Anti-aging sturgeon collagen peptide as well as preparation method and application thereof

Abstract

The invention provides an anti-aging sturgeon collagen peptide as well as a preparation method and application thereof, and belongs to the technical field of biological medicines and sturgeon byproduct processing. The preparation method comprises the following steps: mixing sturgeons with water, adding chlorine dioxide and citric acid, soaking and cleaning, mixing the obtained cleaned sturgeons with water, adding a sodium hydroxide solution and hydrogen peroxide, soaking and cleaning, mixing the obtained impurity-removed and fishy smell-removed sturgeons with water, heating and stirring to obtain a pulpified material, and discarding grease to obtain the sturgeon feed. Cutting and grinding the degreased material, reacting the obtained pulpified material of the sturgeon at 75-82 DEG C and pH of 9.0-9.6 for 1.5-2.5 hours to obtain a material after primary degradation, adding bromelain and neutral protease for enzymolysis, and adding glycogenase into the obtained material after enzymolysis for enzymolysis. The anti-aging sturgeon collagen peptide contains sodium hyaluronate and glycosaminoglycan, the content of the obtained glycosaminoglycan is high, and the anti-aging sturgeon collagen peptide has an anti-aging effect on zebra fish.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and sturgeon by-product processing technology, and particularly relates to an anti-aging sturgeon collagen peptide, its preparation method and application. Background Technology

[0002] Aging is the most fundamental natural law in nature. It is the result of the combined effects of various physiological and pathological factors on the growth and development of living organisms. This process involves complex biological processes, including the process of material metabolism, hormone regulation, and enzyme regulation. These aspects are interconnected and influence each other. Therefore, anti-aging has become a research hotspot.

[0003] Sturgeon, belonging to the order Acipenseriformes, subclass Actinopterygii, class Osteichthyes, is a cartilaginous fish species and one of the largest and longest-lived freshwater fish. Sturgeon contains a rich variety of chemical components, including unsaturated fatty acids, chondroitin sulfate, amino acids, proteins, and trace elements. These chemical components typically possess various biological activities, giving them high economic and scientific value. However, due to their large size and high fat content, sturgeon are difficult to clean and process. Currently, sturgeon are mainly used for primary processing such as caviar production and food consumption, with limited deep processing. Furthermore, research on the chemical composition and pharmacological effects of sturgeon is still in its early stages; while some research results exist, the depth and breadth of the research are insufficient. The material basis for the medicinal effects of sturgeon is not fully understood, and there is a lack of correlation between chemical composition and efficacy evaluation.

[0004] Collagen peptides have a wide range of raw materials and numerous preparation methods, but the bioavailability of products prepared from different raw material sources and methods varies significantly. Determining which method produces sturgeon collagen peptides with anti-aging effects remains a pressing technical challenge. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an anti-aging sturgeon collagen peptide, its preparation method, and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing anti-aging sturgeon collagen peptides, comprising the following steps: (1) After mixing the sturgeon with water, add chlorine dioxide and citric acid, soak and wash to obtain the cleaned sturgeon; (2) After cleaning, the sturgeon is mixed with water, sodium hydroxide solution and hydrogen peroxide are added, and the mixture is soaked and cleaned to obtain sturgeon that has been cleaned of impurities and deodorized. (3) After removing impurities and fishy smell, the sturgeon is mixed with water, heated and stirred to obtain a slurry. The oil is discarded, and the degreased material is sheared and ground to obtain the sturgeon slurry. (4) The sturgeon pulp material was reacted at 75~82℃ and pH 9.0~9.6 for 1.5~2.5h to obtain the material after primary degradation; (5) Add bromelain and neutral protease to the material after primary degradation for the first enzymatic hydrolysis. Add glycogenase to the hydrolyzed material for the second enzymatic hydrolysis to obtain the material after secondary degradation.

[0007] In this invention, sturgeon is mixed with water, then chlorine dioxide and citric acid are added, and the mixture is soaked and washed to obtain cleaned sturgeon. The mass ratio of sturgeon, water, chlorine dioxide, and citric acid is (90~110):(490~510):(1~1.5):(2~3), more preferably (95~105):(495~505):(1.2~1.5):(2.5~3), and even more preferably 100:500:1.25:2.5 or 100:500:1.5:30. The soaking and washing temperature is preferably 20~25℃, more preferably 23~25℃, and even more preferably 25℃; the soaking and washing time is preferably 17~25h, more preferably 18~24h, and even more preferably 18, 20, 22, or 24h; the soaking and washing is preferably performed 5~7 times, and even more preferably 6 times. In this invention, the sturgeon is a sturgeon that has been defiled. The method for preparing the defiled sturgeon includes breaking the sturgeon into 8-10cm pieces, adding water, soaking and stirring for 10-14 hours, draining the blood and wastewater, and obtaining the defiled sturgeon. The sturgeon is a sturgeon with its internal organs removed.

