Method for extracting elastin peptide from jellyfish

Abstract

The invention relates to a method for extracting elastin peptide from jellyfish. The method comprises the following steps: S1, washing fresh jellyfish with clear water to remove impurities or soaking and salting the jellyfish with cold water containing citric acid / acetic acid to desalt; cutting into small blocks for later use; s2, mixing the jellyfish with a sodium hydroxide solution with the pH value of more than or equal to 11.5, carrying out pressure maintaining and shearing treatment by using a high-pressure shearing machine, centrifuging, and retaining precipitates; s3, mixing the precipitate with pure water, adjusting the pH value to be less than or equal to 1.5 by using hydrochloric acid, reacting for 1-4 hours, and centrifuging to reserve the precipitate; s4, the precipitate is mixed with sodium chloride saline water, the concentration of the sodium chloride saline water is 0.8-1.5 M, a reaction is conducted for 1-4 h, centrifugation is conducted, and the precipitate is reserved; s5, mixing the precipitate with pure water, adjusting the pH value to 6.8-7.4, adding elastinase for enzymolysis, performing high-speed centrifugation, and retaining the precipitate; and S6, collecting the precipitate to obtain the jellyfish elastin peptide. The jellyfish is used as a raw material and has the advantages in the aspects of raw material cost, availability, pretreatment and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of jellyfish processing, and in particular to a method for extracting elastin peptides from jellyfish. Background Art

[0002] Elastin peptides are degradation products of elastin, with many excellent properties and a wide range of applications. Elastin is an important component of the skin, and it only contains 5% in the skin, but it is the key to stimulate the production of collagen and elastic fibers in the skin. 70% of the collagen in the skin determines its softness and fullness, while 90% of the elastic fibers are composed of elastin, which determines the firmness and elasticity of the skin. However, the structure and properties of elastin are very stable and not easily degraded, which increases the difficulty of producing elastin peptides. At present, common sources of elastin include fish, pigs, cattle, etc., among which bonito heart arterial bulb is the main source. However, because different people may react differently to bonito elastin peptides, some people may be allergic to some of the ingredients, and experience allergic symptoms such as skin rashes, itching, and shortness of breath. In addition, the extraction and production process of bonito elastin peptides is relatively complicated, resulting in relatively high product prices.

[0003] To this end, the present invention hopes to propose a new source of elastin peptides to expand the population that the elastin peptides are suitable for, simplify the preparation process, and reduce the cost of the elastin peptides. 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 a method for extracting elastin peptides using jellyfish, which is a new source of elastin peptides, and the raw materials are very abundant and easy to obtain, the preparation process is simple, and it is conducive to reducing costs.

[0006] (II) Technical solution

[0007] A method for extracting elastin peptides using jellyfish, comprising the following steps:

[0008] S1, pretreatment: when the raw material is fresh jellyfish, use clean water to rinse the fresh jellyfish to remove impurities; when the raw material is salted jellyfish, use cold water containing citric acid / acetic acid to soak and desalinate; then, cut the jellyfish raw material into small pieces for standby use;

[0009] S2, mixing the jellyfish cut into small pieces with a sodium hydroxide solution with a pH value of ≥ 11.5, using a high-pressure shearing machine to maintain pressure and shear, and retaining the precipitate after centrifugation;

[0010] S3, mix the precipitate with pure water, adjust the pH to ≤1.5 with hydrochloric acid, react for 1-4h, centrifuge and retain the precipitate;

[0011] S4, mixing the precipitate with sodium chloride brine, the concentration of the sodium chloride brine is 0.8-1.5M, reacting for 1-4h, centrifuging and retaining the precipitate;

[0012] S5, mixing the precipitate with pure water, adjusting the pH to between 6.8-7.4, adding elastase for enzymatic hydrolysis, and after enzymatic hydrolysis, centrifuging at high speed and retaining the precipitate;

[0013] S6. Collect the precipitate and further process it to obtain a jellyfish elastin peptide product.

[0014] According to a preferred embodiment of the present invention, in S1, ultrasonic oscillation treatment is performed during desalination by soaking in cold water containing 1-5% citric acid / acetic acid to accelerate desalination. Citric acid or acetic acid helps soften salted jellyfish. Ultrasonic oscillation can be performed using an ultrasonic oscillation cleaning machine. The size of the small pieces is no more than 1 cm 3 When desalting salted jellyfish, take a small amount of water sample and add silver nitrate. If there is no white turbidity, it means that desalination is complete.

[0015] According to a preferred embodiment of the present invention, in S2, the pressure of the high-pressure shearing machine is 100-200 bar, the shearing speed is 5000-10000 rpm, the processing time is 1.5-4 hours, and after the processing, the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15 minutes and the precipitate is retained.

[0016] According to a preferred embodiment of the present invention, step S2 includes:

[0017] S21: After the pressure of the high-pressure shearing machine is 100-200 bar, the shearing speed is 5000-8000 rpm, and the processing time is 1-2h, the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15min and the primary precipitate is retained; S22: The primary precipitate is mixed with a sodium hydroxide solution with a pH ≥ 11.5, and the treatment is carried out at a pressure of 60-100 bar and a shearing speed of 3000-5000 rpm for 0.5-1h, and then the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15min and the secondary precipitate is retained; S23: The secondary precipitate is mixed with a sodium hydroxide solution with a pH ≥ 11.5, and the treatment is carried out at a pressure of 30-60 bar and a shearing speed of 3000-5000 rpm for 0.5-1h, and the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15min and the third precipitate is retained, and the third precipitate is transferred to the next step.

