Aquatic collagen tripeptide and its processing technology

Abstract

The invention relates to the technical field of collagen peptide extraction, and specifically discloses an aquatic collagen tripeptide and a processing technology thereof. The process comprises the following steps: step S1, pretreating fish skin; step S2, enzymatic hydrolysis; step S3, fermentation; and step S4, separation and purification. The invention proceeds from three aspects: first, a modified degreasing agent is added during the degreasing process, wherein the modified degreasing agent comprises 1-ethyl-3-methylimidazole acetate, zinc phytate and calcium citrate in a specific mass ratio as main components; second, a gel emulsion is added during the enzymatic hydrolysis process, wherein the gel emulsion has an oil gel structure, grape seed oil and sodium lignin sulfonate; and third, a composite strain is added during the fermentation process, wherein the composite strain is prepared by mixing Bacillus natto and Lactobacillus plantarum in a specific ratio. Through the synergistic effect of the above steps, the purity and extraction rate of the aquatic collagen tripeptide are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen peptide extraction, in particular to an aquatic collagen tripeptide and a processing technology thereof. Background Art

[0002] Collagen tripeptide is the smallest active structural unit formed by the hydrolysis of natural collagen or gelatin. Due to its small molecular structure, it can be directly absorbed by the human body, thus exhibiting unique physical and chemical properties and partial biological activities, such as good absorption, lowering blood pressure, lowering blood sugar, anti-oxidation, anti-aging and promoting wound healing. Therefore, it has broad application prospects in the fields of cosmetics, health products and medicine.

[0003] In the prior art, collagen peptide extraction methods mainly include alkaline extraction, acid extraction and bio-enzyme extraction. Among them, bio-enzyme extraction has milder reaction conditions, higher degree of hydrolysis and less damage to amino acids compared with the first two, and has been widely used in the processing of fish by-products. However, bio-protease is expensive. If there is no other auxiliary extraction process, it not only consumes a large amount of protease, but also takes a long time to enzymolysis, and the production cost is high. Currently, commonly used auxiliary processes include ultrasonic treatment, heat treatment, high-pressure shear treatment, etc. However, ultrasonic treatment may destroy the protein structure and reduce its biological activity. When used alone, heat treatment and high-pressure shear treatment are not effective, and the collagen peptide extraction efficiency is low. Although high-temperature heat treatment has high extraction efficiency, it will also reduce the biological activity of the protein.

[0004] Based on the above statements, there is an urgent need to provide an aquatic collagen tripeptide with high extraction rate and purity, good biological activity and a processing technology thereof. Summary of the Invention

[0005] In order to solve the problems mentioned in the above background technology, the present invention provides an aquatic collagen tripeptide and a processing technology thereof.

[0006] The first object of the present invention is to provide a processing technology for aquatic collagen tripeptide, comprising the following steps:

[0007] Step S1, fish skin pretreatment: the fish skin is placed in clean water for washing and shredding, and then defatted, dried, crushed, and sieved to obtain defatted fish skin powder;

[0008] Step S2, enzymatic hydrolysis: adding defatted fish skin powder to deionized water, adjusting the pH to 8.2-8.6, then adding compound enzyme and gel emulsion, enzymatic hydrolysis, inactivating the enzyme, cooling, standing and separating to obtain an enzymatic hydrolysis extract, wherein the mass ratio of defatted fish skin powder, deionized water and compound enzyme is 1:76-82:0.08-0.16;

[0009] Step S3, fermentation: inoculating the activated bacterial solution into the enzymatic extract obtained in step S2 for fermentation, filtering, and collecting the fermentation solution, wherein the mass ratio of the enzymatic extract to the activated bacterial solution is 3-5:0.26-0.48;

[0010] Step S4, separation and purification: the fermentation broth is separated by a primary nanofiltration membrane to obtain a small molecule peptide solution, which is then intercepted and filtered through a secondary nanofiltration membrane to obtain aquatic collagen tripeptide.

[0011] Preferably, in step S1, the degreasing process includes: ultrasonically dispersing the chopped fish skin in a modified degreasing agent at a material-liquid ratio of 1:30-40, and then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 110-130 MPa, the homogenization temperature to 72-76°C, and homogenizing for 8-12 times, with each homogenization time being 6-10 minutes.

[0012] Preferably, in step S1, the fish skin is tilapia skin or cod skin.

[0013] Preferably, the modified degreasing agent is prepared by mixing 1-ethyl-3-methylimidazole acetate, zinc phytate, calcium citrate and ethanol aqueous solution in a mass ratio of 2-7:0.1-0.3:0.4-0.6:32-36.

[0014] Preferably, the mass fraction of the ethanol aqueous solution is 12-16%.

[0015] Preferably, in step S2, the composite enzyme is prepared by mixing papain and alkaline protease in a mass ratio of 3-4:1-1.6.

[0016] Preferably, in step S2, the enzymatic hydrolysis temperature is 52-56° C., and the enzymatic hydrolysis time is 4-5 h.

