A method for preparing high-chelation-rate small molecule chelated peptides in functional foods

Abstract

This invention relates to the field of functional food active ingredient preparation technology, specifically a method for preparing high-chelation-rate small molecule chelated peptides in functional foods, comprising the following steps: taking animal and plant composite protein raw materials, pulverizing and sieving them, and then adding purified water to prepare a protein slurry with a fixed solid content; performing a three-stage targeted enzymatic hydrolysis on the protein slurry, sequentially performing neutral enzymatic hydrolysis, acidic enzymatic hydrolysis, and endopeptidase hydrolysis, with each stage of enzymatic hydrolysis using corresponding buffer solutions to adjust parameters, and the stages being directly connected without static settling. This invention employs a three-stage targeted enzymatic hydrolysis process, using neutral protease, acidic protease, and papain for precise stepwise hydrolysis, specifically breaking protein peptide bonds to achieve targeted enrichment of target peptides. The proportion of high-activity small molecule peptides (500-1000 Da) is ≥82%, an improvement of more than 49% compared to traditional processes, providing sufficient highly active binding sites for the chelation reaction and improving the efficiency of the chelation reaction from the source.

Description

Technical Field

[0001] This invention relates to the field of functional food active ingredient preparation technology, specifically a method for preparing high-chelation-rate small molecule chelated peptides in functional foods. Background Technology

[0002] Small molecule chelated peptides are products formed by chelating small molecule active peptides obtained from the enzymatic hydrolysis of animal and plant proteins with essential trace elements. They combine the advantages of small molecule peptides, such as easy absorption and non-antigenicity, with the physiological activity of trace elements. They can simultaneously supplement amino acids and minerals, and their bioavailability is much higher than that of free inorganic salts. They are widely used in functional foods, dietary supplements and other fields.

[0003] However, existing methods for preparing small molecule chelated peptides generally have significant drawbacks: traditional enzymatic hydrolysis processes often employ single enzymes or simple compound enzymes for one-step hydrolysis, resulting in insufficient protein hydrolysis, a wide molecular weight distribution of peptides, and a proportion of target high-activity small molecule peptides (500-1000 Da) generally below 60%, failing to provide sufficient high-activity sites for the chelation reaction; conventional chelation processes often employ a single stirred reaction, resulting in low chelation efficiency, with the final product chelation rate generally below 40%, and high free metal ion content, which not only reduces bioavailability but also poses food safety risks; existing purification processes are cumbersome, often employing single membrane separation or alcohol precipitation processes, resulting in incomplete impurity removal, low retention of active peptides, and difficulty in achieving continuous, large-scale industrial production; existing process parameter control is crude, batch-to-batch stability is poor, and products are prone to stratification and precipitation during shelf life, failing to meet the standardized quality control requirements of functional foods.

[0004] In summary, a method for preparing high-chelation-rate small molecule chelated peptides in functional foods is needed to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing high-chelation-rate small molecule chelated peptides in functional foods, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a method for preparing high-chelation-rate small molecule chelated peptides in functional foods, comprising the following steps: S1. Take animal and plant composite protein raw materials, crush and sieve them, and then add purified water to prepare a protein slurry with fixed solid content; S2. The protein slurry is subjected to a three-stage directional enzymatic hydrolysis, which is carried out in sequence: neutral enzymatic hydrolysis, acidic enzymatic hydrolysis, and endopeptidase hydrolysis. The parameters of each enzymatic hydrolysis are adjusted by the corresponding buffer solution, and the segments are directly connected without standing. S3. The enzyme hydrolysate is inactivated by high temperature and then rapidly cooled to the chelation reaction temperature. S4. Add chelating agent and metal ion source to the cooled enzymatic hydrolysate, and carry out chelation reaction in a closed reactor with coupled ultrasonic and microwave dual fields. S5. The chelate solution is purified by three-stage membrane separation and cation exchange resin in series to remove impurities and free ions; S6. The purified liquid is subjected to vacuum low-temperature concentration and gradient vacuum freeze-drying in sequence to obtain solid chelated peptides; S7. After sieving the solid chelated peptides, they are aseptically vacuum packaged to obtain a high-chelation-rate small molecule chelated peptide product.

[0007] Preferably, in the raw material pretreatment step S1, the animal and plant composite protein raw material is a blend of soy protein isolate and cod skin collagen in a weight ratio of 70-75 parts and 25-30 parts, respectively, wherein the soy protein isolate has a protein content of ≥90% and the cod skin collagen has a protein content of ≥92%. After the raw materials are pulverized, they are passed through a 100-mesh standard pharmaceutical sieve. The resistivity of the purified water is ≥18.2 MΩ·cm. The amount of purified water added is 480-520 parts of the total mass of the composite protein raw materials. The protein slurry is prepared at a temperature of 45-50℃, with a stirring speed of 200-220 r / min and a mixing time of 30 min. The solid content of the protein slurry is precisely controlled to 15%-16%.

