Method for chelating trace elements after marsh fish skin enzymolysis

Abstract

The invention relates to the technical field of deep processing of aquatic products, and discloses a method for chelating trace elements after enzymolysis of marsh marsh fish skins, which comprises the following steps: S1, removing impurities from marsh marsh fish skins, soaking in a sodium bicarbonate solution, cleaning, freeze-drying, crushing and sieving; s2, mixing the marsh fish skin powder with deionized water, adding an auxiliary agent, adjusting the pH value, adding compound protease, performing enzymolysis, performing enzyme deactivation, and performing centrifugal filtration; s3, mixing the trace element compound with deionized water; s4, mixing the marsh fish skin enzymatic hydrolysate with a trace element solution, and carrying out chelation reaction in stages; and S5, decolorizing, centrifuging, concentrating and spray-drying. The method effectively solves the problems of low enzymolysis efficiency, poor chelation stability, poor product absorption effect and the like in traditional marsh fish skin processing.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology for aquatic products, specifically a method for chelating trace elements after enzymatic hydrolysis of salmon skin. Background Technology

[0002] Salmon skin, a major waste product from aquatic product processing, is rich in high-quality components such as collagen and bioactive peptide precursors. Simultaneously, trace elements (such as iron, zinc, and selenium) are essential nutrients for the human body. Combining these two through chelation technology to prepare functional food ingredients can achieve high-value utilization of aquatic waste and solve the problem of low bioavailability in traditional trace element supplements, possessing significant economic and social value. However, existing enzymatic hydrolysis and chelation processes for salmon skin still face several technical bottlenecks: the enzymatic hydrolysis stage struggles to efficiently disrupt the tight spatial structure of fish skin proteins, resulting in low protein hydrolysis and insufficient yield of bioactive peptides; the chelation stage leads to insufficient exposure of chelation sites on peptide chains, and the lack of optimized control over the ionic state of trace elements results in low chelation rates. Furthermore, the formed chelates are prone to dissociation in simulated gastrointestinal environments, making in vitro absorption rates insufficient for practical applications. Furthermore, the design of each step in the existing process, such as enzymatic hydrolysis and chelation, is independent of each other, which further affects the quality and stability of the final product and limits the large-scale application of salmon skin active peptide-trace element chelate in the field of functional foods. Therefore, developing a process that can achieve efficient enzymatic hydrolysis and stable chelation has become an urgent technical problem to be solved. Summary of the Invention

[0003] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for chelating trace elements after enzymatic hydrolysis of salmon skin.

[0004] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for chelating trace elements after enzymatic hydrolysis of salmon skin, comprising the following steps: S1. Pretreatment of salmon skin: Select salmon skin, remove impurities, soak in sodium bicarbonate solution, wash, freeze dry, pulverize and sieve to obtain salmon skin powder. S2. Enzymatic hydrolysis of salmon skin: Mix salmon skin powder with deionized water, stir evenly, add auxiliary agent, adjust pH value, add compound protease, enzymatic hydrolysis, enzyme inactivation, centrifugation and filtration to obtain salmon skin enzymatic hydrolysate. S3. Preparation of trace element solution: Dissolve the trace element compound in deionized water to obtain a trace element solution; S4. Chelation reaction: Mix the enzymatic hydrolysate of salmon skin with the trace element solution and carry out the chelation reaction in stages. S5. Post-processing of chelated products: The above chelation reaction solution is decolorized, centrifuged, concentrated, and spray-dried to obtain salmon skin active peptide-trace element chelate powder.

[0005] Further, in step S1, the mass concentration of the sodium bicarbonate solution is 0.8~1.2%, the soaking temperature is 25~30℃, the soaking time is 20~30 min, and the solid-liquid ratio is 1:8~12.

[0006] Furthermore, the enzymatic hydrolysis method for salmon skin in step S2 is as follows: A1. Weigh 100-120 parts of salmon skin powder, add 800-1000 parts of deionized water, stir evenly, add 2-5 parts of auxiliary agent, stir and pretreat at 30-35℃ for 20-30 min, adjust the pH of the system to 7.0-7.2, add 4-8 parts of complex protease, place in a constant temperature water bath shaker, and enzymatically hydrolyze at 48℃ and 190 r / min for 3-5 h. A2. After the enzymatic hydrolysis is completed, heat the system to 90~95℃ and keep it at that temperature for 10~15 min to inactivate the enzyme. After cooling to room temperature, centrifuge at 5000~8000 r / min for 10~20 min, take the supernatant, and filter it through a 0.22 μm microporous membrane to obtain salmon skin enzymatic hydrolysate.

