Synthetic process of aquatic collagen tripeptide

Abstract

The application discloses a kind of synthesis process of efficient aquatic collagen tripeptide, belong to collagen tripeptide production technical field.The application is used to solve the technical problem that collagen tripeptide is produced when collagen protein substrate enzymolysis process in prior art needs to be filtered and purified multiple times, operation process is complex, and production cost is high.A kind of synthesis process of efficient aquatic collagen tripeptide, comprising the following steps: S1, pretreated fish skin, deionized water and mixed enzyme are blended, incubated, enzymolysis, and then enzyme is inactivated in deionized water, filtration, and filtrate is collected;S2, filtrate is filtered using composite reverse osmosis membrane, to obtain concentrated collagen tripeptide;The concentrated collagen tripeptide is spray dried, and finally collagen tripeptide is prepared.The application uses fish skin as substrate, and aquatic collagen tripeptide is prepared by once enzymolysis and once reverse osmosis membrane filtration.The collagen tripeptide prepared by the application has the advantages of high product purity and simplified process.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen tripeptide production, and in particular to a synthesis process of high-efficiency aquatic collagen tripeptide. Background Art

[0002] my country boasts abundant marine resources and freshwater fish farming conditions. As aquatic product processing output increases, so too does the amount of waste generated. Fish scales, fish skin, and other waste products are rich in collagen, which can be processed to produce high-value-added collagen tripeptides.

[0003] Collagen peptides are highly soluble, easily digestible, and easily utilized by the human body, making them virtually immune to rejection. Collagen peptides can be used as dietary supplements in foods and beverages, as well as as bioactive ingredients in cosmetics, healthcare, and pharmaceuticals. During enzymatic hydrolysis, collagen macromolecules are broken down into smaller collagen tripeptides, making them more readily available for human use. Furthermore, when collagen is enzymatically hydrolyzed into collagen tripeptides, the hydrophilic groups contained within them are lost, making them more absorbent and water-absorbable than collagen, making them highly sought after in the cosmetics industry.

[0004] Currently, the main method for preparing collagen tripeptides is enzymatic hydrolysis. Key factors in this method include the choice of enzyme, the ratio of substrate to enzyme, and the hydrolysis time and temperature. Patent application CN117946207A discloses a high-purity mineral-containing collagen tripeptide and its preparation method. Using fish skin or scales as raw material, the collagen tripeptide is obtained through pretreatment, extraction, and enzymatic hydrolysis after the addition of mineral salts. The collagen tripeptide is then decolorized through an activated carbon fiber membrane and subjected to two nanofiltration steps. This method is complex and requires multiple filtration and purification steps, significantly increasing production costs.

[0005] In view of the technical defects in this aspect, a solution is now proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a high-efficiency synthesis process of aquatic collagen tripeptide, which is used to solve the technical problems in the prior art of using collagen substrate enzymatic hydrolysis process to produce collagen tripeptide, which requires multiple filtration and purification, has a complex operation process and high production cost.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A process for synthesizing a high-efficiency aquatic collagen tripeptide comprises the following steps:

[0009] S1, pretreated fish skin, deionized water and mixed enzyme blend, 80-85 ℃ of insulation, after enzymolysis at 50-55 ℃, then in 95-100 ℃ deionized water enzyme go out, filter, and collect filtrate;

[0010] S2, filtering the filtrate through a composite reverse osmosis membrane to obtain concentrated collagen tripeptide; spray drying the concentrated collagen tripeptide to obtain collagen tripeptide;

[0011] The preparation method of the composite reverse osmosis membrane comprises the following steps:

[0012] B1, hexachlorocyclotriphosphazene, tetrahydrofuran and sodium ethoxide are mixed, and hydroxyethyl acrylate is added dropwise to obtain a reaction system; the reaction system is placed in a nitrogen atmosphere and refluxed at 70-80°C to obtain a product; the product is rotary evaporated to obtain intermediate 1;

[0013] Hydroxyethyl acrylate and hexachlorocyclotriphosphazene undergo a nucleophilic substitution reaction, and the reaction formula for synthesizing intermediate 1 is as follows:

[0014]

[0015] B2, intermediate 1 and ammonia are reacted at -5~0°C for 2-3 hours to obtain intermediate 2; intermediate 2 is washed with deionized water and dried to constant weight to obtain a polyphenylamino polymer;

[0016] Intermediate 1 and ammonia are polycondensed under low temperature to synthesize polyphenylamino polymers as follows:

[0017]

[0018] B3. Add polyphenylamino polymer, dimethyldiallylammonium chloride and sodium persulfate into deionized water to obtain a blended aqueous phase solution; add base membrane into the blended aqueous phase solution, first let it stand at 60-70°C, then let it stand at 25-27°C, then naturally cool to room temperature and continue to stand for 1-2 hours, then remove the base membrane, and ventilate and dry to constant weight to obtain reverse osmosis membrane 1.

