Extraction method of artemia cyst yellow protein and application of artemia cyst yellow protein to zebra fish wound repair activity
By extracting and preparing yolk protein from artemia eggs, the problem of unknown nutritional value of artemia is solved, and a significant improvement in wound repair and antioxidant functions of aquatic organisms has been achieved.
Patent Information
- Application Number
- CN202411882129.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-04-29
AI Technical Summary
The nutritional value and efficacy components provided by artemia to aquatic organisms are not yet known, and it is difficult to provide targeted bait components required by aquatic organisms.
Compositions that promote wound repair are prepared by extracting the incubated artemia yolk protein, and using frozen thawing cycle, isoelectroprecipitation and dialysis.
This method effectively promotes tissue wound repair in zebrafish, improves its antioxidant function, significantly increasing the regeneration area and motility vitality of the caudal fin.
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Figure CN119896721A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to an extraction method of Artemia yolk protein and application thereof to the wound repair activity of zebrafish, and belongs to the field of biomedicine. Background Art
[0002] Brine shrimp, also known as brine artemia, is an aquatic animal of the subphylum Crustacea, class Branchiopoda, order Acaridae, family Artemia, genus Artemia. It is mainly distributed in Tibet and Bohai Bay, my country, and is an important local biological resource. Artemia generally survives as dormant eggs, can withstand a variety of extreme external environments, and has a long survival period. Artemia dormant eggs are often used as a starter bait for various aquatic organisms in the aquaculture field and are widely used in the aquaculture industry. They have the advantages of high nutritional value and ease of use.
[0003] Although Artemia is widely used in aquaculture, the nutritional value and efficacy of Artemia as a food source for aquatic organisms after hatching is still unclear. Since different types of aquatic organisms have different nutritional requirements under different physiological conditions, it is necessary to comprehensively evaluate the efficacy of Artemia, find out the content and types of active ingredients of Artemia as a starter bait, and further expand the application scope of Artemia eggs in the field of aquaculture or new resource protein food, so as to prepare the bait ingredients required by aquatic organisms in a targeted manner, and at the same time, fully explore and reasonably use the local Artemia resources for its functional active ingredients. Summary of the invention
[0004] Technical issues: The nutritional value and effective components that Artemia provides to aquatic organisms are still unclear, making it difficult to provide targeted feed ingredients required by aquatic organisms.
[0005] Technical solution: To solve the above problems, the present invention provides the application of Artemia yolk protein after hatching in the wound repair functionality of zebrafish tissues.
[0006] The invention provides application of a composition with egg yolk protein as a main active substance in preparing a product for promoting wound repair.
[0007] In one embodiment, the amino acid sequence of the yolk protein is as shown in SEQ ID NO.1.
[0008] In one embodiment, in the product, the content of the egg yolk protein is not less than 50 mg / L or 0.1 mg / g.
[0009] In one embodiment, the product is for use in aquatic life.
[0010] In one embodiment, the aquatic organism includes but is not limited to zebrafish.
[0011] In one embodiment, the preparation method of the composition with egg yolk protein as the main active substance is:
[0012] (1) dissolving the hatched Artemia in a deep eutectic solvent containing 10-14% w / v sodium hydroxide and 3%-6% w / v urea, and performing 2-4 freeze-thaw cycles to obtain sample 1;
[0013] (2) dissolving the sample 1 obtained in step (1), shaking for at least 2 hours, and collecting the precipitate by centrifugation;
[0014] (3) dissolving the precipitate obtained in step (2) in a sodium chloride solution, shaking it, and centrifuging to collect the filtrate;
[0015] (4) adjusting the pH of the filtrate in step (3) to 3.9-4.7 to obtain egg yolk crude protein;
[0016] (5) dialyzing and drying the crude egg yolk protein in step (4) to obtain egg yolk protein.
[0017] In one embodiment, the product includes feed or medicine, and the medicine further contains conventional carriers and / or excipients.
[0018] In one embodiment, the conventional carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.
[0019] In one embodiment, the auxiliary material comprises an excipient and / or an additive.
[0020] In one embodiment, the dosage form of the drug includes powder, granules, capsules, tablets, pills or oral liquid.
[0021] The present invention also provides the use of egg yolk protein in preparing products with antioxidant function, and the amino acid sequence of the egg yolk protein is shown as SEQ ID NO.1.
[0022] In one embodiment, the product includes feed, medicine or health care product.
[0023] In one embodiment, the antioxidant function includes at least one of the following:
[0024] (1) Improve the activity of superoxide dismutase and catalase;
[0025] (2) Reduce the activity of malondialdehyde.