[0007] Furthermore, the preparation method of the auxiliary agent in step S2 includes the following steps: B1. Add 600-800 parts of deionized water to a four-necked flask, install a mechanical stirrer, thermometer and constant pressure dropping funnel, turn on the stirrer at 200-250 r / min, add 10-20 parts of L-cysteine ​​and 5-10 parts of itaconic acid, stir until completely dissolved, raise the temperature of the constant temperature water bath to 30-35℃, slowly add 15-20% triethanolamine aqueous solution, control the pH of the system to be stable at 8.0-8.5, continue stirring for 20-30 min after the addition is completed to form a homogeneous sodium salt solution; B2. Weigh 4-8 parts of sodium metabisulfite, add 120-150 parts of deionized water, stir to dissolve into sodium metabisulfite solution, maintain the temperature of the above sodium salt solution, reduce the stirring speed to 100-120 r / min, add sodium metabisulfite solution dropwise through a dropping funnel at a rate of 8-10 mL / min, after the addition is complete, raise the temperature to 37-40℃, add 2-5 parts of glycerol at the same time, and keep the reaction at this temperature for 1-1.5 h; B3. Cool the above reaction system to 22-25℃, adjust the stirring speed to 150-180 r / min, adjust the pH to 6.5-7.0, and add 20-30 parts of 15-20% calcium chloride solution dropwise. After the addition is complete, continue stirring for 1-1.5 h. Transfer the reaction solution to a rotary evaporator and concentrate it to 1 / 3 of the original volume at 45℃ and 0.085 MPa. Slowly add 3 times the volume of 95% ethanol to the concentrate while stirring. Then place it in a refrigerator at 4℃ for 1-2 h. Filter it with a Buchner funnel, collect the precipitate, wash it 3 times with anhydrous ethanol, transfer the precipitate to a vacuum drying oven, and dry it at 30-35℃ and 0.09-0.1 MPa for 8-12 h to obtain the auxiliary agent.

[0008] Further, in step S2, the complex protease is a mixture of Bacillus subtilis protease and Aspergillus oryzae protease in a mass ratio of 2:1, wherein the Bacillus subtilis protease has an enzyme activity ≥15000 U / g and the Aspergillus oryzae protease has an enzyme activity ≥10000 U / g.

[0009] Further, in step S3, the trace element compound is selected from at least one of ferrous sulfate, zinc sulfate, and sodium selenite; the concentration of the trace element solution is 50~100 mmol / L, and the dissolution temperature is 30~40℃.

[0010] Furthermore, the chelation reaction in step S4 is performed as follows: C1. The salmon skin enzymatic hydrolysate is subjected to ultrasonic and microwave treatment. The power parameters are set as follows: ultrasonic power 200~300 W, frequency 28~30 kHz, microwave power 200~300 W. The treatment is carried out at 30~35℃ for 10~15 min. 0.1 mol / L citric acid solution is added to the activated enzymatic hydrolysate to adjust the pH to 5.0~5.5 to obtain the activated enzymatic hydrolysate. C2. Add food-grade sodium gluconate to the trace element solution at a mass ratio of 1:1, stir at 30~35℃ for 10~15 min to form a trace element-gluconate intermediate complex. C3. First stage: Mix the activated enzymatic hydrolysate and the pre-complexed trace element solution at a volume ratio of 4:1, start the constant temperature water bath reactor, set the temperature to 35~40℃, the stirring speed to 150~200 r / min, maintain the pH at 5.0~5.5, and the time to 20~30 min; Second stage: Slowly add 0.1 mol / L NaOH solution to raise the pH of the system to 6.5~7.0, and at the same time raise the temperature to 45~50℃, and the time to 30~45 min; Third stage: Add 0.1% of the enzymatic hydrolysate mass of reduced glutathione, maintain the pH at 6.5~7.0 and the temperature at 45~50℃, and continue the reaction for 10~15 min.

[0011] Furthermore, in step S5, food-grade activated carbon is used for decolorization, with an addition amount of 0.3~0.8% of the reaction solution mass, a decolorization temperature of 30~40℃, and a stirring time of 20~40 min.

[0012] Furthermore, in step S5, the concentration is carried out by vacuum rotary evaporation under the following conditions: temperature 35~45℃, vacuum degree 0.08~0.1 MPa, and concentration to a solid content of 25~35%; the spray drying parameters are: inlet air temperature 110~130℃, outlet air temperature 50~60℃, and feed rate 8~12 mL / min.

[0013] (iii) Beneficial technical effects The method for chelating trace elements after enzymatic hydrolysis of salmon skin in this invention achieves synergistic effects through multi-stage process innovation, possessing significant technical advantages and practical value. In the enzymatic hydrolysis stage, an auxiliary agent is introduced. Leveraging its diverse active functional groups and calcium ions, this agent synergistically disrupts the tight spatial structure of the fish skin protein, exposing more enzymatic sites, and activates the activity of complex proteases. Combined with a scientifically formulated ratio of Bacillus subtilis protease and Aspergillus oryzae protease, efficient protein hydrolysis and full generation of small-molecule active peptides are achieved. In the chelation reaction stage, ultrasonic-microwave synergistic activation pretreatment further stretches the peptide chains. A segmented coordination process adapts to the binding characteristics of different functional groups and trace elements, forming a stable multi-component chelate structure. Compared to the traditional one-step chelation process, this significantly improves the binding efficiency of trace elements and the stability of the product in a simulated gastrointestinal environment, ensuring subsequent absorption.