[0008] In this invention, after obtaining the cleaned sturgeon, the cleaned sturgeon is mixed with water, and sodium hydroxide solution and hydrogen peroxide are added. The mixture is then soaked and cleaned to obtain sturgeon that has been cleaned of impurities and fishy smell. The preferred mass ratio of the cleaned sturgeon, water, sodium hydroxide solution, and hydrogen peroxide is (90~110):(490~510):(0.3~0.6):(0.9~1.5), more preferably (95~105):(495~505):(0.4~0.5):(1.0~1.5), and even more preferably 100:500:0.4:1.0 or 100:500:0.5:1.25; the preferred mass fraction of the sodium hydroxide solution is 30%~35%, more preferably 31%~34%, and even more preferably 32% or 33%, wherein the solvent of the sodium hydroxide solution is water; the preferred mass percentage of the hydrogen peroxide is 34%~36%, and even more preferably 35%; the preferred soaking temperature is 20~25℃, more preferably 22~25℃, and even more preferably 25℃. The soaking time is preferably 10-18 hours, more preferably 12-16 hours, and even more preferably 12, 14, or 16 hours. After soaking, the soaking water is drained to obtain the treated sturgeon. The washing process involves 8-12 washes with water, more preferably 9-11 washes, and even more preferably 10 washes. During washing, the mass ratio of the treated sturgeon to water is 1:8-10. This invention achieves the purpose of removing impurities and deodorizing by adding chlorine dioxide, citric acid, sodium hydroxide, and hydrogen peroxide, as well as washing steps.

[0009] In this invention, after obtaining sturgeon that has been cleaned and deodorized, the sturgeon is mixed with water, heated, and stirred to obtain a slurry. The grease is then discarded, and the degreased material is sheared and ground to obtain the sturgeon slurry. The preferred mass ratio of the cleaned and deodorized sturgeon to water is 1:1~2, more preferably 1:1~1.5, and even more preferably 1:1 or 1:1.5. The preferred heating temperature is 74~82℃, more preferably 75~80℃, and even more preferably 75 or 80℃. The stirring is performed at 550~650 rpm / min for 15~30 min, more preferably at 580~630 rpm / min for 20~30 min, and even more preferably at 600 rpm / min. The mixture is stirred for 20 minutes or stirred at 600 rpm for 30 minutes; the shearing is performed at 13000-17000 rpm for 8-12 seconds, more preferably at 14000-16000 rpm for 9-11 seconds, and even more preferably at 15000 rpm for 10 seconds; the grinding is performed at 18000-21000 rpm for 3-7 seconds, more preferably at 19000-20000 rpm for 4-6 seconds, and even more preferably at 20000 rpm for 5 seconds. This invention further refines the degreased material and increases its uniformity through shearing and grinding.

[0010] In this invention, after obtaining the sturgeon pulp material, the sturgeon pulp material is reacted at 75-82℃ and pH 9.0-9.6 for 1.5-2.5 hours to obtain the material after primary degradation. As a preferred embodiment, the sturgeon pulp material is reacted at 76-80℃ and pH 9.2-9.5 for 1.7-2.2 hours; a more preferred embodiment is that the sturgeon pulp material is reacted at 80℃ and pH 9.5 for 2 hours. The pH is adjusted using food-grade calcium hydroxide.

[0011] In this invention, after obtaining the material after primary degradation, bromelain and neutral protease are added to the material for a first enzymatic hydrolysis. Glycogenase is then added to the hydrolyzed material for a second enzymatic hydrolysis to obtain the material after secondary degradation. The mass ratio of the material after primary degradation, bromelain, and neutral protease is 10:(0.01~0.03):(0.03~0.05), more preferably 10:(0.015~0.025):(0.035~0.045), and even more preferably 10:0.02:0.04. The first enzymatic hydrolysis is performed at pH 7.5~8.0 and 55~60℃ for 2~4 hours, more preferably at pH 7.6~7.9 and 56~58℃ for 2.5~3.5 hours, and even more preferably... The first enzymatic hydrolysis is performed at pH 7.8 and 56°C for 3 hours; the second enzymatic hydrolysis is performed at pH 6.5-7.0 and 55-60°C for 0.5-1.5 hours, more preferably at pH 6.6-6.8 and 56-58°C for 0.7-1.2 hours, and even more preferably at pH 6.8 and 56°C for 1 hour; the mass ratio of the material after primary degradation to glycogenase is 10:0.001-0.003, more preferably 10:0.0015-0.0025, and even more preferably 10:0.002. In this invention, the bromelain activity is 800,000 units / g, the neutral protease activity is 150,000 units / g, and the glycogenase activity is 100,000 units / g. This invention does not specifically limit the sources of bromelain, neutral protease, and glycogenase; they can be prepared using methods known in the art or purchased as commercially available products. For example, the bromelain was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.; the neutral protease was purchased from Yantai Maitel Biotechnology Co., Ltd. (catalog number MNP1.0); and the glycogenase was purchased from Novozymes (catalog number N100). This invention utilizes the synergistic enzymatic hydrolysis of bromelain, neutral protease, and glycogenase to improve protein extraction rate while retaining more glycosaminoglycan components.

