Preparation method of anti-fatigue peptide

Abstract

The invention relates to the technical field of anti-fatigue peptide preparation, and discloses an anti-fatigue peptide preparation method, which comprises: 1, raw material pretreatment: selecting fresh deep sea fish skin, washing, dicing, carrying out freeze drying, and carrying out superfine grinding under nitrogen protection to obtain fish skin micro powder; step 2, preparation of a composite enzymatic hydrolysate: mixing trypsin, papain and bromelain according to a ratio, and adding the mixture into a phosphoric acid buffer solution to prepare the composite enzymatic hydrolysate; a compound enzyme and ultrasonic wave synergistic enzymolysis process is adopted, the synergistic effect of the compound enzyme is combined with assistance of ultrasonic waves, full contact of the enzyme and a substrate is promoted, the enzymolysis efficiency is greatly improved, and compared with a traditional single enzymolysis method or a non-ultrasonic-assistance method, the yield of the anti-fatigue peptide is improved by 20% or above; meanwhile, through a macroporous adsorption resin and ultrafiltration membrane two-step purification combination technology, impurities are effectively removed, the product purity is improved by 30% or above, the molecular weight distribution is more uniform, and the product quality is improved.

Description

Technical Field

[0001] This invention relates to the field of anti-fatigue peptide preparation technology, specifically to a method for preparing anti-fatigue peptides. Background Technology

[0002] Anti-fatigue peptides, as a class of small molecule active substances composed of amino acids linked by peptide bonds, have the characteristics of small molecular weight, easy absorption, high biological activity, and good safety. They can exert anti-fatigue effects through multiple pathways such as participating in energy metabolism, scavenging free radicals, and regulating endocrine function, showing broad application prospects in the fields of functional foods, health products, and medicines.

[0003] Currently, the preparation of anti-fatigue peptides mainly uses animal and plant proteins as raw materials, employing methods such as enzymatic hydrolysis, acid hydrolysis, and alkaline hydrolysis for extraction. Among these, enzymatic hydrolysis is widely used due to its advantages such as mild reaction conditions and good preservation of product activity. However, existing enzymatic hydrolysis processes mostly use single proteases, resulting in low hydrolysis efficiency and low product yield. Furthermore, the lack of effective auxiliary methods during enzymatic hydrolysis leads to long reaction times and low substrate conversion rates. Therefore, there is an urgent need for a method to prepare anti-fatigue peptides that can improve both yield and purity. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing anti-fatigue peptides, which solves the problems of low enzymatic hydrolysis efficiency and low product yield caused by the use of a single protease in existing enzymatic hydrolysis processes.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing anti-fatigue peptides, comprising the following steps:

[0008] Step 1: Raw material pretreatment: Select fresh deep-sea fish skin, clean and cut it into pieces, freeze-dry it, and then pulverize it under nitrogen protection to obtain fish skin powder;

[0009] Step 2: Preparation of compound enzymatic hydrolysate: Mix trypsin, papain and bromelain in a certain proportion and add them to phosphate buffer solution to prepare compound enzymatic hydrolysate;

[0010] Step 3: Enzymatic hydrolysis: Add fish skin powder to the compound enzymatic hydrolysate and carry out the enzymatic hydrolysis reaction with the assistance of ultrasound within a specific temperature range;

[0011] Step 4: Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is subjected to high-temperature enzyme inactivation and then rapidly cooled;

[0012] Step 5: Centrifugation: Centrifuge the enzyme-inactivated reaction solution and collect the supernatant;

[0013] Step 6: Preliminary purification: The supernatant is preliminarily purified by passing it through a macroporous adsorption resin column, and the eluent is collected;

[0014] Step 7: Ultrafiltration purification: The eluent is purified by ultrafiltration through an ultrafiltration membrane, and the permeate is collected;

[0015] Step 8: Decolorization treatment: Add activated carbon to the ultrafiltration permeate for decolorization, and filter to remove the activated carbon;

[0016] Step 9: Concentration and Drying: Concentrate the decolorized solution and freeze-dry it to obtain the anti-fatigue peptide product;

[0017] Step 10: Quality Inspection: Test the finished product for indicators such as peptide content, molecular weight distribution, and anti-fatigue activity.

[0018] Preferably, the freeze-drying conditions in step one are as follows: first, pre-freeze at -42 to -38°C for 1.5 to 2.5 hours, then freeze-dry at a vacuum of 8 to 12 Pa for 10 to 18 hours until the moisture content is less than 5%; the rotation speed of the ultrafine pulverizer is 2800 to 3200 r / min, the time is 12 to 25 minutes, the resulting fish skin micro powder has a particle size of 4 to 12 μm, and is screened through an 1800 to 2200 mesh sieve.