[0018] According to a preferred embodiment of the present invention, in S3, the precipitate is first mixed with pure water and centrifuged, and the precipitate is retained to wash away the sodium hydroxide contained in the precipitate from step S2; then the precipitate is mixed with pure water, and hydrochloric acid is added to adjust the pH to ≤ 1.5, and the reaction is carried out under ultrasonic oscillation for 1-2 hours or shaking reaction for 3-4 hours, and then centrifuged at a speed of 10000-12000 rpm for 8-15 minutes to retain the precipitate. The ultrasonic oscillation frequency can be 40kHz-1MHz.

[0019] According to a preferred embodiment of the present invention, step S3 includes:

[0020] S31: first mix the precipitate with pure water, retain the precipitate after centrifugation to wash off the sodium hydroxide; S32: mix the precipitate with pure water, and add hydrochloric acid to adjust the pH to ≤1.5, react under ultrasonic vibration for 1-2h or shake for 3-4h, centrifuge at a speed of 10000-12000rpm for 8-15min, and retain the precipitate; S33: resuspend the precipitate with a hydrochloric acid solution with a pH≤1.5, centrifuge at a speed of 10000-12000rpm for 8-15min, and retain the precipitate; perform this step 1-3 times.

[0021] According to a preferred embodiment of the present invention, step S4 includes: resuspending the precipitate with pure water, centrifuging at a speed of 10000-12000 rpm for 8-15 minutes, and retaining the precipitate; mixing the precipitate with 0.8-1.5M sodium chloride saline, reacting under ultrasonic vibration for 1-2 hours or shaking for 3-4 hours, and centrifuging at a speed of 10000-12000 rpm for 8-15 minutes to retain the precipitate.

[0022] According to a preferred embodiment of the present invention, step S5 includes:

[0023] S51: Mix the precipitate with pure water, resuspend and mix evenly, centrifuge at a speed of 10000-12000 rpm for 8-15 min, and retain the precipitate; perform this step 2-3 times;

[0024] S52: Mix the precipitate with pure water, adjust the pH to between 6.8 and 7.2, add elastase at a rate of 1 g for every 80-200 g of precipitate, perform enzymatic hydrolysis with stirring at 15-35°C for 18-25 h or perform enzymatic hydrolysis with ultrasonic vibration for 2-6 h, microfilter to remove the residue, centrifuge the filtrate at a speed of 10000-12000 rpm for 8-15 min, remove the supernatant, and retain the precipitate; the microfiltration pore size is 1-10 μm.

[0025] Preferably, the elastase is pancreatic elastase or marine microbial elastase, preferably marine microbial elastase.

[0026] The centrifugation treatment in the above steps is carried out at 0-15° C. (lower than room temperature), preferably 0-10° C., and more preferably 4° C. The ultrasonic oscillation frequency can be medium frequency oscillation, with a frequency of 40 kHz-1 MHz.

[0027] According to a preferred embodiment of the present invention, in order to obtain elastin peptides with higher purity, the precipitate from S5 can be mixed with pure water, resuspended evenly, ultrafiltered using an ultrafiltration membrane with a cutoff molecule of 300Da, and concentrated by rotary evaporation (preferably ≤40°C) to obtain an elastin peptide concentrate, thereby obtaining an elastin peptide concentrated stock solution product; or the elastin peptide concentrate can be freeze-dried to obtain an elastin peptide freeze-dried powder. The elastin peptide concentrated stock solution product can be added with preservatives or stabilizers allowed to be added to cosmetics.

[0028] According to a preferred embodiment of the present invention, in S1, the jellyfish include: R. esculentum Kishinouye, R. hispidum Vanhoffen, R. rhopalophorum Haeckel, R. verrilli Fewkos, Nemopilema nomurai Kishinouye, Cyanea spp., Aurelia spp., Lobonema smithi Mayer and Lobonemoides gracilis Light, etc.

[0029] (III) Beneficial effects

[0030] 1. The present invention uses jellyfish as raw material to prepare elastin peptides; the water content in the jellyfish is above 95%, and 0.5-1% of the remaining less than 5% of the tissues is jellyfish protein; jellyfish protein is 10-20% of the dry matter, which is a good source of protein, and jellyfish has a large output, abundant raw materials, cheap and easy to obtain. Jellyfish extraction replaces the traditional bonito heart artery bulb extraction, and the prepared elastin peptide has a high yield, safe ingredients, and a wide range of audiences. The present invention has a stable process, simple operation, and low cost. Compared with elastin peptides from terrestrial animals such as pigs and cattle, jellyfish polypeptides are safer.

[0031] Jellyfish have a simple structure and do not have a complex internal organ system such as a heart, lungs or digestive tract. The body of a jellyfish is mainly composed of two layers of cells (ectoderm and endoderm) and the mesoglea between these two layers. The mesoglea is a gelatinous substance composed mainly of water and some protein. The only raw material for the preparation of bonito is the bonito heart arterial bulb; therefore, the use of jellyfish has great advantages in terms of raw material acquisition and pretreatment.