[0012] In this invention, after obtaining the material after secondary degradation, enzyme inactivation is performed to obtain the inactivated material. The pH is adjusted, a filter aid is added, and plate and frame filtration is performed. The first filtrate is collected, centrifuged, and the collected clear filtrate is filtered using a nanofiltration membrane. The retained material is collected, and activated carbon fiber is used to adsorb the retained material. The material not adsorbed by the activated carbon fiber is collected. The deodorized material is filtered and dried to obtain anti-aging sturgeon collagen peptides. The enzyme inactivation method is to adjust the pH of the secondary degradation material to 4.5-5.0, heat it to 90-98℃ and hold it for 10-20 minutes to inactivate the enzyme, and obtain the inactivated material. More preferably, the pH of the secondary degradation material is adjusted to 4.8, heated to 95℃ and held for 15 minutes to inactivate the enzyme, and obtain the inactivated material. The pH adjustment to 6.0-7.0 is more preferably 6.2-6.8, and more preferably 6.5. The filter aid is perlite and / or diatomaceous earth; the mass ratio of the inactivated material to the filter aid is preferably 1:1.5~2.5, more preferably 1:1.7~2.2, and even more preferably 1:2; the mass ratio of perlite to diatomaceous earth is 1:1. The centrifugation can be performed at 30000 r / min for 4 s, collecting the clarified filtrate. This centrifugation can remove oil particles and fine insoluble suspended matter from the first filtrate, further improving clarity. The nanofiltration membrane is preferably a 150 Dalton nanofiltration membrane; the permeated portion is discarded, and the retained material is collected. This invention achieves concentration and desalination through nanofiltration. The mass ratio of activated carbon fiber to the retained material is 1:15~25, more preferably 1:17~22, and even more preferably 1:20; the adsorption temperature is 60~65℃, such as 61, 62, 63, or 64℃; and the adsorption time is 30~60 min. This invention utilizes activated carbon fiber to achieve deodorization. Compared to activated carbon powder, using activated carbon fiber significantly reduces solid waste (activated carbon residue) generation while improving production efficiency. The deodorized material is filtered and dried, with the filtration including two-stage paperboard filtration and 0.22-micron membrane filtration. After the two-stage paperboard filtration, filtrate a is collected to obtain a clear concentrate, thus removing all insoluble matter. The clear concentrate is then filtered through a 0.22-micron membrane, and filtrate b is collected to obtain sterilized material. This invention achieves sterilization through 0.22-micron membrane filtration. After sterilization, the material is spray-dried at an inlet temperature of 160-170°C and an outlet temperature of 90-95°C to obtain sturgeon collagen peptides. In this invention, the two-stage paperboard consists of paperboard with a pore size of 4 microns and paperboard with a pore size of 2 microns.The preparation method of the secondary paperboard is as follows: paperboard with a pore size of 4 micrometers and paperboard with a pore size of 2 micrometers are stacked sequentially from top to bottom (that is, the deodorized material first passes through the 4-micrometer paperboard and then through the 2-micrometer paperboard during filtration); the 4-micrometer paperboard and the 2-micrometer paperboard are purchased from Shenyang Great Wall Filter Paperboard Co., Ltd., and the 4-micrometer paperboard is model SCP-311 and the 2-micrometer paperboard is model SCP-321.

[0013] The present invention has found that the sturgeon collagen peptide contains sodium hyaluronate and glycosaminoglycans, and the glycosaminoglycans prepared by the present invention have a high content, above 5.95%.

[0014] This invention provides an application of the above-mentioned anti-aging sturgeon collagen peptide in the preparation of anti-aging products.

[0015] In this invention, the product includes reagents or drugs, and the anti-aging sturgeon collagen peptide significantly promotes the expression of the Sirt1 gene in zebrafish, thus exhibiting an anti-aging effect on zebrafish. The zebrafish are 5-day-old zebrafish. In this invention, the product also includes pharmaceutically acceptable excipients, such as excipients, antioxidants, or flavoring agents. In this invention, the anti-aging sturgeon collagen peptide is the sole active ingredient for anti-aging, but it can also be combined with other anti-aging active ingredients to achieve the same anti-aging purpose.