[0014] The entire process parameters of this invention are coordinated and adapted, and each step, from substrate pretreatment to final product drying, is closely linked. This effectively solves the problems of low enzymatic hydrolysis efficiency, poor chelation stability, and poor product absorption in traditional salmon skin processing. The resulting salmon skin active peptide-trace element chelate has stable quality, good nutritional value, and promising application prospects, providing an efficient and feasible technical solution for the high-value utilization of aquatic waste and the preparation of functional food raw materials. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, all components of the salmon skin active peptide-trace element chelate formulation of this invention are commercially available. All parts used in this invention are parts by weight; Example

[0017] A method for chelating trace elements after enzymatic hydrolysis of salmon skin includes the following steps: S1. Pretreatment of salmon skin: Select salmon skin, remove impurities, soak in sodium bicarbonate solution, wash, freeze dry, pulverize and sieve to obtain salmon skin powder. S2. Enzymatic hydrolysis of salmon skin: Mix salmon skin powder with deionized water, stir evenly, add auxiliary agent, adjust pH value, add compound protease, enzymatic hydrolysis, enzyme inactivation, centrifugation and filtration to obtain salmon skin enzymatic hydrolysate. S3. Preparation of trace element solution: Dissolve the trace element compound in deionized water to obtain a trace element solution; S4. Chelation reaction: Mix the enzymatic hydrolysate of salmon skin with the trace element solution and carry out the chelation reaction in stages. S5. Post-processing of chelated products: The above chelation reaction solution is decolorized, centrifuged, concentrated, and spray-dried to obtain salmon skin active peptide-trace element chelate powder.

[0018] In step S1, the mass concentration of the sodium bicarbonate solution is 0.8%, the soaking temperature is 25℃, the soaking time is 20 min, and the solid-liquid ratio is 1:8.

[0019] The enzymatic hydrolysis method for salmon skin in step S2 is as follows: A1. Weigh 100 parts of salmon skin powder, add 800 parts of deionized water, stir evenly, add 2 parts of auxiliary agent, stir at 30℃ for 20 min for pretreatment, adjust the pH of the system to 7.0, add 4 parts of complex protease, place in a constant temperature water bath shaker, and enzymatically hydrolyze at 48℃ and 190 r / min for 3 h. A2. After the enzymatic hydrolysis is completed, the system is heated to 90℃ and kept at this temperature for 10 min to inactivate the enzyme. After cooling to room temperature, it is centrifuged at 5000 r / min for 10 min. The supernatant is collected and filtered through a 0.22 μm microporous membrane to obtain salmon skin enzymatic hydrolysate.

[0020] The preparation method of the auxiliary agent in step S2 includes the following steps: B1. Add 600 parts of deionized water to a four-necked flask, install a mechanical stirrer, thermometer and constant pressure dropping funnel, turn on the stirrer at 200 r / min, add 10 parts of L-cysteine ​​and 5 parts of itaconic acid, stir until completely dissolved, raise the temperature of the constant temperature water bath to 30℃, slowly add 15% triethanolamine aqueous solution, control the pH of the system to be stable at 8.0, continue stirring for 20 min after the addition is completed to form a uniform sodium salt solution; B2. Weigh 4 parts of sodium metabisulfite, add 120 parts of deionized water, stir to dissolve into sodium metabisulfite solution, keep the temperature of the above sodium salt solution, reduce the stirring speed to 100 r / min, add sodium metabisulfite solution dropwise through a dropping funnel at a rate of 8 mL / min, after the addition is complete, raise the temperature to 37℃, add 2 parts of glycerol at the same time, and keep the reaction at the temperature for 1 h. B3. Cool the above reaction system to 22℃, adjust the stirring speed to 150 r / min, adjust the pH to 6.5, and add 20 parts of 15% calcium chloride solution dropwise. After the addition is complete, continue stirring for 1 h. Transfer the reaction solution to a rotary evaporator and concentrate it to 1 / 3 of the original volume at 45℃ and 0.085 MPa. Slowly add 3 times the volume of 95% ethanol to the concentrate while stirring. Then place it in a refrigerator at 4℃ for 1 h, filter it with a Buchner funnel, collect the precipitate, wash it 3 times with anhydrous ethanol, transfer the precipitate to a vacuum drying oven, and dry it at 30℃ and 0.09 MPa for 8 h to obtain the auxiliary agent.

[0021] In step S2, the complex protease is a mixture of Bacillus subtilis protease and Aspergillus oryzae protease in a mass ratio of 2:1, wherein the Bacillus subtilis protease has an enzyme activity ≥15000 U / g and the Aspergillus oryzae protease has an enzyme activity ≥10000 U / g.

[0022] In step S3, the trace element compound is selected from ferrous sulfate; the concentration of the trace element solution is 50 mmol / L, and the dissolution temperature is 30℃.