[0019] Preferably, the mass ratio of trypsin, papain and bromelain in step two is 2.8-3.2:1.8-2.2:1, the pH of the phosphate buffer solution is 6.8-7.2, the total enzyme concentration in the compound enzymatic hydrolysate is 2.2-2.8%, and the enzymatic hydrolysate is refrigerated at 2-6°C for no more than 24 hours.

[0020] Preferably, in step three, the mass-to-volume ratio of fish skin powder to the compound enzymatic hydrolysate is 1:13-17 (g / mL), the enzymatic hydrolysis temperature is 42-48℃, and the total enzymatic hydrolysis time is 2.5-3.5h; the ultrasonic auxiliary parameters are: power 280-320W, ultrasonic time 4-6s, interval time 8-12s, and the pH is adjusted to 6.8-7.2 every 25-35min during the reaction.

[0021] Preferably, the conditions for high-temperature enzyme inactivation in step four are heating in a water bath at 88-92°C for 12-18 minutes, followed by rapid cooling in an ice bath to room temperature of 23-27°C, with continuous stirring during the cooling process.

[0022] Preferably, the parameters for centrifugation in step five are: rotation speed 7500-8500 r / min, time 18-22 min, temperature 23-27℃, and the volume of the reaction liquid in the centrifuge tube does not exceed 2 / 3 of its capacity.

[0023] Preferably, the macroporous adsorption resin in step six is ​​type AB-8, with a column diameter to column height ratio of 1:7-9; the sample loading flow rate is 1.8-2.2 BV / h, and the sample loading volume is 1 / 4-1 / 2 of the column volume; first, it is rinsed with deionized water at a flow rate of 2.8-3.2 BV / h until the absorbance of the effluent at 280 nm is less than 0.02, and then eluted with 28-32% ethanol solution at a flow rate of 1.8-2.2 BV / h.

[0024] Preferably, the ultrafiltration membrane described in step seven has a molecular weight cutoff of 2.5-3.5 kDa, an ultrafiltration operating pressure of 0.18-0.22 MPa, and a temperature of 28-32°C; when the volume of the concentrate is 1 / 5-1 / 3 of the original volume, an equal amount of deionized water is added to continue ultrafiltration, and the operation is repeated 1-3 times.

[0025] Preferably, the activated carbon in step eight has a particle size of 180-220 mesh, a specific surface area ≥900-1100 m² / g, and is added at 1.2-1.8% of the mass of the ultrafiltration permeate; the decolorization conditions are stirring at 48-52℃ for 25-35 min at a stirring speed of 130-170 r / min, and filtration is performed sequentially using qualitative filter paper and a 0.2-0.24 μm microporous filter membrane;

[0026] The concentration in step nine is carried out by rotary evaporation at 58-62℃ and a vacuum of 0.07-0.09MPa, concentrating to 1 / 6-1 / 4 of the original volume; the freeze-drying conditions are -52 to -48℃ and a vacuum of 4-6Pa, drying to constant weight, and the resulting product is vacuum-sealed after passing through a 90-110 mesh sieve.

[0027] (III) Beneficial Effects

[0028] Compared with the prior art, the present invention provides a method for preparing anti-fatigue peptides, which has the following beneficial effects:

[0029] 1. The preparation method of this anti-fatigue peptide adopts a synergistic enzymatic hydrolysis process using a compound enzyme and ultrasound. The synergistic effect of the compound enzyme combined with the assistance of ultrasound promotes full contact between the enzyme and the substrate, significantly improving the hydrolysis efficiency. Compared with traditional single enzymatic hydrolysis or methods without ultrasound assistance, the yield of anti-fatigue peptide is increased by more than 20%. At the same time, the two-step purification technology of macroporous adsorption resin and ultrafiltration membrane effectively removes impurities, increasing the product purity by more than 30% and making the molecular weight distribution more uniform, thus improving the product quality.

[0030] 2. The preparation method of this anti-fatigue peptide adopts a combination of low-temperature freeze-drying and ultra-fine pulverization with nitrogen protection in the raw material pretreatment stage, which avoids the damage of active ingredients in the raw materials by high temperature and the oxidation and deterioration during the pulverization process, and improves the retention rate of active ingredients by more than 25%. In subsequent steps such as enzymatic hydrolysis and purification, parameters such as temperature and time are strictly controlled to reduce the loss of active ingredients. The anti-fatigue peptide prepared by this method can significantly prolong the swimming exhaustion time of mice by more than 35%, thus improving the product quality.