[0016] This invention provides an application of the above-mentioned anti-aging sturgeon collagen peptide in the preparation of anti-aging products.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an anti-aging sturgeon collagen peptide, its preparation method, and its application. The invention prepares the anti-aging sturgeon collagen peptide containing sodium hyaluronate and glycosaminoglycans through methods such as impurity removal and deodorization, pulping, primary degradation, and enzymatic secondary degradation, achieving a high glycosaminoglycan content. The anti-aging results of this invention demonstrate that the anti-aging sturgeon collagen peptide significantly promotes Sirt1 gene expression in zebrafish, exhibiting an anti-aging effect on zebrafish. Attached Figure Description

[0018] Figure 1 The results show the relative expression levels of the Sirt1 gene in zebrafish treated with different concentrations of sturgeon collagen peptides, compared with the blank control group. <0.05. Detailed Implementation

[0019] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] In the following embodiments, the whole sturgeon refers to a fresh frozen whole sturgeon after the internal organs have been removed.

[0022] The 32% food-grade sodium hydroxide solution was prepared by mixing 32g of sodium hydroxide with 68g of water.

[0023] The paperboard with a pore size of 4 micrometers and the paperboard with a pore size of 2 micrometers were both purchased from Shenyang Great Wall Filter Paperboard Co., Ltd., and the models were SCP-311 and SCP-321, respectively.

[0024] Example 1 A method for preparing anti-aging sturgeon collagen peptides, comprising the following steps: (1) Raw material pretreatment (removal of impurities and deodorization): S1. 100kg of whole sturgeon is cut into 8-10cm pieces using cutting equipment and a coarse crusher. 1000kg of water is added, and the mixture is soaked at 25℃ with intermittent stirring (intermittent stirring means stirring at 12rpm / min for 3 minutes after soaking for 60 minutes). After 12 hours, the blood and wastewater are drained to obtain the whole sturgeon after wastewater discharge. S2. Add 500 kg of water to 100 kg of sturgeon after sewage discharge, add 1.25 kg of food-grade chlorine dioxide (the food-grade chlorine dioxide is a powdered solid with an effective ClO2 concentration of 45%) and 2.5 kg of food-grade citric acid, soak at 25℃ for 24 hours, then drain the soaking water to obtain the soaked sturgeon. S3. Wash 100kg of soaked sturgeon six times in the manner described in step S2 to obtain the cleaned sturgeon. S4. Add 500 kg of water to 100 kg of cleaned sturgeon, add 0.4 kg of 32% food-grade sodium hydroxide solution and 1 kg of food-grade hydrogen peroxide (hydrogen peroxide concentration 35%), soak at 25℃ for 16 h, then drain the soaking water to obtain the treated sturgeon. S5. Add 900 kg of water to 100 kg of treated sturgeon and wash for 2 hours each time. Stir for 5 minutes every 60 minutes during washing. Repeat the washing with clean water 10 times to obtain sturgeon that has been cleaned of impurities and deodorized. (2) Ultrafine crushing (pulping): 100 kg of sturgeon whole fish after impurity removal and deodorization is added to 100 kg of purified water to obtain a solid-liquid mixture. The mixture is heated to 80℃ and stirred at 600 rpm / min for 20 min using a tank-mounted high-speed stirring device to obtain pulverized material. The oil is skimmed off to obtain the degreased material. The degreased material is sheared at 15000 rpm for 10 s using a shear pump and then ground at 20000 rpm for 5 s using a grinding pump to further refine the degreased material and increase its uniformity, thus obtaining the sturgeon pulverized material. (3) Low-temperature primary degradation: 100 kg of sturgeon pulp was reacted at 80 °C and pH 9.5 (the pH was adjusted to 9.5 using food-grade calcium hydroxide) for 2 h to allow the macromolecules to undergo primary degradation, resulting in the primary degradation material. (4) Enzymatic secondary degradation (multi-enzyme): Add 0.02 kg of bromelain (with an activity of 800,000 units / g) and 0.04 kg of neutral protease (with an activity of 150,000 units / g) to 10 kg of material after primary degradation. Enzymatically hydrolyze at pH 7.8 and 56℃ for 3 h. Add 0.002 kg of glycogenase (with an activity of 100,000 units / g) to the hydrolyzed material and enzymatically hydrolyze at pH 6.8 and 56℃ for 1 h to obtain the material after secondary degradation. (5) Enzyme inactivation: Adjust the pH of the material after secondary degradation to 4.8, heat to 95℃ and hold for 15 min to inactivate the enzyme and obtain the inactivated material; (6) Primary filtration (solid-liquid separation): Adjust the pH of 10 kg of inactivated material to 6.5, add 10 kg of perlite and 10 kg of diatomaceous earth, use a press-type plate and frame filter for primary filtration, separate and discard the filter residue, and obtain the first filtrate; (7) Secondary clarification (oil and fine suspended matter): The oil particles and fine insoluble suspended matter in the first filtrate are removed by centrifuging at 30,000 r / min for 4 s using an ultra-high speed tubular centrifuge to further improve the clarification and obtain a clear filtrate. (8) Nanofiltration membrane (concentration and desalination): The clarified filtrate is dehydrated and concentrated using a 150 Dalton nanofiltration membrane device. The permeated portion is discarded and the retained material is collected (the concentration of dehydration and concentration is 22%). (9) Adsorption and deodorization: At 63°C, 1 kg of activated carbon fiber is used to continuously remove pigments and unpleasant flavors from 20 kg of retained material for 30 min. The material that is not adsorbed by the activated carbon fiber (i.e. the material that passes through the activated carbon fiber) is collected to obtain the deodorized material. (10) Tertiary clarification (all insoluble matter): The deodorized material is filtered using a two-stage paperboard, and the filtrate a is collected to obtain a clarified concentrate; the preparation method of the two-stage paperboard is as follows: paperboard with a pore size of 4 micrometers and paperboard with a pore size of 2 micrometers are stacked from top to bottom (that is, the deodorized material passes through the 4-micrometer paperboard and then the 2-micrometer paperboard during filtration).

