Method for preparing high antioxidant activity fish skin collagen peptide by DHPM synergistic UIO-66 molecular imprinting

Abstract

The application provides a method for preparing fish skin collagen peptides with high antioxidant activity by DHPM and UIO-66 molecular imprinting, and relates to the technical fields of aquatic product deep processing and bioactive peptides. The method comprises the following steps: fish skin pretreatment, DHPM-induced collagen protein unfolding, endogenous enzyme autolysis, crude separation, selective target enrichment by using UIO-66 material with histidine as a molecular imprinting template, and drying collection. The application destroys the dense triple helix structure of collagen protein by using the physical field effect of dynamic high pressure microfluidization, exposes the enzyme cutting site, uses the endogenous enzyme of fish skin for autolysis, and then uses the histidine molecular imprinting UIO-66 material to accurately capture the high antioxidant activity peptide segment, so that the enzymolysis efficiency and the antioxidant activity of the product are significantly improved. The application does not need to add exogenous enzymes, and has the advantages of green process, low cost and high product purity. The obtained fish skin collagen peptides can be widely applied to the fields of functional food, cosmetics and medicine.

Description

Technical Field

[0001] This invention relates to the fields of deep processing of aquatic products and bioactive peptide technology, and in particular to a method for preparing highly antioxidant fish skin collagen peptides by DHPM synergistically imprinting UIO-66. Background Technology

[0002] Fish skin is a major byproduct of aquatic product processing, with a huge output but low comprehensive utilization rate. Fish skin is rich in collagen, making it a high-quality raw material for preparing bioactive peptides. Numerous studies have shown that collagen peptides possess various physiological functions, including antioxidant, blood pressure-lowering, antibacterial, wound-healing-promoting, and skin-elasticity-improving effects, thus showing broad application prospects in functional foods, health products, cosmetics, and pharmaceuticals. Developing efficient, green, and low-cost fish skin collagen peptide preparation technologies is of great significance for increasing the added value of aquatic byproducts and promoting the development of a circular economy.

[0003] Currently, the industrial preparation of collagen peptides mainly adopts the exogenous protease hydrolysis method, and commonly used enzymes include alkaline protease, trypsin, papain, etc. This method has the following shortcomings: (1) the cost of exogenous enzyme preparations is high, accounting for more than 30% of the production cost; (2) the hydrolysis process is not easy to control, and the batch-to-batch differences of products are large; (3) after hydrolysis, an additional step is required to remove the exogenous enzyme, which increases the complexity of the process.

[0004] Fish skin tissue naturally contains various endogenous enzymes (such as cathepsins B, L, and S, matrix metalloproteinases, etc.), which can undergo autolysis under certain conditions to release collagen peptides. However, natural collagen has a tight triple helix structure, and the enzyme cleavage sites are encapsulated inside the molecule, making the hydrolysis efficiency of endogenous enzymes extremely low. This has prevented the autolysis method from being used industrially for a long time.

[0005] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing highly antioxidant fish skin collagen peptides using DHPM synergistically with UIO-66 molecular imprinting. This aims to address the problems of high enzyme preparation costs, low enzymatic hydrolysis efficiency due to the dense structure of natural collagen, and insufficient antioxidant activity of the products in existing fish skin collagen peptide preparation methods. The invention provides a green, efficient, low-cost, and easily industrially scalable method for preparing highly antioxidant fish skin collagen peptides.

[0007] In a first aspect, the present invention provides a method for preparing highly antioxidant fish skin collagen peptides, comprising the following steps:

[0008] (1) Pretreatment: The fish skin raw material is pretreated to remove impurities, proteins and fats, and pretreated fish skin is obtained;

[0009] (2) Dynamic high-pressure microjet treatment: The pretreated fish skin is treated with dynamic high-pressure microjet to induce collagen to unfold, and an unfolded collagen suspension is obtained.

[0010] (3) Endogenous enzyme autolysis: The unfolded collagen suspension is placed in a constant temperature reactor, and the endogenous enzyme carried by the fish skin is used to autolyze the unfolded collagen suspension to obtain the enzymatic hydrolysate;

[0011] (4) Crude separation: The enzymatic hydrolysate is centrifuged and microfiltered to remove macromolecular impurities and obtain crude collagen peptide extract;

[0012] (5) Targeted enrichment of UIO-66 molecularly imprinted material: The antioxidant active peptides in the crude collagen peptide extract were selectively enriched by using UIO-66 material with histidine as a molecular imprint template, and then eluted and collected to obtain the enriched solution.

[0013] (6) Drying: The enriched solution is concentrated, desalted and dried to obtain fish skin collagen peptides with high antioxidant activity.

[0014] Optionally, the fish skin mentioned in step (1) is freshwater fish skin.