[0008] Preferably, in step S2, the multi-stage directional enzymatic hydrolysis step, the neutral enzymatic hydrolysis section uses a neutral protease with an enzyme activity ≥100,000 U / g, the enzyme addition amount is 2.2%-2.4% of the dry weight of the composite protein raw material, the pH of the system is adjusted to 7.2-7.3 using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, the enzymatic hydrolysis temperature is 52-54℃, and the enzymatic hydrolysis time is 100-110 min; The acidic enzymatic hydrolysis section uses acidic protease with an enzyme activity ≥80,000 U / g. The amount of enzyme added is 1.7%-1.9% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 4.2-4.3 using citrate-sodium citrate buffer solution. The enzymatic hydrolysis temperature is 47-49℃ and the enzymatic hydrolysis time is 80-90 min. The endopeptidase hydrolysis segment uses papain with an enzyme activity ≥120,000 U / g. The enzyme addition amount is 1.2%-1.4% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 5.7-5.8 using an acetate-sodium acetate buffer solution. The hydrolysis temperature is 57-59℃ and the hydrolysis time is 70-80 min.

[0009] Preferably, the enzymatic hydrolysis and inactivation step S3 uses a shell-and-tube heat exchanger to raise the temperature, with the heating rate controlled at 8-10℃ / min, and the system is heated to 93-95℃ and held for 13-15 minutes to complete the inactivation. After inactivation, a plate cooler is used for rapid cooling, with the cooling rate controlled at 12-15℃ / min, until the system temperature drops to 42-44℃.

[0010] Preferably, in step S4, the dual-field coupling chelation step, the chelation aid is a compound of sodium phytate and anhydrous citric acid in a ratio of 35-38 parts by mass and 22-25 parts by mass, wherein the purity of sodium phytate is ≥98%, the purity of anhydrous citric acid is ≥99%, and the total amount of the chelation aid added is 3.2%-3.4% of the dry weight of the enzymatic hydrolysate. The metal ion source is ferrous gluconate with a purity ≥98.5%, and the amount added is 4.2%-4.4% of the dry weight of the enzymatic hydrolysate. The reactor is airtight and isolated from air. The ultrasonic power is controlled at 300-320W and the ultrasonic frequency is 26-27kHz. The microwave power is controlled at 120-140W and the microwave frequency is 2450MHz. The chelation system temperature is 57-59℃ and the chelation reaction time is 110-120min.

[0011] Preferably, in the purification step S5, the components used in the three-stage membrane separation are all made of polyvinylidene fluoride, namely a 100nm microfiltration membrane, a 500Da ultrafiltration membrane, and a 200Da nanofiltration membrane. The microfiltration membrane operates at a pressure of 0.35-0.4 MPa and a temperature of 37-40℃. The ultrafiltration membrane operates at a pressure of 0.65-0.7 MPa and a temperature of 32-35℃. The nanofiltration membrane operates at a pressure of 1.1-1.2 MPa and a temperature of 27-30°C. The membrane separation retentate is passed into a styrene-based cation exchange resin column with a height-to-diameter ratio of 8:1 and a flow rate of 2-3 BV / h to complete the removal of free ions.

[0012] Preferably, in step S6, the gradient low-temperature drying step, the reduced-pressure low-temperature concentration is performed using a rotary evaporator with a vacuum degree of -0.075 MPa to -0.07 MPa, a concentration temperature of 47-49°C, and concentration until the solid content of the liquid is 32%-34%. The gradient vacuum freeze drying uses a vacuum freeze dryer. The pre-freezing stage temperature is -55℃ to -53℃, the pre-freezing time is 3h, the sublimation stage vacuum degree is 16-18Pa, the sublimation temperature is -20℃ to -15℃, the desorption stage temperature is 25-30℃, and the total drying time is 13-14h.

[0013] Preferably, the aseptic sieving and packaging step S7 uses a 120-mesh stainless steel standard sieve with a sieving speed of 150-160 r / min. The packaging uses aluminum-plastic composite film with a vacuum degree of -0.09MPa to -0.085MPa. The packaging environment is a Class 10,000 clean area with an ambient temperature of 22-24℃ and an ambient humidity of 45%-50%.

[0014] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention adopts a three-stage directional enzymatic hydrolysis process, which precisely hydrolyzes proteins stepwise with neutral protease, acidic protease, and papain, specifically breaking protein peptide bonds to achieve targeted enrichment of target peptides. The proportion of highly active small molecule peptides with 500-1000 Da is ≥82%, which is more than 49% higher than traditional processes, providing sufficient highly active binding sites for chelation reactions and improving chelation reaction efficiency from the source. The invention employs an ultrasonic-microwave dual-field coupled chelation process, which breaks the reaction mass transfer barrier through the cavitation effect of the ultrasonic field, and the thermal and non-thermal effects of the microwave field synergistically activate peptide chelation sites, increasing the chelation rate to more than 45%, which is more than 18% higher than traditional processes. At the same time, it significantly shortens the reaction time and reduces energy consumption. The invention uses a three-stage membrane separation and cation exchange resin series purification process to remove large molecule proteins, undigested impurities, and free metal ions in stages, with a free ion removal rate of ≥95%, resulting in high product purity. At the same time, the entire process is treated at low temperatures, and the active peptide retention rate is ≥90%. The process can be operated continuously and is suitable for industrial scale-up.

[0015] The three-stage membrane separation utilizes different molecular weight cutoff gradients to remove large protein molecules, peptide fragments, and some free ions in a phased manner, avoiding membrane pore blockage or loss of active peptides caused by an excessively wide cutoff range, as seen in single-membrane separation. Subsequent tandem cation exchange resins utilize ion exchange to specifically remove residual free metal ions. Compared to single-membrane separation or single resin columns, this tandem process achieves deep and synergistic removal of impurities and free ions while ensuring high retention of small-molecule active peptides.