[0031] 3. The entire preparation process is clearly defined, with specific parameters for each step. The equipment used is commonly used in the field of bioprocessing (such as freeze dryers and high-speed centrifuges), making it easy to operate and control. At the same time, the raw materials used in the process (deep-sea fish skin) are widely available, and the reagent costs are low. The subsequent concentration and drying steps can yield stable solid products that are easy to store and transport, providing a technical solution for large-scale industrial production. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the structure of a method for preparing an anti-fatigue peptide according to the present invention. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Please see Figure 1 This invention provides a new technical solution: a method for preparing an anti-fatigue peptide, comprising the following steps:

[0035] Step 1: Raw material pretreatment: Select fresh deep-sea fish skin (cod skin or salmon skin), requiring that the surface of the fish skin be undamaged and odorless, and the collection time not exceed 24 hours;

[0036] Rinse the fish skin surface with deionized water to remove blood and impurities, repeating the rinsing 3-5 times until the rinsing solution is clear;

[0037] Cut the cleaned fish skin into uniform 1cm x 1cm pieces with a stainless steel knife, avoiding the use of iron knives to prevent metal ion contamination.

[0038] Spread the cut fish skin pieces evenly on the freeze-drying tray, with a thickness of 0.5-1cm, and place them in the freeze dryer;

[0039] Set the freeze-drying parameters: first pre-freeze at -40℃ for 2 hours, then maintain a vacuum of 10Pa and continue freeze-drying for 12-16 hours until the moisture content of the fish skin is below 5%;

[0040] The dried fish skin was transferred to the feeding chamber of the ultrafine pulverizer, and nitrogen was introduced to replace the air in the chamber three times (each time for 30 seconds, with a 1-minute interval), and pulverized under a nitrogen protective atmosphere.

[0041] During the pulverization process, the pulverizer speed is controlled at 3000 r / min and the pulverization time is 15-20 min. After screening through a 2000-mesh sieve, fish skin micro powder with a particle size of 5-10 μm is obtained and sealed and stored in a desiccator for later use. This step combines ultra-low temperature freeze drying and ultra-fine pulverization with nitrogen protection to retain the active ingredients in the raw materials to the greatest extent and avoid oxidation and deterioration.

[0042] Step 2: Preparation of compound enzymatic hydrolysate: After completing the pretreatment of raw materials and obtaining fish skin powder, the compound enzymatic hydrolysate is prepared; accurately weigh 3g of trypsin (activity unit ≥2500U / mg), 2g of papain (activity unit ≥600U / mg) and 1g of bromelain (activity unit ≥1000U / mg), place them in a sterile beaker, and gently stir and mix them evenly with a glass rod;

[0043] To prepare a phosphate buffer solution with a pH of 7.0: Weigh 0.68 g of potassium dihydrogen phosphate, dissolve it in 500 mL of deionized water, adjust the pH to 7.0 with 0.1 mol / L sodium hydroxide solution, and bring the volume to 1000 mL.

[0044] The mixed complex enzyme was slowly added to the above phosphate buffer solution while stirring with a magnetic stirrer (200 r / min) for 30 min until the enzyme was completely dissolved, resulting in a complex enzyme hydrolysate with a total enzyme concentration of 2.5%.

[0045] Transfer the enzymatic hydrolysate to a brown reagent bottle and store at 4°C for later use (storage time not exceeding 24 hours); the synergistic effect of the complex enzyme can more fully decompose proteins and increase the yield of peptides.

[0046] Step 3: Enzymatic hydrolysis reaction: Once the compound enzymatic hydrolysate is prepared, the enzymatic hydrolysis reaction can be carried out. According to the mass-to-volume ratio of fish skin powder to compound enzymatic hydrolysate of 1:15 (g / mL), slowly add fish skin powder to the compound enzymatic hydrolysate, place it in a constant temperature water bath, and turn on the magnetic stirrer (150 r / min).

[0047] When the temperature rises to 45℃, start the ultrasonic generator, set the ultrasonic power to 300W, the ultrasonic time to 5s, and the interval time to 10s, and start the enzymatic hydrolysis reaction.

[0048] During the reaction, a sample was taken every 30 minutes to measure the pH value of the reaction solution. If the pH value deviated from 7.0±0.2, it was adjusted to the set value with 0.1mol / L hydrochloric acid or sodium hydroxide solution.

[0049] The total enzymatic hydrolysis time was controlled at 3 hours, and the ultrasonic treatment was stopped 10 minutes before the end of the reaction. Ultrasonic assistance can promote the contact between enzyme and substrate and accelerate the reaction.

