A low-fat, low-cost, high-purity and high-quality elastin peptide and its preparation method

Abstract

The present invention belongs to the technical field of active substance extraction, and specifically relates to a low-fat, low-cost, high-purity and high-quality elastin peptide and a preparation method thereof. The present invention uses a mixture of alkali solution and sodium carbonate solution, the overall concentration is low, it is more environmentally friendly, and the degreasing effect is better using microwave heating technology. The present invention uses microsphere immobilized protease, and the enzyme preparation can be fully recovered and reused multiple times, which greatly reduces production costs. The elastase hydrolysate is separated, purified and refined using ion exchange resin series chromatography technology to efficiently remove other impurities, and the prepared elastin peptide has a higher purity. The present invention uses high-voltage pulsed electric field technology (PEF) for sterilization, which has a better sterilization effect, avoids the influence of traditional heat sterilization methods on the color and taste of the product, and better maintains the quality of the elastin peptide.

Description

Technical Field

[0001] The present invention belongs to the technical field of active substance extraction, and in particular relates to a low-fat, low-cost, high-purity and high-quality elastin peptide and a preparation method thereof. Background Art

[0002] Elastin is an important extracellular matrix protein and the primary component of elastic fibers. It is found in large quantities in tissues and organs that are frequently subjected to stress and deformation, such as the lungs, aorta, skin, and ligaments of mammals, and the bulbus arteriosus of fish. Elastin contains a unique cross-linked structure—desmosin and isodesmosin. Tropoelastin, the soluble precursor of elastin, is cross-linked by desmosin and isodesmosin to form a dense protein structure. Both proteins are water-insoluble. Due to their compact structure and hydrophobicity, they possess excellent ductility and elasticity and are resistant to hydrolysis by most hydrolytic enzymes. The levels of desmosin and isodesmosin, amino acids unique to elastin, have a significant impact on the biosynthesis and physiological functions of elastin.

[0003] Elastin tissue is often rich in lipids, which seriously affects its effectiveness. Degreasing can improve the efficiency and purity of elastin enzymatic hydrolysis. Currently, raw materials for preparing elastin peptides are generally pretreated with alkaline solutions, but the concentrations used are relatively high. Chinese patent application CN106519020A uses 0.5% sodium hydroxide solution at 85-95°C to treat elastin-containing raw materials, and Chinese patent CN108977488B uses 3-6% sodium carbonate solution to treat fresh bovine neck ligament to remove fat and connective tissue. However, the wastewater generated by these methods places significant environmental strain. Furthermore, current preparations of elastin peptides typically utilize free protease in liquid or powder form for enzymatic hydrolysis. These enzymes have poor resistance to adverse environmental conditions, easily lose activity, and are difficult to recycle and reuse, resulting in relatively high production costs. Furthermore, conventional methods lack purification and refining processes, resulting in low levels of desmosin and isodesmosin in the prepared elastin peptides. Traditional heat sterilization methods can easily induce glycation reactions in the feed solution, affecting the product's color and taste, leading to reduced product quality.

[0004] Therefore, how to further reduce the fat content of elastin peptide products and reduce costs while retaining the activity of elastin peptides during the preparation of elastin peptides is an urgent problem to be solved in this field. Summary of the Invention

[0005] The present invention addresses the defects and shortcomings of the prior art and provides a low-fat, low-cost, high-purity and high-quality elastin peptide and a preparation method thereof.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a low-fat, low-cost, high-purity and high-quality elastin peptide, the method comprising the following steps:

[0008] (1) Pretreatment: Cut the cleaned elastin-rich animal tissue into small pieces, add 8-12 times its weight of alkaline solution, soak at room temperature for 2-6 hours, and rinse with purified water until the conductivity of the effluent is within 1000 μs / cm, thereby obtaining pretreated elastin tissue, wherein the alkaline solution is a mixed solution of alkali and sodium carbonate, and the mass concentration ratio of alkali to sodium carbonate is (0.05-0.1%): (0.5-1%);

[0009] (2) heating aging: adding 1-3 times the weight of purified water to the pretreated elastin tissue prepared in step (1) after cleaning, and then heating the material using a microwave heating method, with a constant microwave power of 500-800W, and the microwave treatment is performed in an intermittent manner, continuously working for 2-4 minutes, and then resting for 2-4 minutes, with one microwave action and a rest period as one cycle, and performing 6-8 cycles;