[0015] Optionally, the pretreatment in step (1) includes sequentially removing impurities and proteins with an alkaline solution and removing fat with an organic solvent; the alkaline solution is a 0.1-0.3 mol / L NaOH solution, the solid-liquid ratio of the fish skin to the alkaline solution is (1:8)-(1:15) g / mL, the fish skin is soaked in the alkaline solution for 12-36 hours, and the alkaline solution is replaced 1-3 times during the period; the organic solvent is 10-15% n-butanol, the solid-liquid ratio of the fish skin to the organic solvent is (1:10)-(1:15) g / mL, the fish skin is soaked for 12-36 hours, and the solvent is replaced 1-3 times during the period; both the removal of impurities and the removal of fat are carried out at 4°C.

[0016] Optionally, the conditions for the dynamic high-pressure microjet treatment in step (2) are: pressure 100-150MPa, number of cycles 4-8; during the dynamic high-pressure microjet treatment, the pretreated fish skin and deionized water are mixed at a solid-liquid ratio of (1:5)-(1:8) g / mL; the material temperature is controlled below 40℃.

[0017] Optionally, the autolytic enzymatic hydrolysis conditions described in step (3) are: pH 5.0-6.5, temperature 40-55℃, time 12-24h, with stirring at 60-100rpm.

[0018] Optionally, the centrifugation conditions in step (4) are: temperature 4-10℃, rotation speed 8000-12000rpm, time 15-30min; the microfiltration uses a microfiltration membrane with a pore size of 0.45μm.

[0019] Optionally, the preparation method of the UIO-66 material with histidine as a molecular imprint template in step (5) includes: mixing zirconium source, terephthalic acid and histidine in a molar ratio of 1:(1-2):(4-12), and carrying out a solvothermal reaction at 100-150℃ for 12-48h in the presence of an organic solvent and a regulator, and obtaining the material after washing and drying; the zirconium source is at least one of ZrCl4, ZrOCl2·8H2O or Zr(NO3)4·5H2O; the organic solvent is N,N-dimethylformamide; the regulator is glacial acetic acid; and the molar ratio of zirconium source to glacial acetic acid is (1:10)-(1:30).

[0020] Optionally, 2-aminoterephthalic acid is used instead of terephthalic acid to prepare an amino-functionalized UIO-66 material with histidine as a molecular imprint template; wherein the molar ratio of the zirconium source, the 2-aminoterephthalic acid and the histidine is 1:1:(4-12).

[0021] Optionally, the selective targeting enrichment conditions in step (5) are: pH 5.0-7.0, temperature 25-40℃, adsorption time 1-6h; the material-to-liquid ratio of UIO-66 material with histidine as molecular imprint template to crude collagen peptide extract is (1:50)-(1:200) w / v; the elution is carried out using a buffer solution containing 0.1-1.0 mol / L imidazole, and the elution time is 0.5-2h.

[0022] Optionally, the drying in step (6) includes: first concentrating the enriched solution under reduced pressure at 45°C to 1 / 5-1 / 10 of its original volume, then desalting it through an ultrafiltration membrane with a molecular weight cutoff of 1-5 kDa, and finally freeze-drying it.

[0023] Secondly, the present invention provides the application of a highly antioxidant fish skin collagen peptide prepared by any of the above-mentioned optional methods in the preparation of functional foods, health products, cosmetics or pharmaceutical preparations.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The preparation method provided by the present invention combines dynamic high pressure microfluidic (DHPM) physical field treatment with autolytic hydrolysis of fish skin endogenous enzymes. It utilizes high pressure shearing, cavitation and turbulence to destroy the dense triple helix structure of collagen, so that the originally encapsulated enzyme cleavage sites are fully exposed, which significantly improves the accessibility of endogenous enzymes to substrates and hydrolysis efficiency. Compared with direct autolysis without DHPM treatment, the degree of hydrolysis can be increased by more than 50%. Moreover, no exogenous protease needs to be added, which fundamentally reduces the cost of enzyme preparations and simplifies the subsequent separation and purification steps.

[0026] (2) The preparation method provided by the present invention introduces UIO-66 metal-organic framework material (UIO-66-His) with histidine as a molecular imprint template, and utilizes its triple effect of size exclusion, zirconium cluster metal affinity coordination and histidine imprint cavity specific recognition to accurately capture highly antioxidant peptides rich in histidine residues from complex enzymatic hydrolysate. The material has excellent properties of high adsorption capacity, high selectivity and high elution rate, and the purity of the product is significantly improved.