[0019] Under the action of sodium persulfate initiator, the unsaturated double bonds of polyaniline polymer and dimethyldiallylammonium chloride can undergo free radical addition polymerization to obtain polyaniline polymer rich in quaternary ammonium salt side chains. The reaction formula is as follows:

[0020]

[0021] B4. Add trimesoyl chloride to n-hexane to prepare a 0.1-0.2% wt trimesoyl chloride organic phase solution; immerse the reverse osmosis membrane 1 in the trimesoyl chloride organic phase solution, allow to react at room temperature, and then heat and cure at 55-65° C. to obtain a composite reverse osmosis membrane.

[0022] The base film is added to an oven for further heating and curing, promoting further reaction between the acyl chloride groups and excess functional groups, increasing crosslinking and stability, and ultimately producing a single-polymerized reverse osmosis membrane. Trimesoyl chloride is dissolved in an organic solvent, forming a thin nylon (PA) layer on the base. Trimesoyl chloride undergoes an acylation reaction with the amino functional groups in the reverse osmosis membrane, producing a composite reverse osmosis membrane. The reaction equation is as follows:

[0023]

[0024] Furthermore, in step S1, the preparation method of the mixed enzyme is: alkaline protease, ficin and ginger protease are blended in a mass ratio of 10:10:(1-2) to obtain the mixed enzyme.

[0025] Furthermore, in step B1, the amount ratio of hexachlorocyclotriphosphazene, tetrahydrofuran, sodium ethoxide and hydroxyethyl acrylate is 15-25 g:100 mL:0.5-1 g:5-10 mL; the reflux reaction time is 12-18 h; in step B3, the amount ratio of polyphenylamino polymer, dimethyldiallylammonium chloride and deionized water is 0.5-1.5 g:2-4 g:100 mL; the reaction time is 5-10 min, the standing temperature is 25-27 ° C, and the standing time is 2-5 min.

[0026] Furthermore, in step B4, the duration of the static reaction is 5-10 minutes, and the duration of the heating and curing is 10-20 minutes.

[0027] Furthermore, in step S1, the mass ratio of the pretreated fish skin, deionized water and mixed enzyme is (20-30): (60-100): (0.5-1.5); the insulation time is 2-3 hours, the enzymatic hydrolysis time is 5-6 hours, and the enzyme inactivation time is 15-25 minutes.

[0028] Furthermore, in step S1, the preparation method of the pretreated fish skin is as follows: tilapia skin is added to deionized water and washed until no foreign matter is present to obtain washed tilapia skin; the washed tilapia skin is mixed with a NaOH solution, soaked at room temperature for 2-3 hours, and then washed with pure water until neutral to obtain pretreated fish skin.

[0029] Furthermore, in step S2, during the spray drying process, the temperature of the air inlet is 150-160°C, the temperature of the air outlet is 85-95°C, and the spray drying time is 2-3 hours.

[0030] The present invention has the following beneficial effects:

[0031] 1. The present invention uses fish skin as a substrate, undergoes a single enzymatic hydrolysis and a single reverse osmosis membrane filtration, and then prepares high-purity aquatic collagen tripeptides. Fresh tilapia skin is alkali-washed to remove impurities and odors, and the pretreated fish skin is obtained as a substrate. An enzyme is added to the substrate, and then an enzymatic hydrolysis reaction is carried out to prepare a filtrate that is completely hydrolyzed and rich in collagen tripeptides. Typical amino acids present in collagen in aquatic products such as tilapia skin include glycine, hydroxyproline, and proline; among them, ginger protease can specifically hydrolyze the peptide bond of Pro or Hyp at the P2 position, thereby obtaining a filtrate of fully hydrolyzed low-molecular-weight collagen tripeptides.

