Method for extracting elastin from jellyfish

Abstract

The invention relates to the technical field of marine organisms, in particular to a method for extracting elastin from jellyfish. The method comprises the steps of raw material pretreatment, ultrasonic cracking, magnetic nanoparticle adsorption, microwave extraction, multiple times of centrifugal treatment, multi-step purification and concentration, enzymolysis and inactivation, ultrafiltration and the like. The extraction time is shortened through ultrasonic cracking and microwave-assisted extraction, efficient enrichment of target protein is achieved through magnetic nanoparticle adsorption, impurities are removed through multi-step purification, and high purity is ensured. The method reduces the use of harmful chemical substances, is environment-friendly and energy-saving, is simple and convenient to operate, is suitable for automatic and large-scale production, reduces the cost, improves the economic benefit, and provides technical support for large-scale production and application.

Description

Technical Field

[0001] The invention relates to the field of marine biotechnology, in particular to a method for extracting elastin from jellyfish. Background Art

[0002] At present, elastin, as an important biomaterial, is widely used in many fields such as biomedicine, tissue engineering, and cosmetics. Traditional elastin extraction methods mainly rely on the use of chemical reagents, such as acid-base treatment and organic solvent extraction. These methods are not only inefficient, but also prone to environmental pollution and waste of resources. In addition, existing extraction methods often make it difficult to obtain high-purity elastin, which to some extent limits its promotion in high-end application fields.

[0003] With the development of science and technology, researchers have begun to explore more environmentally friendly and efficient elastin extraction technologies. Jellyfish, as a marine biological resource rich in collagen and elastin, has become a new research hotspot. However, there are few literature reports on the efficient and low-cost extraction of elastin from jellyfish, which remains a challenge. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an efficient elastin extraction method, which solves the technical problems of low purity and low elastin content in the prior art.

[0006] (II) Technical solution

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] A method for extracting elastin from jellyfish comprises the following steps:

[0009] Step S1, raw material pretreatment: washing, soaking to remove impurities and salt, and cutting;

[0010] Step S2, ultrasonic lysis: ultrasonic lysis of the cut raw material;

[0011] Step S3, magnetic nanoparticle adsorption: using magnetic nanoparticles to adsorb collagen;

[0012] Step S4, microwave extraction: extracting collagen by microwave assistance;

[0013] Step S5, multiple centrifugation treatments: alkaline treatment and initial centrifugation, pH adjustment and second centrifugation, NaCl saline treatment and third centrifugation;

[0014] Step S6, multi-step purification and concentration: performing delipidation with an organic solvent, further purifying by ethanol precipitation, and separating the target protein by salting out;

[0015] Step S7, enzymatic hydrolysis and inactivation: using elastase for digestion and heating to inactivate the elastase;

[0016] Step S8, ultrafiltration: remove small molecule impurities and concentrate the target protein through ultrafiltration.

[0017] First, the extraction time was significantly shortened and the extraction efficiency was improved by combining ultrasonic lysis and microwave-assisted extraction. Ultrasonic lysis can effectively destroy the cell wall and release more protein, while microwave extraction accelerates the protein dissolution and extraction process, thereby reducing energy consumption. Secondly, the application of magnetic nanoparticle adsorption technology achieves efficient enrichment of the target protein, and a large number of non-target components can be removed by simple magnetic field separation, greatly simplifying the purification steps. The multi-step purification process, including organic solvent degreasing, ethanol precipitation and salting out, further removes non-target proteins and other impurities, ensuring the high purity of the final product. In addition, through the enzymatic hydrolysis and inactivation steps, the purity and activity of the target protein are further improved by using elastase digestion and heat inactivation. The ultrafiltration step effectively removes small molecule impurities and further concentrates the target protein.

[0018] In general, the present invention not only significantly improves the extraction efficiency and purity of elastin, but also reduces the risk of environmental pollution by reducing the use of harmful chemicals, and has higher environmental protection and energy saving properties. In addition, it is easy to operate, has strong applicability, and is easy to realize automation and large-scale production, which helps to reduce production costs and improve economic benefits, and provides reliable technical support for the large-scale production and wide application of elastin.

[0019] Furthermore, the method further comprises the following steps:

[0020] Step S9, multi-step purification: further enzymatic hydrolysis by papain and collagenase to remove non-target proteins, and affinity chromatography to specifically capture the target protein;

[0021] Step S10, freeze drying and storage: freeze drying and storing the purified elastin.

[0022] The present invention further comprises two steps of multi-step purification and freeze-drying and storage, which significantly improves the purity and storage stability of elastin.

[0023] First, in the multi-step purification step (step S9), non-target proteins are further enzymatically removed by papain and collagenase, and the target protein is specifically captured by affinity chromatography. This process can effectively remove the remaining non-target proteins and greatly improve the purity of the final product. The selective enzymatic hydrolysis of papain and collagenase, combined with the highly specific capture of affinity chromatography, ensures the high purity of the target protein, thus meeting the needs of high-end applications.

[0024] Secondly, in the freeze drying and storage step (step S10), the purified elastin is freeze dried and stored, which not only effectively maintains the biological activity of elastin, but also greatly improves its long-term stability. Freeze drying technology can remove moisture, prevent protein denaturation, and reduce the risk of microbial contamination, ensuring the quality stability of the product during long-term storage.

[0025] In general, through multi-step purification, freeze-drying and storage, the present invention not only significantly improves the purity of elastin, but also ensures its long-term stability, providing a strong technical guarantee for the high-quality production and long-term storage of elastin.

[0026] The present invention further describes in detail the process of raw material pretreatment in step S1, which significantly improves the purity of the raw material and the effectiveness of subsequent processing steps.