[0027] (3) The preparation method provided by the present invention, through the synergistic effect of DHPM-induced unfolding, endogenous enzyme autolysis and UIO-66-His targeted enrichment, DHPM treatment not only promotes the enzymatic hydrolysis process, but also causes partial degradation of collagen molecules and exposure of active groups through physical force, which produces a synergistic effect with endogenous enzyme hydrolysis. The collagen peptides prepared after UIO-66-His enrichment have a smaller molecular weight distribution and a higher content of hydrophobic amino acids, which endows them with stronger DPPH free radical and ·OH scavenging ability. The DPPH free radical scavenging rate of the product can be increased by more than 50%, and the ·OH scavenging rate can be increased by more than 38%. The antioxidant activity is significantly better than that of traditional enzymatic hydrolysis methods.

[0028] (4) The preparation method provided by the present invention utilizes the endogenous enzymes carried by the fish skin itself for enzymatic hydrolysis without adding any commercial protease, and there is no need for an additional exogenous enzyme removal step after enzymatic hydrolysis, which simplifies the process flow and avoids the large-scale use of organic solvents in the active peptide separation process. It meets the requirements of green chemistry and clean production, is environmentally friendly, and is easy to scale up for industrial production.

[0029] (5) The preparation method provided by the present invention uses fish skin, a by-product of aquatic product processing, as raw material, realizing the high-value utilization of low-value waste. The obtained fish skin collagen peptide has excellent DPPH free radical and hydroxyl free radical scavenging ability and can be widely used in functional food, health products, cosmetics and pharmaceutical preparations, providing a new way for the resource utilization of aquatic by-products. Attached Figure Description

[0030] Figure 1 This is a comparison chart of the hydrolysis rates of fish skin collagen peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 4 of the present invention;

[0031] Figure 2 This is a comparison chart of the DPPH free radical scavenging rates of fish skin collagen peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 4 of the present invention;

[0032] Figure 3 This is a comparison chart of the ·OH scavenging rates of fish skin collagen peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 4 of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0034] This invention provides a method for preparing fish skin collagen peptides with high antioxidant activity, comprising the following steps:

[0035] (1) Pretreatment: The fish skin raw material is pretreated to remove impurities, proteins and fats, and pretreated fish skin is obtained;

[0036] (2) Dynamic high-pressure microjet treatment: The pretreated fish skin is treated with dynamic high-pressure microjet to induce collagen to unfold, and an unfolded collagen suspension is obtained.

[0037] (3) Endogenous enzyme autolysis: The unfolded collagen suspension is placed in a constant temperature reactor, and the endogenous enzyme carried by the fish skin is used to autolyze the unfolded collagen suspension to obtain the enzymatic hydrolysate;

[0038] (4) Crude separation: The enzymatic hydrolysate is centrifuged and microfiltered to remove macromolecular impurities and obtain crude collagen peptide extract;

[0039] (5) Targeted enrichment of UIO-66 molecularly imprinted material: The antioxidant active peptides in the crude collagen peptide extract were selectively enriched by using UIO-66 material with histidine as a molecular imprint template, and then eluted and collected to obtain the enriched solution.

[0040] (6) Drying: The enriched solution is concentrated, desalted and dried to obtain fish skin collagen peptides with high antioxidant activity.

[0041] Specifically, Dynamic High Pressure Microjet (DHPM) is a novel physical field processing technology that utilizes the ultra-high shear, impact, cavitation and turbulence generated when materials pass through microchannels under high pressure to effectively disrupt the higher-order structure of proteins.

[0042] Specifically, in the separation and purification of bioactive peptides, metal-organic frameworks (MOFs) have attracted much attention due to their high specific surface area, tunable pore structure, and abundant coordination sites. UIO-66, an MOF with zirconium (Zr) as its metal nodes, exhibits excellent chemical and thermal stability. Its Zr-O clusters provide abundant coordination unsaturated sites, making it easier to functionalize. Introducing molecular imprinting technology into the synthesis of UIO-66, by constructing specific recognition cavities within the UIO-66 backbone using template molecules, holds promise for achieving precise targeted enrichment of highly antioxidant peptides.

[0043] In essence, this invention disrupts the dense triple-helix structure of collagen through the physical field of dynamic high-pressure microfluidics (DHPM), fully exposing enzyme cleavage sites. It utilizes the fish skin's own endogenous enzymes for autolytic hydrolysis, eliminating the need for exogenous enzymes. Simultaneously, it leverages the size exclusion, metal affinity coordination, and specific recognition triple effects of histidine-imprinted UIO-66 material (UIO-66-His) to precisely capture highly antioxidant peptides. These three factors synergistically improve hydrolysis efficiency and the antioxidant activity of the product. This invention eliminates the need for exogenous enzymes, resulting in a green, low-cost process with high product purity. The obtained fish skin collagen peptides possess excellent free radical scavenging capabilities and can be widely applied in functional foods, cosmetics, and pharmaceuticals.

[0044] In some embodiments, the fish skin selected in step (1) is freshwater fish skin. Specifically, the selected freshwater fish skin includes, but is not limited to, snakehead, grass carp, silver carp, and bighead carp.