[0023] The chelation reaction method in step S4 is as follows: C1. The salmon skin enzymatic hydrolysate was subjected to ultrasonic and microwave treatment. The power parameters were set as follows: ultrasonic power 200 W, frequency 28 kHz, microwave power 200 W. The hydrolysate was treated at 30℃ for 10 min. 0.1 mol / L citric acid solution was added to the activated enzymatic hydrolysate to adjust the pH to 5.0, thus obtaining the activated enzymatic hydrolysate. C2. Add food-grade sodium gluconate to the trace element solution at a mass ratio of 1:1, stir at 30°C for 10 min to form a trace element-gluconate intermediate complex. C3. First stage: Mix the activated enzymatic hydrolysate and the pre-complexed trace element solution at a volume ratio of 4:1, start the constant temperature water bath reactor, set the temperature to 35℃, the stirring speed to 150 r / min, maintain the pH at 5.0, and the time to 20 min; Second stage: Slowly add 0.1 mol / L NaOH solution to raise the pH of the system to 6.5, and at the same time raise the temperature to 45℃, for 30 min; Third stage: Add 0.1% of the enzymatic hydrolysate mass of reduced glutathione, maintain the pH at 6.5 and the temperature at 45℃, and continue the reaction for 10 min.

[0024] In step S5, food-grade activated carbon is used for decolorization, with an addition amount of 0.3% of the reaction solution mass. The decolorization temperature is 30℃ and the stirring time is 20 min.

[0025] In step S5, the concentration is carried out by vacuum rotary evaporation under the following conditions: temperature 35℃, vacuum degree 0.08 MPa, and concentration to a solid content of 25%; the spray drying parameters are: inlet air temperature 110℃, outlet air temperature 50℃, and feed rate 8 mL / min. Example

[0026] A method for chelating trace elements after enzymatic hydrolysis of salmon skin includes the following steps: S1. Pretreatment of salmon skin: Select salmon skin, remove impurities, soak in sodium bicarbonate solution, wash, freeze dry, pulverize and sieve to obtain salmon skin powder. S2. Enzymatic hydrolysis of salmon skin: Mix salmon skin powder with deionized water, stir evenly, add auxiliary agent, adjust pH value, add compound protease, enzymatic hydrolysis, enzyme inactivation, centrifugation and filtration to obtain salmon skin enzymatic hydrolysate. S3. Preparation of trace element solution: Dissolve the trace element compound in deionized water to obtain a trace element solution; S4. Chelation reaction: Mix the enzymatic hydrolysate of salmon skin with the trace element solution and carry out the chelation reaction in stages. S5. Post-processing of chelated products: The above chelation reaction solution is decolorized, centrifuged, concentrated, and spray-dried to obtain salmon skin active peptide-trace element chelate powder.

[0027] In step S1, the mass concentration of the sodium bicarbonate solution is 1%, the soaking temperature is 28℃, the soaking time is 25 min, and the solid-liquid ratio is 1:10.

[0028] The enzymatic hydrolysis method for salmon skin in step S2 is as follows: A1. Weigh 110 parts of salmon skin powder, add 900 parts of deionized water, stir evenly, add 4 parts of auxiliary agent, stir at 30℃ for 25 min for pretreatment, adjust the pH of the system to 7.0, add 6 parts of complex protease, place in a constant temperature water bath shaker, and enzymatically hydrolyze at 48℃ and 190 r / min for 4 h. A2. After the enzymatic hydrolysis is completed, the system is heated to 90℃ and kept at this temperature for 10 min to inactivate the enzyme. After cooling to room temperature, it is centrifuged at 6000 r / min for 15 min. The supernatant is collected and filtered through a 0.22 μm microporous membrane to obtain salmon skin enzymatic hydrolysate.

[0029] The preparation method of the auxiliary agent in step S2 includes the following steps: B1. Add 700 parts of deionized water to a four-necked flask, install a mechanical stirrer, thermometer and constant pressure dropping funnel, turn on the stirrer at 220 r / min, add 15 parts of L-cysteine ​​and 8 parts of itaconic acid, stir until completely dissolved, raise the temperature of the constant temperature water bath to 35℃, slowly add 20% triethanolamine aqueous solution, control the pH of the system to be stable at 8.5, continue stirring for 30 min after the addition is completed to form a uniform sodium salt solution; B2. Weigh 6 parts of sodium metabisulfite, add 140 parts of deionized water, stir to dissolve into sodium metabisulfite solution, keep the temperature of the above sodium salt solution, reduce the stirring speed to 110 r / min, add sodium metabisulfite solution dropwise through a dropping funnel at a rate of 9 mL / min, after the addition is complete, raise the temperature to 38℃, add 3 parts of glycerol at the same time, and keep the reaction at the temperature for 1.5 h. B3. Cool the above reaction system to 22℃, adjust the stirring speed to 160 r / min, adjust the pH to 6.6, and add 25 parts of 18% calcium chloride solution dropwise. After the addition is complete, continue stirring for 1 h. Transfer the reaction solution to a rotary evaporator and concentrate it to 1 / 3 of the original volume at 45℃ and 0.085 MPa. Slowly add 3 times the volume of 95% ethanol to the concentrate while stirring. Then place it in a refrigerator at 4℃ for 1.5 h, filter it with a Buchner funnel, collect the precipitate, wash it 3 times with anhydrous ethanol, transfer the precipitate to a vacuum drying oven, and dry it at 30℃ and 0.09 MPa for 10 h to obtain the auxiliary agent.