[0016] Meanwhile, the process parameters of this invention are precisely controllable throughout the entire process, with direct connection between stages without static time, avoiding loss of activity and microbial contamination of intermediate products. The batch-to-batch product chelation rate RSD is <5%, the particle size change rate after 30 days of storage at 25℃ in the dark is <10%, the stratification rate is <3%, and the shelf life stability is excellent. All raw materials are food grade and comply with relevant national functional food standards such as GB14880-2012, ensuring high safety. Attached Figure Description

[0017] Figure 1 This is a line graph showing the performance test data of an embodiment of the present invention. Detailed Implementation

[0018] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] I. Raw materials: All raw materials used in this invention are commercially available food-grade and comply with relevant national food quality and safety standards, specifically: Soy protein isolate: food grade, protein content ≥90%, conforming to GB20371-2016; Cod skin collagen: food grade, protein content ≥92%, conforming to GB31645-2018; Neutral protease: food grade, enzyme activity ≥100,000 U / g; Acidic protease: food grade, enzyme activity ≥80,000 U / g; Papain: Food grade, enzyme activity ≥120,000 U / g; Sodium phytate: food grade, purity ≥98%, conforming to GB1886.250-2016; Anhydrous citric acid: food grade, purity ≥99%, conforming to GB1886.235-2016; Ferrous gluconate: food grade, purity ≥98.5%, conforming to GB1903.12-2015; Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium citrate, acetic acid, and sodium acetate: all are food-grade buffers that meet national food additive standards; Polyvinylidene fluoride membrane module: food grade, conforming to GB4806.7-2016; Styrene-based cation exchange resin: food grade, conforming to GB1886.300-2019; Aluminum-plastic composite film: food grade, conforming to GB4806.7-2016.

[0020] II. Process: Based on the above-mentioned raw materials, this invention proposes a method for preparing high-chelation-rate small molecule chelated peptides in functional foods, comprising the following steps: S1. Take animal and plant composite protein raw materials, crush and sieve them, and then add purified water to prepare a protein slurry with fixed solid content; S2. The protein slurry is subjected to a three-stage directional enzymatic hydrolysis, which is carried out in sequence: neutral enzymatic hydrolysis, acidic enzymatic hydrolysis, and endopeptidase hydrolysis. The parameters of each enzymatic hydrolysis are adjusted by the corresponding buffer solution, and the segments are directly connected without standing. S3. The enzyme hydrolysate is inactivated by high temperature and then rapidly cooled to the chelation reaction temperature. S4. Add chelating agent and metal ion source to the cooled enzymatic hydrolysate, and carry out chelation reaction in a closed reactor with coupled ultrasonic and microwave dual fields. S5. The chelate solution is purified by three-stage membrane separation and cation exchange resin in series to remove impurities and free ions; S6. The purified liquid is subjected to vacuum low-temperature concentration and gradient vacuum freeze-drying in sequence to obtain solid chelated peptides; S7. After sieving the solid chelated peptides, they are aseptically vacuum packaged to obtain a high-chelation-rate small molecule chelated peptide product.

[0021] It should also be noted that in step S1, the raw material pretreatment step, the animal and plant composite protein raw material is a blend of soy protein isolate and cod skin collagen in a mass ratio of 70-75 parts and 25-30 parts, respectively, with the soy protein isolate having a protein content of ≥90% and the cod skin collagen having a protein content of ≥92%. After the raw materials are pulverized, they are passed through a 100-mesh standard pharmaceutical sieve. The resistivity of the purified water is ≥18.2 MΩ·cm. The amount of purified water added is 480-520 parts of the total mass of the compound protein raw materials. The temperature for preparing the protein slurry is controlled at 45-50℃, the stirring speed is 200-220r / min, the stirring time is 30min, and the solid content of the protein slurry is precisely controlled to 15%-16%.

[0022] It should also be noted that in step S2, the multi-stage directional enzymatic hydrolysis step, the neutral enzymatic hydrolysis section uses neutral protease with an enzyme activity ≥100,000 U / g. The amount of enzyme added is 2.2%-2.4% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 7.2-7.3 using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, the enzymatic hydrolysis temperature is 52-54℃, and the enzymatic hydrolysis time is 100-110 min. The acidic enzymatic hydrolysis section uses acidic protease with an enzyme activity ≥80,000 U / g. The amount of enzyme added is 1.7%-1.9% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 4.2-4.3 using citrate-sodium citrate buffer solution. The enzymatic hydrolysis temperature is 47-49℃ and the enzymatic hydrolysis time is 80-90 min. The endopeptidation segment uses papain with an enzyme activity ≥120,000 U / g. The amount of enzyme added is 1.2%-1.4% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 5.7-5.8 using an acetate-sodium acetate buffer solution. The enzymatic hydrolysis temperature is 57-59℃ and the enzymatic hydrolysis time is 70-80 min.

[0023] It should also be noted that in step S3, the enzymatic hydrolysis and inactivation step, a shell-and-tube heat exchanger is used to raise the temperature. The heating rate is controlled at 8-10℃ / min. The system is heated to 93-95℃ and held for 13-15 minutes to complete the inactivation. After inactivation, a plate cooler is used for rapid cooling, with the cooling rate controlled at 12-15℃ / min, until the system temperature drops to 42-44℃.