[0017] Preferably, in step S3, the specific conditions of the fermentation treatment are as follows: the temperature is 28-32°C, the fermentation time is 6-8h, oxygen is introduced into the system, and the oxygen ventilation volume is set to 0.5-1m 3 / h, and continue fermenting for 20-30h.

[0018] Preferably, in step S4, the molecular weight of the primary nanofiltration membrane used in the separation process is 620-740 Da, and the molecular weight of the secondary nanofiltration membrane used in the interception filtration process is 160-200 Da.

[0019] Preferably, the gel emulsion is prepared by the following steps:

[0020] Step A1, adding grape seed oil to anhydrous ethanol, raising the temperature to 62-66° C., stirring for 18-24 minutes, and adding dropwise to a mixture of phytosterols, lecithin, and anhydrous ethanol while stirring, controlling the addition to be completed within 15 minutes. After the addition is completed, stirring is continued for 22-26 minutes to obtain an oil phase, wherein the mass ratio of grape seed oil, anhydrous ethanol, and the mixture is 8-12:60-80:36-42, and the mass ratio of phytosterols, lecithin, and anhydrous ethanol in the mixture is 5-6:4:30-40;

[0021] Step A2, adding octenyl succinic anhydride and sodium lignin sulfonate to a buffer solution, heating to 45-55° C., stirring until dissolved, and allowing to stand to obtain an aqueous phase, wherein the mass ratio of octenyl succinic anhydride, sodium lignin sulfonate, and buffer solution is 0.4-0.6:3-5:50-60;

[0022] Step A3: Evenly stir the oil phase and the aqueous phase, raise the temperature to 56-62° C., add the mixture to a high-pressure homogenizer, and homogenize to obtain a gel emulsion, wherein the mass ratio of the oil phase to the aqueous phase is 2-3:8-10. During the above process, a tight three-dimensional network oil gel structure can be formed through homogenization.

[0023] Preferably, in step A2, the buffer solution is prepared by mixing disodium hydrogen phosphate, dipotassium hydrogen phosphate and deionized water in a mass ratio of 7-8:3-5:120.

[0024] Preferably, in step A3, the homogenization pressure is 120-140 MPa, the homogenization temperature is 72-76° C., the homogenization treatment is 3-5 times, and the homogenization treatment time is 5-9 min.

[0025] Preferably, in step S3, the activated bacterial solution is prepared by the following steps:

[0026] Step B1, using beef extract peptone medium as the basal medium, adding 1-3% carbon source, 0.02-0.04% magnesium salt, 0.003-0.005% manganese salt and 0.1-0.12% zinc salt to the basal medium by mass percentage, and adjusting the pH value of the system to 6.8-7.2 to obtain an activated medium;

[0027] Step B2, inoculating the composite strain into the activation medium obtained in step B1 at an inoculum size of 5-7% by volume, setting the culture temperature to 36-40°C, shaking the culture on a shaker, adding an inorganic potassium source at the 6th and 12th hours of fermentation, the shaking rate being 140-220 rpm, and the culture time being 24-30 hours to obtain an activated bacterial solution.

[0028] Preferably, in step B2, the composite bacteria are mixed in a sterile environment with a live bacteria count ratio of 1:0.2-0.5 between Bacillus natto and Lactobacillus plantarum.

[0029] The second object of the present invention is to provide an aquatic collagen tripeptide prepared by the processing technology of the aquatic collagen tripeptide as described above.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] In order to improve the extraction rate and purity of collagen tripeptide, the technical solution of the present invention starts from two aspects. First, a modified degreasing agent is added during the degreasing process. The modified degreasing agent is mainly composed of 1-ethyl-3-methylimidazole acetate, zinc phytate and calcium citrate in a specific mass ratio. 1-ethyl-3-methylimidazole acetate can form hydrogen bonds with ester bonds and carboxylic acid groups in lipids in shredded fish skin, weaken the cohesion between lipid molecules, and promote their dissolution. The presence of zinc phytate and calcium citrate can be tightly combined with collagen by chemical bonds, so that collagen The white surface is rich in zinc and calcium. The two elements work together to enhance the efficiency. Not only can they effectively chelate with collagen, but also enhance the extraction performance of collagen. Secondly, gel emulsion is added during the enzymatic hydrolysis process. The gel emulsion has an oil gel structure, grape seed oil and sodium lignin sulfonate. The gel structure has a large number of micropores and mesopores. More complex enzyme molecules can attach to the internal pores, which improves the extraction rate of collagen tripeptide. The linoleic acid in grape seed oil can stabilize the three-dimensional conformation of the enzyme, and oleic acid can reduce the surface tension of the reaction system, promoting the extraction of collagen in water. The dissolution and dispersion in the phase increases the contact area between the compound enzyme and the defatted fish skin powder. Through their synergistic effect, the extraction rate of collagen tripeptide can be improved. The presence of sodium lignin sulfonate can not only complex the residual calcium ions and zinc ions in the defatted fish skin powder, but also act as a surfactant to prevent the aggregation of compound enzyme molecules, maintain the accessibility of its catalytic site, increase the contact area between the compound enzyme, calcium ions, zinc ions and defatted fish skin powder, improve the activity of the compound enzyme, and further improve the extraction rate and purity of collagen tripeptide. Third, the addition of composite bacteria, composite The strain is prepared by mixing Bacillus natto and Lactobacillus plantarum in a specific ratio. Bacillus natto and Lactobacillus plantarum in the composite strain secrete nattokinase and cellulase, which can efficiently decompose the peptide bonds in collagen and release small-molecule collagen peptides. Lactobacillus plantarum not only lowers the pH of the fermentation system by producing acid and activates the activity of acid protease in Bacillus natto, but also the acid protease and peptidase secreted by it can further hydrolyze collagen peptides to small molecular weight. Through their synergistic effect, the extraction rate and purity of collagen tripeptides are further improved. DETAILED DESCRIPTION