[0027] Further, the raw material pretreatment in step S1: washing, soaking to remove impurities and salt, and cutting; put the fresh jellyfish raw material under running water, and thoroughly wash it three times with tap water to ensure that the dirt, sand and other impurities on the surface are removed. When washing, gently rub all parts of the jellyfish by hand to ensure full coverage; put the cleaned jellyfish raw material into a large container, add 5 times the volume of double distilled water, and soak for 48 hours. During this period, the distilled water is replaced every 2-3 hours to ensure that the salt is completely removed. Before the end of the soaking, take a part of the soaking water, add AgNO3 solution to detect the chloride ion content. If a white precipitate is generated, it means that there are still chloride ions remaining, and it is necessary to continue to replace the distilled water and extend the soaking time. If no white precipitate is generated, it means that the chloride ions have been basically removed and the next step can be entered; cut the soaked jellyfish raw material into small pieces of 1x1 cm to ensure the effectiveness of the subsequent processing steps.

[0028] In general, through detailed cleaning and soaking steps, the present invention significantly improves the purity of the raw materials, removes surface impurities and salts, and provides high-quality starting materials for subsequent ultrasonic lysis and other processing steps, thereby improving the efficiency and effect of the entire extraction process.

[0029] Step S2, ultrasonic lysis: put the 1x1 cm small pieces of jellyfish tissue cut in step 1 into liquid nitrogen, ensure that the tissue blocks are completely immersed in liquid nitrogen, and freeze for 30 minutes to ensure that the tissue is completely frozen; quickly transfer the frozen jellyfish tissue blocks to a pre-cooled stainless steel or glass container, and add a buffer of 25-27mM Tris-HCl buffer with a pH of 7.4-7.6 to the container to cover all tissue blocks; set the power of the ultrasonic lysing machine to 500-600W and the frequency to 20-30kHz, turn on the ultrasonic lysing machine for 30 minutes, stop the ultrasonic lysing machine every 5 minutes during the lysing process and put the container into an ice bath for cooling for 5 minutes to avoid protein denaturation due to overheating; turn off the ultrasonic lysing machine after 30 minutes, and observe whether the jellyfish tissue has been fully lysed. The ideal lysing result should be that the tissue becomes evenly dispersed without obvious complete tissue blocks; transfer the lysed jellyfish tissue homogenate to a new container for standby use to ensure the effectiveness of subsequent processing steps.

[0030] In general, through the detailed ultrasonic lysis steps, the present invention significantly improves the lysis efficiency of jellyfish tissue, avoids protein denaturation due to overheating, thereby ensuring the effectiveness of subsequent processing steps and improving the overall quality and efficiency of elastin extraction.

[0031] Furthermore, the specific steps of adsorbing collagen by magnetic nanoparticles in step 3 are as follows:

[0032] Step S31, functionalizing the surface of magnetic nanoparticles to modify carboxyl or amino groups for binding with collagen;

[0033] Step S32, the magnetic nanoparticles are dispersed in a PBS buffer solution with a pH of 7-8 to prepare a 2-4 mg / mL magnetic nanoparticle suspension;

[0034] Step S33, mixing the magnetic nanoparticle suspension and the jellyfish homogenate in a ratio of 10:1-8:1, stirring at room temperature for 1-2 hours at a stirring speed of 50-100 rpm;

[0035] Step S34, using an external magnetic field to separate the magnetic nanoparticles and letting it stand for 5-10 minutes;

[0036] Step S35, adding PBS buffer to the magnetic nanoparticles, gently stirring or shaking for several times, separating using a magnetic field again, removing the supernatant, and repeating this operation 2-3 times to ensure that all non-specifically adsorbed proteins and other impurities are removed;

[0037] Step S35, adding a NaCl solution with a pH of 7-8 and a concentration of 1-1.8 mol / L to the magnetic nanoparticles as a desorption buffer, ensuring that the desorption buffer fully covers the magnetic nanoparticles;

[0038] Step S36, stirring or shaking several times to ensure that the desorption buffer can be evenly distributed, standing at room temperature for 30 minutes to ensure that the collagen is fully desorbed, using an external magnetic field to separate the magnetic nanoparticles, and removing the supernatant as the desorption solution.

[0039] Through detailed magnetic nanoparticle adsorption steps, the present invention significantly improves the enrichment efficiency and purity of collagen. The surface functionalization treatment of magnetic nanoparticles and the adsorption and desorption under specific conditions effectively remove non-target proteins and other impurities, ensure the high purity of the final product, and provide high-quality raw materials for subsequent extraction steps. This process not only improves the extraction efficiency, but also simplifies the operation process, reduces production costs, and enhances the reliability and practicality of the entire extraction process.

[0040] Further, in step S4, PBS or Tris-HCl buffer is mixed with the desorption solution produced in step S3, the volume ratio of buffer to desorption solution is 2-3:5; the microwave power is 500W-600, and the microwave treatment time is 5-10 minutes. The buffer is preferably 25-27mM Tris-HCl buffer with a pH of 7.4-7.6.

[0041] First, PBS or Tris-HCl buffer is mixed with the desorption solution produced in step S3 at a volume ratio of 2-3:5. This ratio is set to ensure that the collagen in the desorption solution can be fully dissolved and stably present in the buffer. Then, the mixed solution is treated with a microwave power of 500W-600W for 5-10 minutes. Microwave treatment can quickly heat the mixed solution in a short time, accelerate the dissolution and extraction process of collagen, and avoid protein denaturation caused by long-term high temperature treatment.

[0042] In general, the present invention significantly improves the extraction efficiency of collagen through the specific steps of microwave extraction. Microwave treatment not only speeds up the extraction speed and reduces the processing time, but also ensures the structural integrity of collagen and avoids denaturation caused by high temperature. This optimized step not only improves the extraction efficiency, but also ensures the high purity and biological activity of the final product, thereby enhancing the effect and reliability of the entire extraction process.