[0032] 2. The base membrane used in the present invention is a bisphenol A-type polysulfone-based membrane; the bisphenol A-type polysulfone-based membrane is polymerized by bisphenol A and dichlorodiphenyl sulfone, and the main chain contains sulfone groups and arylene groups. As the support layer of the prepared composite reverse osmosis membrane, it has excellent antioxidant, heat resistance, and acid and alkali resistance. Hydroxyethyl acrylate and hexachlorocyclotriphosphazene undergo a nucleophilic substitution reaction to synthesize an intermediate 1 containing unsaturated double bonds; intermediate 1 is polycondensed with ammonia at a low temperature to synthesize a polyphenylamino polymer rich in polyamino groups and multiple double bonds; the polyphenylamino polymer is grafted with quaternary ammonium salt side chains to serve as the first membrane layer for preparing the base membrane; the excess amino functional groups in the first membrane layer react with trimesoyl chloride to form the second membrane layer of the prepared composite reverse osmosis membrane. The composite reverse osmosis membrane prepared by the present invention is prepared by two consecutive interfacial polymerization reactions to prepare a double-layer asymmetric reverse osmosis membrane with a loose interior and a dense exterior. The above-mentioned reverse osmosis membrane can filter out a variety of impurities. High-purity collagen tripeptide can be prepared after only one filtration through the composite reverse osmosis membrane, which has the advantages of high product purity, simplified process and high efficiency. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] The tilapia skin used in Example 1 of the present invention was purchased from Guangzhou Xida Lian Frozen Food Co., Ltd. with the product number A688; the bisphenol A type polysulfone membrane material used in Examples 2-4 of the present invention was purchased from Dongguan Langge Plastic Co., Ltd. with the brand number 2010.

[0035] Example 1

[0036] This embodiment provides a method for preparing pretreated fish skin for use in a synthesis process of an efficient aquatic collagen tripeptide, comprising the following steps:

[0037] A1, fresh tilapia skin is added in deionized water and cleaned to no foreign matter, obtain the tilapia skin after cleaning. According to solid-to-liquid ratio 1:5, the tilapia skin after cleaning and the NaOH solution of 0.5%wt are mixed, at room temperature soaking treatment 2h, after adopting pure water to be cleaned to neutrality, obtain pretreated fish skin.

[0038] Example 2

[0039] This embodiment provides a method for preparing a composite reverse osmosis membrane for a high-efficiency synthesis process of aquatic collagen tripeptide, comprising the following steps:

[0040] B1. Pre-immerse the bisphenol A polysulfone membrane in a 0.5 mol / L sodium bisulfite solution to prevent the growth of bacteria and microorganisms; then cut the bisphenol A polysulfone membrane into 10 cm × 10 cm samples, wash the samples with deionized water, and vacuum dry them at 50°C to constant weight to obtain a base membrane.

[0041] B2. In a 250mL three-necked flask equipped with a mechanical stirrer, thermometer, and condensing reflux device, 15g of hexachlorocyclotriphosphazene, 100mL of tetrahydrofuran, and 0.5g of sodium ethoxide were added in sequence. 5mL of hydroxyethyl acrylate was then added dropwise to the flask over 3 minutes to obtain a reaction system. Under a nitrogen atmosphere, the flask was placed at 70°C and refluxed for 12 hours to obtain the product. The product was rotary evaporated at 50°C for 20 minutes to synthesize intermediate 1. 20g of intermediate 1 was placed in a 250mL three-necked flask. The air in the flask was replaced with ammonia. The flask was then transferred to a water bath and reacted at -5°C for 2 hours to synthesize intermediate 2. Intermediate 2 was washed with deionized water and dried to constant weight to prepare a polyphenylamino polymer.

[0042] B3. Add 0.5 g of polyphenylamino polymer, 2 g of dimethyldiallylammonium chloride, and 0.02 g of sodium persulfate to 100 mL of deionized water to prepare a blended aqueous solution. Add a 10 cm × 10 cm sample of basement membrane to the blended aqueous solution, submerging the membrane. The membrane is first reacted at 60°C for 5 min, then allowed to stand at 25°C for 3 min. The membrane is then removed and hung in a fume hood to air dry to constant weight, yielding reverse osmosis membrane 1.