[0032] 2. The process of the present invention is designed based on the characteristics of jellyfish itself, and its process flow is as follows: the pre-treated jellyfish pieces are mixed with sodium hydroxide solution for reaction, the cell structure is destroyed to denature the protein, and the precipitate is retained by centrifugation; since the specific gravity of jellyfish is very close to that of water, and the specific gravity of protein is generally between 1.3 and 1.4, most of the jellyfish residue will remain in the centrifugal supernatant and be removed (a small part enters the centrifugal precipitate), and the fishy smell of the salted jellyfish is also removed; the precipitate is then placed in a strong acidic environment with a pH of ≤1.5 so that the cell structure that was not destroyed under alkaline conditions before is further destroyed under strong acidic conditions, so that the cell contents flow out, the protein is denatured and a protein precipitate is generated, and the jellyfish residue will remain in the supernatant and be removed (only a small amount enters the centrifugal precipitate); then low-concentration sodium chloride salt water is used for treatment and centrifugation, and the precipitate is collected to further purify the protein; then elastase is used to enzymatically hydrolyze the elastin in the precipitate to generate elastin peptides, and the elastin peptides are collected by centrifugation to obtain a jellyfish elastin peptide product after treatment.

[0033] In the process of the present invention, in addition to enzymatically hydrolyzing the extracted protein to generate elastin peptides, no exogenous enzymes are used in the protein extraction process (the jellyfish contains autolytic enzymes), and only sodium hydroxide solution treatment and centrifugal collection of precipitation, hydrochloric acid solution treatment and centrifugal collection of precipitation, and sodium chloride solution treatment and centrifugal collection of precipitation are performed in sequence. The entire process is simple and easy to operate, and jellyfish elastin peptides with a molecular weight of 100-300 Da can be prepared. For other raw materials (such as pigs and cattle), the target protein cannot be separated by centrifugal collection of precipitation due to the large specific gravity of the residue.

[0034] 3. In order to further improve the purity of the product, after the enzymolysis is completed, microfiltration can be used to remove the residue (including the jellyfish residue that has not been destroyed by the above treatment and remains in the precipitate) and bacteria, and the filtrate is retained. After high-speed centrifugation, the precipitate is retained to obtain the jellyfish elastin peptide. At this time, the precipitate is freeze-dried to obtain a jellyfish elastin peptide freeze-dried powder with high purity; if in order to further improve the purity of the product, ultrafiltration can also be used. Ultrafiltration membrane ultrafiltration, ultrafiltration permeate rotary evaporation concentration, to obtain elastin peptide concentrate, to obtain elastin peptide concentrated stock solution product; or the elastin peptide concentrate is freeze-dried to obtain an elastin peptide freeze-dried powder product.

[0035] 4. The present invention uses jellyfish to extract elastin peptides instead of the traditional method of extracting elastin peptides from fish, pigs, cattle, etc. After repeated trial production verification, the raw material cost is low, the product is white or off-white powder, the product quality is stable, and the product protein polypeptide molecular weight range is 100-300Da; protein peptides of this molecular weight are most easily absorbed by the skin, and they are mainly present in human skin, blood vessels, lungs and other tissues, giving these tissues elasticity and retractility; protein polypeptides are used for skin repair, which helps the skin restore fullness and elasticity. In addition, the product can also be used in many fields such as skin care products, medicine and food processing. The scheme of the present invention provides a new idea for utilizing the rich biological resources in the ocean. DETAILED DESCRIPTION

[0036] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods.

[0037] The process of preparing elastin peptide using jellyfish in the present invention comprises the following steps:

[0038] (1) Processing of jellyfish raw materials

[0039] The jellyfish raw materials include fresh jellyfish or jellyfish preserved by salting. For the newly caught jellyfish, the impurities can be washed away by clean water; if it is salted jellyfish, it needs to be desalted because it contains higher salt and a small amount of preservatives and antioxidants.

[0040] The desalination method can be desalination by soaking in clean water or repeated washing and desalination. During soaking, the clean water is changed every 2-4 hours. The whole process may need to change the water 3-5 times, until the clean water is soaked and added to the silver nitrate until there is no white turbidity, which means that the desalination is completed. Repeated washing and desalination requires the use of flowing / circulating water to desalinate the salted jellyfish for a long time. The above two methods consume a lot of water. In a preferred embodiment of the present invention, cold water soaking desalination containing 1-5% citric acid / acetic acid is used, or accompanied by ultrasonic oscillation treatment; citric acid / acetic acid can help rapid desalination, and ultrasonic oscillation can also accelerate desalination. Ultrasonic oscillation can use a medium-frequency ultrasonic oscillation cleaning machine. The method of desalination by soaking in cold water containing 1-5% citric acid / acetic acid can save more than 30% of water. Generally, after 1-2 soakings, the salted jellyfish can be desalted. Among them, weakly acidic citric acid or acetic acid helps soften the salted jellyfish to facilitate subsequent protein extraction and processing.

[0041] Next, the fresh jellyfish or desalted salted jellyfish treated as above is cut into small pieces of about 1 cm×1 cm×1 cm for later use.