[0010] (3) Two-phase separation: The liquid after heat extraction is allowed to stand and the two phases are separated, with the oil phase in the upper layer and the water phase in the lower layer. The oil phase is fully removed by liquid separation;

[0011] (4) Enzymatic hydrolysis: adding 5-15% of the weight of the pretreated elastin tissue to the aqueous phase of the microsphere-immobilized protease, adjusting the temperature to 50-65°C, and performing enzymatic hydrolysis for 3-8 hours to obtain an enzymatic hydrolyzate containing elastin peptides;

[0012] (5) solid-liquid separation: the enzymatic hydrolysate containing elastin peptides prepared in step (4) is subjected to solid-liquid separation using a 50-100 nm ceramic membrane to obtain a clear and transparent enzymatic hydrolysate;

[0013] (6) Decolorization and purification: The enzymatic hydrolysis solution prepared in step (5) is sequentially passed through a series chromatography column filled with a cation exchange resin, anion exchange resin, and cation exchange resin at a flow rate of 4-6 BV / h to remove other miscellaneous protein peptides including collagen peptides, thereby preparing an elastin peptide solution, wherein the cation exchange resin, anion exchange resin, and cation exchange resin are respectively weak acid type, strong base type, and weak acid type ion exchange resins, and the column volume ratio is 1:1.2:1-1:1.5:1;

[0014] (7) Sterilization: The elastin peptide solution prepared in step (6) is sterilized using a high-voltage pulsed electric field (PEF) technique with an electric field strength of 15-30 kV / cm and a pulse number of 100-350;

[0015] (8) Drying: spray-drying the sterilized elastin peptide solution prepared in step (7) to obtain elastin peptide powder.

[0016] As an optional manner, in the above preparation method, in step (1), the elastin-rich animal tissue is the aorta, skin, ligament of mammals such as cows or pigs, or the bulbus arteriosus of the heart of fish.

[0017] Preferably, the elastin-rich animal tissue is fish bulbus arteriosus.

[0018] Preferably, the elastin-rich animal tissue is cut into blocks of (2-4) mm×(2-4) mm×(2-4) mm.

[0019] As an optional manner, in the above preparation method, in step (1), the base is sodium hydroxide or potassium hydroxide.

[0020] Preferably, the base is sodium hydroxide.

[0021] As an optional mode, in the above preparation method, in step (4), the immobilized protease is selected from one or more of the following: alkaline protease, papain, bromelain, neutral protease, flavor protease, trypsin or pepsin.

[0022] As an optional mode, in the above preparation method, the immobilized protease is selected from alkaline protease and papain.

[0023] As an optional manner, in the above preparation method, the preparation method of the immobilized protease in step (4) comprises the following steps: based on the total mass of the immobilized protease, the mass concentration of sodium alginate is 2-4%, the mass concentration of chitosan is 3-5%, the mass concentration of calcium chloride is 2-4%, the mass concentration of glutaraldehyde is 0.5-1%, the mass concentration of protease is 0.5-0.8%, the cross-linking time is 1-4h, and the immobilization temperature is 35-45°C, thereby preparing the microsphere immobilized protease.

[0024] The microsphere-immobilized protease prepared by the present invention has relatively good thermodynamic stability and enzymatic properties, and the enzyme activity can still be maintained at above 65% after being reused 5 times.

[0025] As an optional method, in the above preparation method, the method for recovering the immobilized protease in step (4) includes the following steps: passing the enzymatic hydrolyzate containing the immobilized protease through a 40-60 mesh standard sieve to recover the microsphere immobilized protease, rinsing with pure water and refrigerating for repeated use.

[0026] In a second aspect, the present invention provides an elastin peptide prepared by the preparation method described in the first aspect.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention uses a mixture of alkali solution and sodium carbonate solution, which has a low overall concentration and is more environmentally friendly, and uses microwave heating technology for better degreasing effect.

[0029] (2) The present invention uses microsphere-immobilized protease, and the enzyme preparation can be fully recovered and reused multiple times, greatly reducing production costs.

[0030] (3) The ion exchange resin serial chromatography technology is used to separate, purify and refine the elastase hydrolysate, effectively removing other impurities, and the prepared elastin peptide has a high purity.