[0024] It should also be noted that in step S4, the dual-field coupling chelation step, the chelation aid is a mixture of sodium phytate and anhydrous citric acid in a mass ratio of 35-38 parts and 22-25 parts, respectively. The purity of sodium phytate is ≥98%, the purity of anhydrous citric acid is ≥99%, and the total amount of chelation aid added is 3.2%-3.4% of the dry weight of the enzymatic hydrolysate. The metal ion source used is ferrous gluconate with a purity ≥98.5%, and the amount added is 4.2%-4.4% of the dry weight of the enzymatic hydrolysate. The reactor is sealed to isolate it from air. The ultrasonic power is controlled at 300-320W and the ultrasonic frequency is 26-27kHz. The microwave power is controlled at 120-140W and the microwave frequency is 2450MHz. The chelation system temperature is 57-59℃ and the chelation reaction time is 110-120min.

[0025] It should also be noted that in step S5, the three-stage membrane separation uses polyvinylidene fluoride components, namely a 100nm microfiltration membrane, a 500Da ultrafiltration membrane, and a 200Da nanofiltration membrane. The microfiltration membrane operates at a pressure of 0.35-0.4 MPa and a temperature of 37-40℃. The ultrafiltration membrane operates at a pressure of 0.65-0.7 MPa and a temperature of 32-35℃. Nanofiltration membranes operate at a pressure of 1.1-1.2 MPa and a temperature of 27-30℃. The membrane separation retentate is passed into a styrene-based cation exchange resin column with a height-to-diameter ratio of 8:1 and a flow rate of 2-3 BV / h to complete the removal of free ions.

[0026] It should also be noted that in step S6, the gradient low-temperature drying step, the reduced pressure low-temperature concentration uses a rotary evaporator with a vacuum degree of -0.075MPa to -0.07MPa, a concentration temperature of 47-49℃, and concentration until the solid content of the liquid is 32%-34%. Gradient vacuum freeze drying uses a vacuum freeze dryer. The pre-freezing stage temperature is -55℃ to -53℃, and the pre-freezing time is 3 hours. The sublimation stage vacuum degree is 16-18 Pa, and the sublimation temperature is -20℃ to -15℃. The desorption stage temperature is 25-30℃, and the total drying time is 13-14 hours.

[0027] It should also be noted that the aseptic sieving and packaging step S7 uses a 120-mesh stainless steel standard sieve with a sieving speed of 150-160 r / min. The packaging uses aluminum-plastic composite film with a vacuum degree of -0.09MPa to -0.085MPa. The packaging environment is a Class 10,000 clean area with an ambient temperature of 22-24℃ and an ambient humidity of 45%-50%.

[0028] Example 1: The formula and process parameters for this example are as follows: S1. Compound protein raw materials: 72 parts soy protein isolate, 28 parts cod skin collagen, purified water added at 500 parts of the total mass of the compound protein raw materials, protein slurry solid content 15.5%, mixing temperature 48℃, stirring speed 210r / min; S2. Neutral enzymatic hydrolysis stage: The amount of neutral protease added is 2.3% of the dry weight of the composite protein raw material, pH 7.25, hydrolysis temperature 53℃, and hydrolysis time 105 min; Acidic enzymatic hydrolysis stage: The amount of acidic protease added is 1.8% of the dry weight of the composite protein raw material, pH 4.25, hydrolysis temperature 48℃, and hydrolysis time 85 min; Endolytic enzymatic hydrolysis stage: The amount of papain added is 1.3% of the dry weight of the composite protein raw material, pH 5.75, hydrolysis temperature 58℃, and hydrolysis time 75 min; S3. Heating rate 9℃ / min, inactivation temperature 94℃, holding time 14min; cooling rate 13℃ / min, cooling down to 43℃; S4. Chelating aids: 36 parts sodium phytate, 24 parts anhydrous citric acid, with a total addition amount of 3.3% of the dry weight of the enzymatic hydrolysate; 4.3% ferrous gluconate addition amount of ferrous gluconate; ultrasonic power 310W, frequency 26.5kHz; microwave power 130W, frequency 2450MHz; chelation temperature 58℃, reaction time 115min; S5. Microfiltration membrane operating pressure 0.38 MPa, temperature 38℃; ultrafiltration membrane operating pressure 0.68 MPa, temperature 33℃; nanofiltration membrane operating pressure 1.15 MPa, temperature 28℃; resin column flow rate 2.5 BV / h; S6. Concentration vacuum degree -0.072MPa, temperature 48℃, concentrated to solid content 33%; freeze-drying pre-freezing temperature -54℃, pre-freezing for 3h; sublimation stage vacuum degree 17Pa, temperature -18℃; desorption stage temperature 28℃, total drying time 13.5h. S7. Screening speed 155r / min, packaging vacuum degree -0.088MPa, packaging ambient temperature 23℃, humidity 48%.