[0043] Further, the multiple centrifugation treatments in step S5 include step S51: alkali treatment and initial centrifugation, step S52: pH adjustment and second centrifugation, and step S53: NaCl saline treatment and third centrifugation;

[0044] The alkali treatment and preliminary centrifugation process of step S51 is as follows: prepare a NaOH solution with a pH of 11-12, mix it with the mixed solution in step S4 in equal volumes, stir for 5-10 minutes, let stand for 20-30 minutes, centrifuge, the centrifugation condition is 10000-12000rpm, 5-10 minutes, collect the supernatant, add deionized water to the precipitate to resuspend it, centrifuge again, collect the supernatant and mix it with the first supernatant;

[0045] The pH adjustment and second centrifugation process of step S52 are as follows: the pH of the supernatant collected in step S51 is adjusted to 1.0-2.0 by HCl, and centrifuged at 10000-12000 rpm for 5-10 minutes, and the supernatant is collected and the precipitate is retained;

[0046] The NaCl salt water treatment and the third centrifugation process of step S53 are as follows: add a NaCl solution with a concentration of 1-2 mol / L to the precipitate in step S52 to fully cover the precipitate, shake the reaction at room temperature and then centrifuge; the shaking speed is 100-110 rpm / min, and the shaking time is 3 hours; the centrifugation condition is 10000-12000 rpm, 5-10 minutes, and the supernatant is collected;

[0047] The supernatants from multiple centrifugations were pooled.

[0048] The present invention significantly improves the purity and extraction efficiency of elastin through detailed multiple centrifugation steps. Multiple centrifugation steps of alkali treatment, pH adjustment and NaCl brine treatment effectively remove non-target proteins and other impurities, ensuring the high purity of the final product. This series of steps not only improves the extraction efficiency, but also ensures the quality and stability of the final product, thereby enhancing the effect and reliability of the entire extraction process.

[0049] Further, the multi-step purification and concentration in step S6 includes S61 organic solvent degreasing, S62 ethanol precipitation and S63 salting out;

[0050] S61: adding an organic solvent to the mixed supernatant obtained in step S5 and mixing the mixture evenly; the organic solvent and the supernatant are added in a ratio of 1:1; centrifuging the mixed solution at a speed of 3000-4000 rpm for 5-10 minutes, and collecting the degreased supernatant; the organic solvent is ether or ethyl acetate;

[0051] S62 ethanol precipitation: add 3 volumes of ethanol at -20°C to the delipidated supernatant, mix thoroughly and place at 4°C overnight to allow protein precipitation to form completely; then centrifuge at 10000-12000 rpm for 5-10 minutes and retain the precipitate;

[0052] Add distilled water to the pellet to resuspend it.

[0053] The mixture was centrifuged again at 10000-12000 rpm for 5-10 minutes to retain the precipitate; the precipitate after the final washing was suspended in a buffer solution, which was a 25-27 mM Tris-HCl buffer solution with a pH of 7.4-7.6.

[0054] S63 salting out: add saturated ammonium sulfate solution to the suspended precipitate, the volume ratio of saturated ammonium sulfate solution to buffer is 1:1, mix well and let stand for more than half an hour to allow the protein precipitate to form completely;

[0055] Centrifuge again at 10000-12000 rpm for 5-10 minutes and retain the precipitate;

[0056] Add distilled water to the pellet to resuspend it.

[0057] The mixture was centrifuged again at 10000-12000 rpm for 5-10 minutes to retain the precipitate; the precipitate after the final washing was suspended in a buffer to obtain a homogenate, wherein the buffer was a 25-27 mM Tris-HCl buffer at a pH of 7.4-7.6.

[0058] First, in step S61, the fat component is effectively removed, reducing the interference factors in the subsequent purification steps. Then, in step S62, the target protein is further purified by ethanol precipitation, which effectively removes non-target proteins and other impurities and improves the purity of the target protein. Finally, in step S63, the target protein is further separated by salting out, which further improves the purity and recovery rate of the target protein.

[0059] In general, the present invention significantly improves the purity and recovery rate of elastin through detailed multi-step purification and concentration steps. The multiple purification steps of organic solvent degreasing, ethanol precipitation and salting out effectively remove non-target proteins and other impurities, ensuring the high purity and high recovery rate of the final product. This series of steps not only improves the extraction efficiency, but also ensures the quality and stability of the final product, thereby enhancing the effect and reliability of the entire extraction process.

[0060] Further, according to the activity of the enzyme, 100 units of elastase are added to the homogenate, the pH is adjusted to 6.8-7.2, and the mixture is shaken at 150-180 rpm for 1-1.5 hours at 25-30°C. The shaken sample is heated in boiling water for 5-10 minutes to inactivate the elastase, cooled to 4-6°C, centrifuged at 10000-12000 rpm for 5-10 minutes, and the supernatant is retained.

[0061] First, according to the activity of the enzyme, add elastase that provides at least 100 units of activity to the homogenate and adjust the pH to 6.8-7.2. This pH range is the optimal activity range of elastase, ensuring the maximum catalytic efficiency of the enzyme. Then, at 25-30°C, shake at a speed of 150-180rpm for 1-1.5 hours to allow elastase to fully digest the target protein and promote its degradation and release.

[0062] Subsequently, the shaken sample is placed in boiling water and heated for 5-10 minutes to inactivate the elastase. This process effectively terminates the action of the enzyme, avoids excessive degradation and nonspecific cleavage, and protects the structural integrity of the target protein. After heating, the sample is cooled to 4-6°C and centrifuged at 10000-12000rpm for 5-10 minutes, and the supernatant is retained. This centrifugation step removes the enzyme and undigested residual substances, ensuring the high purity of the target protein.

[0063] In general, the present invention significantly improves the purity and activity of elastin through detailed enzymatic hydrolysis and inactivation steps. The enzymatic hydrolysis treatment under the conditions of pH and temperature adjustment ensures the effective degradation and release of the target protein, while the steps of boiling water heating and low-temperature centrifugation effectively terminate the activity of the enzyme, protect the structural integrity of the target protein, and remove impurities. This series of steps not only improves the extraction efficiency, but also ensures the high purity and biological activity of the final product, thereby enhancing the effect and reliability of the entire extraction process.

[0064] Furthermore, the ultrafiltration treatment has a molecular weight cut-off of ≤5 kDa.