[0045] In some embodiments, the pretreatment used in step (1) includes sequentially removing impurities and proteins with an alkaline solution and removing fat with an organic solvent; the alkaline solution used is a 0.1-0.3 mol / L NaOH solution, the solid-liquid ratio of fish skin to alkaline solution is (1:8)-(1:15) g / mL, the fish skin is soaked in the alkaline solution for 12-36 hours, and the alkaline solution is replaced 1-3 times during the period; the organic solvent used is 10-15% n-butanol, the solid-liquid ratio of fish skin to organic solvent is (1:10)-(1:15) g / mL, the fish skin is soaked for 12-36 hours, and the solvent is replaced 1-3 times during the period; both the removal of impurities and the removal of fat are carried out at 4°C.

[0046] In some embodiments, the conditions for dynamic high-pressure microfluidic treatment used in step (2) are: pressure 100-150MPa, number of cycles 4-8; during the dynamic high-pressure microfluidic treatment, the pretreated fish skin and deionized water are mixed at a solid-liquid ratio of (1:5)-(1:8) g / mL, stirred overnight to make it uniformly dispersed, and preheated to 25-30℃. After preheating, the dispersion is immediately transferred to the feed container of the dynamic high-pressure microfluidic equipment (M-110EH, Microfluidics, USA). During the treatment, the material temperature is controlled to be <40℃.

[0047] In some embodiments, the autolytic hydrolysis conditions in step (3) are: pH 5.0-6.5, temperature 40-55℃, time 12-24h, supplemented by stirring at 60-100rpm. Specifically, the unfolded collagen suspension obtained by dynamic high-pressure microfluidic treatment in step (2) is preferably treated with a 0.1-0.5mol / L citric acid solution, the pH is precisely adjusted to 5.5-6.0, the temperature is adjusted to 40℃, the autolytic hydrolysis time is 12-24h, and low-speed stirring at 60-100rpm is used during the process.

[0048] In some embodiments, the centrifugation conditions in step (4) are: temperature 4-10℃, rotation speed 8000-12000rpm, time 15-30min; the microfiltration uses a microfiltration membrane with a pore size of 0.45μm.

[0049] Specifically, the enzymatic hydrolysate obtained in step (3) is cooled to room temperature and centrifuged at 10,000 rpm for 15-30 min at 4°C, and the supernatant is collected. The supernatant is then filtered through a 0.45 μm microfiltration membrane to remove residual cell debris, insoluble impurities and macromolecular aggregates, resulting in a clear crude collagen peptide extract.

[0050] In some embodiments, the preparation method of the UIO-66 material (UIO-66-His) with histidine as a molecular imprint template in step (5) includes: mixing zirconium source, terephthalic acid and histidine in a molar ratio of 1:(1-2):(4-12), and carrying out a solvothermal reaction at 100-150°C for 12-48 h in the presence of an organic solvent and a regulator, followed by washing and drying to obtain the final product. Specifically, the zirconium source is at least one of ZrCl4, ZrOCl2·8H2O or Zr(NO3)4·5H2O; the organic solvent is N,N-dimethylformamide (DMF); the regulator is glacial acetic acid or triethylamine; and the molar ratio of zirconium source to regulator is (1:10)-(1:30). Specifically, after the solvothermal reaction is completed, the product is collected by centrifugation and washed repeatedly with DMF and methanol to remove unreacted raw materials and residual template molecules histidine. The product is then vacuum dried at 60-120℃ to obtain the histidine-imprinted UIO-66 material, named UIO-66-His.

[0051] In some embodiments, 2-aminoterephthalic acid is used instead of terephthalic acid to prepare amino-functionalized UIO-66 materials with histidine as a molecular imprint template; wherein the molar ratio of the zirconium source, the 2-aminoterephthalic acid and the histidine is 1:1:(4-12).

[0052] In some embodiments, the selective targeting enrichment conditions described in step (5) are: pH 5.0-7.0, temperature 25-40℃, and adsorption time 1-6h; the ratio of UIO-66 material (UIO-66-His) with histidine as the molecular imprint template to the crude collagen peptide extract is (1:50)-(1:200) w / v; the elution process is performed using PBS buffer (pH 6.5-7.5) containing 0.1-1.0 mol / L imidazole, eluted at 30℃ for 0.5-2h.

[0053] Specifically, the UIO-66 material (UIO-66-His), with histidine as a molecular imprint template, was used to target and enrich highly antioxidant peptides. The process included the following steps: The crude collagen peptide extract was adjusted to pH 5.0-7.0 with buffer solution. UIO-66-His molecularly imprinted material was added at a material-to-liquid ratio of (1:50) to (1:200) w / v. Adsorption was carried out at 25-40℃ with constant shaking for 2-4 hours. After adsorption, the UIO-66-His molecularly imprinted material containing the adsorbed active peptides was collected by centrifugation (8000-12000 rpm, 5-10 min), and the supernatant was discarded. Elution was then performed.