[0032] In order to make the embodiments of the present invention easier to understand, the present invention will be described in detail below with reference to specific examples. These examples are only for illustration and do not limit the scope of application of the present invention.

[0033] The contents of the main raw materials and their components used in the examples and comparative examples are as follows:

[0034] Tilapia and cod were purchased from China National Fisheries Co., Ltd. and stored frozen at -20°C after being transported to the laboratory. Lactobacillus plantarum ATCC 8014 was purchased from Hangzhou Baosai Biotechnology Co., Ltd., Bacillus natto NF01-6 was purchased from China Industrial Microbiological Culture Collection Administration Center, papain and alkaline protease were purchased from Hebei Pengyu Biotechnology Co., Ltd., sodium lignin sulfonate was purchased from Pand (Shanghai) International Trade Co., Ltd. with a CAS number of 8061-51-6, phytosterols were purchased from Guangdong Mingcheng Biotechnology Co., Ltd. with a CAS number of 83-46-5, lecithin was purchased from Shandong Aicai Biotechnology Co., Ltd., and grape seed oil was purchased from Wuhan Kangqiong Biopharmaceutical Technology Co., Ltd. with a CAS number of 85594-37-2.

[0035] The present invention is further described in detail below with reference to Examples and Comparative Examples.

[0036] Preparation Examples 1-3 and Comparative Preparation Examples 1-2 provide a gel emulsion.

[0037] Preparation Example 1

[0038] This preparation example provides a gel emulsion, which is prepared by the following steps:

[0039] Step A1, adding grape seed oil to anhydrous ethanol, raising the temperature to 62° C., controlling the speed to 540 rpm and stirring for 18 minutes, and adding dropwise to the mixture of phytosterols, lecithin and anhydrous ethanol while stirring, and controlling the dripping to be completed within 15 minutes. After the dripping is completed, maintaining the speed unchanged and continuing stirring for 22 minutes to obtain an oil phase, wherein the mass ratio of grape seed oil, anhydrous ethanol and the mixture is 8:60:36, and the mass ratio of phytosterols, lecithin and anhydrous ethanol in the mixture is 5:4:30;

[0040] Step A2, adding octenyl succinic anhydride and sodium lignin sulfonate to a buffer solution, heating to 45°C, stirring at a speed of 460 rpm for 16 minutes until dissolved, and standing for 10 hours to obtain an aqueous phase, wherein the mass ratio of octenyl succinic anhydride, sodium lignin sulfonate and buffer solution is 0.4:3:50, and the buffer solution is a mixture of disodium hydrogen phosphate, dipotassium hydrogen phosphate and deionized water in a mass ratio of 7:3:120;

[0041] Step A3, stirring the oil phase and the aqueous phase at a speed of 560 rpm for 16 minutes until uniform, raising the temperature to 56°C, controlling the homogenization pressure to 120 MPa, the homogenization temperature to 72°C, and homogenizing for 3 times, each homogenization time being 5 minutes, to obtain a gel emulsion, wherein the mass ratio of the oil phase to the aqueous phase is 2:8.

[0042] Preparation Example 2

[0043] This preparation example provides a gel emulsion, which is prepared by the following steps:

[0044] Step A1, adding grape seed oil to anhydrous ethanol, raising the temperature to 64° C., controlling the speed to 560 rpm and stirring for 21 minutes, and adding dropwise to the mixture of phytosterols, lecithin and anhydrous ethanol while stirring, and controlling the dripping to be completed within 15 minutes. After the dripping is completed, maintaining the speed unchanged and continuing stirring for 24 minutes to obtain an oil phase, wherein the mass ratio of grape seed oil, anhydrous ethanol and the mixture is 10:70:39, and the mass ratio of phytosterols, lecithin and anhydrous ethanol in the mixture is 5.5:4:35;

[0045] Step A2, adding octenyl succinic anhydride and sodium lignin sulfonate to a buffer solution, heating to 50°C, stirring at a speed of 500 rpm for 18 minutes until dissolved, and standing for 12 hours to obtain an aqueous phase, wherein the mass ratio of octenyl succinic anhydride, sodium lignin sulfonate and buffer solution is 0.5:4:55, and the buffer solution is a mixture of disodium hydrogen phosphate, dipotassium hydrogen phosphate and deionized water in a mass ratio of 7.5:4:120;

[0046] Step A3, stirring the oil phase and the aqueous phase at a speed of 560 rpm for 18 minutes until uniform, heating to 59°C, adding them to a high-pressure homogenizer, controlling the homogenization pressure to 130 MPa, the homogenization temperature to 74°C, and homogenizing for 4 times, each homogenization time being 7 minutes, to obtain a gel emulsion, wherein the mass ratio of the oil phase to the aqueous phase is 2.5:9.