[0065] In the ultrafiltration treatment step, an ultrafiltration membrane with a molecular weight cutoff of ≤5kDa is used for treatment, which significantly improves the purity and recovery rate of the target protein. Specifically, the supernatant after enzymatic hydrolysis and inactivation treatment is introduced into an ultrafiltration device, and by selecting an ultrafiltration membrane with a molecular weight cutoff of ≤5kDa, it is ensured that small molecule impurities are effectively removed, and the target protein is retained. The ultrafiltration process effectively removes incompletely digested enzymes, salts and other small molecule residues, thereby ensuring the high purity of the target protein. In addition, the ultrafiltration treatment not only concentrates the target protein, but also improves the recovery rate, making the concentration of the final product higher, which is convenient for subsequent treatment and application. This process not only maintains the structural integrity of the target protein, improves the biological activity of the final product, but also simplifies the operating process, is easy to automate and scale production, and reduces production costs. In general, by selecting an ultrafiltration membrane with a molecular weight cutoff of ≤5kDa for treatment, the present invention significantly improves the purity and recovery rate of the target protein, and enhances the effect and reliability of the entire extraction process.

[0066] Furthermore, the total protein extract was detected by ultrafiltration solution, and papain and collagenase were added successively to remove non-target proteins, and the amount of papain and collagenase added was 2-3% and 0.1-0.3% of the total protein crude extract weight, respectively;

[0067] The pH of papain was adjusted to 6-8, and the temperature was 45-50°C, and the temperature was shaken at 150-180 rpm for 3-5 hours;

[0068] Collagenase was hydrolyzed at pH 7-7.8, 35-40°C, and shaking at 150-180 rpm for 2-4 hours;

[0069] The solution after enzymatic hydrolysis is centrifuged at 10000-12000 rpm for 5-10 minutes, and the precipitate is collected to obtain a crude elastin extract.

[0070] Through the above detailed multi-step purification steps, the present invention significantly improves the purity and recovery rate of elastin. Specifically, the precise addition amount of papain and collagenase and the optimized enzymatic hydrolysis conditions effectively remove non-target proteins and ensure the high purification of the target protein (elastin).

[0071] First, the amount of papain added is 2-3% of the total protein crude extract weight, and the enzymatic hydrolysis is carried out at 150-180 rpm for 3-5 hours at a pH of 6-8 and at 45-50°C. Under this condition, papain efficiently hydrolyzes non-target proteins, ensuring the thoroughness and efficiency of the enzymatic hydrolysis. Under suitable pH and temperature conditions, papain can efficiently remove a variety of non-target proteins and improve the purity of the target protein.

[0072] Next, collagenase is added in an amount of 0.1-0.3% of the total protein crude extract weight, and the pH is adjusted to 7-7.8, 35-40°C, and the enzyme is hydrolyzed at 150-180 rpm for 2-4 hours. Collagenase continues to hydrolyze under mild conditions, ensuring the structural integrity of the target protein (elastin) and avoiding losses caused by excessive degradation. Through the synergistic action of these two enzymes, non-target proteins are further removed, while the target protein is retained.

[0073] Finally, the enzymatically hydrolyzed solution is centrifuged at 10,000-12,000 rpm for 5-10 minutes, and the precipitate is collected to obtain a high-purity crude elastin extract. This centrifugation step effectively separates the enzymatically hydrolyzed product, ensuring the high purity and high recovery rate of the target protein.

[0074] This series of steps not only improves the purification efficiency, but also ensures the high quality and activity of the final product. By precisely controlling the amount of enzyme added and the enzymatic conditions, the purity and structural integrity of the target protein are ensured, enhancing the effect and reliability of the entire extraction process.

[0075] Further, the crude elastin extract was dissolved in a 25-27 mM Tris-HCl buffer at pH 7.4-7.6 and passed through an affinity chromatography column loaded with anti-elastin antibody at a rate of 0.5-1.0 mL / min, ensuring that the sample remained for 10-15 minutes.

[0076] The column is washed with 25-27 mM Tris-HCl buffer at pH 7.4-7.6 at a flow rate of 1-2 mL / min, followed by elution of the target protein with 1 M NaCl, pH 7.4-7.6 Tris-HCl buffer at a flow rate of 1-2 mL / min, and the eluent is neutralized to pH 7.0-7.4 with, for example, 1 M, pH 8.0 Tris-HCl.

[0077] This series of steps not only significantly improves the purity of elastin, but also ensures the structural integrity of the target protein. Through the specific binding and precise elution conditions of affinity chromatography, non-target proteins and other impurities are effectively removed, ensuring the high purity and high activity of the final product. This optimized purification step not only improves the purification efficiency, but also ensures the high quality and reliability of the final product.

[0078] In the freeze drying and storage step, the purified elastin is freeze dried and stored to ensure its long-term stability and biological activity.

[0079] The specific steps are as follows:

[0080] Prepare samples: Adjust the elastin solution purified by affinity chromatography to an appropriate concentration to ensure that there are no impurities in the solution.

[0081] Pre-freezing: Place the sample in a freeze dryer and first pre-freeze it, lowering the solution temperature to -40℃ to -50℃ to ensure that the sample is completely frozen.

[0082] Primary drying: Under vacuum conditions, gradually raise the sample temperature to around -20°C, maintain this temperature and continue to evacuate the sample to sublime the water in the sample and remove most of the water.

[0083] Secondary drying: Further increase the sample temperature to 0°C to +10°C and continue to vacuum to remove the remaining adsorbed water and bound water to ensure that the sample is completely dry.

[0084] Sealed Storage: Transfer the freeze-dried elastin powder into a sterile dry glass or plastic bottle and seal it with a rubber stopper to prevent moisture from entering.

[0085] Through freeze-drying, the purified elastin is converted into a stable dry powder, which not only greatly reduces the moisture content and improves the stability of long-term storage, but also facilitates transportation and storage. Finally, the freeze-dried elastin powder is stored in an environment of 4-8°C under sealed conditions to ensure its long-term stability and biological activity.

[0086] This process not only improves the storage stability of elastin, but also ensures its reliability and effectiveness in subsequent applications.