[0054] In some embodiments, the drying in step (6) includes: first concentrating the enriched solution under reduced pressure at 45°C to 1 / 5-1 / 10 of its original volume, then desalting it through an ultrafiltration membrane with a molecular weight cutoff of 1-5 kDa, and finally freeze-drying it.

[0055] The present invention also provides the application of the highly antioxidant fish skin collagen peptide prepared by any of the above optional embodiments in the preparation of functional foods, health products, cosmetics or pharmaceutical preparations.

[0056] Example 1

[0057] This embodiment 1 provides a method for preparing highly antioxidant fish skin collagen peptides by DHPM synergistically UIO-66 molecular imprinting, including the following steps:

[0058] (1) Fish skin pretreatment

[0059] Take an appropriate amount of fresh blackfish skin, remove the scales and flesh, wash it clean, and cut it into small pieces of 1cm × 1cm. Soak the fish skin pieces in a 0.1mol / L NaOH solution at a solid-liquid ratio of 1:10g / mL for 24 hours, changing the alkali solution twice during this period, at a treatment temperature of 4℃. After removing impurities and proteins, wash the fish skin three times with distilled water, then soak it in 10% n-butanol at a solid-liquid ratio of 1:10g / mL for 24 hours, changing the solvent twice during this period, at a treatment temperature of 4℃. After defatting, wash with distilled water until there is no n-butanol odor, obtaining pretreated fish skin.

[0060] (2) DHPM-induced substrate unfolding

[0061] The pretreated fish skin was mixed with water at a solid-liquid ratio of 1:6 g / mL, stirred overnight to ensure uniform dispersion, and preheated to 25-30℃. The mixture was then circulated four times at 100 MPa using a dynamic high-pressure microfluidic apparatus (M-110EH, Microfluidics, USA) to obtain an unfolded collagen suspension; the material temperature was controlled below 40℃ during the treatment process.

[0062] (3) Heat-induced autolysis of endogenous enzymes

[0063] The resulting suspension was transferred to a constant temperature reactor, the pH was adjusted to 5.5, the temperature was adjusted to 45°C, and autolysis was carried out for 12 hours with low-speed stirring (80 rpm). The resulting hydrolysate was then heated to 95°C and kept at 15 minutes to inactivate the enzyme.

[0064] (4) Coarse separation

[0065] The enzyme-inactivated hydrolysate was cooled to room temperature and centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was then collected. The supernatant was filtered through a 0.45 μm microfiltration membrane to obtain a clear crude collagen peptide extract.

[0066] (5) UIO-66-His targeted enrichment

[0067] The crude collagen peptide extract was adjusted to pH 6.0 with 0.1 mol / L PBS, and UIO-66-His material was added at a ratio of 1:50 w / v. Adsorption was carried out at 30°C with shaking for 1 hour. After adsorption, the sample was centrifuged at 8000 rpm for 5 minutes and the precipitate was collected. The precipitate was dispersed in PBS buffer (pH 7.4) containing 0.5 mol / L imidazole, and eluted at 30°C with shaking for 1 hour. The supernatant was collected by centrifugation at 8000 rpm.

[0068] (6) Drying

[0069] The eluent was concentrated to 1 / 5 of its original volume under reduced pressure at 45°C, and then desalted through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected. The desalted solution was freeze-dried to obtain fish skin collagen peptide powder with high antioxidant activity.

[0070] Example 2

[0071] Example 2 provides a method for preparing highly antioxidant fish skin collagen peptides using DHPM-co-UIO-66 molecular imprinting, comprising the following steps:

[0072] (1) Fish skin pretreatment

[0073] Take an appropriate amount of fresh blackfish skin, remove the scales and flesh, clean it thoroughly, and cut it into small pieces of 1cm × 1cm. Soak the fish skin pieces in a 0.2mol / L NaOH solution at a solid-liquid ratio of 1:15g / mL for 36 hours, changing the alkali solution twice during this period, at a treatment temperature of 4℃. After removing impurities and proteins, wash the fish skin three times with distilled water, then soak it in 12% n-butanol at a solid-liquid ratio of 1:10g / mL for 24 hours, changing the solvent twice during this period, at a treatment temperature of 4℃. After defatting, wash with distilled water until there is no n-butanol odor, obtaining the pretreated fish skin.

[0074] (2) DHPM-induced substrate unfolding

[0075] The pretreated fish skin was mixed with water at a solid-liquid ratio of 1:6 g / mL, stirred overnight to ensure uniform dispersion, and preheated to 25-30℃. The mixture was then circulated 6 times at 120 MPa using a dynamic high-pressure microfluidic apparatus (M-110EH, Microfluidics, USA) to obtain an unfolded collagen suspension; the material temperature was controlled below 40℃ during the treatment process.