[0042] (2) Sodium hydroxide solution treatment

[0043] In this step, the jellyfish raw material cut into small pieces in the previous step is mixed with a sodium hydroxide solution with a pH ≥ 11.5 (preferably pH = 12) for soaking reaction, and after high-speed centrifugation, the precipitate is retained. Preferably, the soaking reaction is pressurized and sheared in a high-pressure shearing machine. In this process, the jellyfish raw material is beaten into a finer shred state, and at the same time, the alkaline sodium hydroxide solution is used to destroy the cell structure of the jellyfish shreds, so that the cell contents flow out, and the protein is denatured under strong alkalinity to produce protein precipitation. In addition, salted jellyfish has a strong fishy smell. When treated with a sodium hydroxide solution, some volatile organic compounds (such as amines, sulfides, etc.) may undergo chemical changes, thereby reducing the fishy smell.

[0044] Preferably, the processing method in the high-pressure shearing machine may be one of the following methods:

[0045] Method 1: The pressure of the high-pressure shearing machine is 100-200 bar, and the shearing speed is 5000-10000 rpm. After the treatment, centrifuge at a speed of 10000-12000 rpm for 8-15 minutes and retain the precipitate. The high-pressure shearing machine can continue to treat for 1.5-4 hours. Alternatively, the three treatments can be repeated as follows.

[0046] Method 2: First, after the high-pressure shearing machine has a pressure of 100-200 bar, a shearing speed of 5000-8000 rpm, and a processing time of 1-2 hours, the mixture is centrifuged at a speed of 10000-12000 rpm for 8-15 minutes and a primary precipitate is retained; then, the primary precipitate is mixed with a sodium hydroxide solution with a pH value of ≥ 11.5, treated at a pressure of 60-100 bar and a shearing speed of 3000-5000 rpm for 0.5-1 hour, and then centrifuged at a speed of 10000-12000 rpm for 8-15 minutes and a secondary precipitate is retained; finally, the secondary precipitate is mixed with a sodium hydroxide solution with a pH value of ≥ 11.5, treated at a pressure of 30-60 bar and a shearing speed of 3000-5000 rpm for 0.5-1 hour, and then centrifuged at a speed of 10000-12000 rpm for 8-15 minutes and a third precipitate is retained, and the third precipitate is transferred to the next step. Compared with method 1, method 2 has lower energy consumption, but method 1 is beneficial to improve product yield.

[0047] Since the specific gravity of jellyfish is very close to that of water, and the specific gravity of protein is generally between 1.3-1.4, most of the residue and impurities of jellyfish will remain in the centrifugal supernatant and be removed, and a small part of the residue and denatured protein will enter the centrifugal precipitation.

[0048] (3) Hydrochloric acid solution treatment

[0049] In this step, the precipitate obtained in the previous step is mixed with pure water, adjusted to pH ≤ 1.5 (preferably pH = 1) with hydrochloric acid, reacted for 1-4 hours, centrifuged and the precipitate is retained. During the sodium hydroxide solution treatment, some cell structures that are not destroyed by the hydroxide solution are further destroyed under strong acid conditions, releasing proteins, and the proteins are denatured under strong acidity and produce protein precipitation.

[0050] Preferably, the hydrochloric acid solution treatment process may be one of the following methods:

[0051] Method (A): First, the precipitate is mixed with pure water and centrifuged, and the precipitate is retained to wash away the sodium hydroxide adsorbed by the precipitate; then, the precipitate is mixed with pure water, and hydrochloric acid is added to adjust the pH to ≤1.5 (preferably pH=1), and the mixture is reacted under ultrasonic oscillation for 1-2 hours or shaken for 3-4 hours, and then centrifuged at a speed of 10000-12000 rpm for 8-15 minutes to retain the precipitate. The ultrasonic oscillation frequency can be medium frequency oscillation.

[0052] Method (B): firstly, the precipitate is mixed with pure water, and the precipitate is retained after centrifugation to wash away the sodium hydroxide; then, the precipitate is mixed with pure water, and hydrochloric acid is added to adjust the pH to ≤1.5 (preferably pH=1), and the reaction is carried out under ultrasonic vibration for 1-2 hours or shaking reaction for 3-4 hours, and then the precipitate is centrifuged at a speed of 10000-12000rpm for 8-15 minutes, and the precipitate is retained; finally, the precipitate is resuspended once with a hydrochloric acid solution of pH≤1.5 (preferably pH=1), and the precipitate is centrifuged at a speed of 10000-12000rpm for 8-15 minutes, and the precipitate is retained; this step is performed 1-3 times. After the above treatment, most of the jellyfish residues and impurities are removed in the supernatant of the centrifugation, and the denatured protein and a small amount of jellyfish residues enter the centrifugal precipitate.

[0053] Ultrasonic vibration is mainly used to accelerate the destruction speed and destruction of the hydrochloric acid solution on the jellyfish cell structure and accelerate the release of protein. In some embodiments, ultrasonic vibration may not be applied, but by extending the duration of the hydrochloric acid solution soaking reaction and stirring / applying a certain oscillation speed, a considerable effect can be achieved. For fresh jellyfish raw materials, production needs to be completed in a relatively short time. Ultrasonic vibration is conducive to greatly shortening the production cycle, which is more in line with the needs of mass production and industrial production.