[0031] (4) The present invention uses high-voltage pulsed electric field technology (PEF) for sterilization, which has a better sterilization effect, avoids the influence of traditional heat sterilization on product color and taste, and better maintains the quality of elastin peptide. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0035] Preparation Example:

[0036] Example 1

[0037] A method for preparing low-fat, low-cost, high-purity and high-quality elastin peptide comprises the following steps:

[0038] (1) Pretreatment: adding 8 times the weight of an alkaline solution to the cleaned tilapia cardiac arterial bulb, soaking the solution at room temperature for 4 hours, and washing the solution with purified water until the conductivity of the effluent is within 1000 μs / cm, thereby obtaining the pretreated tilapia cardiac arterial bulb, wherein the alkaline solution is a mixed solution of sodium hydroxide and sodium carbonate, and the mass concentration ratio of sodium hydroxide to sodium carbonate is 0.05%:1%;

[0039] (2) Heat aging: After cleaning, the tilapia cardiac artery bulb was added with 2 times its weight of purified water, and then the material was heated using a microwave heating method. The microwave power was constant at 500 W, and the microwave treatment was performed in an intermittent manner. After continuous operation for 4 minutes, the microwave treatment was rested for 4 minutes. The execution of one microwave action and rest period was one cycle, and 6 cycles were performed.

[0040] (3) Two-phase separation: The liquid after heat extraction is allowed to stand and the two phases are separated, with the oil phase in the upper layer and the water phase in the lower layer. The oil phase is fully removed by liquid separation;

[0041] (4) Enzymatic hydrolysis: adding 5% of the weight of the pretreated tilapia cardiac arterial bulb microsphere-immobilized alkaline protease and 10% of the weight of the pretreated tilapia cardiac arterial bulb microsphere-immobilized papain to the aqueous phase, adjusting the temperature to 55° C., and performing enzymatic hydrolysis for 5 h to obtain an enzymatic hydrolyzate containing elastin peptides;

[0042] (5) solid-liquid separation: The enzymatic hydrolysate containing elastin peptide prepared in step (4) is subjected to solid-liquid separation using a ceramic membrane (100 nm) to prepare a clear and transparent enzymatic hydrolysate;

[0043] (6) Decolorization and purification: The enzymatic hydrolysis solution prepared in step (5) is sequentially passed through a series chromatography column filled with a cation exchange resin, anion exchange resin, and cation exchange resin at a flow rate of 4 BV / h to remove other miscellaneous protein peptides including collagen peptides, thereby preparing an elastin peptide solution, wherein the cation exchange resin, anion exchange resin, and cation exchange resin are respectively weak acid type, strong base type, and weak acid type ion exchange resins, and the column volume ratio is 1:1.2:1;

[0044] (7) Sterilization: The elastin peptide solution prepared in step (6) was sterilized using a high-voltage pulsed electric field technique (PEF) with an electric field strength of 20 kV / cm and a pulse number of 200;

[0045] (8) Drying: spray-drying the sterilized elastin peptide solution prepared in step (7) to obtain elastin peptide powder.

[0046] The method for preparing the immobilized protease in step (4) comprises the following steps: based on the total mass of the immobilized protease, the mass concentration of sodium alginate is 3%, the mass concentration of chitosan is 3%, the mass concentration of calcium chloride is 3.5%, the mass concentration of glutaraldehyde is 1%, the mass concentration of protease is 0.6%, the cross-linking time is 1 hour, and the immobilization temperature is 40° C., thereby preparing the microsphere-immobilized protease. The obtained microsphere-immobilized protease has good thermodynamic stability and enzymatic properties, and the enzyme activity can still be maintained at above 65% after repeated use five times.

[0047] The method for recovering the immobilized protease in step (4) comprises the following steps: passing the enzymatic hydrolysate containing the immobilized protease through a 40-mesh standard sieve to recover the microsphere-immobilized protease, rinsing with pure water and refrigerating for repeated use.

[0048] Example 2

[0049] A method for preparing low-fat, low-cost, high-purity and high-quality elastin peptide comprises the following steps:

[0050] (1) Pretreatment: adding 8 times the weight of an alkaline solution to the cleaned tilapia cardiac arterial bulb and soaking it at room temperature for 4 hours, and then washing it with purified water until the conductivity of the effluent is within 1000 μs / cm, thereby obtaining the pretreated tilapia cardiac arterial bulb, wherein the alkaline solution is a mixed solution of sodium hydroxide and sodium carbonate, and the mass concentration ratio of sodium hydroxide to sodium carbonate is 0.1%:0.5%;

[0051] (2) Heat aging: After cleaning, the tilapia cardiac artery bulb was added with 2 times its weight of purified water, and then the material was heated using a microwave heating method. The microwave power was constant at 800 W, and the microwave treatment was performed in an intermittent manner. After continuous operation for 2 minutes, the microwave treatment was rested for 2 minutes. The execution of one microwave action and a rest period was considered as one cycle, and 6 cycles were performed.