[0076] (3) Heat-induced autolysis of endogenous enzymes

[0077] The resulting suspension was transferred to a constant temperature reactor, the pH was adjusted to 6.0, the temperature was adjusted to 50°C, and autolysis was carried out for 18 hours with low-speed stirring (80 rpm). The resulting hydrolysate was then heated to 95°C and kept at 15 minutes to inactivate the enzyme.

[0078] (4) Coarse separation

[0079] The enzyme-inactivated hydrolysate was cooled to room temperature and centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was then collected. The supernatant was filtered through a 0.45 μm microfiltration membrane to obtain a clear crude collagen peptide extract.

[0080] (5) UIO-66-His targeted enrichment

[0081] The crude collagen peptide extract was adjusted to pH 6.0 with 0.1 mol / L PBS, and UIO-66-His material was added at a ratio of 1:100 w / v. Adsorption was carried out at 30°C with shaking for 2 hours. After adsorption, the sample was centrifuged at 8000 rpm for 5 minutes and the precipitate was collected. The precipitate was dispersed in PBS buffer (pH 7.4) containing 1.0 mol / L imidazole, and eluted at 30°C with shaking for 1 hour. The supernatant was collected by centrifugation at 8000 rpm. The precipitate was then dispersed in PBS buffer (pH 7.4) containing 0.5 mol / L imidazole, and eluted at 30°C with shaking for 1 hour. The supernatant was collected by centrifugation at 8000 rpm.

[0082] (6) Drying

[0083] The eluent was concentrated to 1 / 5 of its original volume under reduced pressure at 45°C, and then desalted through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected. The desalted solution was freeze-dried to obtain fish skin collagen peptide powder with high antioxidant activity.

[0084] Example 3

[0085] Example 3 provides a method for preparing highly antioxidant fish skin collagen peptides using DHPM-assisted UIO-66 molecular imprinting, comprising the following steps:

[0086] (1) Fish skin pretreatment

[0087] Take an appropriate amount of fresh blackfish skin, remove the scales and flesh, wash it clean, and cut it into small pieces of 1cm × 1cm. Soak the fish skin pieces in a 0.2mol / L NaOH solution at a solid-liquid ratio of 1:8g / mL for 36 hours, changing the alkali solution three times during this period, at a treatment temperature of 4℃. After removing impurities and proteins, wash the fish skin three times with distilled water, then soak it in 15% n-butanol at a solid-liquid ratio of 1:10g / mL for 36 hours, changing the solvent three times during this period, at a treatment temperature of 4℃. After defatting, wash with distilled water until there is no n-butanol odor, obtaining pretreated fish skin.

[0088] (2) DHPM-induced substrate unfolding

[0089] The pretreated fish skin was mixed with water at a solid-liquid ratio of 1:6 g / mL, stirred overnight to ensure uniform dispersion, and preheated to 25-30℃. The mixture was then circulated 8 times at 150 MPa using a dynamic high-pressure microfluidic apparatus (M-110EH, Microfluidics, USA) to obtain an unfolded collagen suspension; the material temperature was controlled below 40℃ during the treatment process.

[0090] (3) Heat-induced autolysis of endogenous enzymes

[0091] The resulting suspension was transferred to a constant temperature reactor, the pH was adjusted to 6.0, the temperature was adjusted to 55°C, and autolysis was carried out for 24 hours with low-speed stirring (80 rpm). The resulting hydrolysate was then heated to 95°C and kept at 15 minutes to inactivate the enzyme.

[0092] (4) Coarse separation

[0093] The enzyme-inactivated hydrolysate was cooled to room temperature and centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was then collected. The supernatant was filtered through a 0.45 μm microfiltration membrane to obtain a clear crude collagen peptide extract.

[0094] (5) UIO-66-His targeted enrichment

[0095] The crude collagen peptide extract was adjusted to pH 6.0 with 0.1 mol / L PBS, and UIO-66-His material was added at a ratio of 1:200 w / v. Adsorption was carried out at 30°C with shaking for 4 hours. After adsorption, the sample was centrifuged at 8000 rpm for 5 minutes and the precipitate was collected. The precipitate was dispersed in PBS buffer (pH 7.4) containing 1.0 mol / L imidazole, and eluted at 30°C with shaking for 1 hour. The supernatant was collected by centrifugation at 8000 rpm. The precipitate was then dispersed in PBS buffer (pH 7.4) containing 0.5 mol / L imidazole, and eluted at 30°C with shaking for 1 hour. The supernatant was collected by centrifugation at 8000 rpm.