[0026] Beneficial effects:
[0027] The present invention extracts yolk protein from Artemia eggs, and in vivo experiments confirm that the yolk protein is beneficial to the tissue wound repair of zebrafish and improves the anti-oxidation function of zebrafish. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 The protein images of egg yolk protein stained with Coomassie brilliant blue are (a) non-reduced form and (b) reduced form. M is the protein molecular weight standard. 1, 2 and 3 are three parallel samples respectively.
[0029] Figure 2 The electrophoresis of yolk protein stained with periodic acid-Schiff is (a) non-reduced form and (b) reduced form. M is the protein molecular weight standard. 1, 2 and 3 are three parallel samples.
[0030] Figure 3 The electrophoresis diagram of phosphoprotein stained with methyl green of egg yolk protein (a) non-reduced form (b) reduced form, M is the protein molecular weight standard, 1, 2 and 3 are three parallel samples respectively;
[0031] Figure 4 The electrophoresis of yolk protein stained with Sudan Black B is (a) non-reduced form and (b) reduced form. M is the protein molecular weight standard. 1, 2 and 3 are three parallel samples.
[0032] Figure 5 This is the spectrum of the characteristic sequence of yolk protein identified by mass spectrometry;
[0033] Figure 6 This is the result of homology comparison between Artemia yolk protein and yolk protein from other sources;
[0034] Figure 7 Quantitative graph of the effect of Artemia yolk protein on the wound repair area of zebrafish tail fin;
[0035] Figure 8 Typical pictures of Artemia yolk protein repairing the wound on the tail fin of zebrafish;
[0036] Fig. 9 Effects of yolk protein on the motor function of zebrafish;
[0037] Fig.10 Effect of Artemia salina yolk protein on antioxidant activity of zebrafish caudal fin wound repair. DETAILED DESCRIPTION
[0038] The present invention is further described in detail below in conjunction with implementation examples and drawings, but the implementation of the present invention is not limited thereto.
[0039] The present invention uses an alkali solution combined with a urea solution to destroy the chitin in the shell of the Artemia, and then uses an isoelectric precipitation method to extract the Artemia yolk protein. According to the characteristics of the protein, the protein components, sugar groups, phosphorus groups and lipid groups are specifically electrophoresed and stained, and a combined mass spectrometry analysis method is performed to confirm that the protein extracted by the salt dissolution method is the yolk protein.
[0040] Functional activity of Artemia egg yolk protein wound repair: 120 zebrafish larvae at the developmental stage of 3dpf were randomly divided into four groups, namely M (model) group, 25mg / L low-dose yolk protein group, 50mg / LVg medium-dose yolk protein group and 100mg / L high-dose yolk protein group, 30 in each group. Each time, a rubber-tipped dropper was used to draw one fish and placed it on a glass slide, and a 0.2mg / mL tricaine solution was added for anesthesia. After about 30s, the anesthetic solution was sucked away, and the glass slide was placed under a stereo microscope. The tail of the zebrafish was cut under the microscope using a pre-sterilized razor blade. After completion, a photo was taken and then placed in a 6-well plate with E3 water or different concentrations of yolk protein solution added in advance, and the culture medium was changed regularly every day.
[0041] Example 1: Extraction and identification of Artemia yolk protein
[0042] 1. Destroy the chitin on the outer wall of Artemia after hatching
[0043] Experimental method: Weigh a certain amount of Artemia egg powder, soak it in 2% salinity for 2 hours, and then incubate it with aeration at a certain temperature. Then dissolve the hatched Artemia in a low eutectic solvent containing 3%-6% urea in 10-14% sodium hydroxide, and undergo 2-4 ultra-low temperature freeze-thaw cycles during the process. The process is constantly stirred to completely destroy the chitin in the Artemia outer wall, and then separate and extract the target Artemia egg yolk protein source.
[0044] 2. Extraction of Artemia yolk protein by isoelectric precipitation after hatching Artemia eggs
[0045] Experimental method: Weigh a certain amount of Artemia sample with chitin removed from the outer wall, add ultrapure water at a solid-liquid ratio of 1:20-1:30, shake at 4°C for 2h, centrifuge at a speed of 6000r / min for 15min, repeat twice, collect Artemia egg residue, add 1mol / L NaCl solution at a solid-liquid ratio of 1:20, shake overnight at 4°C, centrifuge at a speed of 6000r / min for 15min, repeat twice, collect filtrate, and adjust the isoelectric point pH of the filtrate to 3.9-4.7 to obtain crude yolk protein. The crude yolk protein is dialyzed, the yolk protein solution obtained after dialysis is freeze-dried, and the resulting powder is collected, i.e., yolk protein, for subsequent animal experiments.