[0052] (3) Two-phase separation: The liquid after heat extraction is allowed to stand and the two phases are separated, with the oil phase in the upper layer and the water phase in the lower layer. The oil phase is fully removed by liquid separation;

[0053] (4) Enzymatic hydrolysis: adding 5% of the weight of the pretreated tilapia cardiac arterial bulb microsphere-immobilized alkaline protease and 10% of the weight of the pretreated tilapia cardiac arterial bulb microsphere-immobilized papain to the aqueous phase, adjusting the temperature to 55° C., and performing enzymatic hydrolysis for 5 h to obtain an enzymatic hydrolyzate containing elastin peptides;

[0054] (5) solid-liquid separation: The enzymatic hydrolyzate containing elastin peptide prepared in step (4) is subjected to solid-liquid separation using a ceramic membrane (100 nm) to obtain a clear and transparent enzymatic hydrolyzate solution;

[0055] (6) Decolorization and purification: The enzymatic hydrolysis solution prepared in step (5) is sequentially passed through a series chromatography column filled with a cation exchange resin, anion exchange resin, and cation exchange resin at a flow rate of 4 BV / h to remove other miscellaneous protein peptides including collagen peptides, thereby preparing an elastin peptide solution, wherein the cation exchange resin, anion exchange resin, and cation exchange resin are respectively weak acid type, strong base type, and weak acid type ion exchange resins, and the column volume ratio is 1:1.2:1;

[0056] (7) Sterilization: The elastin peptide solution prepared in step (6) was sterilized using a high-voltage pulsed electric field technique (PEF) with an electric field strength of 30 kV / cm and a pulse number of 100;

[0057] (8) Drying: spray-drying the sterilized elastin peptide solution prepared in step (7) to obtain elastin peptide powder.

[0058] The method for preparing the immobilized protease in step (4) comprises the following steps: based on the total mass of the immobilized protease, the mass concentration of sodium alginate is 3%, the mass concentration of chitosan is 3%, the mass concentration of calcium chloride is 3.5%, the mass concentration of glutaraldehyde is 1%, the mass concentration of protease is 0.6%, the cross-linking time is 1 hour, and the immobilization temperature is 40° C., thereby preparing the microsphere-immobilized protease. The obtained microsphere-immobilized protease has good thermodynamic stability and enzymatic properties, and the enzyme activity can still be maintained at above 65% after repeated use five times.

[0059] The method for recovering the immobilized protease in step (4) comprises the following steps: passing the enzymatic hydrolysate containing the immobilized protease through a 40-mesh standard sieve to recover the microsphere-immobilized protease, rinsing with pure water and refrigerating for repeated use.

[0060] Comparative Example 1

[0061] The step (2) in Example 1 was changed to heat aging: 2 times the weight of purified water was added to the cleaned tilapia cardiac arterial bulb and stirred at 90° C. for 4 h.

[0062] Comparative Example 2

[0063] Step (4) in Example 1 was changed to enzymatic hydrolysis: 0.5% alkaline protease by weight of the tilapia arterial bulb and 1.0% papain by weight of the tilapia arterial bulb were added to the aqueous phase, the temperature was adjusted to 55° C., and enzymatic hydrolysis was performed for 5 hours to obtain an enzymatic hydrolyzate containing elastin peptides.

[0064] Step (5) was changed to enzyme inactivation: the enzymatic hydrolysate was heated to 90°C and kept warm for 15 min.

[0065] Comparative Example 3

[0066] The step (6) in Example 1 was changed to decolorization and nanofiltration: the clarified enzymatic hydrolysate was decolorized by an activated carbon fiber membrane and desalted by a nanofiltration membrane with a molecular weight of 200D.

[0067] Comparative Example 4

[0068] Step (7) in Example 1 was changed to sterilization: the enzymatic hydrolyzate was treated at 121° C. for 5 s using an ultra-high temperature instantaneous sterilization (UHT) device.

[0069] Effect embodiment:

[0070] Effect Example 1

[0071] The test indicators and their detection and analysis methods involved in the various examples and comparative examples prepared in the above preparation examples of the present invention are as follows:

[0072] The present invention uses the second method of GB5009.6-2016 for the determination of fat in foods to determine the fat content in elastin peptides.