[0054] (4) Sodium chloride solution treatment

[0055] In this step, the precipitate obtained in the previous step is mixed with sodium chloride salt water, the concentration of which is 0.8-1.5 M, and the reaction is carried out for 1-4 hours, and the precipitate is retained after centrifugation. In the above treatment process, some cell structures that have not been destroyed are further destroyed under the action of sodium salt, releasing proteins, and the proteins are denatured under sodium salt and produce protein precipitation.

[0056] Preferably, the process of the sodium chloride solution treatment can be specifically as follows: resuspend the precipitate with pure water, centrifuge at a speed of 10000-12000rpm for 8-15min, retain the precipitate, and wash off the hydrochloric acid introduced in the previous step through this process; then mix the precipitate with 0.8-1.5M sodium chloride brine, react under ultrasonic vibration for 1-2h or shake for 3-4h, and then centrifuge at a speed of 10000-12000rpm for 8-15min, retain the precipitate. The remaining jellyfish residue and impurities are removed in the supernatant of the centrifugation, and the denatured protein and a very small amount of jellyfish residue enter the centrifugal precipitate. After salting out, the protein is further purified.

[0057] (5) Elastase enzymatic treatment

[0058] In this step, the precipitate obtained in the previous step is mixed with pure water, adjusted to 6.8-7.4 (preferably adjusted to 7) using hydrochloric acid or NaOH solution, and elastase is added, enzymatically hydrolyzed at 15-35° C., and then high-speed centrifugation is performed after enzymatic hydrolysis to retain the precipitate. The elastase is pancreatic elastase or marine microbial elastase, preferably marine microbial elastase.

[0059] Preferably, the process of the above-mentioned elastase enzymatic hydrolysis treatment can be specifically as follows: the precipitate is mixed with pure water, resuspended and mixed, centrifuged at a speed of 10000-12000rpm for 8-15min, and the precipitate is retained; this step is performed 2-3 times; then, the precipitate is mixed with pure water, the pH is adjusted to between 6.8-7.2, and elastase is added in an amount of 1g of elastase per 80-200g of precipitate, stirred at 15-35°C for 18-25h or ultrasonic vibration enzymatic hydrolysis for 2-6h, microfiltration is performed to remove the residue, the filtrate is centrifuged at a speed of 10000-12000rpm for 8-15min, the supernatant is removed, and the precipitate is retained; the microfiltration pore size is 1-10μm. Through this microfiltration operation, microorganisms that may be introduced during the production process and jellyfish tissues that have not been destroyed after the aforementioned multiple treatments are mainly removed. The pore size of 1-10 μm can effectively remove bacteria, suspended solids and other relatively large colloidal particles, while too fine a pore size not only results in slow filtration, high energy consumption, but also leads to the retention of target proteins. In some embodiments, the operation of "microfiltration to remove residues" is not necessary.

[0060] (6) Treating the centrifugal precipitate after enzymatic hydrolysis to obtain a jellyfish elastin peptide product

[0061] In this step, the precipitate obtained in the previous step is placed in a freeze dryer for freeze drying to obtain a jellyfish elastin peptide product. The product is in the form of white or off-white powder, the purity of the protein polypeptide in the product reaches more than 75%, and the molecular weight of most polypeptides is concentrated between 100-300Da.

[0062] In some embodiments, in order to obtain a higher purity elastin peptide, the centrifugal precipitate after enzymatic hydrolysis can be further purified and resuspended, and then ultrafiltration is performed using an ultrafiltration membrane with a cutoff molecule of 300Da, and the permeate is concentrated by rotary evaporation (preferably ≤40°C) to obtain an elastin peptide concentrate, and an elastin peptide concentrated stock solution product is obtained; or the elastin peptide concentrate is freeze-dried to obtain an elastin peptide freeze-dried powder, and the purity of the protein polypeptide in the product reaches more than 95%, and most of the polypeptide molecular weights are concentrated between 100-300Da. Among them, some preservatives or stabilizers allowed to be added to cosmetics can be added to the elastin peptide concentrated stock solution product.

[0063] The centrifugation in each of the above steps is carried out at 0-15° C. (lower than room temperature), and a refrigerated centrifuge can be used, preferably at 0-10° C., more preferably at 4° C. The ultrasonic oscillation frequency can be medium frequency oscillation, with a frequency of 40 kHz-1 MHz.

[0064] The method of the present invention can be used to treat various types of jellyfish, including but not limited to R. esculentum Kishinouye, R. hispidum Vanhoffen, R. rhopalophorum Haeckel, R. verrilli Fewkos, Nemopilema nomurai Kishinouye, Cyanea spp., Aurelia spp., Lobonema smithi Mayer and Lobonemoides gracilis Light, etc.

[0065] The following is a description of the preferred embodiments of the present invention.

[0066] Example 1

[0067] This embodiment is a method for preparing elastin peptide using fresh jellyfish, and the steps are as follows:

[0068] (1) Rinse fresh jellyfish (fresh yellow spot jellyfish) with clean water to remove impurities, drain the water, weigh 1 kg and cut into 1 cm 3 Small pieces for later use.