[0087] (III) Beneficial effects

[0088] The beneficial effects of the present invention are as follows: the present invention relates to a method for extracting elastin from jellyfish, comprising the steps of raw material pretreatment, ultrasonic lysis, magnetic nanoparticle adsorption, microwave extraction, multiple centrifugal treatments, multi-step purification, enzymatic hydrolysis and inactivation, and ultrafiltration. The extraction efficiency and purity of elastin are significantly improved, and the extraction time is effectively shortened by combining ultrasonic lysis and microwave-assisted extraction; the magnetic nanoparticle adsorption technology is used to achieve efficient enrichment of the target protein, and the multi-step purification process further removes non-target proteins and other impurities, ensuring the high purity of the final product; at the same time, the method of the present invention reduces the use of harmful chemicals, reduces the risk of environmental pollution, and has higher environmental protection and energy saving; in addition, the method is simple to operate, has strong applicability, is easy to realize automation and large-scale production, helps to reduce production costs, improve economic benefits, and provides reliable technical support for the large-scale production and wide application of elastin. DETAILED DESCRIPTION

[0089] Unless otherwise specified, "parts" in the following examples refer to parts by mass.

[0090] Embodiment 1:

[0091] Step S1: Raw material pretreatment

[0092] Cleaning:

[0093] Place the fresh jellyfish material (moon jellyfish) under running water and wash it thoroughly three times with tap water, making sure to remove any dirt, sand and other impurities on the surface. Gently rub all parts of the jellyfish with your hands while washing, ensuring full coverage.

[0094] soak:

[0095] Put the cleaned jellyfish raw materials into a large container, add 5 times the volume of double distilled water, and soak for 48 hours. During this period, change the distilled water every 2 hours to ensure that the salt is completely removed. Before the end of the soaking, take a part of the soaking water and add AgNO3 solution to test the chloride ion content. If white precipitate is generated, it means that there are still chloride ions remaining, and it is necessary to continue to change the distilled water and extend the soaking time; if no white precipitate is generated, it means that the chloride ions have been basically removed.

[0096] Cutting:

[0097] The soaked jellyfish raw materials were cut into small pieces of 1 × 1 cm to ensure the effectiveness of subsequent processing steps.

[0098] Step S2: Ultrasonic lysis

[0099] freezing:

[0100] Place the 1×1 cm small pieces of jellyfish tissue cut in step S1 into liquid nitrogen, making sure that the tissue block is completely immersed in liquid nitrogen. The freezing time is 30 minutes to ensure that the tissue is completely frozen.

[0101] Transfer:

[0102] The frozen jellyfish tissue blocks were quickly transferred to a pre-cooled stainless steel container, and 26 mM Tris-HCl buffer, pH 7.5, was added to the container to cover all tissue blocks.

[0103] Ultrasonic lysis:

[0104] Set the power of the ultrasonic lysing machine to 550W and the frequency to 25kHz, and turn on the ultrasonic lysing machine for 30 minutes. During the lysis process, stop the ultrasonic lysing machine every 5 minutes and put the container in an ice bath to cool for 5 minutes to avoid protein denaturation due to overheating. After 30 minutes, turn off the ultrasonic lysing machine and observe whether the jellyfish tissue has been fully lysed. The ideal lysis result should be that the tissue becomes evenly dispersed without obvious intact tissue blocks.

[0105] Transfer:

[0106] The lysed jellyfish tissue homogenate was transferred to a new container for later use.

[0107] Step S3: Magnetic Nanoparticle Adsorption

[0108] Surface functionalization treatment:

[0109] The magnetic nanoparticles are subjected to surface functionalization treatment to modify the carboxyl group or the amino group for binding with collagen. The magnetic nanoparticles of the present application are magnetic ferroferric oxide, and the surface functionalized magnetic nanoparticles are commercially available.

[0110] Preparation of suspension:

[0111] The magnetic nanoparticles were dispersed in a PBS buffer solution at pH 7.5 to prepare a 3 mg / mL magnetic nanoparticle suspension.

[0112] Adsorption:

[0113] The magnetic nanoparticle suspension and the jellyfish homogenate were mixed in a ratio of 10:1 and stirred at room temperature for 1 hour at a stirring speed of 75 rpm.

[0114] Separation:

[0115] Use an external magnetic field to separate the magnetic nanoparticles and let it stand for 5 minutes.

[0116] washing:

[0117] Add PBS buffer to the magnetic nanoparticles, gently stir or shake several times, separate using a magnetic field again, remove the supernatant, and repeat this operation three times to ensure that all non-specifically adsorbed proteins and other impurities are removed.

[0118] Desorption:

[0119] Add a 1.4 mol / L NaCl solution with a pH of 7.5 to the magnetic nanoparticles as a desorption buffer to ensure that the desorption buffer fully covers the magnetic nanoparticles. Stir or shake several times to ensure that the desorption buffer is evenly distributed, and let it stand at room temperature for 30 minutes to ensure that the collagen is fully desorbed. Use an external magnetic field to separate the magnetic nanoparticles and remove the supernatant as the desorption solution.

[0120] Step S4: Microwave extraction

[0121] Mixing buffer:

[0122] A 26 mM Tris-HCl buffer solution having a pH of 7.5 was mixed with the desorption solution produced in step S3 at a volume ratio of 2:5.

[0123] Microwave treatment:

[0124] The mixed solution was treated with a microwave power of 550 W for 8 minutes.

[0125] Step S5: Multiple centrifugation

[0126] Alkali treatment and initial centrifugation:

[0127] Prepare a NaOH solution with a pH of 11.5, and mix it with the mixed solution in step S4 in equal volumes, stir for 8 minutes, and then let stand for 25 minutes. Centrifuge at 10,000 rpm for 8 minutes, and collect the supernatant. Add deionized water to the precipitate and resuspend it, centrifuge again, collect the supernatant, and mix it with the first supernatant.