[0047] Preparation Example 3

[0048] This preparation example provides a gel emulsion, which is prepared by the following steps:

[0049] Step A1, adding grape seed oil to anhydrous ethanol, raising the temperature to 66° C., controlling the speed to 580 rpm and stirring for 24 minutes, and adding dropwise to the mixture of phytosterols, lecithin and anhydrous ethanol while stirring, and controlling the dripping to be completed within 15 minutes. After the dripping is completed, maintaining the speed unchanged and continuing stirring for 26 minutes to obtain an oil phase, wherein the mass ratio of grape seed oil, anhydrous ethanol and the mixture is 12:80:42, and the mass ratio of phytosterols, lecithin and anhydrous ethanol in the mixture is 6:4:40;

[0050] Step A2, adding octenyl succinic anhydride and sodium lignin sulfonate to a buffer solution, raising the temperature to 55°C, stirring at a speed of 540 rpm for 20 minutes until dissolved, and standing for 14 hours to obtain an aqueous phase, wherein the mass ratio of octenyl succinic anhydride, sodium lignin sulfonate and buffer solution is 0.6:5:60, and the buffer solution is a mixture of disodium hydrogen phosphate, dipotassium hydrogen phosphate and deionized water in a mass ratio of 8:5:120;

[0051] Step A3, stirring the oil phase and the aqueous phase at a speed of 600 rpm for 20 minutes until uniform, heating to 62°C, adding them to a high-pressure homogenizer, controlling the homogenization pressure to 140 MPa, the homogenization temperature to 76°C, and homogenizing for 5 times, each homogenization time being 9 minutes, to obtain a gel emulsion, wherein the mass ratio of the oil phase to the aqueous phase is 3:10.

[0052] Comparative Preparation Example 1

[0053] This comparative preparation example provides a gel emulsion, which is prepared by the following steps:

[0054] Step A1, adding oleic acid to anhydrous ethanol, raising the temperature to 62°C, controlling the speed to 540 rpm and stirring for 18 minutes, and adding dropwise to the mixture of phytosterols, lecithin and anhydrous ethanol while stirring, and controlling the dripping to be completed within 15 minutes. After the dripping is completed, maintaining the speed unchanged and continuing stirring for 22 minutes to obtain an oil phase, wherein the mass ratio of oleic acid, anhydrous ethanol and the mixture is 8:60:36, and the mass ratio of phytosterols, lecithin and anhydrous ethanol in the mixture is 5:4:30;

[0055] Step A2, adding octenyl succinic anhydride and sodium lignin sulfonate to a buffer solution, heating to 45°C, stirring at a speed of 460 rpm for 16 minutes until dissolved, and standing for 10 hours to obtain an aqueous phase, wherein the mass ratio of octenyl succinic anhydride, sodium lignin sulfonate and buffer solution is 0.4:3:50, and the buffer solution is a mixture of disodium hydrogen phosphate, dipotassium hydrogen phosphate and deionized water in a mass ratio of 7:3:120;

[0056] Step A3, stirring the oil phase and the aqueous phase at a speed of 560 rpm for 16 minutes until uniform, raising the temperature to 56°C, controlling the homogenization pressure to 120 MPa, the homogenization temperature to 72°C, and homogenizing for 3 times, each homogenization time being 5 minutes, to obtain a gel emulsion, wherein the mass ratio of the oil phase to the aqueous phase is 2:8.

[0057] Comparative Preparation Example 2

[0058] This comparative preparation example provides a gel emulsion, which is prepared by the following steps:

[0059] Step A1, adding grape seed oil to anhydrous ethanol, raising the temperature to 62° C., controlling the speed to 540 rpm and stirring for 18 minutes, and adding dropwise to the mixture of phytosterols, lecithin and anhydrous ethanol while stirring, and controlling the dripping to be completed within 15 minutes. After the dripping is completed, maintaining the speed unchanged and continuing stirring for 22 minutes to obtain an oil phase, wherein the mass ratio of grape seed oil, anhydrous ethanol and the mixture is 8:60:36, and the mass ratio of phytosterols, lecithin and anhydrous ethanol in the mixture is 5:4:30;

[0060] Step A2, adding octenyl succinic anhydride and dodecyl dimethyl betaine to a buffer solution, heating to 45°C, stirring at a speed of 460 rpm for 16 minutes until dissolved, and standing for 10 hours to obtain an aqueous phase, wherein the mass ratio of octenyl succinic anhydride, dodecyl dimethyl betaine and buffer solution is 0.4:3:50, and the buffer solution is a mixture of disodium hydrogen phosphate, dipotassium hydrogen phosphate and deionized water in a mass ratio of 7:3:120;

[0061] Step A3, stirring the oil phase and the aqueous phase at a speed of 560 rpm for 16 minutes until uniform, raising the temperature to 56°C, controlling the homogenization pressure to 120 MPa, the homogenization temperature to 72°C, and homogenizing for 3 times, each homogenization time being 5 minutes, to obtain a gel emulsion, wherein the mass ratio of the oil phase to the aqueous phase is 2:8.