[0069] (2) The jellyfish raw material was mixed with 1L of sodium hydroxide solution with a pH of 12 for soaking reaction, stirred and soaked at room temperature for 6 hours, centrifuged at 12000rpm for 10 minutes at 4°C, and the precipitate was retained; the precipitate was mixed with 0.8L of sodium hydroxide solution with a pH of 12 for soaking reaction, stirred and soaked at room temperature for 5 hours, centrifuged at 12000rpm for 10 minutes at 4°C, and the precipitate was retained; the precipitate was mixed with 0.6L of sodium hydroxide solution with a pH of 12 for soaking reaction, stirred and soaked at room temperature for 3 hours, centrifuged at 12000rpm for 10 minutes at 4°C, and the precipitate was retained.

[0070] (3) Mix the precipitate obtained in the previous step with 1L of pure water, centrifuge at 12000rpm for 10min at 4℃, and keep the precipitate. Mix the precipitate with 1.5L of pure water, adjust the pH to 1 with 1M hydrochloric acid, soak and react for 2h at a shaking frequency of 70 times / min, centrifuge at 12000rpm for 10min at 4℃, and keep the precipitate. Add 1L of 1M hydrochloric acid solution to the precipitate to resuspend it, centrifuge at 12000rpm for 10min at 4℃, and keep the precipitate; repeat this process twice.

[0071] (4) The precipitate obtained in the previous step was resuspended in 1.5 L of purified water and centrifuged at 12,000 rpm for 10 min at 4°C. 1.5 L of 1 M NaCl solution was added to the precipitate and the mixture was shaken at 70 times / min for 3 h. The mixture was then centrifuged at 12,000 rpm for 15 min at 4°C and the precipitate was retained.

[0072] (5) The precipitate obtained in the previous step was mixed evenly with 1 L of pure water, resuspended, and centrifuged at 12,000 rpm for 15 min at 4°C to retain the precipitate; this process was repeated twice. After that, the precipitate was mixed with 1.5 L of pure water, the pH was adjusted to 7, 5 g of elastase (commercially available) was added, and after enzymatic hydrolysis at room temperature for 24 h, the precipitate was centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was removed, and the precipitate was retained.

[0073] (6) placing the precipitate in a freeze dryer for freeze drying to obtain jellyfish elastin peptide.

[0074] Example 2

[0075] This embodiment is a method for preparing elastin peptide using salted jellyfish, and the steps are as follows:

[0076] (1) Take the salted jellyfish (salt-pickled jellyfish) and rinse with clean water to remove impurities, soak it in cold water with 1% citric acid for 2 hours, and use a 40kHz medium-frequency ultrasonic oscillator to treat it intermittently. Then change to clean water and soak it for 3 hours (no more ultrasound), and the desalination is successful. Take 1 kg of desalinated salted jellyfish and cut it into 1 cm 3If using clean water for desalination, it is necessary to change the water every 2-4 hours, and the desalination purpose can be achieved after 3-5 water changes.

[0077] (2) The jellyfish raw material was mixed with 1 L of sodium hydroxide solution with a pH of 12 for soaking reaction, stirred and soaked at room temperature for 6 hours, and centrifuged at 12000 rpm for 10 minutes at 4°C to retain the precipitate; this process was repeated 3 times.

[0078] (3) Mix the precipitate obtained in the previous step with 1L of pure water, centrifuge at 12000rpm for 10min at 4℃, and keep the precipitate. Mix the precipitate with 1.5L of pure water, adjust the pH to 1 with 1M hydrochloric acid, soak and react for 2h at a shaking frequency of 70 times / min, centrifuge at 12000rpm for 10min at 4℃, and keep the precipitate. Add 1L of 1M hydrochloric acid solution to the precipitate to resuspend it, centrifuge at 12000rpm for 10min at 4℃, and keep the precipitate; repeat this process 3 times.

[0079] (4) The precipitate obtained in the previous step was resuspended in 1.5 L of purified water and centrifuged at 12,000 rpm for 10 min at 4°C. 1.5 L of 1 M NaCl solution was added to the precipitate and the mixture was shaken at 70 times / min for 3 h. The mixture was then centrifuged at 12,000 rpm for 15 min at 4°C and the precipitate was retained.

[0080] (5) The precipitate obtained in the previous step was mixed evenly with 1 L of pure water, resuspended, and centrifuged at 12,000 rpm for 15 min at 4°C to retain the precipitate; this process was repeated twice. After that, the precipitate was mixed with 1.5 L of pure water, the pH was adjusted to 7, 4 g of elastase (commercially available) was added, and after enzymatic hydrolysis at room temperature for 24 h, the precipitate was centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was removed, and the precipitate was retained.

[0081] (6) placing the precipitate in a freeze dryer for freeze drying to obtain jellyfish elastin peptide.

[0082] Example 3

[0083] The difference between this embodiment and embodiment 1 is the processing conditions of step (2). In this embodiment, step (2) is changed to: treat the jellyfish raw material with 1L of sodium hydroxide solution with a pH of 12 in a high-pressure shearing machine at a pressure of 120 bar and a rotation speed of 8000 rpm for 3 hours, and then centrifuge at 12000 rpm for 10 minutes at 4°C to retain the precipitate. Compared with step (2) of embodiment 1, this embodiment can save 11.3 hours of time by performing the soaking reaction of the sodium hydroxide solution in the high-pressure shearing machine, and only centrifuge once. The remaining steps of this embodiment are the same as those of embodiment 1.