[0046] 3. Staining and identification of Artemia yolk protein based on specific staining method
[0047] The protein band changes were detected by specific staining electrophoresis, and Coomassie brilliant blue staining was used for protein components ( Figure 1 ), and the sugar groups were stained by periodic acid and Schiff's reagent ( Figure 2 ), and methyl green-molybdate was used for staining of phosphorus groups ( Figure 3 ), Sudan black was used for dyeing lipid groups ( Figure 4 ), and finally the decolorized film was photographed using a gel imager. The obtained egg yolk protein was analyzed by mass spectrometry. The results are as follows Figure 5 shown.
[0048] The results showed that after staining with Coomassie brilliant blue, periodic acid-Schiff reagent, methyl green-molybdate and Sudan black B, positive reactions appeared at the same position, proving that the extracted protein was indeed glycophospholipid protein. By comparing the difference in electrophoresis bands between the addition of β-ME (β-mercaptoethanol) in the loading buffer, it was proved that the protein existed as a polymer, and each monomer also contained glycophospholipid groups. Finally, the protein mainly aggregated around 110 kDa and 70 kDa, which was consistent with the molecular weight of 113.57 kDa of vitogenin-3 and 247.64 kDa of vitogenin-superoxide dismutase fusion protein found in the Artemia egg protein group by mass spectrometry (as shown in Table 1). It was preliminarily determined that the 110 kDa band on the film was vitogenin with a molecular weight of 113.57 kDa, and the remaining bands were vitogenin-superoxide dismutase fusion proteins with a molecular weight of 247.64 kDa.
[0049] Table 1 Mass spectrometry identification of the composition of Artemia egg proteins after hatching
[0050]
[0051] 4. Homology comparison between Artemia yolk protein and yolk protein from other sources
[0052] The amino acid sequence of Artemia egg vitellogenin (shown in SEQ ID No. 1) was analyzed by BLAST using UniProt database. The results showed that the vitellogenin from Artemia egg had poor homology with other species. The analysis of the overall sequence showed that it was closest to superoxide dismutase, but the similarity was only 29.9%. Therefore, this protein is a new type of protein, which differs greatly from other proteins in sequence, and also differs greatly from vitellogenin from other sources in structure and function ( Figure 6 ).
[0053] Example 2: Artemia yolk protein promotes wound repair in zebrafish
[0054] Take zebrafish larvae 72 hours after fertilization, place the larvae in a 0.2mg / mL tricaine solution for anesthesia for a few seconds, and then remove 5mm of the tail fin under a microscope with a sterilized blade. The larvae after tail cutting are placed in E3 culture solution containing different concentrations (25mg / L, 50mg / L, 100mg / L) of Artemia protein, and the control group (M) places the larvae after tail cutting in E3 culture solution. The tail-cut zebrafish are observed for the growth and repair of the tail fin at 24hpa, 48hpa, 72hpa, and 96hpa. The tail tissue repair is observed and photographed under an inverted microscope, and then the regeneration area of the tail of each larva is measured using image processing software.
[0055] Experimental results: Quantitative analysis of the repair area of the tail fin tissue found that ( Figure 7 and Figure 8 ), Artemia yolk protein treatment group significantly increased the regeneration area of the tail fin at 24hpa and 48hpa, at 48hpa, when the protein concentration was 50mg / L, the tail fin regeneration ability was the strongest, at 96hpa, there was no statistical difference in the regeneration area of the tail fin between 50mg / L and 100mg / L. In short, 50mg / L Artemia yolk protein can promote the regeneration area of zebrafish tail fin by 24% and shorten the repair time of tail fin tissue.
[0056] Example 3: Artemia yolk protein improves the athletic ability of zebrafish
[0057] The tail-cut juvenile zebrafish were raised under standard conditions, with a water temperature of 28-29℃ and a light cycle of 14 hours light / 10 hours dark. The E3 culture medium containing 50 mg / L Artemia protein was replaced regularly to keep it clean. At the same time, the model group was set without Artemia protein, and the zebrafish without tail cut were set as the blank group. The observation room and temperature control system were turned on 30 minutes in advance to stabilize the temperature control system. The zebrafish were washed three times in a 6-well plate with E3 culture medium and then transferred to a 96-well plate, with one zebrafish in each well, and the E3 culture medium was filled to the same height to reduce the interference of the group effect. The zebrafish was placed in the experimental container and adapted to the environment for 30 minutes. After the temperature was constant, the display status would change to Ready. Put in a 96-well plate and check if there were bubbles at the bottom. The movement behavior of the zebrafish was recorded from above using a high-resolution camera, and the recording time was usually 10-30 minutes.