[0062] Preparation Examples 4-6 and Comparative Preparation Examples 3-4 provide an activated bacterial solution.

[0063] Preparation Example 4

[0064] This preparation example provides an activated bacterial solution, which is prepared by the following steps:

[0065] Step B1, using beef extract peptone medium as the basal medium, supplementing the basal medium with 1% glucose, 0.02% magnesium sulfate, 0.003% manganese sulfate, and 0.1% zinc sulfate, by mass percentage, and adjusting the pH of the system to 6.8 with a 1.0% mass fraction of sodium bicarbonate aqueous solution to obtain an activated medium;

[0066] Step B2, inoculating the composite strain into the activation medium obtained in step B1 at an inoculum size of 5% by volume, setting the culture temperature to 36° C., shaking culture, adding potassium sulfate at the 6th and 12th hours of fermentation, the shaking rate was 140 rpm, and the culture time was 24 hours to obtain an activated bacterial solution, and the composite strain was mixed in a sterile environment with Bacillus natto NF01-6 and Lactobacillus plantarum ATCC 8014 at a viable cell count ratio of 1:0.2.

[0067] Preparation Example 5

[0068] This preparation example provides an activated bacterial solution, which is prepared by the following steps:

[0069] Step B1, using beef extract peptone medium as the basal medium, supplementing the basal medium with 2% sucrose, 0.03% manganese nitrate, 0.004% manganese nitrate, and 0.11% zinc nitrate, by mass percentage, and adjusting the pH of the system to 7.0 with a 1.0% mass fraction of sodium bicarbonate aqueous solution to obtain an activated medium;

[0070] Step B2, inoculating the composite strain into the activation medium obtained in step B1 at an inoculum size of 6% by volume, setting the culture temperature to 38° C., shaking culture, adding potassium chloride at the 6th and 12th hours of fermentation, the shaking rate was 180 rpm, and the culture time was 27 hours to obtain an activated bacterial solution, and the composite strain was mixed in a sterile environment with Bacillus natto NF01-6 and Lactobacillus plantarum ATCC 8014 at a viable cell count ratio of 1:0.35.

[0071] Preparation Example 6

[0072] This preparation example provides an activated bacterial solution, which is prepared by the following steps:

[0073] Step B1, using beef extract peptone medium as the basal medium, supplementing the basal medium with 3% fructose, 0.04% magnesium sulfate, 0.005% manganese sulfate, and 0.12% zinc sulfate, by mass percentage, and adjusting the pH of the system to 7.2 with a 2.5% mass fraction of sodium bicarbonate aqueous solution to obtain an activated medium;

[0074] Step B2, inoculating the composite strain into the activation medium obtained in step B1 at an inoculum rate of 7% by volume, setting the culture temperature to 40° C., shaking culture, adding potassium sulfate at the 6th and 12th hours of fermentation, the shaking rate was 220 rpm, and the culture time was 30 hours to obtain an activated bacterial solution, and the composite strain was mixed in a sterile environment with Bacillus natto NF01-6 and Lactobacillus plantarum ATCC 8014 at a viable cell count ratio of 1:0.5.

[0075] Comparative Preparation Example 3

[0076] This comparative preparation example provides an activated bacterial solution, which is prepared by the following steps:

[0077] Step B1, using beef extract peptone medium as the basal medium, supplementing the basal medium with 1% glucose, 0.02% magnesium sulfate, 0.003% manganese sulfate, and 0.1% zinc sulfate, by mass percentage, and adjusting the pH of the system to 6.8 with a 1.0% mass fraction of sodium bicarbonate aqueous solution to obtain an activated medium;

[0078] Step B2, inoculating Bacillus natto NF01-6 into the activation medium obtained in step B1 at an inoculum size of 5% by volume, setting the culture temperature to 36 ° C, shaking culture on a shaker, adding potassium sulfate at the 6th and 12th hours of fermentation, the shaking rate was 140 rpm, and the culture time was 24 hours to obtain an activated bacterial solution.

[0079] Comparative Preparation Example 4

[0080] This comparative preparation example provides an activated bacterial solution, which is prepared by the following steps:

[0081] Step B1, using beef extract peptone medium as the basal medium, supplementing the basal medium with 1% glucose, 0.02% magnesium sulfate, 0.003% manganese sulfate, and 0.1% zinc sulfate, by mass percentage, and adjusting the pH of the system to 6.8 with a 1.0% mass fraction of sodium bicarbonate aqueous solution to obtain an activated medium;

[0082] Step B2, inoculating Lactobacillus plantarum ATCC 8014 into the activation medium obtained in step B1 at an inoculum size of 5% by volume, setting the culture temperature to 36°C, shaking culture on a shaking table, adding potassium sulfate at the 6th and 12th hours of fermentation, the shaking rate being 140 rpm, and the incubation time being 24 hours to obtain an activated bacterial solution.