[0043] B4. Add trimesoyl chloride to n-hexane to prepare a 0.1% wt. organic phase solution of trimesoyl chloride. Add reverse osmosis membrane 1 to the organic phase solution until the trimesoyl chloride solution completely submerges the membrane. Allow the membrane to react at room temperature for 5 minutes, then remove it and heat-cure it in a 55°C oven for 10 minutes to produce a composite reverse osmosis membrane.

[0044] Example 3

[0045] This embodiment provides a method for preparing a composite reverse osmosis membrane for a high-efficiency synthesis process of aquatic collagen tripeptide, comprising the following steps:

[0046] B1. Pre-immerse the purchased bisphenol A polysulfone membrane material in a 0.8 mol / L sodium bisulfite solution to prevent the growth of bacteria and microorganisms; then cut the bisphenol A polysulfone membrane material into 10 cm × 10 cm samples, wash the samples with deionized water, and vacuum dry them at 55°C to constant weight to obtain a base membrane.

[0047] B2. To a 250mL three-necked flask equipped with a mechanical stirrer, thermometer, and reflux apparatus, 20g of hexachlorocyclotriphosphazene, 100mL of tetrahydrofuran, and 0.8g of sodium ethoxide were added sequentially. 6mL of hydroxyethyl acrylate was then added dropwise to the flask over 5 minutes to obtain a reaction system. Under a nitrogen atmosphere, the flask was placed at 75°C and refluxed for 16 hours to obtain the product. The product was rotary evaporated at 55°C for 25 minutes to synthesize Intermediate 1. 25g of Intermediate 1 was placed in a 250mL three-necked flask. The air in the flask was replaced with ammonia. The flask was then transferred to a water bath and reacted at -2°C for 2.2 hours to synthesize Intermediate 2. Intermediate 2 was washed with deionized water and dried to constant weight to obtain a polyphenylamino polymer.

[0048] B3. Add 1 g of polyphenylamino polymer, 3 g of dimethyldiallylammonium chloride, and 0.025 g of sodium persulfate to 100 mL of deionized water to prepare a blended aqueous solution. Add a 10 cm × 10 cm sample of basement membrane to the blended aqueous solution, submerging the membrane. The membrane is first reacted at 65°C for 8 min, then allowed to stand at 26°C for 5 min. The membrane is then removed and hung in a fume hood to air dry to constant weight, yielding reverse osmosis membrane 1.

[0049] B4. Add trimesoyl chloride to n-hexane to prepare a 0.15% wt. organic phase solution of trimesoyl chloride. Add reverse osmosis membrane 1 to the organic phase solution until the trimesoyl chloride solution completely submerges the membrane. Allow the membrane to react at room temperature for 6 minutes, then remove it and heat-cure it in a 60°C oven for 15 minutes to produce a composite reverse osmosis membrane.

[0050] Example 4

[0051] This embodiment provides a method for preparing a composite reverse osmosis membrane for a high-efficiency synthesis process of aquatic collagen tripeptide, comprising the following steps:

[0052] B1. Pre-immerse the purchased bisphenol A polysulfone membrane material in a 1 mol / L sodium bisulfite solution to prevent the growth of bacteria and microorganisms; then cut the bisphenol A polysulfone membrane material into 10 cm × 10 cm samples, wash the samples with deionized water, and vacuum dry them at 60°C to constant weight to obtain a base membrane.

[0053] B2. In a 250mL three-necked flask equipped with a mechanical stirrer, thermometer, and reflux apparatus, 25g of hexachlorocyclotriphosphazene, 100mL of tetrahydrofuran, and 1g of sodium ethoxide were added sequentially. 10mL of hydroxyethyl acrylate was then added dropwise to the flask over 5 minutes to obtain a reaction system. Under a nitrogen atmosphere, the flask was placed at 80°C and refluxed for 18 hours to obtain the product. The product was rotary evaporated at 60°C for 30 minutes to synthesize intermediate 1. 30g of intermediate 1 was placed in a 250mL three-necked flask. The air in the flask was replaced with ammonia. The flask was then transferred to a water bath and reacted at -5°C for 2 hours to synthesize intermediate 2. Intermediate 2 was washed with deionized water and dried to constant weight to obtain a polyphenylamino polymer.