[0030] In step S2, the complex protease is a mixture of Bacillus subtilis protease and Aspergillus oryzae protease in a mass ratio of 2:1, wherein the Bacillus subtilis protease has an enzyme activity ≥15000 U / g and the Aspergillus oryzae protease has an enzyme activity ≥10000 U / g.

[0031] In step S3, the trace element compound is selected from zinc sulfate; the concentration of the trace element solution is 60 mmol / L, and the dissolution temperature is 35℃.

[0032] The chelation reaction method in step S4 is as follows: C1. The salmon skin enzymatic hydrolysate was subjected to ultrasonic and microwave treatment. The power parameters were set as follows: ultrasonic power 250 W, frequency 28 kHz, and microwave power 250 W. The hydrolysate was treated at 35℃ for 15 min. 0.1 mol / L citric acid solution was added to the activated enzymatic hydrolysate to adjust the pH to 5.0, thus obtaining the activated enzymatic hydrolysate. C2. Add food-grade sodium gluconate to the trace element solution at a mass ratio of 1:1, stir at 35℃ for 10 min to form a trace element-gluconate intermediate complex. C3. First stage: Mix the activated enzymatic hydrolysate and the pre-complexed trace element solution at a volume ratio of 4:1, start the constant temperature water bath reactor, set the temperature to 38℃, the stirring speed to 180 r / min, maintain the pH at 5.2, and the time to 20 min; Second stage: Slowly add 0.1 mol / L NaOH solution to raise the pH of the system to 6.6, and at the same time raise the temperature to 45℃, for 30 min; Third stage: Add 0.1% of the enzymatic hydrolysate mass of reduced glutathione, maintain the pH at 6.5 and the temperature at 48℃, and continue the reaction for 10 min.

[0033] In step S5, food-grade activated carbon is used for decolorization, with an addition amount of 0.5% of the reaction solution mass. The decolorization temperature is 35℃ and the stirring time is 30 min.

[0034] In step S5, the concentration is carried out by vacuum rotary evaporation under the following conditions: temperature 40℃, vacuum degree 0.09 MPa, and concentration to a solid content of 30%; the spray drying parameters are: inlet air temperature 120℃, outlet air temperature 55℃, and feed rate 10 mL / min. Example

[0035] A method for chelating trace elements after enzymatic hydrolysis of salmon skin includes the following steps: S1. Pretreatment of salmon skin: Select salmon skin, remove impurities, soak in sodium bicarbonate solution, wash, freeze dry, pulverize and sieve to obtain salmon skin powder. S2. Enzymatic hydrolysis of salmon skin: Mix salmon skin powder with deionized water, stir evenly, add auxiliary agent, adjust pH value, add compound protease, enzymatic hydrolysis, enzyme inactivation, centrifugation and filtration to obtain salmon skin enzymatic hydrolysate. S3. Preparation of trace element solution: Dissolve the trace element compound in deionized water to obtain a trace element solution; S4. Chelation reaction: Mix the enzymatic hydrolysate of salmon skin with the trace element solution and carry out the chelation reaction in stages. S5. Post-processing of chelated products: The above chelation reaction solution is decolorized, centrifuged, concentrated, and spray-dried to obtain salmon skin active peptide-trace element chelate powder.

[0036] In step S1, the mass concentration of the sodium bicarbonate solution is 1.2%, the soaking temperature is 30℃, the soaking time is 30 min, and the solid-liquid ratio is 1:12.

[0037] The enzymatic hydrolysis method for salmon skin in step S2 is as follows: A1. Weigh 120 parts of salmon skin powder, add 1000 parts of deionized water, stir evenly, add 5 parts of auxiliary agent, stir at 35℃ for 30 min for pretreatment, adjust the pH of the system to 7.2, add 8 parts of complex protease, place in a constant temperature water bath shaker, and enzymatically hydrolyze at 48℃ and 190 r / min for 5 h. A2. After the enzymatic hydrolysis is completed, the system is heated to 95℃ and kept at this temperature for 15 min to inactivate the enzyme. After cooling to room temperature, it is centrifuged at 8000 r / min for 20 min. The supernatant is collected and filtered through a 0.22 μm microporous membrane to obtain salmon skin enzymatic hydrolysate.