[0029] Example 2: The formula and process parameters for this example are as follows: S1. Compound protein raw materials: 70 parts soy protein isolate, 30 parts cod skin collagen, purified water added at 480 parts of the total mass of the compound protein raw materials, protein slurry solid content 15%, mixing temperature 45℃, stirring speed 200r / min; S2. Neutral enzymatic hydrolysis stage: The amount of neutral protease added is 2.2% of the dry weight of the composite protein raw material, pH 7.2, enzymatic hydrolysis temperature 52℃, and enzymatic hydrolysis time 100 min; Acidic enzymatic hydrolysis stage: The amount of acidic protease added is 1.7% of the dry weight of the composite protein raw material, pH 4.2, enzymatic hydrolysis temperature 47℃, and enzymatic hydrolysis time 80 min; Endopeptidase enzymatic hydrolysis stage: The amount of papain added is 1.2% of the dry weight of the composite protein raw material, pH 5.7, enzymatic hydrolysis temperature 57℃, and enzymatic hydrolysis time 70 min; S3. Heating rate 8℃ / min, inactivation temperature 93℃, holding time 13min; cooling rate 12℃ / min, cooling down to 42℃; S4. Chelating aids: 35 parts sodium phytate, 25 parts anhydrous citric acid, with a total addition amount of 3.2% of the dry weight of the enzymatic hydrolysate; 4.2% of the dry weight of the enzymatic hydrolysate added; ultrasonic power 300W, frequency 26kHz; microwave power 120W, frequency 2450MHz; chelation temperature 57℃, reaction time 110min; S5. Microfiltration membrane operating pressure 0.35MPa, temperature 37℃; ultrafiltration membrane operating pressure 0.65MPa, temperature 32℃; nanofiltration membrane operating pressure 1.1MPa, temperature 27℃; resin column flow rate 2BV / h; S6. Concentration vacuum degree -0.075MPa, temperature 47℃, concentration to solid content 32%; freeze drying pre-freezing temperature -55℃, pre-freezing for 3h; sublimation stage vacuum degree 16Pa, temperature -20℃; desorption stage temperature 25℃, total drying time 13h. S7. Screening speed 150r / min, packaging vacuum degree -0.09MPa, packaging ambient temperature 22℃, humidity 45%.

[0030] Example 3: The formula and process parameters for this example are as follows: S1. Compound protein raw materials: 75 parts soy protein isolate, 25 parts cod skin collagen, purified water added at 520 parts of the total mass of the compound protein raw materials, protein slurry solid content 16%, mixing temperature 50℃, stirring speed 220r / min; S2. Neutral enzymatic hydrolysis stage: The amount of neutral protease added is 2.4% of the dry weight of the composite protein raw material, pH 7.3, enzymatic hydrolysis temperature 54℃, and enzymatic hydrolysis time 110 min; Acidic enzymatic hydrolysis stage: The amount of acidic protease added is 1.9% of the dry weight of the composite protein raw material, pH 4.3, enzymatic hydrolysis temperature 49℃, and enzymatic hydrolysis time 90 min; Endopeptidase hydrolysis stage: The amount of papain added is 1.4% of the dry weight of the composite protein raw material, pH 5.8, enzymatic hydrolysis temperature 59℃, and enzymatic hydrolysis time 80 min; S3. Heating rate 10℃ / min, inactivation temperature 95℃, holding time 15min; cooling rate 15℃ / min, cooling down to 44℃; S4. Chelating aids: 38 parts sodium phytate, 22 parts anhydrous citric acid, with a total addition amount of 3.4% of the dry weight of the enzymatic hydrolysate; 4.4% of the dry weight of the enzymatic hydrolysate added; ultrasonic power 320W, frequency 27kHz; microwave power 140W, frequency 2450MHz; chelation temperature 59℃, reaction time 120min; S5. Microfiltration membrane operating pressure 0.4 MPa, temperature 40℃; ultrafiltration membrane operating pressure 0.7 MPa, temperature 35℃; nanofiltration membrane operating pressure 1.2 MPa, temperature 30℃; resin column flow rate 3 BV / h; S6. Concentration vacuum degree -0.07MPa, temperature 49℃, concentration to solid content 34%; freeze drying pre-freezing temperature -53℃, pre-freezing for 3h; sublimation stage vacuum degree 18Pa, temperature -15℃; desorption stage temperature 30℃, total drying time 14h. S7. Screening speed 160r / min, packaging vacuum degree -0.085MPa, packaging ambient temperature 24℃, humidity 50%.

[0031] Example 4: The formula and process parameters for this example are as follows: S1. Compound protein raw materials: 73 parts soy protein isolate, 27 parts cod skin collagen, purified water added at 490 parts of the total mass of the compound protein raw materials, protein slurry solid content 15.2%, mixing temperature 46℃, stirring speed 205r / min; S2. Neutral enzymatic hydrolysis stage: The amount of neutral protease added is 2.25% of the dry weight of the composite protein raw material, pH 7.22, hydrolysis temperature 52.5℃, and hydrolysis time 102 min; Acidic enzymatic hydrolysis stage: The amount of acidic protease added is 1.75% of the dry weight of the composite protein raw material, pH 4.22, hydrolysis temperature 47.5℃, and hydrolysis time 82 min; Endolytic enzymatic hydrolysis stage: The amount of papain added is 1.25% of the dry weight of the composite protein raw material, pH 5.72, hydrolysis temperature 57.5℃, and hydrolysis time 72 min; S3. Heating rate 8.5℃ / min, inactivation temperature 93.5℃, holding time 13.5min; cooling rate 12.5℃ / min, cooling to 42.5℃; S4. Chelating aids: 35.5 parts sodium phytate and 24.5 parts anhydrous citric acid, with a total addition amount of 3.25% of the dry weight of the enzymatic hydrolysate; 4.25% ferrous gluconate was added; ultrasonic power 305W, frequency 26.2kHz; microwave power 125W, frequency 2450MHz; chelation temperature 57.5℃, reaction time 112min; S5. Microfiltration membrane operating pressure 0.36 MPa, temperature 37.5℃; ultrafiltration membrane operating pressure 0.66 MPa, temperature 32.5℃; nanofiltration membrane operating pressure 1.12 MPa, temperature 27.5℃; resin column flow rate 2.2 BV / h; S6. Concentration vacuum degree -0.074MPa, temperature 47.5℃, concentrated to solid content 32.5%; freeze-drying pre-freezing temperature -54.5℃, pre-freezing for 3h; sublimation stage vacuum degree 16.5Pa, temperature -19℃; desorption stage temperature 26℃, total drying time 13.2h; S7. Screening speed 152 r / min, packaging vacuum degree -0.089 MPa, packaging ambient temperature 22.5℃, humidity 46%.