[0058] Experimental results: This experiment selected three indicators, namely, moving distance, moving speed and maximum acceleration, to explore the effect of yolk protein treatment on the movement ability of zebrafish (see Fig. 9). At 48hpa, the total movement distance of zebrafish in a fixed time was reduced by 26.3% compared with the blank group in the model group, while the total movement distance of zebrafish treated with Artemia egg yolk protein was significantly increased by 24.0% when the 50mg / L yolk protein group was compared with the model group. By observing the changes in movement speed, at 48hpa, the average movement speed of zebrafish in the model group was significantly reduced by 25.0% compared with the blank group, and the average movement speed of zebrafish treated with Artemia egg yolk protein was significantly increased by 15.0% compared with the model group. By observing the changes in maximum acceleration, at 48hpa, the maximum acceleration of zebrafish in the model group was reduced by 41.6% compared with the blank group, and the maximum acceleration of zebrafish treated with Artemia egg yolk protein was significantly increased by 32.6% compared with the model group.
[0059] Example 4: Artemia yolk protein increases the antioxidant capacity of wounds
[0060] The zebrafish tissues at 48 hpa and a protein concentration of 50 mg / L in Example 2 were collected, and the zebrafish fry tissues of each group were ground in a freezing grinder, centrifuged to obtain the supernatant, transferred to a new centrifuge tube, and placed in an ice box for later use. The BCA protein concentration was first determined, and then the SOD, CAT and MDA levels were determined according to the requirements in the kit instructions.
[0061] The results show that ( Fig.10 ), at 48hpa, compared with the blank group, the MDA level of zebrafish in the model group increased, and there was a significant difference. Compared with the model group, the MDA level of zebrafish treated with Artemia egg vitellogenin decreased, and there was a significant difference. After treatment with Artemia egg vitellogenin at a concentration of 50mg / L, compared with the model group, the SOD activity of zebrafish larvae increased by 27%, the CAT activity increased by 51%, and the MDA activity decreased significantly by 37%, which improved the level of antioxidant enzymes in zebrafish.
[0062] This method confirmed that Artemia protein has the biological functions of promoting tissue wound repair, improving motor activity and anti-oxidation.
[0063] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. Use of a composition with egg yolk protein as the main active ingredient in the preparation of a product that promotes wound repair, characterized in that: The amino acid sequence of the yolk protein is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that: In the product, the content of the yolk protein is not less than 50 mg / L or 0.1 mg / g.
3. The use according to claim 2, characterized in that: The product is applied to aquatic organisms, including but not limited to zebrafish.
4. The use according to claim 3, characterized in that: The preparation method of the composition is: (1) dissolving the hatched Artemia in a deep eutectic solvent containing 10-14% w / v sodium hydroxide and 3%-6% w / v urea, and performing 2-4 freeze-thaw cycles to obtain sample 1; (2) dissolving the sample 1 obtained in step (1), shaking for at least 2 hours, and collecting the precipitate by centrifugation; (3) dissolving the precipitate obtained in step (2) in a sodium chloride solution, shaking it, and centrifuging to collect the filtrate; (4) adjusting the pH of the filtrate in step (3) to 3.9-4.7 to obtain egg yolk crude protein; (5) dialyzing and drying the crude egg yolk protein described in step (4) to obtain a composition with the egg yolk protein as the main active ingredient.
5. The use according to claim 4, characterized in that: The product includes feed or medicine; the medicine also contains conventional carriers and / or auxiliary materials.
6. The use according to claim 5, characterized in that The conventional carriers include microcapsules, microspheres, nanoparticles and / or liposomes.
7. The use according to claim 6, characterized in that The dosage form of the medicine includes powder, granule, capsule, tablet, pill or oral solution.
8. The use of egg yolk protein in the preparation of products with antioxidant function, characterized in that: The amino acid sequence of the yolk protein is shown in SEQ ID NO.
1.
9. The product according to claim 8, characterized in that The products include feed, medicine or health products.
10. The use according to claim 9, characterized in that: The antioxidant function includes at least one of the following: (1) Improve the activity of superoxide dismutase and catalase; (2) Reduce the activity of malondialdehyde.