[0038] The preparation method of the auxiliary agent in step S2 includes the following steps: B1. Add 800 parts of deionized water to a four-necked flask, install a mechanical stirrer, thermometer and constant pressure dropping funnel, turn on the stirrer at 250 r / min, add 20 parts of L-cysteine ​​and 10 parts of itaconic acid, stir until completely dissolved, raise the temperature of the constant temperature water bath to 35℃, slowly add 20% triethanolamine aqueous solution, control the pH of the system to be stable at 8.5, continue stirring for 30 min after the addition is completed to form a uniform sodium salt solution; B2. Weigh 8 parts of sodium metabisulfite, add 150 parts of deionized water, stir to dissolve into sodium metabisulfite solution, keep the temperature of the above sodium salt solution, reduce the stirring speed to 120 r / min, add sodium metabisulfite solution dropwise through a dropping funnel at a rate of 10 mL / min, after the addition is complete, raise the temperature to 40℃, add 5 parts of glycerol at the same time, and keep the reaction at the temperature for 1.5 h. B3. Cool the above reaction system to 25℃, adjust the stirring speed to 180 r / min, adjust the pH to 7.0, and add 30 parts of 20% calcium chloride solution dropwise. After the addition is complete, continue stirring for 1.5 h. Transfer the reaction solution to a rotary evaporator and concentrate it to 1 / 3 of the original volume at 45℃ and 0.085 MPa. Slowly add 3 times the volume of 95% ethanol to the concentrate while stirring. Then place it in a refrigerator at 4℃ for 2 h. Filter it with a Buchner funnel, collect the precipitate, wash it 3 times with anhydrous ethanol, transfer the precipitate to a vacuum drying oven, and dry it at 35℃ and 0.1 MPa for 12 h to obtain the auxiliary agent.

[0039] In step S2, the complex protease is a mixture of Bacillus subtilis protease and Aspergillus oryzae protease in a mass ratio of 2:1, wherein the Bacillus subtilis protease has an enzyme activity ≥15000 U / g and the Aspergillus oryzae protease has an enzyme activity ≥10000 U / g.

[0040] In step S3, the trace element compound is selected from sodium selenite; the concentration of the trace element solution is 100 mmol / L, and the dissolution temperature is 40℃.

[0041] The chelation reaction method in step S4 is as follows: C1. The salmon skin enzymatic hydrolysate was subjected to ultrasonic and microwave treatment. The power parameters were set as follows: ultrasonic power 300 W, frequency 30 kHz, microwave power 300 W. The hydrolysate was treated at 35℃ for 15 min. 0.1 mol / L citric acid solution was added to the activated enzymatic hydrolysate to adjust the pH to 5.5 to obtain the activated enzymatic hydrolysate. C2. Add food-grade sodium gluconate to the trace element solution at a mass ratio of 1:1, stir at 35℃ for 15 min to form a trace element-gluconate intermediate complex. C3. First stage: Mix the activated enzymatic hydrolysate and the pre-complexed trace element solution at a volume ratio of 4:1, start the constant temperature water bath reactor, set the temperature to 40℃, the stirring speed to 200 r / min, maintain the pH at 5.5, and the time to 30 min; Second stage: Slowly add 0.1 mol / L NaOH solution to raise the pH of the system to 7.0, and simultaneously raise the temperature to 50℃, for 45 min; Third stage: Add 0.1% of the enzymatic hydrolysate mass of reduced glutathione, maintain the pH at 7.0 and the temperature at 50℃, and continue the reaction for 15 min.

[0042] In step S5, food-grade activated carbon is used for decolorization, with an addition amount of 0.8% of the reaction solution mass. The decolorization temperature is 40℃ and the stirring time is 40 min.

[0043] In step S5, the concentration is carried out by vacuum rotary evaporation under the following conditions: temperature 45℃, vacuum degree 0.1 MPa, and concentration to a solid content of 35%; the spray drying parameters are: inlet air temperature 130℃, outlet air temperature 60℃, and feed rate 12 mL / min.

[0044] Comparative Example 1: Step S2 does not use any auxiliary agents, and the rest of the process is the same as in Example 1.

[0045] Comparative Example 2: The auxiliary agent was replaced with an equal mass of 1% sodium sulfite solution, and the rest of the process was the same as in Example 1.

[0046] Comparative Example 3: Step S4 chelation process was changed to mixing enzymatic hydrolysate and trace element solution at a ratio of 4:1, adjusting pH to 6.5, and reacting at 45℃ for 60 min; the rest of the process was the same as in Example 1.

[0047] 1. Degree of protein hydrolysis (DH) determination Formaldehyde titration method: Take 5 mL of enzymatic hydrolysate, dilute with 20 mL of deionized water, and adjust the pH to 8.2 with 0.1 mol / L NaOH; add 10 mL of neutral formaldehyde solution (mass fraction 36%), shake well, and titrate with 0.1 mol / L NaOH to pH 8.2, and record the volume consumed V1; for the blank group, take 5 mL of deionized water, repeat the above steps, and record the volume consumed V0; Calculate: DH (%) = (V1 - V0) × C × 10 -3 ×7.5×100 / (m×α×h0), where C is the NaOH concentration (mol / L), m is the protein content in the enzymatic hydrolysate (g), α is the amino nitrogen coefficient (0.89), and h0 is the total amino nitrogen content in the protein (mmol / g, salmon skin protein is calculated as 8.6).