[0025] (11) Low-temperature non-destructive sterilization: The clarified concentrate is filtered through a 0.22-micron filter membrane, and the filtrate b is collected to obtain the sterilized material; (12) Spray drying: The sterilized material is dried using a spray drying tower (inlet air 170℃, outlet air 95℃) to remove moisture and obtain sturgeon collagen peptide powder.

[0026] Performance verification of sturgeon collagen peptide powder: 1. The sturgeon collagen peptide powder prepared in this embodiment was sent to Hubei Ruibang Biotechnology Co., Ltd. to test the content of sodium hyaluronate and glycosaminoglycans (GAGs) (calculated as chondroitin sulfate) using QB / T 4576-2013 and GB / T 20365-2006 respectively (report number: A2240264181101001C). The test results are shown in Table 1.

[0027] Table 1. Sodium hyaluronate and glycosaminoglycans (GAGs) content of sturgeon collagen peptide powder

[0028] The results in Table 1 show that the sturgeon collagen peptide powder prepared by the method of the present invention contains 2.27g of sodium hyaluronate per 100g, and contains 6.63% glycosaminoglycans (GAGs).

[0029] 2. The sturgeon collagen peptide powder prepared in this embodiment was diluted with water to 0.71 g / L, 0.36 g / L, and 0.14 g / L, respectively. Thirty-six 5-day-old zebrafish were randomly divided into three groups. The 5-day-old zebrafish were cultured in fish embryo culture medium and exposed to sturgeon collagen peptide sample solutions at concentrations of 0.71 g / L, 0.36 g / L, and 0.14 g / L, respectively. A blank control group was also set up: 12 5-day-old zebrafish were exposed to fish embryo culture medium with the same concentrations as the sample treatment groups. After 24 hours of exposure, RNA was extracted from the zebrafish, cDNA was synthesized, and real-time PCR amplification was performed. β-actin was used as the housekeeping gene, and Ct was used as the amplification result. The relative expression level of Sirt1 (senescence regulatory gene) was calculated and statistically analyzed. The results are shown in [Figure number missing]. Figure 1 As shown.

[0030] The fish embryo culture medium is prepared by weighing 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride and dissolving them in 10 L of water.

[0031] Figure 1 The results showed that sturgeon collagen peptide solutions of 0.71 g / L, 0.36 g / L, and 0.14 g / L significantly promoted the expression of the Sirt1 gene in zebrafish, with promotion rates of 11% (p=0.021), 17% (p=0.017), and 14% (p=0.015), respectively. Therefore, sturgeon collagen peptides have an anti-aging effect on zebrafish.

[0032] 3. The cystine and tryptophan content in the sturgeon collagen peptide powder prepared in this embodiment was tested by Hubei Ruibang Biotechnology Co., Ltd. (report number A225005669610100102C). The results are shown in Table 2. At the same time, Hubei Ruibang Biotechnology Co., Ltd. was also commissioned to test the histidine, tyrosine, isoleucine, valine, alanine, proline, arginine, lysine, serine, glutamic acid, glycine, methionine, leucine, phenylalanine, threonine, and aspartic acid content in the sturgeon collagen peptide powder prepared in this embodiment using the GB 5009.124-2016 method (report number A225005669610100101C). The results are shown in Table 3.