[0054] B3. Add 1.5 g of polyphenylamino polymer, 4 g of dimethyldiallylammonium chloride, and 0.03 g of sodium persulfate to 100 mL of deionized water to prepare a blended aqueous phase solution. Immerse a 10 cm × 10 cm sample of basement membrane in 100 mL of the blended aqueous phase solution. First, react at 70°C for 10 min, then let stand at 27°C for 5 min. Then, remove the basement membrane and hang it in a fume hood to air-dry until constant weight is obtained, thereby obtaining reverse osmosis membrane 1.

[0055] B4. Add trimesoyl chloride to n-hexane to prepare a 0.2% wt. trimesoyl chloride organic phase solution. Immerse reverse osmosis membrane 1 in 100 mL of the trimesoyl chloride organic phase solution, allow to react at room temperature for 10 minutes, then remove and heat-cure in a 65°C oven for 20 minutes to produce a composite reverse osmosis membrane.

[0056] Example 5

[0057] This embodiment provides a synthesis process of a high-efficiency aquatic collagen tripeptide, comprising the following steps:

[0058] S1, alkaline protease, ficin, and ginger protease were blended in a mass ratio of 10:10:1 to produce an enzyme mixture. Pretreated fish skin, deionized water, and the enzyme mixture were blended in a mass ratio of 20:60:0.5, incubated at 80°C for 2 hours, and then subjected to enzymatic hydrolysis at 50°C for 5 hours to produce an enzymatic hydrolyzate. The enzymatic hydrolyzate was inactivated in deionized water at 95°C for 15 minutes, filtered, and the filtrate collected.

[0059] S2. The filtrate is filtered using the composite reverse osmosis membrane prepared in Example 2 to remove macromolecular peptides, impurities and deionized water to obtain concentrated collagen tripeptide. The concentrated collagen tripeptide is added to a spray dryer for spray drying, wherein the air inlet temperature is 150°C, the air outlet temperature is 85°C, and the spray drying time is 2 hours, and finally a solid aquatic collagen tripeptide is prepared.

[0060] Example 6

[0061] This embodiment provides a synthesis process of a high-efficiency aquatic collagen tripeptide, comprising the following steps:

[0062] S1, alkaline protease, ficin, and ginger protease were blended in a mass ratio of 10:10:1 to obtain an enzyme mixture. The pretreated fish skin, deionized water, and the enzyme mixture were blended in a mass ratio of 25:80:1, incubated at 82°C for 3 hours, and then enzymatically hydrolyzed at 52°C for 5.5 hours. After completion, the enzyme was inactivated in deionized water at 98°C for 20 minutes, filtered, and the filtrate was collected.

[0063] S2. The filtrate was filtered using the composite reverse osmosis membrane prepared in Example 3 to remove macromolecular peptides, impurities, and deionized water to obtain concentrated collagen tripeptide. The concentrated collagen tripeptide was added to a spray dryer for spray drying, wherein the air inlet temperature was 155°C, the air outlet temperature was 90°C, and the spray drying time was 3 hours, and finally a solid aquatic collagen tripeptide was prepared.

[0064] Example 7

[0065] This embodiment provides a synthesis process of a high-efficiency aquatic collagen tripeptide, comprising the following steps:

[0066] S1, alkaline protease, ficin, and ginger protease were blended in a mass ratio of 10:10:2 to obtain an enzyme mixture. The pretreated fish skin, deionized water, and the enzyme mixture were blended in a mass ratio of 30:100:1.5, incubated at 85°C for 3 hours, and then enzymatically hydrolyzed at 55°C for 6 hours. After completion, the enzyme was inactivated in deionized water at 100°C for 25 minutes, filtered, and the filtrate was collected.

[0067] S2. The filtrate was filtered using the composite reverse osmosis membrane prepared in Example 4 to remove macromolecular peptides, impurities, and deionized water to obtain concentrated collagen tripeptide. The concentrated collagen tripeptide was added to a spray dryer for spray drying, wherein the air inlet temperature was 160°C, the air outlet temperature was 95°C, and the spray drying time was 3 hours, and finally a solid aquatic collagen tripeptide was prepared.

[0068] Comparative Example 1

[0069] The difference between this comparative example and Example 7 is that, when preparing the composite reverse osmosis membrane, step B2 is omitted and an equal mass of m-phenylenediamine is used instead of the polyphenylamino polymer.