[0048] 2. Determination of bioactive peptide yield High Performance Liquid Chromatography (HPLC): Chromatographic conditions: C18 column (250 mm × 4.6 mm), mobile phase acetonitrile-0.1% phosphoric acid water (10:90), flow rate 1 mL / min, detection wavelength 220 nm, column temperature 30℃; Standard curve: Prepare glycine-proline-hydroxyproline standard solutions of 1, 2, 5, 10, and 20 mg / mL, and plot the peak area-concentration standard curve; Sample determination: Take the enzymatic hydrolysate and filter it through a 0.22 μm filter membrane, inject 20 μL, calculate the concentration of active peptides based on the peak area, and the yield (%) = (mass of active peptides / amount of protein in fish skin powder) × 100. 3. Determination of trace element chelation rate Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES): Take 10 mL of chelation reaction solution, add 10 mL of anhydrous ethanol to precipitate the chelate, and centrifuge at 5000 r / min for 15 min; take the supernatant, dilute it 10 times with 5% nitric acid solution, and determine the free trace element concentration C1 by ICP-OES; for the total concentration group, take 10 mL of chelation reaction solution, digest it with 5% nitric acid, and then determine the total trace element concentration C. 总 ; Calculate: Chelation rate (%) = (C 总 -C1)×100 / C 总 .

[0049] 4. Determination of in vitro absorption rate of chelates Simulated gastrointestinal digestion method: Simulated gastric juice digestion: Take 0.5 g of chelate powder, add 20 mL of artificial gastric juice (pH 1.2, containing 0.32% pepsin), and digest at 37℃ with shaking for 2 h; Simulated intestinal fluid digestion: Adjust the pH to 7.0 with 0.1 mol / L NaOH, add 20 mL of artificial intestinal fluid (containing 1% trypsin), and digest at 37℃ with shaking for 4 h; Centrifuge (5000 r / min, 15 min), collect the supernatant, and determine the concentration of soluble trace elements C by ICP-OES. 溶 ; Calculation: In vitro absorption rate (%) = (C 溶 × digestion liquid volume) × 100 / (total mass of trace elements in chelates).

[0050] Table 1 Results of protein hydrolysis degree and bioactive peptide yield test

[0051] Table 2 Results of Trace Element Chelation Rate and In Vitro Absorption Rate of Chelates

[0052] As can be seen from the data in Tables 1 and 2, all indicators of Examples 1-3 are better than those of Comparative Examples 1-3.

[0053] This invention demonstrates the synergistic effect of the auxiliary agent and the segmented chelation process, which can significantly enhance the enzymatic hydrolysis. Furthermore, the auxiliary agent produced by the process of this invention is more effective than sodium sulfite. At the same time, the segmented chelation process can form a stable multi-component coordination structure, and the chelation rate and in vitro absorption rate remain at a high level, effectively improving the stability and utilization rate of the final product.

[0054] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method of chelating trace elements after enzymatic hydrolysis of salmonid fish skin, characterized by, The method comprises the following steps: S1, mahi mahi skin pretreatment: select mahi mahi skin, remove impurities, soak in sodium bicarbonate solution, clean, freeze dry, crush and sieve, and obtain mahi mahi skin powder; S2, mahi mahi skin enzymolysis: mix the mahi mahi skin powder with deionized water, stir uniformly, add auxiliary agent, adjust the pH value, add compound protease, and perform enzymolysis, enzyme inactivation, centrifugal filtration, and obtain mahi mahi skin enzymolysis liquid; S3, trace element solution preparation: mix trace element compounds with deionized water to dissolve, and obtain trace element solution; S4, chelation reaction: mix the mahi mahi skin enzymolysis liquid with the trace element solution, and perform chelation reaction in stages; S5, chelation product post-treatment: perform decolorization, centrifugal, concentration, and spray drying on the chelation reaction liquid, and obtain mahi mahi skin active peptide-trace element chelate powder.

2. A method of chelating trace elements after enzymatic hydrolysis of a salmonid skin according to claim 1, characterized in that, In step S1, the mass concentration of the sodium bicarbonate solution is 0.8-1.2%, the soaking temperature is 25-30 DEG C, the soaking time is 20-30 min, and the solid-liquid ratio is 1:8-12.

3. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and the chelated trace elements are in the form of a powder. In step S2, the mahi mahi skin enzymolysis method is as follows: A1, weigh 100-120 parts of mahi mahi skin powder, add 800-1000 parts of deionized water, stir uniformly, add 2-5 parts of auxiliary agent, 30-35 DEG C stirring pretreatment 20-30 min, adjust the system pH to 7.0-7.2, add 4-8 parts of compound protease, place in constant temperature water bath shaker, 48 DEG C, 190 r / min enzymolysis 3-5 h; A2, after the enzymolysis is completed, the system is heated to 90-95 DEG C, and enzyme inactivation is performed for 10-15 min; after cooling to room temperature, centrifugal filtration is performed at 5000-8000 r / min for 10-20 min; the supernatant is taken, filtered through a 0.22 mu m microporous filter membrane, and the mahi mahi skin enzymolysis liquid is obtained.

4. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and the chelated trace elements are in the form of a powder. The preparation method of the auxiliary agent in step S2 comprises the following steps: B1, add 600-800 parts of deionized water to a four-necked flask, install a mechanical stirrer, a thermometer and a constant pressure dropping funnel, start stirring, the stirring speed is 200-250 r / min, add 10-20 parts of L-cysteine and 5-10 parts of itaconic acid, stir until completely dissolved, then heat the constant temperature water bath to 30-35 DEG C, slowly add 15-20% triethanolamine aqueous solution, control the system pH to be stable at 8.0-8.5, continue stirring for 20-30 min after the addition is completed, and a uniform sodium salt solution is formed; B2, weigh 4-8 parts of sodium metabisulfite, add 120-150 parts of deionized water, stir to dissolve into sodium metabisulfite solution, keep the temperature of the above-mentioned sodium salt solution, reduce the stirring speed to 100-120 r / min, add the sodium metabisulfite solution through the dropping funnel at a speed of 8-10 mL / min, after the addition is completed, heat to 37-40 DEG C, and add 2-5 parts of glycerol at the same time, and react for 1-1.5 h under heat preservation; B3. Cool the above reaction system to 22-25℃, adjust the stirring speed to 150-180 r / min, adjust the pH to 6.5-7.0, and add 20-30 parts of 15-20% calcium chloride solution dropwise. After the addition is complete, continue stirring for 1-1.5 h. Transfer the reaction solution to a rotary evaporator and concentrate it to 1 / 3 of the original volume at 45℃ and 0.085 MPa. Slowly add 3 times the volume of 95% ethanol to the concentrate while stirring. Then place it in a refrigerator at 4℃ for 1-2 h. Filter it with a Buchner funnel, collect the precipitate, wash it 3 times with anhydrous ethanol, transfer the precipitate to a vacuum drying oven, and dry it at 30-35℃ and 0.09-0.1 MPa for 8-12 h to obtain the auxiliary agent.

5. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and the chelated trace elements are in the form of a powder. In step S2, the complex protease is a mixture of Bacillus subtilis protease and Aspergillus oryzae protease in a mass ratio of 2:1, wherein the Bacillus subtilis protease has an enzyme activity ≥15000 U / g and the Aspergillus oryzae protease has an enzyme activity ≥10000 U / g.

6. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and the chelated trace elements are in the form of a powder. In step S3, the trace element compound is selected from at least one of ferrous sulfate, zinc sulfate, and sodium selenite; the concentration of the trace element solution is 50~100 mmol / L, and the dissolution temperature is 30~40℃.

7. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and the chelated trace elements are in the form of a powder. The chelation reaction method in step S4 is as follows: C1. The salmon skin enzymatic hydrolysate is subjected to ultrasonic and microwave treatment. The power parameters are set as follows: ultrasonic power 200~300 W, frequency 28~30 kHz, microwave power 200~300 W. The treatment is carried out at 30~35℃ for 10~15 min. 0.1 mol / L citric acid solution is added to the activated enzymatic hydrolysate to adjust the pH to 5.0~5.5 to obtain the activated enzymatic hydrolysate. C2. Add food-grade sodium gluconate to the trace element solution at a mass ratio of 1:1, stir at 30~35℃ for 10~15 min to form a trace element-gluconate intermediate complex. C3. First stage: Mix the activated enzymatic hydrolysate and the pre-complexed trace element solution at a volume ratio of 4:1, start the constant temperature water bath reactor, set the temperature to 35~40℃, the stirring speed to 150~200 r / min, maintain the pH at 5.0~5.5, and the time to 20~30 min; Second stage: Slowly add 0.1 mol / L NaOH solution to raise the pH of the system to 6.5~7.0, and at the same time raise the temperature to 45~50℃, and the time to 30~45 min; Third stage: Add 0.1% of the enzymatic hydrolysate mass of reduced glutathione, maintain the pH at 6.5~7.0 and the temperature at 45~50℃, and continue the reaction for 10~15 min.

8. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and wherein the chelated trace elements are selected from the group consisting of calcium, magnesium, potassium, sodium, phosphorus, zinc, copper, manganese, iron, iodine, selenium, and combinations thereof. In step S5, food-grade activated carbon is used for decolorization, with an addition amount of 0.3~0.8% of the reaction solution mass. The decolorization temperature is 30~40℃, and the stirring time is 20~40 min.

9. The method of claim 1, wherein the Mahi-mahi skin is enzymatically hydrolyzed and chelated with trace elements, and the chelated trace elements are in the form of a powder. In step S5, the concentration is performed by vacuum rotary evaporation under the conditions of a temperature of 35-45°C, a vacuum degree of 0.08-0.1 MPa, and a solid content of 25-35%; and the spray drying parameters are as follows: an inlet air temperature of 110-130°C, an outlet air temperature of 50-60°C, and a feeding speed of 8-12 mL / min.

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