[0050] Step 4: Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, enzyme inactivation treatment is required to terminate the enzyme activity; transfer the reaction solution to a three-necked flask, place it in a constant temperature water bath at 90℃, and start stirring at 100 r / min for 15 min.

[0051] After enzyme inactivation is complete, immediately place the three-necked flask in an ice bath and rapidly cool it to room temperature (25±2℃). Stir continuously during the cooling process to ensure a uniform temperature drop. High-temperature enzyme inactivation can quickly terminate the reaction and avoid excessive enzymatic hydrolysis.

[0052] Step 5: Centrifugation: After enzyme inactivation and cooling to room temperature, centrifuge the reaction solution to remove impurities; dispense the cooled reaction solution into centrifuge tubes, each tube not exceeding 2 / 3 of the tube's volume, and place them into a high-speed centrifuge after equilibration.

[0053] Set the centrifuge parameters as follows: speed 8000 r / min, centrifugation time 20 min, temperature 25℃;

[0054] After centrifugation, carefully remove the centrifuge tube and use a pipette to transfer the supernatant into a beaker. Discard the precipitate at the bottom (the precipitate can be collected for component analysis or other purposes). Centrifugation can separate most of the impurities, resulting in a relatively pure enzymatic hydrolysate.

[0055] Step 6: Preliminary purification: After centrifugation to obtain the supernatant, perform preliminary purification; Pretreatment of macroporous adsorption resin column: Soak AB-8 type macroporous adsorption resin in 95% ethanol for 24 hours, then pack it into the chromatography column (column diameter to column height ratio 1:8), wash with 95% ethanol at a flow rate of 3 BV / h until the effluent mixed with distilled water (1:5) is not turbid, and then wash with deionized water at a flow rate of 5 BV / h until there is no ethanol smell;

[0056] Sample loading: Filter the supernatant after centrifugation through a 0.45μm microporous membrane, and then pump it into the pretreated macroporous adsorption resin column at a flow rate of 2BV / h. The sample loading volume is 1 / 3 of the column volume.

[0057] Rinsing: After sample loading, rinse the resin column with deionized water at a flow rate of 3 BV / h until the absorbance of the effluent at 280 nm is less than 0.02 (considered colorless).

[0058] Elution: Use a 30% ethanol solution (prepared with deionized water) to elute at a flow rate of 2 BV / h, and collect the eluent (starting from when the effluent has obvious absorbance at 280 nm and ending when the absorbance value is below 0.05); macroporous adsorption resin can effectively adsorb target peptides and remove some small molecule impurities.

[0059] Step 7: Ultrafiltration purification: After obtaining the eluent through preliminary purification, further ultrafiltration purification is carried out; Ultrafiltration system pretreatment: The surface of the ultrafiltration membrane with a molecular weight cutoff of 3kDa is first rinsed with deionized water, then soaked in 0.5% sodium hydroxide solution for 30 minutes, and then rinsed with deionized water until neutral;

[0060] Pour the eluent into the ultrafiltration cup, install the ultrafiltration membrane, set the operating pressure to 0.2 MPa and the temperature to 30°C, and start stirring (300 r / min) to perform ultrafiltration;

[0061] During ultrafiltration, when the volume of the concentrate is 1 / 4 of the original volume, add an equal amount of deionized water and continue ultrafiltration. Repeat the operation twice and collect the permeate.

[0062] After ultrafiltration, the ultrafiltration membrane is rinsed with deionized water and stored in 0.1% formaldehyde solution for later use. Ultrafiltration can further separate substances of different molecular weights to obtain peptide solutions with more uniform molecular weight.

[0063] Step 8: Decolorization treatment: After ultrafiltration purification, the permeate is decolorized to remove pigments and other impurities; accurately weigh activated carbon (particle size 200 mesh, specific surface area ≥1000m² / g), add it to the permeate at 1.5% of the mass of the ultrafiltration permeate, and place it on a constant temperature water bath.

[0064] Set the temperature to 50℃, turn on the stirrer (150 r / min), and time for 30 minutes to ensure that the activated carbon is fully dispersed;

[0065] After decolorization, the product is filtered using a Buchner funnel. First, it is filtered with qualitative filter paper, and then with a 0.22μm microporous membrane to remove activated carbon particles. Activated carbon can adsorb pigments and other impurities, thus improving product purity.