[0032] Example 5: The formula and process parameters for this example are as follows: S1. Composite protein raw materials: 74 parts soy protein isolate, 26 parts cod skin collagen, purified water added at 510 parts of the total mass of composite protein raw materials, protein slurry solid content 15.8%, mixing temperature 49℃, stirring speed 215r / min; S2. Neutral enzymatic hydrolysis stage: The amount of neutral protease added is 2.35% of the dry weight of the composite protein raw material, pH 7.28, hydrolysis temperature 53.5℃, and hydrolysis time 108 min; Acidic enzymatic hydrolysis stage: The amount of acidic protease added is 1.85% of the dry weight of the composite protein raw material, pH 4.28, hydrolysis temperature 48.5℃, and hydrolysis time 88 min; Endolytic enzymatic hydrolysis stage: The amount of papain added is 1.35% of the dry weight of the composite protein raw material, pH 5.78, hydrolysis temperature 58.5℃, and hydrolysis time 78 min; S3. Heating rate 9.5℃ / min, inactivation temperature 94.5℃, holding time 14.5min; cooling rate 14℃ / min, cooling to 43.5℃; S4. Chelating aids: 37.5 parts sodium phytate and 22.5 parts anhydrous citric acid, with a total addition amount of 3.35% of the dry weight of the enzymatic hydrolysate; 4.35% ferrous gluconate was added; ultrasonic power 315W, frequency 26.8kHz; microwave power 135W, frequency 2450MHz; chelation temperature 58.5℃, reaction time 118min; S5. Microfiltration membrane operating pressure 0.39 MPa, temperature 39℃; ultrafiltration membrane operating pressure 0.69 MPa, temperature 34℃; nanofiltration membrane operating pressure 1.18 MPa, temperature 29℃; resin column flow rate 2.8 BV / h; S6. Concentration vacuum degree -0.071MPa, temperature 48.5℃, concentrated to solid content 33.5%; freeze-drying pre-freezing temperature -53.5℃, pre-freezing for 3h; sublimation stage vacuum degree 17.5Pa, temperature -16℃; desorption stage temperature 29℃, total drying time 13.8h; S7. Screening speed 158r / min, packaging vacuum degree -0.086MPa, packaging ambient temperature 23.5℃, humidity 49%.

[0033] Comparative Example 1: This comparative example omits the three-stage directional enzymatic hydrolysis process and uses a neutral protease for single-step enzymatic hydrolysis. The amount of enzyme added is 5.4% of the dry weight of the composite protein raw material, the hydrolysis temperature is 53℃, the pH is 7.25, and the total hydrolysis time is 265 min. All other parameters are completely consistent with those of Example 1.

[0034] Comparative Example 2: This comparative example omits the ultrasonic-microwave dual-field coupling chelation process and only uses conventional mechanical stirring chelation. The stirring speed is 350 r / min, the chelation temperature is 58℃, and the reaction time is 115 min. The remaining parameters are completely consistent with those of Example 1.

[0035] Comparative Example 3: This comparative example omits the three-stage membrane separation and cation exchange resin series purification process, and only uses a 500Da ultrafiltration membrane for single-stage purification. The operating pressure is 0.68MPa and the temperature is 33℃. All other parameters are completely consistent with those of Example 1.

[0036] Comparative Example 4: The composite protein raw materials of this comparative example are 90 parts of soy protein isolate and 10 parts of cod skin collagen, and the remaining parameters are completely consistent with those of Example 1.

[0037] Comparative Example 5: The chelation reaction temperature of this comparative example was set to 70°C, and the other parameters were completely consistent with those of Example 1.

[0038] III. Performance Testing: The performance of the small molecule chelate peptides prepared in Examples 1-5 and Comparative Examples 1-5 was tested using the following methods: a. Percentage of target peptides in the range of 500-1000 Da: Detected using high-performance gel filtration chromatography (GPC), the detection method was performed according to Appendix A of GB / T22729-2008 "Marine Fish Oligopeptide Powder" for determination of molecular weight distribution; b. Chelation rate: The 1,000-phenanthroline colorimetric method was used for detection, following the spectrophotometric method of GB5009.90-2016 "National Food Safety Standard - Determination of Iron in Food". The formula for calculating the chelation rate is: Chelation rate = (chelated iron content / total iron content) × 100%; c. Free ferrous ion content: Detected by atomic absorption spectrophotometry, in accordance with GB5009.90-2016 "National Food Safety Standard - Determination of Iron in Food" atomic absorption spectrometry. d. Active peptide retention rate: The content of active peptides before and after drying was determined using the Folin-phenol method. The detection method was performed according to Appendix B of GB / T22729-2008 "Marine Fish Oligopeptide Powder" for the determination of oligopeptide content. The formula for calculating the active peptide retention rate is as follows: Active peptide retention rate = (active peptide content after drying / active peptide content before drying) × 100%; e. 30d Particle Size Change Rate: The particle size before and after 30 days of storage at 25℃ in the dark was measured using a dynamic light scattering particle size analyzer (DLS). The measurement method was performed according to GB / T19627-2005 "Particle Size Analysis - Photon Correlation Spectroscopy". The formula for calculating the particle size change rate is as follows: Particle size change rate = |Average particle size after placement - Average particle size before placement| / Average particle size before placement × 100%; f. 30d Separation Rate: The finished product was prepared into a 10mg / mL aqueous solution, centrifuged at 3000rpm for 15min, and the percentage of separated volumes was measured. The detection method was performed according to the stability-related testing requirements of GB7101-2021 "National Food Safety Standard for Beverages". The formula for calculating the separation rate is: Separation rate = (Separation volume / Total solution volume) × 100%.