[0033] Table 2 Results of cystine and tryptophan content in sturgeon collagen peptide powder

[0034] Table 3. Results of amino acid content in sturgeon collagen peptide powder

[0035] The results in Tables 2 and 3 show that the sturgeon collagen peptide powder prepared by this invention contains cystine, tryptophan, histidine, tyrosine, isoleucine, valine, alanine, proline, arginine, lysine, serine, glutamic acid, glycine, methionine, leucine, phenylalanine, threonine, and aspartic acid. The total amount of each amino acid involved in Tables 2 and 3 in every 100g of sturgeon collagen peptide powder is as high as 89.78g.

[0036] Example 2 A method for preparing anti-aging sturgeon collagen peptides, comprising the following steps: (1) Raw material pretreatment (removal of impurities and deodorization): S1. 100kg of whole sturgeon is cut into 8-10cm pieces using cutting equipment and a coarse crusher. 1000kg of water is added, and the mixture is soaked at 25℃ with intermittent stirring (intermittent stirring means stirring at 12rpm / min for 3 minutes after soaking for 60 minutes). After 12 hours, the blood and wastewater are drained to obtain the whole sturgeon after wastewater discharge. S2. Add 500 kg of water to 100 kg of sturgeon after sewage discharge, add 1.5 kg of food-grade chlorine dioxide (the food-grade chlorine dioxide is a powdered solid with an effective ClO2 concentration of 45%) and 3 kg of food-grade citric acid, soak at 25℃ for 18 hours, then drain the soaking water to obtain the soaked sturgeon. S3. Wash the soaked sturgeon six times in the manner described in step S2 to obtain the cleaned sturgeon. S4. Add 500 kg of water to 100 kg of cleaned sturgeon, add 0.5 kg of 32% food-grade sodium hydroxide solution and 1.25 kg of food-grade hydrogen peroxide (hydrogen peroxide concentration 35%), soak at 25℃ for 12 h, then drain the soaking water to obtain the treated sturgeon. S5. Add 1000kg of water to 100kg of treated sturgeon and wash for 2 hours each time. Repeat the washing with clean water 8 times to obtain the sturgeon after removing impurities and fishy smell. (2) Ultrafine crushing (pulping): 100 kg of sturgeon whole fish after impurity removal and deodorization is added to 150 kg of purified water to obtain a solid-liquid mixture. The mixture is heated to 75°C and stirred at 600 rpm / min for 30 min using a tank-mounted high-speed stirring device to obtain pulverized material. The oil is skimmed off to obtain the degreased material. The degreased material is sheared at 16000 rpm for 10 s using a shear pump and then ground at 18000 rpm for 5 s using a grinding pump to further refine the degreased material and increase its uniformity, thus obtaining sturgeon pulverized material. (3) Low-temperature primary degradation: 100 kg of sturgeon pulp was reacted at 75°C and pH 10 (pH was adjusted to 9.5 using food-grade calcium hydroxide) for 2 h to allow the macromolecules to undergo primary degradation, resulting in the primary degradation material. (4) Enzymatic secondary degradation (multi-enzyme): Add 0.024 kg of bromelain (with an activity of 800,000 units / g) and 0.04 kg of neutral protease (with an activity of 150,000 units / g) to 10 kg of material after primary degradation. Enzymatically hydrolyze at pH 7.5 and 56℃ for 4 h. Add 0.001 kg of glycogenase (with an activity of 100,000 units / g) to the hydrolyzed material and enzymatically hydrolyze at pH 6.5 and 56℃ for 1 h to obtain the material after secondary degradation. (5) Enzyme inactivation: Adjust the pH of the material after secondary degradation to 4.6, heat to 95℃ and hold for 15 min to inactivate the enzyme and obtain the inactivated material; (6) Primary filtration (solid-liquid separation): Adjust the pH of 10 kg of inactivated material to 6.4, add 20 kg of perlite, and use a press-type plate and frame filter for primary filtration. Separate and discard the filter residue to obtain the first filtrate. (7) Secondary clarification (oil and fine suspended matter): The oil particles and fine insoluble suspended matter in the first filtrate are removed by centrifuging at 30,000 r / min for 4 s using an ultra-high speed tubular centrifuge to further improve the clarification and obtain a clear filtrate. (8) Nanofiltration membrane (concentration and desalination): The clarified filtrate is dehydrated and concentrated to 20% using a 150 Dalton nanofiltration membrane device. The permeated portion is discarded and the retained material is collected. (9) Adsorption and deodorization: At 60℃, 1kg of activated carbon fiber is used to continuously remove pigments and unpleasant flavors from 20kg of retained material for 30min. The material that is not adsorbed by the activated carbon fiber (i.e. the material that passes through the activated carbon fiber) is collected to obtain the deodorized material. (10) Tertiary clarification (all insoluble matter): The deodorized material is filtered using a two-stage paperboard, and the filtrate a is collected to obtain a clarified concentrate; the preparation method of the two-stage paperboard is as follows: paperboard with a pore size of 4 micrometers and paperboard with a pore size of 2 micrometers are stacked from top to bottom (that is, the deodorized material passes through the 4-micrometer paperboard and then the 2-micrometer paperboard during filtration).