[0128] pH adjustment and second centrifugation:

[0129] The supernatant collected in step S51 was adjusted to pH 1.5 by HCl, and centrifuged at 10,000 rpm for 8 minutes to collect the supernatant and retain the precipitate.

[0130] NaCl saline treatment and third centrifugation:

[0131] Add 1.5 mol / L NaCl solution to the precipitate in step S52 to fully cover the precipitate, shake the reaction at room temperature for 3 hours at a shaking speed of 105 rpm / min. Centrifuge at 10000 rpm for 8 minutes, and collect the supernatant. Mix the supernatants after multiple centrifugations.

[0132] Step S6: Multi-step purification and concentration

[0133] Organic solvent degreasing:

[0134] An organic solvent (diethyl ether) was added to the mixed supernatant in step S5 and mixed evenly, and the volume of the organic solvent and the supernatant were added in a ratio of 1: 1. The mixed solution was centrifuged at 3500 rpm for 8 minutes, and the delipidated supernatant was collected.

[0135] Ethanol precipitation:

[0136] Add 3 volumes of ethanol at -20°C to the delipidated supernatant, mix thoroughly and place overnight at 4°C to allow protein precipitation to form completely. Centrifuge at 10,000 rpm for 8 minutes and retain the precipitate. Add distilled water to the precipitate to resuspend it, centrifuge again at 10,000 rpm for 8 minutes and retain the precipitate. Suspend the final washed precipitate in 26 mM Tris-HCl buffer at pH 7.5.

[0137] Salting out:

[0138] Add saturated ammonium sulfate solution to the suspended precipitate, the volume ratio of saturated ammonium sulfate solution to buffer is 1:1, mix well and let stand for more than 30 minutes to allow protein precipitation to form completely. Centrifuge again at 10000rpm for 8 minutes and retain the precipitate. Add distilled water to the precipitate to resuspend it, centrifuge again at 10000rpm for 8 minutes and retain the precipitate. Suspend the final washed precipitate in 26mM Tris-HCl buffer at pH 7.5.

[0139] Step S7: Enzymatic hydrolysis and inactivation

[0140] Add elastase:

[0141] According to the activity of the enzyme, 100 units of elastase were added to the homogenate, and the pH was adjusted to 7.0. The mixture was shaken at 165 rpm for 1.5 hours at 28°C to allow the elastase to fully digest the target protein and promote its degradation and release.

[0142] Inactivation:

[0143] The shaken sample was placed in boiling water and heated for 7 minutes to inactivate elastase. After heating, the sample was cooled to 5°C and centrifuged at 10,000 rpm for 8 minutes, and the supernatant was retained.

[0144] Step S8: Ultrafiltration

[0145] Ultrafiltration treatment:

[0146] The supernatant after enzymatic hydrolysis and inactivation is introduced into the ultrafiltration device. By selecting an ultrafiltration membrane with a molecular weight cutoff of ≤5kDa, small molecule impurities are effectively removed while the target protein is retained. The ultrafiltration process effectively removes incompletely digested enzymes, salts and other small molecule residues, thereby ensuring the high purity of the target protein.

[0147] Step S9: Multi-step purification

[0148] Papain and collagenase were added: the activity of papain was set to 250 units / mg, and the activity of collagenase was set to 100 units / mg.

[0149] The total protein extract was detected by ultrafiltration solution, and papain and collagenase were added successively to remove non-target proteins. The amount of papain added was 2% of the total protein crude extract weight, and the enzymatic hydrolysis was carried out at 165 rpm for 4 hours at pH adjusted to 7.0 and 48°C. The amount of collagenase added was 0.2% of the total protein crude extract weight, and the enzymatic hydrolysis was carried out at 165 rpm for 3 hours at pH adjusted to 7.5 and 38°C.

[0150] Centrifugation:

[0151] The solution after enzymatic hydrolysis was centrifuged at 10000 rpm for 8 minutes, and the precipitate was collected to obtain a high-purity crude elastin extract.

[0152] Step S10: Affinity chromatography

[0153] Dissolve the sample:

[0154] The crude elastin extract was dissolved in a Tris-HCl buffer at a pH of 7.5 and a concentration of 26 mM.

[0155] Through affinity chromatography:

[0156] Pass the sample through an affinity chromatography column loaded with anti-elastin antibody (ab217356) at a flow rate of 0.75 mL / min, ensuring that the sample stays for 12 minutes.

[0157] washing:

[0158] The column was washed with a 26 mM Tris-HCl buffer at pH 7.5 at a flow rate of 1.5 mL / min.

[0159] Elution:

[0160] The target protein was then eluted with 1 M NaCl, pH 7.5 Tris-HCl buffer at a flow rate of 1.5 mL / min, and the eluate was neutralized to pH 7.2 with 1 M Tris-HCl buffer, pH 8.0.

[0161] Step S11: Freeze drying and storage

[0162] Pre-freeze:

[0163] The samples were placed in a freeze dryer and first pre-frozen to reduce the solution temperature to -45°C to ensure that the samples were completely frozen.

[0164] Primary drying:

[0165] Under vacuum conditions, the sample temperature was gradually raised to -18°C, and the temperature was maintained while the vacuum was continuously drawn to allow the water in the sample to sublime and remove most of the water.

[0166] Secondary drying:

[0167] The sample temperature was further increased to +5°C and vacuum was continued to remove the remaining adsorbed and bound water to ensure that the sample was completely dry.

[0168] Sealed storage:

[0169] The freeze-dried elastin powder was transferred into sterile dry glass bottles and sealed with rubber stoppers to prevent moisture ingress.

[0170] Step S1 is the amount of jellyfish raw material initially treated, and step S10 is to detect the elastin content and purity of the neutralized eluate. In this application, the elastin purity is 70% (elastin amount / protein amount in the eluate), and the elastin content is 0.5% (elastin amount / elastin in the jellyfish raw material)

[0171] Comparative Example 1

[0172] The difference between this comparative example and Example 1 is that step S3: magnetic nanoparticle adsorption operation is omitted. The remaining steps and conditions of this comparative example are the same as those of Example 1.