[0073] Desmosin / isodesmosin content was determined according to the method in Appendix A of T / CHC 1010-2023 Elastin Peptide. The specific method is as follows: Samples were hydrolyzed under acidic conditions and determined using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Desmosin and isodesmosin standards were used for qualitative and confirmatory analysis, and external standard analysis was used for quantification. Chromatographic conditions: Atlantis dC18 (3 μm, 150 mm × 2.1 mm), column temperature 30°C, flow rate 0.3 mL / min, mobile phase A: aqueous solution (containing 7 mM heptafluorobutyric anhydride and 5 mM ammonium acetate), mobile phase B: 80% acetonitrile (containing 7 mM heptafluorobutyric anhydride and 5 mM ammonium acetate); elution program: gradient elution from 100% to 30% mobile phase A over 0-14 min, elution back to 100% mobile phase A over 14-22 min. Mass spectrometry conditions: electrospray ionization source, positive ion mode, ion source spray voltage 3.5 kV, capillary temperature 320 ° C, collision energy 35 eV, desmosine parent ion (m / z) 526.3, daughter ion (m / z) 481.3, isodesmosine parent ion (m / z) 526.3, daughter ion

[0074] (m / z)397.3.

[0075] Desmosin content (%) = desmosin mass in sample / sample mass × 100.

[0076] Isodesmosin content (%) = mass of isodesmosin in sample / mass of sample × 100.

[0077] The total colony count was determined according to GB 4789.2.

[0078] The whiteness was measured using a JC-BD1A benchtop whiteness meter.

[0079] The determination of ash content shall refer to GB 5009.4-2016.

[0080] Table 1 shows a comparison of the fat content percentage, desmosin content %, isodesmosin content %, total bacterial count, whiteness, and ash content of the samples prepared in Examples 1-2 and Comparative Examples 1-4.

[0081] Table 1 Physical and chemical indicators of samples prepared in various embodiments and comparative examples

[0082]

[0083] As shown in Table 1, the samples prepared in the Examples have relatively low fat contents, all less than 0.5%, while the fat content in Comparative Example 1 is higher, indicating that microwave heating can better disrupt the natural bond between fat and protein components in the bulbus arteriosus, resulting in more efficient oil removal. The data from Examples and Comparative Example 2 show that, with the same enzyme dosage, the immobilized enzyme and free protease preparations achieve the same experimental results. Furthermore, the immobilized enzyme can be recycled and reused multiple times, eliminating the need for heating to inactivate the enzyme, significantly reducing production time and costs. The data from Examples and Comparative Example 3 show that the desmosin / isodesmosin content in the samples from Comparative Example 3 is significantly lower, and the whiteness is significantly lower than that of the Examples, demonstrating that ion exchange resins are more effective in adsorbing pigments, separating impurities, reducing ash content, and effectively improving the purity of elastin peptides. The data from Examples and Comparative Example 4 show that the elastin peptides sterilized at high temperatures have a darker color and significantly reduced whiteness, suggesting that high-temperature sterilization may damage the elastin peptides, resulting in a decrease in desmosin and isodesmosin content. Compared to traditional high-temperature sterilization, high-voltage pulsed electric field (PEF) technology offers superior sterilization efficacy and a higher whiteness.

[0084] Effect Example 2

[0085] Test method: Recruitment of participants aged 35-45 years with a BMI of 18.5-27.9 kg / m 2 A total of 120 healthy female volunteers were randomly divided into four groups, namely control group, experimental group 1, experimental group 2 and experimental group 3, with 30 people in each group. The control group ate a normal diet and did not take any additional elastin peptide products. In addition to the normal diet, each person in the experimental group 1 orally added 3g of a commercially available elastin peptide every day, with the contents of desmosin and isodesmosin being 0.07% and 0.13% respectively. Each person in the experimental group 2 orally added 3g of the elastin peptide product prepared in comparative example 3 every day, and each person in the experimental group 3 orally added 3g of the elastin peptide product prepared in example 1 every day. All were taken with warm water for a total of 12 weeks. On the 0th day from the beginning of the experiment and the 90th day after the end of the experiment, CORTEX ( The skin physiological index tester (Combo) was used to detect the moisture content of the stratum corneum and skin elasticity of the volunteers' skin.