[0083] Examples 1-3 and Comparative Examples 1-5 provide an aquatic collagen tripeptide and a processing technology thereof.

[0084] Example 1

[0085] This embodiment provides a processing technology for aquatic collagen tripeptide, comprising the following steps:

[0086] Step S1, fish skin pretreatment: tilapia fish skin is placed in clean water, washed and chopped, then degreased, dried to constant weight at 50° C., crushed, and passed through a 300-mesh sieve to obtain defatted fish skin powder, wherein the degreasing process comprises: according to a material-liquid ratio of 1:30, ultrasonically dispersing the chopped tilapia fish skin in a modified degreasing agent, controlling the ultrasonic frequency to 30 kHz, the ultrasonic power to 500 w, and the ultrasonication for 12 min, then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 110 MPa, the homogenization temperature to 72° C., and homogenizing 8 times, each homogenization time being 6 min, and the modified degreasing agent is formed by mixing 1-ethyl-3-methylimidazole acetate, zinc phytate, calcium citrate, and an ethanol aqueous solution with a mass fraction of 12% in a mass ratio of 2:0.1:0.4:32;

[0087] Step S2, enzymatic hydrolysis: adding defatted fish skin powder to deionized water, adjusting the pH to 8.2 with a 10% by mass sodium bicarbonate aqueous solution, then adding the composite enzyme and the gel emulsion prepared in Preparation Example 1, controlling the hydrolysis temperature to 52° C., the enzymatic hydrolysis time to 4 h, treating at 121° C. for 20 min to inactivate the enzyme, cooling, standing and separating, to obtain an enzymatic extract, wherein the mass ratio of defatted fish skin powder, deionized water and composite enzyme is 1:76:0.08, and the composite enzyme is prepared by mixing papain and alkaline protease in a mass ratio of 3:1;

[0088] Step S3, fermentation: The activated bacterial solution prepared in Preparation Example 4 was inoculated into the enzymatic extract obtained in Step S2 for fermentation treatment, filtered, and the fermentation solution was collected. The mass ratio of the activated bacterial solution enzyme to the enzymatic extract was 0.26:3. The specific conditions for the fermentation treatment were as follows: the temperature was 28°C, the fermentation time was 6 hours, oxygen was introduced into the system, and the oxygen ventilation rate was set to 0.5m 3 / h, and continue to ferment for 20h;

[0089] Step S4, separation and purification: the fermentation broth is separated using a 620Da polyethersulfone nanofiltration membrane to obtain a small molecule peptide solution, which is then filtered through a polyamide nanofiltration membrane with a molecular weight cutoff of 160Da to obtain aquatic collagen tripeptide.

[0090] Example 2

[0091] This embodiment provides a processing technology for aquatic collagen tripeptide, comprising the following steps:

[0092] Step S1, fish skin pretreatment: cod skin is placed in clean water, washed and chopped, then degreased, dried to constant weight at 55°C, crushed, and passed through a 320-mesh sieve to obtain defatted fish skin powder, wherein the degreasing process comprises: according to a material-liquid ratio of 1:35, ultrasonically dispersing the chopped tilapia skin in a modified degreasing agent, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, and the ultrasonication for 14min, then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 120MPa, the homogenization temperature to 72°C, and homogenizing 8 times, each homogenization time being 8min, and the modified degreasing agent is composed of 1-ethyl-3-methylimidazole acetate, zinc phytate, calcium citrate, and an ethanol aqueous solution with a mass ratio of 4.5:0.2:0.5:34, and a mass fraction of 14%;

[0093] Step S2, enzymolysis: adding defatted fish skin powder to deionized water, adjusting the pH to 8.4 with a 15% mass fraction of sodium bicarbonate aqueous solution, then adding the composite enzyme and the gel emulsion prepared in Preparation Example 2, controlling the decomposition temperature to 54° C., the enzymolysis time to 4.5 h, treating at 121° C. for 18 min to inactivate the enzyme, cooling, standing and separating to obtain an enzymatic extract, wherein the mass ratio of defatted fish skin powder, deionized water and composite enzyme is 1:79:0.12, and the composite enzyme is prepared by mixing papain and alkaline protease in a mass ratio of 3.5:1.3;

[0094] Step S3, fermentation: The activated bacterial solution prepared in Preparation Example 5 was inoculated into the enzymatic extract obtained in Step S2 for fermentation treatment, filtered, and the fermentation solution was collected. The mass ratio of the activated bacterial solution enzyme to the enzymatic extract was 0.37:4. The specific conditions for the fermentation treatment were as follows: temperature 30°C, fermentation time 7h, oxygen was introduced into the system, and the oxygen ventilation rate was set to 0.75m 3 / h, and continue to ferment for 25h;

[0095] Step S4, separation and purification: the fermentation broth is separated using a 680Da polyethersulfone nanofiltration membrane to obtain a small molecule peptide solution, which is then filtered through a polyamide nanofiltration membrane with a molecular weight cutoff of 200Da to obtain aquatic collagen tripeptide.