[0039] The test results are shown in Table 1: Table 1: Performance Test Data of Examples and Comparative Examples IV. Analysis Conclusion: Based on the test data in Table 1, we can see that: Comparative Example 1 used a single-step enzymatic hydrolysis, and the proportion of the target peptide was only 52.3%, which was 37.9% lower than that of Example 1. This proves that the three-stage targeted enzymatic hydrolysis process of the present invention can significantly improve the enrichment efficiency of the target small molecule peptides and provide sufficient highly active sites for the chelation reaction.

[0040] Comparative Example 2 used conventional stirring chelation, and the chelation rate was only 32.5%, which was 30.6% lower than that of Example 1. The content of free ferrous ions increased by 369.9%, which proves that the ultrasonic-microwave dual-field coupling chelation process of the present invention can significantly improve the chelation efficiency and reduce the content of free metal ions.

[0041] Comparative Example 3, which used single-stage ultrafiltration purification, showed a 7.5% decrease in active peptide retention and a 267.2% increase in free ferrous ion content, resulting in a significant decrease in product stability. This demonstrates that the three-stage membrane separation and resin tandem purification process of this invention can effectively improve product purity and activity retention, and enhance product stability.

[0042] In Comparative Example 4, the raw material ratio exceeded the range, and the proportion of the target peptide and the chelation rate both decreased significantly. In Comparative Example 5, the chelation temperature exceeded the range, and the chelation rate decreased by 17.3%. The activity retention rate and product stability were significantly reduced, which verified the scientific validity and necessity of the parameter range defined in this invention.

[0043] The products prepared in Examples 1-5 of this invention all have a target peptide ratio of ≥82%, a chelation rate of ≥45%, a free ferrous ion content of ≤22.5mg / kg, an active peptide retention rate of ≥91%, a 30-day particle size change rate of ≤9%, and a stratification rate of ≤3%. All of these properties are far superior to those of the comparative examples, verifying the stability and superiority of the process of this invention. Among them, Example 1 has the best parameters and is the best example of this invention.

[0044] In summary, the three-stage enzymatic hydrolysis sequence of this invention exhibits a synergistic effect: neutral protease first cleaves the flexible region on the surface of large protein molecules, exposing their internal structure; acidic protease then hydrolyzes the peptide bonds of acidic amino acid residues, reducing peptide size and increasing carboxyl group density; finally, the endonuclease precisely cleaves the target small peptide. If the sequence is reversed, the folded state of the large molecule hinders enzymatic cleavage, significantly reducing hydrolysis efficiency. The ultrasonic-microwave dual-field coupling chelation mechanism: ultrasonic cavitation breaks down mass transfer barriers, accelerating collisions between peptide sites and metal ions; microwaves induce conformational perturbations in the peptide chain, exposing chelating groups and lowering activation energy. Currently, there is no complete process in the field of "neutral → acidic → endonuclease continuous enzymatic hydrolysis + dual-field coupling chelation + tertiary membrane separation and cation exchange resin tandem," and this invention fills this gap.

[0045] This invention's process is highly adaptable to large-scale food factory production: the three enzymatic hydrolysis stages are directly connected without static settling, eliminating the need for intermediate enzyme inactivation and cooling steps, enabling continuous pipeline production, reducing equipment footprint and energy consumption. High-temperature instantaneous inactivation combined with rapid cooling significantly shortens the reaction time compared to traditional stirring chelation, resulting in a significant reduction in overall energy consumption. Three-stage membrane separation and resin series connection ensure high levels of active peptide retention and free ion removal, significantly improving overall product yield compared to traditional processes and reducing raw material costs. The entire process is solvent-free, reducing wastewater discharge, and the membranes and resins can be reused. Rapid technical upgrades can be achieved simply by adding ultrasonic and microwave devices to existing production lines, facilitating stable large-scale production.

[0046] 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 high-chelation-rate small molecule chelated peptides in functional foods, characterized in that, Includes the following steps: S1. Take animal and plant composite protein raw materials, crush and sieve them, and then add purified water to prepare a protein slurry with fixed solid content; S2. The protein slurry is subjected to a three-stage directional enzymatic hydrolysis, which is carried out in sequence: neutral enzymatic hydrolysis, acidic enzymatic hydrolysis, and endopeptidase hydrolysis. The parameters of each enzymatic hydrolysis are adjusted by the corresponding buffer solution, and the segments are directly connected without standing. S3. The enzyme hydrolysate is inactivated by high temperature and then rapidly cooled to the chelation reaction temperature. S4. Add chelating agent and metal ion source to the cooled enzymatic hydrolysate, and carry out chelation reaction in a closed reactor with coupled ultrasonic and microwave dual fields. S5. The chelate solution is purified by three-stage membrane separation and cation exchange resin in series to remove impurities and free ions; S6. The purified liquid is subjected to vacuum low-temperature concentration and gradient vacuum freeze-drying in sequence to obtain solid chelated peptides; S7. After sieving the solid chelated peptides, they are aseptically vacuum packaged to obtain a high-chelation-rate small molecule chelated peptide product.