[0084] Example 4

[0085] The difference between this embodiment and embodiment 1 is the processing conditions of step (2). In this embodiment, step (2) is changed to: the jellyfish raw material is treated with 1L of sodium hydroxide solution with a pH of 12 in a high-pressure shearing machine at a pressure of 100120 bar and a rotation speed of 8000 rpm for 1 hour, and then centrifuged at 12000 rpm for 10 minutes at 4°C, retaining a primary precipitate; then, the primary precipitate is mixed with a sodium hydroxide solution with a pH of 12, treated at a pressure of 80 bar and a shear speed of 5000 rpm for 1 hour, centrifuged at 12000 rpm for 10 minutes at 4°C, and retaining a secondary precipitate; finally, the secondary precipitate is mixed with a sodium hydroxide solution with a pH of 12, treated at a pressure of 60 bar and a shear speed of 5000 rpm for 1 hour, centrifuged at 12000 rpm for 10 minutes at 4°C, retaining a third precipitate, and transferring the third precipitate to the next step.

[0086] The remaining steps of this embodiment are the same as those of Embodiment 1.

[0087] Example 5

[0088] The difference between this embodiment and embodiment 1 lies in the processing conditions of step (3). In this embodiment, step (3) is changed to: mix the precipitate obtained in the previous step with 1L of pure water, centrifuge at 12000rpm for 10min at 4°C, and retain the precipitate. Mix the precipitate with 1.5L of pure water, adjust the pH to 1 with 1M hydrochloric acid, react under 40KHZ ultrasonic vibration for 1h, centrifuge at 12000rpm for 10min at 4°C, and retain the precipitate. Add 1L of 1M hydrochloric acid solution to the precipitate to resuspend it, centrifuge at 12000rpm for 10min at 4°C, and retain the precipitate; repeat this process twice. The remaining steps of this embodiment are the same as those of embodiment 1.

[0089] Example 6

[0090] The difference between this embodiment and embodiment 1 is the processing conditions of step (4). In this embodiment, step (4) is changed to: add 1.5L of pure water to resuspend the precipitate obtained in the previous step, centrifuge at 12000rpm for 15min at 4°C, add 1.5L of 1M NaCl solution to the obtained precipitate, react under 40KHZ ultrasonic vibration for 1.5h, then centrifuge at 12000rpm for 15min at 4°C, and retain the precipitate. The remaining steps of this embodiment are the same as those of embodiment 1.

[0091] Example 7

[0092] The difference between this embodiment and embodiment 1 is the processing conditions of step (5). In this embodiment, step (5) is changed to: the precipitate obtained in the previous step is mixed evenly with 1L of pure water, resuspended, centrifuged at 12000rpm for 15min at 4°C, and the precipitate is retained; this process is repeated twice. After that, the precipitate is mixed with 1.5L of pure water, the pH is adjusted to 7, 5g of elastase (commercially available product) is added, and the enzymatic hydrolysis reaction is carried out under ultrasonic vibration of 50KHZ for 5h, and the precipitate is centrifuged at 12000rpm for 10min at 4°C, and the supernatant is removed and the precipitate is retained. The remaining steps of this embodiment are the same as those of embodiment 1.

[0093] Example 8

[0094] The difference between this example and Example 1 is the processing conditions of step (5). In this example, step (5) is changed to: the precipitate obtained in the previous step is mixed evenly with 1L of pure water, resuspended, centrifuged at 12000rpm for 15min at 4°C, and the precipitate is retained; this process is repeated twice. After that, the precipitate is mixed with 1.5L of pure water, the pH is adjusted to 7, 5g of elastase (commercially available product) is added, and after enzymatic hydrolysis at room temperature for 24h, vacuum filtration is performed using a microfiltration membrane (pore size of 5-10μm), the filtrate is centrifuged at 12000rpm for 10min at 4°C, the supernatant is removed, and the precipitate is retained.

[0095] Example 9

[0096] The difference between this example and example 1 is the processing conditions of step (6). In this example, step (6) is changed to: resuspend the precipitate with 5 L of pure water, then ultrafilter using an ultrafiltration membrane with a cutoff molecular weight of 300 Da, and concentrate the permeate by rotary evaporation at 30° C. to obtain an elastin peptide concentrate, which is then placed in a freeze dryer for freeze drying to obtain jellyfish elastin peptide.

[0097] The quality and purity of the jellyfish elastin peptides prepared in the above examples are as follows:

[0098] Group Protein peptide grams purity Example 1 49.3g 76.4％ Example 2 43.5g 75.6％ Example 3 53.7g 76.2％ Example 4 50.2g 76.7％ Example 5 49.0g 76.0％ Example 6 49.8g 77.1％ Example 7 49.2g 77.4％ Example 8 42.7g 80.3％ Example 9 35.6g 92.4％

[0099] After testing, the protein peptides prepared in the above embodiments are basically consistent with the chromatographic analysis results of the current elastin peptide standard material from fish. The polypeptide products were sent to a third-party testing agency for testing, and the molecular weight of the elastin peptide was concentrated between 100-300Da. Peptides with a molecular weight of 100-300Da can be better absorbed by the skin, and the molecules are small, so they can penetrate into the deep layers of the skin more easily through the skin barrier (mainly the stratum corneum).