[0066] Step 9: Concentration and Drying: After the decolorization process, the solution is concentrated and dried to obtain a solid product; Rotary Evaporation Concentration: The decolorized solution is transferred into the evaporation flask of a rotary evaporator, and the water bath temperature is set to 60℃, the vacuum degree to 0.08MPa, and the rotation speed to 50r / min for reduced pressure concentration;

[0067] When the solution volume is concentrated to 1 / 5 of the original volume, stop the concentration and transfer the concentrated solution to a clean petri dish, with the thickness controlled at 0.3-0.5 cm.

[0068] Freeze-drying: Place the petri dish in a freeze dryer, pre-freeze at -50℃ for 3 hours, then maintain a vacuum of 5Pa and freeze-dry for 24-30 hours until constant weight;

[0069] The dried solid is taken out, ground into fine powder with an agate mortar, and passed through a 100-mesh sieve to obtain the anti-fatigue peptide product, which is then vacuum-sealed in an aluminum foil bag; concentration and drying can yield a solid product, which is convenient for storage and transportation.

[0070] Step 10: Quality Inspection: After obtaining the anti-fatigue peptide product, quality inspection is required to ensure product quality; Peptide content determination: The Coomassie Brilliant Blue method is used, with bovine serum albumin as the standard, to determine the mass fraction of peptides in the finished product;

[0071] Molecular weight distribution determination: High-performance gel permeation chromatography (GPC) was used with a TSK gel G2000SWxl column, a mobile phase of 0.1 mol / L phosphate buffer (pH 7.0), a flow rate of 0.5 mL / min, and a column temperature of 30 °C. The molecular weight distribution range was determined by comparison with standards.

[0072] Anti-fatigue activity test: The weighted swimming test of mice was used. Kunming mice were randomly divided into groups and administered different doses of anti-fatigue peptide solution by gavage. The control group was given physiological saline. After gavage for 7 consecutive days, the time to exhaustion of the mice was measured and the rate of extension of exhaustion time was calculated.

[0073] Other indicators: The moisture content (Kal Fischer method), ash content (muffle furnace ignition method), and heavy metal content (atomic absorption spectrometry) of the finished product are tested to ensure compliance with relevant quality standards; various indicators of the obtained anti-fatigue peptide products are tested, including peptide content, molecular weight distribution, and anti-fatigue activity, to ensure that the product quality meets the requirements.

[0074] Example 1:

[0075] Raw material pretreatment: Select 100g of fresh cod skin, wash it with deionized water, cut it into small pieces of 1cm×1cm, put it into a freeze dryer, freeze dry it at -40℃ and vacuum degree of 10Pa until the moisture content is 4%, and then put the dried fish skin into an ultra-micro pulverizer and pulverize it to a particle size of 5μm under nitrogen protection to obtain fish skin powder.

[0076] Preparation of compound enzymatic hydrolysate: Weigh 3g of trypsin, 2g of papain and 1g of bromelain, mix them evenly, add them to 200mL of phosphate buffer solution with pH 7.0, stir evenly to obtain compound enzymatic hydrolysate.

[0077] Enzymatic hydrolysis: Fish skin powder was added to the compound enzymatic hydrolysate at a mass-to-volume ratio of 1:15 (g / mL). The mixture was placed in a constant temperature water bath at 45℃ and magnetic stirring was turned on (150 r / min). Ultrasonic assisted treatment was also used, with an ultrasonic power of 300W, an ultrasonic time of 5s, and an interval of 10s. The total enzymatic hydrolysis time was 3h. During this period, the pH value was adjusted to 7.0±0.2 every 30min.

[0078] Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is transferred to a three-necked flask, placed in a constant temperature water bath at 90°C, stirred (100 r / min) for 15 min, and then placed in an ice bath to cool rapidly to room temperature.

[0079] Centrifugation: The cooled reaction solution was dispensed into centrifuge tubes, and after equilibration, it was placed in a high-speed centrifuge and centrifuged at 8000 r / min and 25℃ for 20 min. The supernatant was then collected.

[0080] Preliminary purification: After filtering the supernatant through a 0.45 μm microporous membrane, it was pumped into a pretreated AB-8 macroporous adsorption resin column (column diameter to column height ratio 1:8) at a flow rate of 2 BV / h, with a sample loading volume of 1 / 3 of the column volume; after sample loading, it was washed with deionized water at a flow rate of 3 BV / h until the absorbance of the eluent at 280 nm was less than 0.02; then it was eluted with 30% ethanol solution at a flow rate of 2 BV / h, and the eluent was collected.

[0081] Ultrafiltration purification: Pour the eluent into the ultrafiltration cup, use the pretreated 3kDa ultrafiltration membrane, and perform ultrafiltration at an operating pressure of 0.2MPa and 30℃. When the volume of the concentrate is 1 / 4 of the original volume, add an equal amount of deionized water and continue ultrafiltration. Repeat twice and collect the permeate.