[0096] (6) Drying

[0097] The eluent was concentrated to 1 / 5 of its original volume under reduced pressure at 45°C, and then desalted through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected. The desalted solution was freeze-dried to obtain fish skin collagen peptide powder with high antioxidant activity.

[0098] Comparative Example 1

[0099] Step (5) was not performed; the remaining steps are the same as in Example 3.

[0100] Comparative Example 2

[0101] Steps (2) and (5) were not performed; the remaining steps were the same as in Example 3.

[0102] Comparative Example 3

[0103] Step (2) was not performed; the remaining steps are the same as in Example 3.

[0104] Comparative Example 4

[0105] Comparative Example 4 provides a method for preparing antioxidant active fish skin collagen peptides using a conventional enzymatic hydrolysis method, including the following steps:

[0106] (1) Fish skin pretreatment

[0107] Take an appropriate amount of fresh blackfish skin, remove the scales and flesh, wash it clean, and cut it into small pieces of 1cm × 1cm. Soak the fish skin pieces in a 0.2mol / L NaOH solution at a solid-liquid ratio of 1:8g / mL for 36 hours, changing the alkali solution three times during this period, at a treatment temperature of 4℃. After removing impurities and proteins, wash the fish skin three times with distilled water, then soak it in 15% n-butanol at a solid-liquid ratio of 1:10g / mL for 36 hours, changing the solvent three times during this period, at a treatment temperature of 4℃. After defatting, wash with distilled water until there is no n-butanol odor, obtaining pretreated fish skin.

[0108] (2) Enzymatic hydrolysis

[0109] The obtained pretreated fish skin was mixed with water at a solid-liquid ratio of 1:6 (g / mL) and stirred overnight to ensure uniform dispersion. Then, a commercially available alkaline protease was used for treatment at an enzyme addition of 6000 U / g, and enzymatic hydrolysis was performed at 50℃ and pH 9.0 for 6 h.

[0110] (3) Collection

[0111] The resulting hydrolysate was heated to 95°C and kept at that temperature for 15 minutes to inactivate the enzyme. Then it was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected.

[0112] The fish skin collagen peptide powders obtained in the above examples and comparative examples were subjected to the following measurements:

[0113] Degree of hydrolysis determination: Prepare OPA reagent by mixing 100 μL of standard working solutions of different concentrations with 1 mL of o-phthalaldehyde, reacting in the dark for 4 min, and measuring the absorbance at 340 nm. Establish an NH2 standard curve using leucine as the standard solution, determine the free amino group concentration, and calculate the degree of hydrolysis of fish skin collagen using the following formula:

[0114]

[0115] Where A represents the concentration of free amino groups in the sample, A 总 A0 represents the free amino concentration after complete hydrolysis of the sample, while A0 represents the free amino concentration in the unhydrolyzed blank group.

[0116] DPPH free radical scavenging rate determination: Equal volumes of sample solution (5 mg / mL) and DPPH solution (0.0394 mg / mL, dissolved in anhydrous ethanol) were mixed thoroughly and reacted in the dark for 20 min. The absorbance was measured at 517 nm and recorded as A2. The reaction system using distilled water instead of the sample solution was the control group, recorded as A1; the reaction system using anhydrous ethanol instead of the DPPH solution was the blank group, recorded as A3. The DPPH scavenging rate was calculated using the following formula:

[0117]

[0118] • OH scavenging capacity determination: 8 mmol / L ferrous sulfate solution and 3% hydrogen peroxide solution were thoroughly mixed. Then, 500 μL of 15 mg / mL sample solution and 500 μL of 3 mM salicylic acid solution were added sequentially. After thorough mixing, the mixture was reacted at 37°C in the dark for 20 min. The absorbance was measured at 510 nm and recorded as A5. The reaction system using 500 μL of distilled water instead of the sample solution served as the control group and was recorded as A4. The reaction system using distilled water instead of 3% hydrogen peroxide solution served as the blank group and was recorded as A6. The OH scavenging rate was calculated using the following formula:

[0119]

[0120] Depend on Figure 1 As can be seen, compared with the untreated (Comparative Example 2), Example 3 of the present invention increases the degree of hydrolysis of fish skin collagen peptides by 1.04 times through DHPM-induced substrate unfolding and synergistic endogenous enzyme autolysis. Compared with the commonly used alkaline protease hydrolysis (Comparative Example 4), the degree of hydrolysis of Example 3 is significantly improved. Figure 2 and Figure 3 As can be seen from the comparison, compared with the method without UIO-66-His enrichment (Comparative Example 1), the DPPH radical scavenging rate of Example 3 increased by 51.11%, and the ·OH radical scavenging rate increased by 38.93%; compared with Comparative Example 2, the DPPH radical scavenging rate and ·OH radical scavenging rate of Example 3 increased by 60.71% and 44.86%, respectively; compared with Comparative Example 3, the DPPH radical scavenging rate and ·OH radical scavenging rate of Example 3 increased by 38.93% and 19.52%, respectively. Furthermore, compared with Comparative Example 4, the DPPH radical scavenging rate and ·OH radical scavenging rate of Example 3 increased by 45.22% and 29.90%, respectively, indicating that the method of the present invention has significant technical advantages.