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting elastin peptides using jellyfish, characterized in that: Includes steps: S1, pretreatment: when the raw material is fresh jellyfish, use clean water to rinse the fresh jellyfish to remove impurities; when the raw material is salted jellyfish, use cold water containing citric acid / acetic acid to soak and desalinate; then, cut the jellyfish raw material into small pieces for standby use; S2, mixing the jellyfish cut into small pieces with a sodium hydroxide solution with a pH value of ≥ 11.5, using a high-pressure shearing machine to maintain pressure and shear, and retaining the precipitate after centrifugation; S3, mix the precipitate with pure water, adjust the pH to ≤1.5 with hydrochloric acid, react for 1-4h, centrifuge and retain the precipitate; S4, mixing the precipitate with sodium chloride brine, the concentration of the sodium chloride brine is 0.8-1.5M, reacting for 1-4h, centrifuging and retaining the precipitate; S5, mixing the precipitate with pure water, adjusting the pH to between 6.8-7.4, adding elastase for enzymatic hydrolysis, and after enzymatic hydrolysis, centrifuging at high speed and retaining the precipitate; S6. Collect the precipitate and further process it to obtain a jellyfish elastin peptide product.

2. The method according to claim 1, characterized in that In S1, ultrasonic vibration treatment is performed during desalination by soaking in cold water containing citric acid / acetic acid; the size of the jellyfish pieces does not exceed 1 cm 3 .

3. The method according to claim 1, characterized in that In S2, the pressure of the high-pressure shearing machine is 100-200 bar, the shearing speed is 5000-10000 rpm, the processing time is 1.5-4 hours, and after the processing, the centrifugal treatment is performed at a speed of 10000-12000 rpm for 8-15 minutes and the precipitate is retained.

4. The method according to claim 1, characterized in that Step S2 includes: S21: After the pressure of the high-pressure shearing machine is 100-200 bar, the shearing speed is 5000-8000 rpm, and the processing time is 1-2h, the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15min and the primary precipitate is retained; S22: The primary precipitate is mixed with a sodium hydroxide solution with a pH ≥ 11.5, and the treatment is carried out at a pressure of 60-100 bar and a shearing speed of 3000-5000 rpm for 0.5-1h, and then the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15min and the secondary precipitate is retained; S23: The secondary precipitate is mixed with a sodium hydroxide solution with a pH ≥ 11.5, and the treatment is carried out at a pressure of 30-60 bar and a shearing speed of 3000-5000 rpm for 0.5-1h, and the centrifugal treatment is carried out at a speed of 10000-12000 rpm for 8-15min and the third precipitate is retained, and the third precipitate is transferred to the next step.

5. The method according to claim 1, characterized in that In S3, the precipitate is first mixed with pure water and centrifuged, and the precipitate is retained to wash away the sodium hydroxide contained in the precipitate from step S2; then the precipitate is mixed with pure water, and hydrochloric acid is added to adjust the pH to ≤ 1.

5. After reacting under ultrasonic vibration for 1-2 hours or shaking for 3-4 hours, the mixture is centrifuged at a speed of 10000-12000 rpm for 8-15 minutes, and the precipitate is retained.

6. The method according to claim 1, characterized in that Step S3 includes: S31: first mix the precipitate with pure water, retain the precipitate after centrifugation to wash off the sodium hydroxide; S32: mix the precipitate with pure water, and add hydrochloric acid to adjust the pH to ≤1.5, react under ultrasonic vibration for 1-2h or shake for 3-4h, centrifuge at a speed of 10000-12000rpm for 8-15min, and retain the precipitate; S33: resuspend the precipitate with hydrochloric acid solution with a pH≤1.5, centrifuge at a speed of 10000-12000rpm for 8-15min, and retain the precipitate; perform this step 1-3 times.

7. The method according to claim 1, characterized in that Step S4 includes: resuspending the precipitate with pure water, centrifuging at a speed of 10000-12000 rpm for 8-15 minutes, and retaining the precipitate; mixing the precipitate with 0.8-1.5M sodium chloride saline, reacting under ultrasonic vibration for 1-2 hours or shaking for 3-4 hours, and centrifuging at a speed of 10000-12000 rpm for 8-15 minutes to retain the precipitate.

8. The method according to claim 1, characterized in that Step S5 includes: S51: Mix the precipitate with pure water, resuspend and mix evenly, centrifuge at a speed of 10000-12000 rpm for 8-15 min, and retain the precipitate; perform this step 2-3 times; S52: Mix the precipitate with pure water, adjust the pH to between 6.8 and 7.2, add elastase at a rate of 1 g for every 80-200 g of precipitate, perform enzymatic hydrolysis with stirring at 15-35°C for 18-25 h or perform enzymatic hydrolysis with ultrasonic vibration for 2-6 h, microfilter to remove the residue, centrifuge the filtrate at a speed of 10000-12000 rpm for 8-15 min, remove the supernatant, and retain the precipitate; the microfiltration pore size is 1-10 μm.

9. The method according to claim 8, characterized in that The elastase is pancreatic elastase or marine microbial elastase.

10. The method according to claim 1, characterized in that Step S6 is: mixing the precipitate from S5 with pure water, resuspending evenly, ultrafiltration using an ultrafiltration membrane with a cutoff molecule of 300Da, and concentrating by liquid rotary evaporation to obtain an elastin peptide concentrate to obtain an elastin peptide concentrated stock solution product; or freeze-drying the elastin peptide concentrate to obtain an elastin peptide freeze-dried powder.