[0082] Decolorization treatment: Add 1.5% activated carbon (particle size 200 mesh, specific surface area ≥1000m² / g) to the ultrafiltration permeate, and treat for 30 min at 50℃ and stirring speed 150r / min. Then remove the activated carbon by filtering with qualitative filter paper and 0.22μm microporous membrane in sequence.

[0083] Concentration and drying: The decolorized solution was concentrated to 1 / 5 of its original volume by rotary evaporation at 60℃ and 0.08MPa vacuum. The solution was then transferred to a petri dish (0.3-0.5cm thick) and freeze-dried to constant weight at -50℃ and 5Pa vacuum. The powder was then passed through a 100-mesh sieve to obtain 8.6g of the anti-fatigue peptide product.

[0084] Quality testing: The peptide content was determined to be 92% by the Coomassie brilliant blue method; the molecular weight was mainly distributed in the range of 1-3 kDa by the GPC method; the mouse weight-bearing swimming experiment showed that it could prolong the time of exhaustion of mice swimming by 35%; the moisture content was 2.1%, the ash content was 0.8%, and the heavy metal content all met the standards.

[0085] Example 2:

[0086] Raw material pretreatment: Select 100g of fresh salmon skin, wash it with deionized water, cut it into small pieces of 1cm×1cm, put it into a freeze dryer, freeze dry it at -40℃ and vacuum degree of 10Pa until the moisture content is 3%, and then put the dried fish skin into an ultra-micro pulverizer and pulverize it to a particle size of 10μm under nitrogen protection to obtain fish skin micro powder.

[0087] Preparation of compound enzymatic hydrolysate: Weigh 6g of trypsin, 4g of papain and 2g of bromelain, mix them evenly, and add them to 400mL of phosphate buffer solution with pH 7.0. Stir evenly to obtain compound enzymatic hydrolysate.

[0088] Enzymatic hydrolysis: Fish skin powder was added to the compound enzymatic hydrolysate at a mass-to-volume ratio of 1:15 (g / mL). The mixture was placed in a constant temperature water bath at 45℃ and magnetic stirring was turned on (150 r / min). Ultrasonic assisted treatment was also used, with an ultrasonic power of 300W, an ultrasonic time of 5s, and an interval of 10s. The total enzymatic hydrolysis time was 3h. During this period, the pH value was adjusted to 7.0±0.2 every 30min.

[0089] Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is transferred to a three-necked flask, placed in a constant temperature water bath at 90°C, stirred (100 r / min) for 15 min, and then placed in an ice bath to cool rapidly to room temperature.

[0090] Centrifugation: The cooled reaction solution was dispensed into centrifuge tubes, and after equilibration, it was placed in a high-speed centrifuge and centrifuged at 8000 r / min and 25℃ for 20 min. The supernatant was then collected.

[0091] Preliminary purification: After filtering the supernatant through a 0.45 μm microporous membrane, it was pumped into a pretreated AB-8 macroporous adsorption resin column (column diameter to column height ratio 1:8) at a flow rate of 2 BV / h, with a sample loading volume of 1 / 3 of the column volume; after sample loading, it was washed with deionized water at a flow rate of 3 BV / h until the absorbance of the eluent at 280 nm was less than 0.02; then it was eluted with 30% ethanol solution at a flow rate of 2 BV / h, and the eluent was collected.

[0092] Ultrafiltration purification: Pour the eluent into the ultrafiltration cup, use the pretreated 3kDa ultrafiltration membrane, and perform ultrafiltration at an operating pressure of 0.2MPa and 30℃. When the volume of the concentrate is 1 / 4 of the original volume, add an equal amount of deionized water and continue ultrafiltration. Repeat twice and collect the permeate.

[0093] Decolorization treatment: Add 1.5% activated carbon (particle size 200 mesh, specific surface area ≥1000m² / g) to the ultrafiltration permeate, and treat for 30 min at 50℃ and stirring speed 150r / min. Then remove the activated carbon by filtering with qualitative filter paper and 0.22μm microporous membrane in sequence.

[0094] Concentration and drying: The decolorized solution was concentrated to 1 / 5 of its original volume by rotary evaporation at 60℃ and 0.08MPa vacuum. The solution was then transferred to a petri dish (0.3-0.5cm thick) and freeze-dried to constant weight at -50℃ and 5Pa vacuum. The powder was then passed through a 100-mesh sieve to obtain 9.2g of the anti-fatigue peptide product.