[0037] (11) Low-temperature non-destructive sterilization: The clarified concentrate is filtered through a 0.22-micron filter membrane, and the filtrate b is collected to obtain the sterilized material; (12) Spray drying: The sterilized material is dried using a spray drying tower (inlet air 165℃, outlet air 90℃) to remove moisture and obtain sturgeon collagen peptide powder.

[0038] Performance verification of sturgeon collagen peptide powder: The sturgeon collagen peptide powder prepared in this embodiment was sent to Hubei Ruibang Biotechnology Co., Ltd. to test the content of glycosaminoglycans (GAGs) (calculated as chondroitin sulfate) using GB / T 20365-2006 (report number: 2024-L04228). The test results are shown in Table 4.

[0039] Table 4. Glycosaminoglycan (GAGs) content of sturgeon collagen peptide powder

[0040] The results in Table 4 show that the sturgeon collagen peptide powder obtained by the preparation method of the present invention contains 5.95% glycosaminoglycans (GAGs).

[0041] Comparative Example 1 A method for preparing sturgeon collagen peptides, comprising the following steps: (1) Raw material pretreatment (removal of impurities and deodorization): S1. 100kg of whole sturgeon is cut into 8-10cm pieces using cutting equipment and a coarse crusher. 1000kg of water is added, and the mixture is soaked at 25℃ with intermittent stirring (intermittent stirring means stirring at 12rpm / min for 3 minutes after soaking for 60 minutes). After 12 hours, the blood and wastewater are drained to obtain the whole sturgeon after wastewater discharge. S2. Add 500 kg of water to the whole sturgeon after sewage discharge, add 0.5 kg of sodium hydroxide solution with a mass fraction of 30% and 1 kg of food-grade hydrogen peroxide (hydrogen peroxide concentration of 35%), soak at 25℃ for 18 hours, then drain the soaking water to obtain the treated whole sturgeon. S3. Add 1000kg of water to 100kg of treated sturgeon and wash for 2 hours each time. Repeat the washing with clean water 6 times to obtain the sturgeon after removing impurities and fishy smell. (2) Heat treatment (denaturation): 150 kg of purified water was added to 100 kg of whole sturgeon after impurity removal and deodorization to obtain a solid-liquid mixture. The pH was adjusted to 10 using food-grade calcium hydroxide, the temperature was raised to 95℃, and the mixture was boiled for 4 hours to make the material into a pulp, thus obtaining the sturgeon pulped material. (3) Enzymatic hydrolysis: After 10 kg of sturgeon pulp was cooled to 60°C, 0.03 kg of alkaline protease (the activity of the alkaline protease was 350,000 units) was added and enzymatic hydrolysis was carried out at pH 8.3 for 4 h to obtain the enzymatically hydrolyzed material; (4) Enzyme inactivation: After the enzymatic hydrolysis is completed, the hydrolyzed material is heated to 100℃ and boiled for 20 minutes to inactivate the enzyme and obtain the inactivated material. (5) Primary filtration (solid-liquid separation): Adjust the pH of 10 kg of inactivated material to 6.0, add 20 kg of perlite, and use a press-type plate and frame filter for primary filtration. Separate and discard the filter residue to obtain the first filtrate. (6) Secondary clarification (oil and fine suspended matter): The filtrate is centrifuged at 30,000 r / min for 4 s using an ultra-high speed tubular centrifuge to remove oil particles and fine insoluble suspended matter from the initial filtrate, thereby further improving the clarification and obtaining a clear filtrate. (7) Nanofiltration membrane (concentration and desalination): The clarified filtrate is dehydrated and concentrated to 20% using a 150 Dalton nanofiltration membrane device. The permeated portion is discarded and the retained material is collected. (8) Adsorption and deodorization: Add 0.5 kg of activated carbon fiber to 10 kg of retained material, react at pH 4.5 and 80℃ for 1.5 h, collect the material that was not adsorbed by the activated carbon fiber (i.e. the material that passed through the activated carbon fiber), and obtain the deodorized material. (9) Tertiary clarification (all insoluble matter): The deodorized material is filtered using a two-stage paperboard, and the filtrate a is collected to obtain a clear concentrate; (10) Low-temperature non-destructive sterilization: The clarified concentrate is passed through a 0.22μm microporous membrane, and the filtrate b is collected to obtain the sterilized material; (11) Spray drying: The sterilized material is dried using a spray drying tower (inlet air 180℃, outlet air 95℃) to remove moisture and obtain sturgeon collagen peptide powder.