[0173] The final elastin purity detected was 65% (amount of elastin / amount of protein in the eluate), and the elastin content was 0.2% (amount of elastin / amount of elastin in the jellyfish raw material).

[0174] Comparative Example 2

[0175] The difference between this comparative example and Example 1 is that step S9: multi-step purification is omitted, but the operation of detecting the total protein extract of the solution after ultrafiltration is retained. The remaining steps and conditions of this comparative example are the same as those of Example 1.

[0176] The final elastin purity detected was 60% (amount of elastin / amount of protein in the eluate), and the elastin content was 0.3% (amount of elastin / amount of elastin in the jellyfish raw material).

[0177] Comparative Example 3

[0178] The difference between this comparative example and Example 1 is that step S5: multiple centrifugation treatments are omitted, and the remaining steps and conditions of this comparative example are the same as those of Example 1.

[0179] The final elastin purity detected was 55% (amount of elastin / amount of protein in the eluate), and the elastin content was 0.2% (amount of elastin / amount of elastin in the jellyfish raw material).

[0180] In Comparative Example 1, the important step of magnetic nanoparticle adsorption is omitted, which plays a key role in enriching the target protein and removing impurities in the embodiment. The main function of magnetic nanoparticle adsorption in Comparative Example 1 is to enrich the target protein from the complex jellyfish homogenate and remove non-specific proteins and other impurities. Omitting this step means that more non-target proteins will enter the subsequent processing steps, resulting in a decrease in purity. Magnetic nanoparticles reduce the interference of impurities in subsequent steps by specifically adsorbing the target protein. Therefore, omitting this step will lead to an increase in impurities, thereby affecting purity. In the absence of magnetic nanoparticle adsorption, the concentration of the target protein in the jellyfish homogenate is not improved. This means that in the subsequent processing steps, the proportion of the target protein is relatively low, resulting in a decrease in the absolute amount of elastin in the final extract. In the absence of specific adsorption, the target protein is more susceptible to non-specific adsorption, such as adsorption to the container wall or other macromolecules, which can also lead to a decrease in content.

[0181] The reason why the elastin content and yield in Comparative Example 2 are low is mainly attributed to the omission of the multi-step purification step (step S9). In Example 1, by adding papain and collagenase for enzymolysis, non-target proteins can be effectively removed, thereby improving the purity and recovery rate of the target protein (elastin). Omitting this step results in the failure of background protein to be effectively removed. The presence of non-target proteins not only increases the proportion of impurities in the extract, but also occupies the binding sites in the subsequent processing steps, thereby reducing the purity and recovery rate of the target protein; at the same time, the enzymolysis process helps to release those elastins wrapped in complex protein structures. The lack of this step makes some elastins fail to be fully released and recovered, further leading to a decrease in the elastin content and yield in the final extract.

[0182] The reason for the low elastin content and yield in Comparative Example 3 is mainly attributed to the omission of multiple centrifugation steps (step S5). In Example 1, various impurities in the jellyfish homogenate, such as salt, non-specific proteins and other non-target components, can be effectively removed by multiple centrifugation treatments, including alkali treatment, pH adjustment and NaCl brine treatment, thereby gradually improving the purity and recovery rate of the target protein (elastin). Omitting this step results in the failure to effectively remove these impurities, resulting in a high content of non-target proteins and other impurities in the extract, thereby reducing the purity of the target protein; at the same time, the presence of impurities occupies binding sites in subsequent processing steps (such as ultrafiltration and enzymatic hydrolysis), affecting the recovery efficiency of the target protein, and ultimately leading to a significant decrease in the elastin content and yield.

[0183] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may alter, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for extracting elastin from jellyfish, characterized in that: The following steps are involved: Step S1, raw material pretreatment: washing, soaking to remove impurities and salt, and cutting; Step S2, ultrasonic lysis: ultrasonic lysis of the cut raw material; Step S3, magnetic nanoparticle adsorption: adding a solution of magnetic nanoparticles to the cracked raw material, and using the magnetic nanoparticles to adsorb collagen; Step S4, microwave extraction: using endogenous enzymes contained in jellyfish combined with microwave-assisted extraction of collagen; Step S5, multiple centrifugation treatment: performing alkaline treatment and initial centrifugation, adjusting the pH value and performing a second centrifugation, treating the centrifugal precipitate with NaCl saline and performing a third centrifugation, and retaining the supernatant; Step S6, multi-step purification: adding an organic solvent to the supernatant to perform organic solvent separation and delipidation, purifying the aqueous phase by ethanol precipitation, and separating the target protein by salting out; Step S7, enzymatic hydrolysis and inactivation: using elastase for digestion and heating to inactivate the elastase; Step S8, ultrafiltration: remove small molecule impurities by ultrafiltration.

2. The method for extracting elastin according to claim 1, characterized in that: The following steps are also included: Step S9, multi-step purification: further enzymatic hydrolysis by papain and collagenase to remove non-target proteins, and affinity chromatography to specifically capture the target protein; Step S10, freeze drying and storage: freeze drying and storing the purified elastin.

3. The method for extracting elastin according to claim 1, characterized in that: The specific steps of magnetic nanoparticles adsorbing collagen in step S3 are as follows: Step S31, functionalizing the surface of magnetic nanoparticles to modify carboxyl or amino groups for binding with collagen; Step S32, the magnetic nanoparticles are dispersed in a PBS buffer solution with a pH of 7-8 to prepare a 2-4 mg / mL magnetic nanoparticle suspension; Step S33, mixing the magnetic nanoparticle suspension and the jellyfish homogenate in a ratio of 10:1-8:1, stirring at room temperature for 1-2 hours at a stirring speed of 50-100 rpm; Step S34, using an external magnetic field to separate the magnetic nanoparticles and letting it stand for 5-10 minutes; Step S35, adding PBS buffer to the magnetic nanoparticles, gently stirring or shaking for several times, and then using the magnetic field for separation again to remove the supernatant, and repeating this operation 2-3 times to remove non-specifically adsorbed proteins and other impurities; Step S35, adding a NaCl solution with a pH of 7-8 and a concentration of 1-1.8 mol / L to the magnetic nanoparticles as a desorption buffer, ensuring that the desorption buffer fully covers the magnetic nanoparticles; Step S36, stirring or shaking several times to ensure that the desorption buffer can be evenly distributed, standing at room temperature for 30 minutes to ensure that the collagen is fully desorbed, using an external magnetic field to separate the magnetic nanoparticles, and the collected supernatant is the desorption solution.