[0095] Quality testing: The peptide content was determined to be 93% by the Coomassie brilliant blue method; the molecular weight was mainly distributed in the range of 1-3 kDa by the GPC method; the mouse weight-bearing swimming experiment showed that it could prolong the time of exhaustion of mice swimming by 38%; the moisture content was 1.8%, the ash content was 0.7%, and the heavy metal content all met the standards.

[0096] The following is a brief description of the equipment used in this article, including its structure: Freeze dryer: mainly composed of a freeze drying chamber, condenser, vacuum system, and refrigeration system; the freeze drying chamber is a sealed container used to hold the material to be dried; the condenser is used to capture sublimated water and prevent water from entering the vacuum system; the vacuum system consists of a vacuum pump, etc., to maintain the vacuum environment inside the chamber; the refrigeration system uses a compressor to achieve low-temperature refrigeration, meeting the temperature requirements for pre-freezing and sublimation drying of the material.

[0097] Ultrafine pulverizer: It includes a feeding hopper, a pulverizing chamber, a pulverizing device, and a screening system; the feeding hopper is used to store the material to be pulverized and can be vented with protective gas; the pulverizing chamber has built-in high-speed rotating pulverizing components (such as blades or hammers) that pulverize the material through high-speed impact; the screening system is equipped with a screen of a specific mesh size to screen out micro powder that meets the particle size requirements.

[0098] Thermostatic water bath: It consists of a water bath tank, a heating device, a temperature control system and a stirring device; the water bath tank is a container for holding water and is used to place the reaction vessel; the heating device provides heat to raise the water temperature; the temperature control system maintains a constant water temperature through sensors and controllers; the stirring device can make the water temperature in the tank uniform.

[0099] Ultrasonic generator: mainly includes an ultrasonic transducer, an amplitude transformer, and a control system; the ultrasonic transducer converts electrical energy into mechanical energy to generate ultrasonic vibrations; the amplitude transformer is used to amplify the amplitude and enhance the effect of the ultrasonic waves; the control system can adjust parameters such as ultrasonic power, working time, and interval time; High-speed centrifuge: consists of a rotor, centrifuge chamber, drive system, and control system; the rotor is used to hold centrifuge tubes and can rotate at high speed; the centrifuge chamber is a sealed space that provides the centrifugal environment; the drive system drives the rotor to rotate, generating centrifugal force; the control system is used to set parameters such as speed, centrifugation time, and temperature.

[0100] Chromatography column: It is usually a cylindrical glass or stainless steel container with inlet and outlet and sieve plate at both ends; the sieve plate is used to support the resin and allow liquid to pass through, and the column is used to fill the adsorption resin to achieve the separation and purification of materials.

[0101] Ultrafiltration system: It consists of an ultrafiltration membrane module, a pressurizing device, and inlet / outlet pipelines; the ultrafiltration membrane module is the core, containing an ultrafiltration membrane that retains a specific molecular weight; the pressurizing device provides the operating pressure to allow the liquid to pass through the ultrafiltration membrane; the inlet / outlet pipelines are used for the transport and collection of the solution.

[0102] Rotary evaporator: It consists of an evaporation flask, a heating water bath, a rotating mechanism, a vacuum system, and a condenser. The evaporation flask can rotate to increase the evaporation area; the heating water bath provides heat to promote solution evaporation; the rotating mechanism drives the evaporation flask to rotate; the vacuum system lowers the evaporation temperature and accelerates the evaporation rate; the condenser is used to condense the evaporated vapor and collect it back.

[0103] Buchner funnel and suction flask: The Buchner funnel is a ceramic or glass funnel with many small holes at the bottom, on which filter paper can be placed; the suction flask is a glass bottle connected to the Buchner funnel, and a negative pressure is generated by a suction device to accelerate the filtration speed.

[0104] High-performance gel permeation chromatography (GPC) mainly consists of a solvent delivery system, an injector, a chromatographic column, a detector, and a data processing system. The solvent delivery system delivers the mobile phase; the injector introduces the sample into the system; the chromatographic column is filled with gel packing material to achieve the separation of substances with different molecular weights; the detector detects the separated substances; and the data processing system analyzes and processes the detection results.

