was calculated to obtain ΔAUC%.

[0062] The results are shown in Table 1. Figure 2 The greater the ΔAUC% indicates that the polypeptide increases the survival time of the nematode and the stronger the antioxidant capacity. Finally, the best antioxidant sequence AbaPep#383 (sequence number: AbaPep#383) was screened out.

[0063] Example 4 AbaPep#383 Abalone Polypeptide Improves Swing Frequency

[0064] (1) Wild-type C. elegans N2 was cultured in a liquid 96-well plate to the L4 stage to the early adult stage, and the worm liquid was taken into a 1.5 mL centrifuge tube, washed 2-3 times with S Medium culture solution, and the residual bacterial liquid was removed. After adjusting the worm density, the nematodes were divided into a blank group and a polypeptide group for experiment in a new 96-well plate. Specifically, 5-FUdR, AMP, concentrated NA22 bacterial liquid, polypeptide sample, nematodes and S Medium were added to each well, wherein the final concentration of 5-FUdR was 75 μg / mL, the final concentration of AMP was 100 μg / mL, the final concentration of polypeptide sample was 1 or 2 mM, the OD 570 of the system was controlled to be 0.5 after adding the concentrated NA22 bacterial liquid, the worm density was controlled to be 30-40 per well, and S Medium was added to 100 μL; the control group used an equal amount of S Medium instead of the polypeptide sample. At this time, it was recorded as the 0th day of the adult, and was placed in a 120 r / min, 20℃ constant temperature incubator shaker for culture to the 10th day.

[0065] (2) C. elegans N2 was taken out of the well plate and placed in a 1.5 mL centrifuge tube, washed 2-3 times with S Medium culture solution, and 50 μL of worm liquid (5-10 nematodes were appropriate) was taken and placed on a glass slide. After about 10 s of adaptation, a 30 s swimming analysis video was recorded. The "BBPS" parameter in the output data was the swing frequency, which was recorded and analyzed by the wrMTrck plug-in of the motion tracker software ImageJ.

[0066] The experimental results are shown in Table 2. Figure 3 Compared with the control, AbaPep#383 polypeptide significantly improved the swing frequency of C. elegans.

[0067] Example 5 AbaPep#383 Abalone Polypeptide Improves Turning Ability

[0068] (1) The specific operation is the same as that in Example 4;

[0069] (2) The wild-type C. elegans N2 was taken out from the well plate, placed in a 1.5 mL centrifuge tube, washed 2-3 times with S Medium culture solution, and 20 μL of worm solution (controlling the worm amount to be 30-50) was placed on a 3.5 cm NGM agar plate without food.

[0070] (3) After 10 min (3-5 min were needed for the liquid in the worm solution to be absorbed by the agar plate and for the C. elegans to change from an aggregated state to a dispersed state, and then to freely crawl to adapt to the new environment and enter a relatively stable movement state, the determination could be started), a 30 s crawling analysis video was recorded, the wrMTrck plug-in in the movement tracker software ImageJ was used to record and process and analyze the video file, and the "Direction" parameter in the output data was the number of turns and the turning index was calculated:

[0071] Turning index = N 多肽组低于平均 / N 对照组总体 ; wherein N 多肽组低于平均 is the number of C. elegans with the number of turns less than the average number of turns of the control group in the polypeptide group, and N 对照组总体 is the total number of worms in the control group.

[0072] The results are shown in Table 1. Figure 4 Compared with the control, the AbaPep#383 polypeptide significantly increased the turning index of C. elegans and improved the turning ability.

[0073] Example 6 AbaPep#383 Abalone Polypeptide Reduces Paralysis Ratio

[0074] (1) The specific operation is the same as that in Example 4;

[0075] (2) The wild-type C. elegans N2 was taken out from the well plate, placed in a 1.5 mL centrifuge tube, washed 2-3 times with S Medium culture solution, and 20 μL of worm solution (controlling the worm amount to be 30-50) was placed on a 3.5 cm NGM agar plate without food.

[0076] (3) After 10 min (3-5 min were needed for the liquid in the worm solution to be absorbed by the agar plate and for the C. elegans to change from an aggregated state to a dispersed state, and then to freely crawl to adapt to the new environment and enter a relatively stable movement state, the determination could be started), a 30 s crawling analysis video was recorded, the wrMTrck plug-in in the movement tracker software ImageJ was used to record and process and analyze the video file, and the "Fraction Paralyzed" parameter in the output data was the paralysis ratio.

[0077] Results as shown in Table 1, AbaPep#383 polypeptide significantly reduced the paralysis rate of C. elegans compared with the control. Figure 5

[0078] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.​

Claims

1. An abalone polypeptide that enhances balance, characterized in that... Its amino acid sequence is NFLVWVNEEDHLR.

2. The method for preparing the abalone polypeptide for enhancing balance as described in claim 1, characterized in that... It includes the following steps: The above-mentioned abalone polypeptides can be prepared directly through solid-phase synthesis, or by using abalone as raw material and obtaining abalone polypeptides through combined enzymatic hydrolysis and purification with trypsin and papain.

3. The method for preparing abalone polypeptides that enhance balance according to claim 2, characterized in that: The purification process includes alcohol extraction, membrane ultrafiltration, and gel chromatography.

4. The application of the abalone polypeptide for enhancing balance as described in claim 1 in the preparation of antioxidant health products.

5. An antioxidant health product, characterized in that... It contains the abalone polypeptide as described in claim 1.

6. A health product, characterized in that... It contains the abalone polypeptide as described in claim 1.

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