[0096] Example 3

[0097] This embodiment provides a processing technology for aquatic collagen tripeptide, comprising the following steps:

[0098] Step S1, fish skin pretreatment: tilapia fish skin is placed in clean water, washed and chopped, then degreased, dried to constant weight at 60°C, crushed, and passed through a 340-mesh sieve to obtain defatted fish skin powder, wherein the degreasing process comprises: according to a material-liquid ratio of 1:40, ultrasonically dispersing the chopped tilapia fish skin in a modified degreasing agent, controlling the ultrasonic frequency to 40kHz, the ultrasonic power to 600w, and the ultrasonication for 16min, then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 130MPa, the homogenization temperature to 76°C, and homogenizing 12 times, each homogenization time being 10min, and the modified degreasing agent is composed of 1-ethyl-3-methylimidazole acetate, zinc phytate, calcium citrate, and an ethanol aqueous solution with a mass fraction of 16% in a mass ratio of 7:0.3:0.6:36;

[0099] Step S2, enzymolysis: add defatted fish skin powder to deionized water, adjust the pH to 8.6 with a 20% mass fraction of sodium bicarbonate aqueous solution, then add the composite enzyme and the gel emulsion prepared in Preparation Example 3, control the decomposition temperature to 56 ° C, the enzymolysis time to 5 h, treat at 121 ° C for 20 min to inactivate the enzyme, cool, and stand for separation to obtain an enzymatic extract, wherein the mass ratio of defatted fish skin powder, deionized water and composite enzyme is 1:82:0.16, and the composite enzyme is composed of papain and alkaline protease in a mass ratio of 4:1.6. The specific conditions of the fermentation treatment are as follows: the temperature is 32 ° C, the fermentation time is 8 h, oxygen is introduced into the system, and the oxygen ventilation volume is set to 1m 3 / h, and continue to ferment for 30h;

[0100] Step S3, fermentation: The activated bacterial solution prepared in Preparation Example 6 was inoculated into the enzymatic extract obtained in Step S2 for fermentation, filtered, and the fermentation solution was collected. The mass ratio of the activated bacterial solution enzyme to the enzymatic extract was 0.48:5:

[0101] Step S4, separation and purification: the fermentation broth is separated using a 740Da polyethersulfone nanofiltration membrane to obtain a small molecule peptide solution, which is then filtered through a polyamide nanofiltration membrane with a molecular weight cutoff of 200Da to obtain aquatic collagen tripeptide.

[0102] Comparative Example 1

[0103] Comparative Example 1 is the same as Example 1, except that the gel emulsion in Example 1 is replaced by the gel emulsion prepared in Comparative Preparation Example 1.

[0104] Comparative Example 2

[0105] Comparative Example 2 is the same as Example 1, except that the gel emulsion in Example 1 is replaced by the gel emulsion prepared in Comparative Preparation Example 2.

[0106] Comparative Example 3

[0107] Comparative Example 3 is the same as Example 1, except that the activated bacterial solution in Example 1 is replaced by the activated bacterial solution prepared in Comparative Preparation Example 3.

[0108] Comparative Example 4

[0109] Comparative Example 4 is the same as Example 1, except that the activated bacterial solution in Example 1 is replaced by the activated bacterial solution prepared in Comparative Preparation Example 4.

[0110] Comparative Example 5

[0111] Comparative Example 5 is the same as Example 1, except that 1-ethyl-3-methylimidazole acetate in the modified degreasing agent of Example 1 is replaced by sodium acetate.

[0112] Performance testing

[0113] 1. Test of Purity, Extraction Rate and Hydroxyproline Content of Aquatic Collagen Tripeptide

[0114] The hydroxyproline content in the collagen tripeptide in Examples 1-3 and Comparative Examples 1-5 was determined using an acid hydrolysis method hydroxyproline kit;

[0115] With reference to the collagen peptide detection standard: YY / T 1805.3-2022 "Collagen for Tissue Engineering Medical Devices Part 3: Detection of Collagen Content Based on Characteristic Peptide Assay-Liquid Chromatography-Mass Spectrometry", the purity and extraction rate of the collagen tripeptides in Examples 1-3 and Comparative Examples 1-5 were determined;

[0116] The above specific test results are shown in Table 1 below;

[0117] Table 1 Performance parameters of aquatic collagen tripeptides prepared in Examples 1-3 and Comparative Examples 1-5

[0118]

[0119] It can be seen from Table 1 that compared with Comparative Examples 1-5, the collagen tripeptide prepared by the method provided in Examples 1-3 has a higher extraction rate and purity.

[0120] 2. Molecular weight distribution test of aquatic collagen tripeptide

[0121] The molecular weight distribution of the aquatic collagen tripeptide obtained in Example 2 was determined by size exclusion chromatography, and the results are shown in Table 2.