[0105] 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 for preparing an anti-fatigue peptide, characterized in that: Includes the following steps: Step 1: Raw material pretreatment: Select fresh deep-sea fish skin, clean and cut it into pieces, freeze-dry it, and then pulverize it under nitrogen protection to obtain fish skin powder; Step 2: Preparation of compound enzymatic hydrolysate: Mix trypsin, papain and bromelain in a certain proportion and add them to phosphate buffer solution to prepare compound enzymatic hydrolysate; Step 3: Enzymatic hydrolysis: Add fish skin powder to the compound enzymatic hydrolysate and carry out the enzymatic hydrolysis reaction with the assistance of ultrasound within a specific temperature range; Step 4: Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is subjected to high-temperature enzyme inactivation and then rapidly cooled; Step 5: Centrifugation: Centrifuge the enzyme-inactivated reaction solution and collect the supernatant; Step 6: Preliminary purification: The supernatant is preliminarily purified by passing it through a macroporous adsorption resin column, and the eluent is collected; Step 7: Ultrafiltration purification: The eluent is purified by ultrafiltration through an ultrafiltration membrane, and the permeate is collected; Step 8: Decolorization treatment: Add activated carbon to the ultrafiltration permeate for decolorization, and filter to remove the activated carbon; Step 9: Concentration and Drying: Concentrate the decolorized solution and freeze-dry it to obtain the anti-fatigue peptide product; Step 10: Quality Inspection: Test the finished product for indicators such as peptide content, molecular weight distribution, and anti-fatigue activity.

2. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: The freeze-drying conditions described in step one are as follows: first, pre-freeze at -42 to -38℃ for 1.5-2.5 hours, then freeze-dry under a vacuum of 8-12 Pa for 10-18 hours until the moisture content is less than 5%; the rotation speed of the ultrafine pulverizer is 2800-3200 r / min, and the time is 12-25 min. The resulting fish skin micro powder has a particle size of 4-12 μm and is screened through an 1800-2200 mesh sieve.

3. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: In step two, the mass ratio of trypsin, papain, and bromelain is 2.8-3.2:1.8-2.2:1, the pH of the phosphate buffer solution is 6.8-7.2, the total enzyme concentration in the compound enzymatic hydrolysate is 2.2-2.8%, and the enzymatic hydrolysate is stored at 2-6℃ for no more than 24 hours.

4. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: In step three, the mass-to-volume ratio of fish skin powder to compound enzymatic hydrolysate is 1:13-17 (g / mL), the enzymatic hydrolysis temperature is 42-48℃, and the total enzymatic hydrolysis time is 2.5-3.5h. The ultrasonic auxiliary parameters are: power 280-320W, ultrasonic time 4-6s, interval time 8-12s, and the pH is adjusted to 6.8-7.2 every 25-35min during the reaction.

5. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: The conditions for high-temperature enzyme inactivation in step four are heating in a water bath at 88-92℃ for 12-18 minutes, followed by rapid cooling in an ice bath to room temperature of 23-27℃, with continuous stirring during the cooling process.

6. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: The parameters for centrifugation in step five are: rotation speed 7500-8500 r / min, time 18-22 min, temperature 23-27℃, and the volume of the reaction liquid in the centrifuge tube does not exceed 2 / 3 of its capacity.

7. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: The macroporous adsorption resin mentioned in step six is ​​type AB-8, with a column diameter to column height ratio of 1:7-9; the sample loading flow rate is 1.8-2.2 BV / h, and the sample loading volume is 1 / 4-1 / 2 of the column volume; first, rinse with deionized water at a flow rate of 2.8-3.2 BV / h until the absorbance of the effluent at 280 nm is less than 0.02, and then elute with 28-32% ethanol solution at a flow rate of 1.8-2.2 BV / h.

8. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: The ultrafiltration membrane described in step seven has a molecular weight cutoff of 2.5-3.5 kDa, an ultrafiltration operating pressure of 0.18-0.22 MPa, and a temperature of 28-32℃. When the volume of the concentrate is 1 / 5-1 / 3 of the original volume, add an equal amount of deionized water and continue ultrafiltration. Repeat the operation 1-3 times.

9. The method for preparing an anti-fatigue peptide according to claim 1, characterized in that: The activated carbon mentioned in step eight has a particle size of 180-220 mesh and a specific surface area ≥900-1100 m² / g, and the amount added is 1.2-1.8% of the mass of the ultrafiltration permeate; the decolorization conditions are stirring at 48-52℃ for 25-35 min at a stirring speed of 130-170 r / min, and the filtration is carried out sequentially using qualitative filter paper and 0.2-0.24 μm microporous filter membrane; The concentration in step nine is carried out by rotary evaporation at 58-62℃ and a vacuum of 0.07-0.09MPa, concentrating to 1 / 6-1 / 4 of the original volume; the freeze-drying conditions are -52 to -48℃ and a vacuum of 4-6Pa, drying to constant weight, and the resulting product is vacuum-sealed after passing through a 90-110 mesh sieve.

Images