[0121] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing fish skin collagen peptides with high antioxidant activity, characterized in that, Includes the following steps: (1) Pretreatment: The fish skin raw material is pretreated to remove impurities, proteins and fats, and pretreated fish skin is obtained; (2) Dynamic high pressure microjets: The pretreated fish skin is treated with dynamic high pressure microjets to induce collagen to unfold, and an unfolded collagen suspension is obtained. (3) Endogenous enzyme autolysis: The unfolded collagen suspension is placed in a constant temperature reactor, and the endogenous enzyme carried by the fish skin is used to autolyze the unfolded collagen suspension to obtain the enzymatic hydrolysate; (4) Crude separation: The enzymatic hydrolysate is centrifuged and microfiltered to remove macromolecular impurities and obtain crude collagen peptide extract; (5) Targeted enrichment of UIO-66 molecularly imprinted material: The antioxidant active peptides in the crude collagen peptide extract were selectively enriched by using UIO-66 material with histidine as a molecular imprint template, and then eluted and collected to obtain the enriched solution. (6) Drying: The enriched solution is concentrated, desalted and dried to obtain fish skin collagen peptides with high antioxidant activity.

2. The method according to claim 1, characterized in that, The fish skin mentioned in step (1) is freshwater fish skin; and / or, the pretreatment includes sequentially removing impurities and proteins with an alkaline solution and removing fat with an organic solvent; the alkaline solution is a 0.1-0.3 mol / L NaOH solution, the solid-liquid ratio of the fish skin to the alkaline solution is (1:8)-(1:15) g / mL, the fish skin is soaked in the alkaline solution for 12-36 hours, and the alkaline solution is replaced 1-3 times during the period; the organic solvent is 10-15% n-butanol, the solid-liquid ratio of the fish skin to the organic solvent is (1:10)-(1:15) g / mL, the fish skin is soaked for 12-36 hours, and the solvent is replaced 1-3 times during the period; both the removal of impurities and the removal of fat are carried out at 4°C.

3. The method according to claim 1, characterized in that, The conditions for dynamic high-pressure microjet treatment in step (2) are: pressure 100-150MPa, number of cycles 4-8; during the dynamic high-pressure microjet treatment, the pretreated fish skin and deionized water are mixed at a solid-liquid ratio of (1:5)-(1:8) g / mL; the material temperature is controlled below 40℃.

4. The method according to claim 1, characterized in that, The conditions for autolytic enzymatic hydrolysis described in step (3) are: pH 5.0-6.5, temperature 40-55℃, time 12-24h, with stirring at 60-100rpm.

5. The method according to claim 1, characterized in that, The centrifugation conditions in step (4) are: temperature 4-10℃, rotation speed 8000-12000rpm, time 15-30min; the microfiltration uses a microfiltration membrane with a pore size of 0.45μm.

6. The method according to claim 1, characterized in that, The preparation method of UIO-66 material with histidine as molecular imprint template in step (5) includes: mixing zirconium source, terephthalic acid and histidine in a molar ratio of 1:(1-2):(4-12), and carrying out a solvothermal reaction at 100-150℃ for 12-48h in the presence of organic solvent and regulator, and obtaining the material after washing and drying; the zirconium source is at least one of ZrCl4, ZrOCl2·8H2O or Zr(NO3)4·5H2O; the organic solvent is N,N-dimethylformamide; the regulator is glacial acetic acid; the molar ratio of zirconium source to glacial acetic acid is (1:10)-(1:30).

7. The method according to claim 1, characterized in that, The selective targeting enrichment conditions described in step (5) are: pH 5.0-7.0, temperature 25-40℃, and adsorption time 1-6h; the ratio of UIO-66 material with histidine as molecular imprint template to crude collagen peptide extract is (1:50)-(1:200) w / v; the elution is carried out using a buffer solution containing 0.1-1.0 mol / L imidazole for 0.5-2h.

8. The method according to claim 1, characterized in that, The drying process described in step (6) includes: first, concentrating the enriched solution under reduced pressure at 45°C to 1 / 5-1 / 10 of its original volume, then desalting it through an ultrafiltration membrane with a molecular weight cutoff of 1-5 kDa, and finally freeze-drying it.

9. The use of a highly antioxidant fish skin collagen peptide prepared by the method according to any one of claims 1-8 in the preparation of functional foods, health products, cosmetics or pharmaceutical preparations.

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