4. The method for extracting elastin according to claim 1, characterized in that: In step S4, PBS or Tris-HCl buffer is mixed with the desorption liquid produced in step S3, and the volume ratio of the buffer to the desorption liquid is 2-3:5; the microwave power is 500W-600, and the microwave treatment time is 5-10 minutes.

5. The method for extracting elastin according to claim 1, characterized in that: The multiple centrifugation treatments in step S5 include step S51: alkali treatment and initial centrifugation, step S52: pH adjustment and second centrifugation, and step S53: NaCl saline treatment and third centrifugation; The alkali treatment and preliminary centrifugation process of step S51 is as follows: prepare a NaOH solution with a pH of 11-12, mix it with the mixed solution in step S4 in equal volumes, stir for 5-10 minutes, let stand for 20-30 minutes, centrifuge, the centrifugation condition is 10000-12000rpm, 5-10 minutes, collect the supernatant, add deionized water to the precipitate to resuspend it, centrifuge again, collect the supernatant and mix it with the first supernatant; The pH adjustment and second centrifugation process of step S52 are as follows: the pH of the supernatant collected in step S51 is adjusted to 1.0-2.0 by HCl, and centrifuged at 10000-12000 rpm for 5-10 minutes, and the supernatant is collected and the precipitate is retained; The NaCl salt water treatment and the third centrifugation process of step S53 are as follows: add a NaCl solution with a concentration of 1-2 mol / L to the precipitate in step S52 to fully cover the precipitate, shake the reaction at room temperature and then centrifuge; the shaking speed is 100-110 rpm / min, and the shaking time is 3 hours; the centrifugation condition is 10000-12000 rpm, 5-10 minutes, and the supernatant is collected; The supernatants from multiple centrifugations were pooled.

6. The method for extracting elastin according to claim 1, characterized in that: The multi-step purification and concentration in step S6 includes S61 organic solvent degreasing, S62 ethanol precipitation and S63 salting out; S61: adding an organic solvent to the mixed supernatant obtained in step S5 and mixing the mixture evenly; the organic solvent and the supernatant are added in a ratio of 1:1; centrifuging the mixed solution at a speed of 3000-4000 rpm for 5-10 minutes, and collecting the degreased supernatant; the organic solvent is ether or ethyl acetate; S62 ethanol precipitation: add 3-4 times the volume of ethanol at -20℃ to the delipidated supernatant, mix thoroughly and leave overnight at 2-4℃ to allow protein precipitation to form completely; then centrifuge at 10000-12000rpm for 5-10 minutes and retain the precipitate; Add distilled water to the sediment to resuspend it; Centrifuge again at 10000-12000 rpm for 5-10 minutes to retain the precipitate; suspend the precipitate after the final wash in a buffer solution, wherein the buffer solution is a 25-27 mM Tris-HCl buffer solution at a pH of 7.4-7.6; S63 salting out: add saturated ammonium sulfate solution to the suspended precipitate, the volume ratio of saturated ammonium sulfate solution to buffer is 1:1, mix well and let stand for more than half an hour to allow the protein precipitate to form completely; Centrifuge again at 10000-12000 rpm for 5-10 minutes and retain the precipitate; Add distilled water to the sediment to resuspend it; Centrifuge again at 10000-12000 rpm for 5-10 minutes and retain the precipitate; The pellet after the final wash was suspended in a buffer solution of 25-27 mM Tris-HCl buffer at pH 7.4-7.6 to obtain a homogenate.

7. The method for extracting elastin according to claim 1, characterized in that: According to the activity of the enzyme, 100 units of elastase are added to the homogenate, the pH is adjusted to 6.8-7.2, and the mixture is shaken at 150-180 rpm for 1-1.5 hours at 25-30°C. The shaken sample is heated in boiling water for 5-10 minutes to inactivate the elastase, cooled to 4-6°C, centrifuged at 10000-12000 rpm for 5-10 minutes, and the supernatant is retained.

8. The method for extracting elastin according to claim 1, characterized in that: Ultrafiltration treatment molecular weight cut-off ≤ 5kDa.

9. The method for extracting elastin according to claim 1, characterized in that: The total protein extract was detected by ultrafiltration solution, and papain and collagenase were added successively to remove non-target proteins, and the amount of papain and collagenase added was 2-3% and 0.1-0.3% of the total protein crude extract weight respectively; Papain was enzymolyzed at pH 6-8, at 45-50°C, and at 150-180 rpm for 3-5 hours; Collagenase was hydrolyzed at pH 7-7.8, 35-40°C, and shaking at 150-180 rpm for 2-4 hours; The solution after enzymatic hydrolysis is centrifuged at 10000-12000 rpm for 5-10 minutes, and the precipitate is collected to obtain a crude elastin extract.

10. The method for extracting elastin according to claim 1, characterized in that: The crude elastin extract was dissolved in a 25-27 mM Tris-HCl buffer at pH 7.4-7.6 and passed through an affinity chromatography column loaded with anti-elastin antibody at 0.5-1.0 mL / min, ensuring that the sample remained for 10-15 minutes; The column is washed with 25-27 mM Tris-HCl buffer at pH 7.4-7.6 at a flow rate of 1-2 mL / min, followed by elution of the target protein with 1 M NaCl, pH 7.4-7.6 Tris-HCl buffer at a flow rate of 1-2 mL / min, and the pH of the eluate is neutralized to 7.0-7.2 with, for example, 1 M, pH 6.5-6.8 Tris-HCl.