[0070] Comparative Example 2

[0071] The difference between this comparative example and Example 7 is that, when preparing the composite reverse osmosis membrane, in step B3, when preparing the blended aqueous phase solution, no dimethyldiallylammonium chloride is added.

[0072] Comparative Example 3

[0073] The difference between this comparative example and Example 7 is that the method for preparing the mixed enzyme is different, and specifically comprises the following steps:

[0074] Alkaline protease and ficin are blended in a mass ratio of 1:1 to obtain a mixed enzyme.

[0075] Performance testing:

[0076] 1. The water flux and salt rejection of the composite reverse osmosis membranes prepared in Examples 5-7 and Comparative Examples 1-3 were measured using a cross-flow filtration membrane evaluation instrument. The prepared polyamide membranes to be tested were placed in a membrane pool with an effective area of ​​25 cm 2 Keep the water tank temperature at around 25°C, adjust the pressure to 1.5 MPa, and the flow rate to 1.5 LPM. Pre-pressurize for 1 hour to ensure the membrane structure is in a stable state. Once the flux stabilizes, begin measuring the water flux and desalination rate. The desalination rate is calculated as follows:

[0077] R=【1-C p / C f 】×100%

[0078] Where, R=salt rejection rate,%;C p = osmotic salt concentration; C f = stock solution salt concentration;

[0079] The calculation formula for water flux is as follows:

[0080]

[0081] Where, Jw = water flux, L / m 2 h; V = volume of solution per unit time per unit time per effective membrane area, L; = sampling time interval, h; A = effective membrane area, m 2 The specific test results are shown in Table 1.

[0082] Table 1. Performance test data of samples

[0083]

[0084] Data analysis: The composite reverse osmosis membranes used in Examples 5-7 of the present invention have a high degree of crosslinking, which promotes the performance improvement of the membrane, as shown by the composite reverse osmosis membranes prepared in Examples 5-7 all having high water flux and salt rejection values. However, in Comparative Example 1, when preparing the composite reverse osmosis membrane, m-phenylenediamine of equal mass was used to replace the polyphenylamino polymer. The polyphenylamino polymer can be side-linked to graft unsaturated quaternary ammonium salts, thereby improving its own electrostatic adsorption and crosslinking bonding capabilities, resulting in a decrease in the degree of crosslinking of the composite reverse osmosis membrane prepared in Comparative Example 1, as shown by an increase in the water flux and a decrease in the salt rejection of the composite reverse osmosis membrane prepared in Comparative Example 1 and the composite reverse osmosis membrane prepared in Comparative Example 2.

[0085] 2. Prepare 1 mg / mL solutions of the high-efficiency aquatic collagen tripeptides prepared in Examples 5-7 and Comparative Examples 1-3 using deionized water. Liquid chromatography was performed according to Q / HNHY0110S-2021. The solutions were filtered through a 0.22 μm organic system filter and analyzed using HPLC to determine the collagen tripeptide and hydroxyproline contents. Collagen tripeptides are tripeptides beginning with glycine. The relative molecular mass of the prepared small-molecule collagen peptides was determined by mass spectrometry, and the relative content was calculated based on the area. Next, weigh 0.4 mg of the collagen polypeptides prepared in Examples 5-7 and add them to 400 μL of DPPH solution (DPPH concentration 0.2 mmmol / L, methanol solvent). Mix thoroughly, insulate in the dark for 30 minutes, and then centrifuge for 1 minute. Using distilled water as a control, the absorbance of the samples was measured at 5°C and the absorbance of the reagent sample was measured at a wavelength of 517 nm. The DPPH scavenging ability was calculated using the formula.

[0086] DPPH free radical scavenging rate (%) = [1-(As-Ab / Ac)] × 100%

[0087] Where As is the absorbance of the sample to be tested after reacting with DPPH solution, Ab is the absorbance of anhydrous ethanol instead of DPPH at a wavelength of 517 nm, and Ac is the absorbance of the control group.