[0086] Table 2 Changes in moisture content of the skin stratum corneum before and after the test

[0087] Grouping 0d(μS) 90d(μS) Rate of change (%) control group 182.50±16.09 193.14±14.15 5.83 Experimental group 1 189.15±19.13 235.03±20.01 24.26 Experimental Group 2 190.50±21.07 233.33±24.61 22.48 Experimental group 3 198.50±20.11 276.30±29.90 39.19

[0088] As shown in Table 2, the subjects' skin moisture content increased after ingesting the commercially available elastin peptide and the lower-purity elastin peptide prepared in Comparative Example 3. Ingestion of the high-purity elastin peptide prepared in Example 1 significantly increased the moisture content of the subjects' stratum corneum. This indicates that the high-purity elastin peptide prepared by the present method has a more significant effect on increasing skin moisture content.

[0089] Table 3 Changes in skin elasticity before and after the test

[0090]

[0091]

[0092] As can be seen from Table 3, the subjects' skin elasticity improved after supplementing with the elastin peptide prepared in Comparative Example 3, while the improvement in skin elasticity was even greater after supplementing with the elastin peptide with high desmosin and isodesmosin content prepared in Example 1. This indicates that the high-purity elastin peptide prepared by the method of the present invention is more conducive to improving and enhancing skin elasticity than ordinary commercially available elastin peptides, and also shows that the method of the present invention has higher health care and socioeconomic value.

[0093] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing an elastin peptide having high desmosine and isodesmosine contents and improving skin moisture content and skin elasticity, characterized by: The steps of the preparation method are as follows: (1) Pretreatment: adding 8 times the weight of alkaline solution to the cleaned tilapia cardiac arterial bulb, soaking for 4 hours at room temperature, and washing with purified water until the conductivity of the effluent is within 1000 μs / cm, thereby pre-treating the tilapia cardiac arterial bulb, wherein the alkaline solution is a mixed solution of sodium hydroxide and sodium carbonate with a mass concentration ratio of 0.05%:1%; (2) Heat aging: After cleaning, the tilapia cardiac artery bulb was added with 2 times its weight of purified water, and then the material was heated using a microwave heating method. The microwave power was constant at 500 W, and the microwave treatment was performed in an intermittent manner. After continuous operation for 4 minutes, the microwave treatment was rested for 4 minutes. The execution of one microwave action and rest period was one cycle, and 6 cycles were performed. (3) Two-phase separation: The liquid after heat extraction is allowed to stand and the two phases are separated, with the oil phase in the upper layer and the water phase in the lower layer. The oil phase is fully removed by liquid separation; (4) Enzymatic hydrolysis: adding 5% of the weight of the pretreated tilapia cardiac arterial bulb microsphere-immobilized alkaline protease and 10% of the weight of the pretreated tilapia cardiac arterial bulb microsphere-immobilized papain to the aqueous phase, adjusting the temperature to 55° C., and performing enzymatic hydrolysis for 5 h to obtain an enzymatic hydrolyzate containing elastin peptides; (5) solid-liquid separation: The enzymatic hydrolyzate containing elastin peptide prepared in (4) is subjected to solid-liquid separation using a ceramic membrane to prepare a clear and transparent enzymatic hydrolyzate solution; (6) Decolorization and purification: The enzymatic hydrolyzed solution prepared in (5) was sequentially passed through a series of chromatography columns filled with weak acid-strong base-weak acid ion exchange resins at a flow rate of 4 BV / h to remove other impurity peptides including collagen peptides, thereby preparing an elastin peptide solution. The column volume ratio was 1:1.2:

1. (7) Sterilization: The elastin peptide solution prepared in (6) was sterilized using a high-voltage pulsed electric field technique with an electric field strength of 20 kV / cm and a pulse number of 200; (8) Drying: The sterilized elastin peptide solution prepared in (7) was spray-dried to obtain an elastin peptide powder, wherein the elastin peptide had a desmosin content of 0.121% and an isodesmosin content of 0.333%.

2. The preparation method according to claim 1, wherein: The preparation method of the immobilized protease in (4) comprises the following steps: based on the total mass of the immobilized protease, the mass concentration of sodium alginate is 3%, the mass concentration of chitosan is 3%, the mass concentration of calcium chloride is 3.5%, the mass concentration of glutaraldehyde is 1%, the mass concentration of protease is 0.6%, the cross-linking time is 1 hour, and the immobilization temperature is 40°C, thereby preparing the microsphere immobilized protease.

3. The preparation method according to claim 1, wherein: The method for recovering the immobilized protease in (4) comprises the following steps: passing the enzymatic hydrolysate containing the immobilized protease through a 40-mesh standard sieve to recover the microsphere-immobilized protease, rinsing it with pure water, and storing it in a refrigerator for repeated use.

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