2. The preparation method according to claim 1, characterized in that, In step S1, the raw material pretreatment step, the animal and plant composite protein raw material is a mixture of soy protein isolate and cod skin collagen in 70-75 parts by weight and 25-30 parts by weight. After the raw materials are pulverized, they are passed through a 100-mesh standard pharmaceutical sieve. The resistivity of the purified water is ≥18.2 MΩ·cm. The amount of purified water added is 480-520 parts of the total mass of the composite protein raw materials. The protein slurry is prepared at a temperature of 45-50℃, with a stirring speed of 200-220 r / min and a mixing time of 30 min. The solid content of the protein slurry is adjusted to 15%-16%.

3. The preparation method according to claim 2, characterized in that, In step S2, the multi-stage directional enzymatic hydrolysis step, the neutral enzymatic hydrolysis section uses neutral protease with an enzyme activity ≥100,000 U / g. The amount of enzyme added is 2.2%-2.4% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 7.2-7.3 using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer. The enzymatic hydrolysis temperature is 52-54℃, and the enzymatic hydrolysis time is 100-110 min. The acidic enzymatic hydrolysis section uses acidic protease with an enzyme activity ≥80,000 U / g. The amount of enzyme added is 1.7%-1.9% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 4.2-4.3 using citrate-sodium citrate buffer solution. The enzymatic hydrolysis temperature is 47-49℃ and the enzymatic hydrolysis time is 80-90 min. The endopeptidase hydrolysis segment uses papain with an enzyme activity ≥120,000 U / g. The enzyme addition amount is 1.2%-1.4% of the dry weight of the composite protein raw material. The pH of the system is adjusted to 5.7-5.8 using an acetate-sodium acetate buffer solution. The hydrolysis temperature is 57-59℃ and the hydrolysis time is 70-80 min.

4. The preparation method according to claim 3, characterized in that, The enzymatic hydrolysis and inactivation step S3 uses a shell-and-tube heat exchanger to raise the temperature. The heating rate is controlled at 8-10℃ / min. The system is heated to 93-95℃ and held for 13-15min to complete the inactivation. After inactivation, a plate cooler is used for rapid cooling, with the cooling rate controlled at 12-15℃ / min, until the system temperature drops to 42-44℃.

5. The preparation method according to claim 4, characterized in that, In step S4, the dual-field coupling chelation step, the chelation aid is a compound of sodium phytate and anhydrous citric acid in a ratio of 35-38 parts by weight and 22-25 parts by weight, and the total amount of the chelation aid added is 3.2%-3.4% of the dry weight of the enzymatic hydrolysate. The metal ion source is ferrous gluconate, and the amount added is 4.2%-4.4% of the dry weight of the enzymatic hydrolysate. The reactor is airtight and isolated from air. The ultrasonic power is controlled at 300-320W and the ultrasonic frequency is 26-27kHz. The microwave power is controlled at 120-140W and the microwave frequency is 2450MHz. The chelation system temperature is 57-59℃ and the chelation reaction time is 110-120min.

6. The preparation method according to claim 5, characterized in that, In step S5, the tandem purification step, the components used in the three-stage membrane separation are all made of polyvinylidene fluoride, namely a 100nm microfiltration membrane, a 500Da ultrafiltration membrane, and a 200Da nanofiltration membrane. The microfiltration membrane operates at a pressure of 0.35-0.4 MPa and a temperature of 37-40℃. The ultrafiltration membrane operates at a pressure of 0.65-0.7 MPa and a temperature of 32-35℃. The nanofiltration membrane operates at a pressure of 1.1-1.2 MPa and a temperature of 27-30°C. The membrane separation retentate is passed into a styrene-based cation exchange resin column with a height-to-diameter ratio of 8:1 and a flow rate of 2-3 BV / h to complete the removal of free ions.

7. The preparation method according to claim 6, characterized in that, In step S6, the gradient low-temperature drying step, the reduced-pressure low-temperature concentration is carried out using a rotary evaporator with a vacuum degree of -0.075 MPa to -0.07 MPa and a concentration temperature of 47-49°C, until the solid content of the liquid is 32%-34%. The gradient vacuum freeze drying uses a vacuum freeze dryer. The pre-freezing stage temperature is -55℃ to -53℃, the pre-freezing time is 3h, the sublimation stage vacuum degree is 16-18Pa, the sublimation temperature is -20℃ to -15℃, the desorption stage temperature is 25-30℃, and the total drying time is 13-14h.

8. The preparation method according to claim 7, characterized in that, The aseptic sieving and packaging step S7 uses a 120-mesh stainless steel standard sieve with a sieving speed of 150-160 r / min. The packaging uses aluminum-plastic composite film, with a vacuum degree of -0.09MPa to -0.085MPa, an ambient temperature of 22-24℃, and an ambient humidity of 45%-50%.

Images