[0088] Table 2. Performance test data of samples

[0089]

[0090] Data Analysis: Comparative analysis of the data in Table 2 shows that the collagen tripeptides synthesized in Examples 5-7 of the present invention all possess high purity, as determined by the hydroxyproline and collagen tripeptide contents. Correspondingly, the high-efficiency aquatic collagen tripeptides prepared in Examples 5-7 of the present invention all possess high DPPH radical scavenging ability. However, in Comparative Example 3, ginger protease was not added during enzyme preparation, resulting in a lower concentration of the collected aquatic collagen tripeptides, indicating a decrease in DPPH radical scavenging ability.

[0091] The above contents are merely examples and explanations of the structure of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the structure of the invention or exceed the scope defined by the claims, they shall fall within the scope of protection of the present invention.

[0092] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0093] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to specific embodiments. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A process for synthesizing aquatic collagen tripeptide, characterized in that: The following steps are involved: S1, pretreated fish skin, deionized water and mixed enzyme blend, 80-85 ℃ of insulation, after enzymolysis at 50-55 ℃, then in 95-100 ℃ deionized water enzyme go out, filter, and collect filtrate; S2, filtering the filtrate through a composite reverse osmosis membrane to obtain concentrated collagen tripeptide; spray drying the concentrated collagen tripeptide to obtain collagen tripeptide; The preparation method of the composite reverse osmosis membrane comprises the following steps: B1, hexachlorocyclotriphosphazene, tetrahydrofuran and sodium ethoxide are mixed, and hydroxyethyl acrylate is added dropwise to obtain a reaction system; the reaction system is placed in a nitrogen atmosphere and refluxed at 70-80°C to obtain a product; the product is rotary evaporated to obtain intermediate 1; B2, intermediate 1 and ammonia are reacted at -5~0°C for 2-3h to obtain intermediate 2; intermediate 2 is washed with deionized water and dried to constant weight to obtain a polyphenylamino polymer; B3, blending a polyphenylamino polymer, dimethyldiallylammonium chloride, sodium persulfate, and deionized water to obtain a blended aqueous solution; immersing a base membrane in the blended aqueous solution, reacting, allowing to stand, and then drying under ventilation to a constant weight to obtain a reverse osmosis membrane 1; B4, adding trimesoyl chloride to n-hexane to prepare a 0.1-0.2%wt trimesoyl chloride organic phase solution; immersing the reverse osmosis membrane 1 in the trimesoyl chloride organic phase solution, allowing it to react at room temperature, and heating to cure to obtain a composite reverse osmosis membrane; In step S1, the preparation method of the mixed enzyme is: alkaline protease, ficin and ginger protease are blended in a mass ratio of 10:10:(1-2) to obtain the mixed enzyme.

2. The synthesis process of aquatic collagen tripeptide according to claim 1, characterized in that: In step B1, the ratio of hexachlorocyclotriphosphazene, tetrahydrofuran, sodium ethoxide and hydroxyethyl acrylate is 15-25 g:100 mL:0.5-1 g:5-10 mL; the reflux reaction time is 12-18 h; in step B3, the ratio of polyphenylamino polymer, dimethyldiallylammonium chloride and deionized water is 0.5-1.5 g:2-4 g:100 mL; the standing temperature is 25-27 ° C, and the standing time is 2-5 min.

3. The synthesis process of aquatic collagen tripeptide according to claim 1, characterized in that: In step B4, the standing reaction time is 5-10 minutes, and the heating and curing time is 10-20 minutes.

4. The synthesis process of aquatic collagen tripeptide according to claim 1, characterized in that: In step S1, the mass ratio of the pretreated fish skin, deionized water, and mixed enzyme is (20-30): (60-100): (0.5-1.5); the insulation time is 2-3 hours, the enzymatic hydrolysis time is 5-6 hours, and the enzyme inactivation time is 15-25 minutes.

5. The synthesis process of aquatic collagen tripeptide according to claim 1, characterized in that: In step S1, the preparation method of the pretreated fish skin is as follows: tilapia skin is added to deionized water and washed until no foreign matter is present to obtain washed tilapia skin; the washed tilapia skin is mixed with a NaOH solution, soaked at room temperature, and then washed with pure water until neutral to obtain pretreated fish skin.

6. The synthesis process of aquatic collagen tripeptide according to claim 1, characterized in that: In step S2, during the spray drying process, the temperature of the air inlet is 150-160°C, the temperature of the air outlet is 85-95°C, and the spray drying time is 2-3 hours.

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