[0042] The sturgeon collagen peptide powder prepared in this comparative example was sent to Hubei Ruibang Biotechnology Co., Ltd. to detect the content of sodium hyaluronate (report No. B8E531010B8F1098555) and glycosaminoglycans (GAGs) (calculated as chondroitin sulfate) (report No. B1E604018B1F10A7321) using QB / T 4576-2013 and GB / T 20365-2006, respectively. The test results are shown in Table 5.

[0043] Table 5. Sodium hyaluronate and glycosaminoglycans (GAGs) content of sturgeon collagen peptide powder

[0044] The results in Table 5 show that the sturgeon collagen peptide powder prepared in Comparative Example 1 does not contain sodium hyaluronate, and the content of glycosaminoglycans (GAGs) is low, at 0.368%. It is evident that different methods of preparing sturgeon collagen peptides result in sturgeon collagen peptide powders with different compositions or contents. Compared with Comparative Example 1, the sturgeon collagen peptide powder prepared by the method of this invention has a higher content of glycosaminoglycans (GAGs).

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing anti-aging sturgeon collagen peptides, characterized in that, Includes the following steps: (1) After mixing the sturgeon with water, add chlorine dioxide and citric acid, soak and wash to obtain the cleaned sturgeon; (2) After cleaning, the sturgeon is mixed with water, sodium hydroxide solution and hydrogen peroxide are added, and the mixture is soaked and cleaned to obtain sturgeon that has been cleaned of impurities and deodorized. (3) After removing impurities and fishy smell, the sturgeon is mixed with water, heated and stirred to obtain a slurry. The oil is discarded, and the degreased material is sheared and ground to obtain the sturgeon slurry. (4) The sturgeon pulp material was reacted at 75~82℃ and pH 9.0~9.6 for 1.5~2.5h to obtain the material after primary degradation; (5) Add bromelain and neutral protease to the material after primary degradation for the first enzymatic hydrolysis. Add glycogenase to the hydrolyzed material for the second enzymatic hydrolysis to obtain the material after secondary degradation.

2. The preparation method according to claim 1, characterized in that, After obtaining the material after secondary degradation, enzyme inactivation is performed to obtain the inactivated material. The pH is adjusted, a filter aid is added, and plate and frame filtration is performed to collect the first filtrate. The filtrate is centrifuged, and the collected clear filtrate is filtered using a nanofiltration membrane. The retained material is collected, and activated carbon fiber is used to adsorb the retained material. The material not adsorbed by the activated carbon fiber is collected. The deodorized material is filtered and dried to obtain anti-aging sturgeon collagen peptides.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of sturgeon, water, chlorine dioxide and citric acid is (90~110):(490~510):(1~1.5):(2~3); the soaking and cleaning temperature is 20~25℃, the soaking and cleaning time is 17~25h, and the soaking and cleaning is repeated 5~7 times.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the cleaned sturgeon, water, sodium hydroxide solution, and hydrogen peroxide is (90~110):(490~510):(0.3~0.6):(0.9~1.5); the soaking temperature is 20~25℃, and the soaking time is 10~18h; the mass fraction of the sodium hydroxide solution is 30%~35%; the mass percentage of the hydrogen peroxide is 34%~36%; and the cleaning is performed by 8~12 water rinses.

5. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the sturgeon after impurity removal and deodorization to water is 1:1~2; the heating temperature is 74~82℃; the stirring is at 550~650rpm / min for 15~20min; the shearing is at 13000~17000rpm for 8~12s; and the grinding is at 18000~21000rpm for 3~7s.

6. The preparation method according to claim 1, characterized in that, In step (4), the mass ratio of the primary degraded material, bromelain, and neutral protease is 10:(0.01~0.03):(0.03~0.05); the first enzymatic hydrolysis is performed at pH 7.5~8.0 and 55~60℃ for 2~4 hours; the second enzymatic hydrolysis is performed at pH 6.5~7.0 and 55~60℃ for 0.5~1.5 hours; the mass ratio of the primary degraded material to glycogenase is 10:0.001~0.

003.

7. The preparation method according to claim 2, characterized in that, The pH is adjusted to 6.0-7.0; the filter aid is perlite and / or diatomaceous earth; the mass ratio of the inactivated material to the filter aid is 1:1.5-2.5; the mass ratio of perlite to diatomaceous earth is 1:

1.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The sturgeon collagen peptide contains sodium hyaluronate and glycosaminoglycans.

9. The anti-aging sturgeon collagen peptide prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the anti-aging sturgeon collagen peptide according to claim 9 in the preparation of anti-aging products.

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