[0122] Table 2 Molecular weight distribution of aquatic collagen tripeptides prepared in Example 2

[0123]

[0124] As can be seen from Table 2, 100% of the aquatic collagen tripeptides obtained in Example 2 were below 2.0 kDa, and more than 80% were below 0.5 kDa, which met the standards of collagen tripeptides in the prior art, indicating that the processing technology of the aquatic collagen tripeptide provided by the present invention has extremely high feasibility.

[0125] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A processing technology for aquatic collagen tripeptide, characterized in that: The following steps are involved: Step S1, fish skin pretreatment: the fish skin is placed in clean water for cleaning and shredded, then subjected to a degreasing treatment, dried, pulverized, and sieved to obtain a defatted fish skin powder, wherein the degreasing treatment process comprises: ultrasonically dispersing the shredded fish skin in a modified degreasing agent at a material-liquid ratio of 1:30-40, then adding the shredded fish skin into a high-pressure homogenizer, controlling the homogenization pressure to 110-130 MPa, the homogenization temperature to 72-76° C., and homogenizing for 8-12 times, with each homogenization treatment time being 6-10 min; Step S2, enzymolysis: adding defatted fish skin powder to deionized water, adjusting the pH to 8.2-8.6, then adding compound enzyme and gel emulsion, enzymolysis, inactivating the enzyme, cooling, standing and separating to obtain an enzymatic extract, wherein the mass ratio of defatted fish skin powder, deionized water and compound enzyme is 1:76-82:0.08-0.16, the compound enzyme is prepared by mixing papain and alkaline protease in a mass ratio of 3-4:1-1.6, the enzymolysis temperature is 52-56° C., and the enzymolysis time is 4-5 hours; Step S3, fermentation: inoculating the activated bacterial solution into the enzymatic extract obtained in step S2 for fermentation, filtering, and collecting the fermentation solution; Step S4, separation and purification: the fermentation broth is separated using a primary nanofiltration membrane to obtain a small molecule peptide solution, which is then intercepted and filtered using a secondary nanofiltration membrane to obtain aquatic collagen tripeptide; The modified degreasing agent is prepared by mixing 1-ethyl-3-methylimidazolium acetate, zinc phytate, calcium citrate and ethanol aqueous solution in a mass ratio of 2-7:0.1-0.3:0.4-0.6:32-36; The gel emulsion is prepared by the following steps: Step A1: add grape seed oil to anhydrous ethanol, heat to 62-66° C., stir for 18-24 minutes, and add dropwise to the mixture of phytosterols, lecithin, and anhydrous ethanol while stirring. The addition is controlled to be completed within 15 minutes. After the addition is completed, continue stirring for 22-26 minutes to obtain an oil phase; Step A2: add octenylsuccinic anhydride and sodium lignin sulfonate to a buffer solution, heat to 45-55° C., stir until dissolved, and allow to stand to obtain an aqueous phase; Step A3: Stir the oil phase and the water phase evenly, raise the temperature to 56-62° C., add the mixture into a high-pressure homogenizer, and homogenize to obtain a gel emulsion; The activated bacterial solution is prepared by the following steps: Step B1, using beef extract peptone medium as the basal medium, adding 1-3% carbon source, 0.02-0.04% magnesium salt, 0.003-0.005% manganese salt and 0.1-0.12% zinc salt to the basal medium by mass percentage, and adjusting the pH value of the system to 6.8-7.2 to obtain an activated medium; Step B2, inoculating the composite bacteria into the activation culture medium obtained in step B1 at an inoculum size of 5-7% by volume, setting the culture temperature to 36-40°C, shaking and culturing on a shaker, adding an inorganic potassium source at the 6th and 12th hours of fermentation, the shaking rate being 140-220 rpm, and the culture time being 24-30 hours to obtain an activated bacterial solution, wherein the composite bacteria are mixed in a sterile environment with Bacillus natto and Lactobacillus plantarum at a live cell count ratio of 1:0.2-0.

5.

2. The processing technology of aquatic collagen tripeptide according to claim 1, characterized in that: In step S3, the specific conditions of the fermentation treatment are as follows: the temperature is 28-32°C, the fermentation time is 6-8h, oxygen is introduced into the system, and the oxygen ventilation volume is set to 0.5-1m 3 / h, and then continue fermentation for 20-30h. In step S4, the molecular weight of the primary nanofiltration membrane used in the separation process is 620-740Da, and the molecular weight of the secondary nanofiltration membrane used in the interception filtration process is 160-200Da.

3. The processing technology of aquatic collagen tripeptide according to claim 1, characterized in that: In step A1, the mass ratio of grape seed oil, anhydrous ethanol and the mixed liquid is 8-12:60-80:36-42; in the mixed liquid, the mass ratio of phytosterols, lecithin and anhydrous ethanol is 5-6:4:30-40; in step A2, the mass ratio of octenylsuccinic anhydride, sodium lignin sulfonate and buffer is 0.4-0.6:3-5:50-60; and in step A3, the mass ratio of the oil phase to the aqueous phase is 2-3:8-10.

4. An aquatic collagen tripeptide prepared by the processing process of the aquatic collagen tripeptide according to any one of claims 1 to 3.

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