Preparation process of placenta polypeptide freeze-dried powder by enzymatic hydrolysis

Abstract

The application relates to the polypeptide preparation technical field, and discloses a preparation process of placenta polypeptide freeze-dried powder by an enzymatic hydrolysis method, raw material pretreatment; broken pretreatment, ultrasonic treatment and high-pressure microjet homogenization treatment are sequentially carried out on placenta homogenate liquid; repeated freeze-thawing is carried out to break the wall; immobilized enzyme step-by-step enzymolysis is carried out, magnetic separation recovery is carried out after the first-stage enzymolysis by using magnetic nano-carrier immobilized trypsin, second-stage enzymolysis is carried out by using a composite enzyme of magnetic nano-carrier immobilized neutral protease and flavor protease; centrifugal separation and membrane separation are carried out; chromatography enrichment is carried out; a freeze-drying protective agent is added; and freeze-drying is carried out. The application forms a synergistic wall-breaking system by combining the broken pretreatment of ultrasonic and high-pressure microjet homogenization with repeated freeze-thawing, the freeze-thawing times are effectively reduced, the step-by-step enzymolysis and magnetic separation technology of the magnetic nano-carrier immobilized enzyme are used, the damage of polypeptide activity caused by enzyme inactivation by heating is avoided, the enzyme can be reused, and the obtained placenta polypeptide freeze-dried powder has higher quality.

Description

Technical Field

[0001] This invention relates to the field of polypeptide preparation technology, and more specifically to a process for preparing placental polypeptide lyophilized powder using an enzymatic hydrolysis method. Background Technology

[0002] The placenta contains abundant bioactive substances such as proteins, polypeptides, amino acids, hormones, cytokines, and nucleic acids, which have various physiological functions such as immune regulation, anti-oxidation, and anti-aging. Extracting polypeptide active ingredients from the placenta is an important way to develop bioactive products. Currently, the main methods for preparing placental peptides include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Among these, enzymatic hydrolysis has become a research hotspot due to its advantages such as mild reaction conditions, good preservation of product activity, and high safety. However, the current lyophilized powder preparation process for placental peptides has the following problems: In current placental peptide preparation processes, the cell wall disruption efficiency is low, resulting in numerous freeze-thaw cycles, long production cycles, and reduced overall production efficiency. Existing stepwise enzymatic hydrolysis processes use heating to inactivate enzymes during step switching, leading to peptide activity loss, and the inability to recover and reuse free enzymes, resulting in high costs. Furthermore, existing placental peptide products exhibit low purity and dispersed molecular weight of antioxidant peptides, with significant activity loss due to the freeze-drying process. Summary of the Invention

[0003] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a process for preparing placental polypeptide freeze-dried powder using enzymatic hydrolysis, so as to solve the technical problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis, comprising the following steps: Step S1: Raw material pretreatment. Remove the fascia and blood vessels from the animal placenta, wash it, cut it into small pieces, add physiological saline or deionized water to homogenize it, and obtain placental homogenate. Step S2, pre-treatment of crushing: The placental homogenate is subjected to ultrasonic treatment and high-pressure micro-jet homogenization treatment in sequence. Step S3: Repeated freeze-thaw cycle to break the cell wall. The placental homogenate after the crushing and pretreatment in step S2 is frozen at -20℃ to -40℃ for 12-24 hours, and then frozen and thawed repeatedly 2-4 times. Step S4: Stepwise enzymatic hydrolysis of immobilized enzyme. The trypsin immobilized on the magnetic nanocarrier is added to the placental homogenate obtained in step S3 after freeze-thaw to carry out the first enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the immobilized trypsin is separated and recovered by an external magnetic field. The composite enzyme of neutral protease and flavor protease immobilized on the magnetic nanocarrier is added to the recovered enzymatic hydrolysate to carry out the second enzymatic hydrolysis. Step S5: Centrifugation and membrane separation. Centrifuge the enzymatic hydrolysate after step S4, collect the supernatant, and filter it through a microfiltration membrane and an ultrafiltration membrane in sequence. Collect the ultrafiltration permeate. Step S6: Chromatographic enrichment. The ultrafiltration permeate obtained in step S5 is passed through a macroporous adsorption resin column for chromatographic separation. The ethanol eluent is collected and concentrated under reduced pressure to obtain placental polypeptide concentrate. Step S7: Add freeze-drying protectant. Add freeze-drying protectant to the placental polypeptide concentrate obtained in step S6. Step S8: Freeze-drying. The concentrate obtained in step S7 is freeze-dried to obtain placental polypeptide freeze-dried powder.

[0005] In a preferred embodiment, in step S1, the animal placenta is any one of sheep placenta, human placenta, bovine placenta, and camel placenta. In step S1, homogenization is carried out at 4-10°C, and washing is performed repeatedly with 0.9% physiological saline 2-4 times. The placenta is then cut into small pieces of 0.5cm-2cm × 0.5cm-2cm.

[0006] In a preferred embodiment, during step S2, the placental homogenate is first ultrasonically treated for 10-30 minutes at an ultrasonic power of 200-500W and a frequency of 20-40kHz, and then subjected to high-pressure micro-jet homogenization treatment at a pressure of 100-200MPa, and the process is repeated 2-4 times.

[0007] In a preferred embodiment, in step S4, the enzymatic hydrolysis conditions for the first stage are: pH 7.0-8.5, hydrolysis temperature 35-45℃, and hydrolysis time 2-6 hours, with the amount of trypsin immobilized on the magnetic nanocarrier added being 1%-5% of the substrate protein mass based on trypsin; the enzymatic hydrolysis conditions for the second stage are: pH 6.0-7.5, hydrolysis temperature 45-55℃, and hydrolysis time 2-4 hours, with the mass ratio of neutral protease to flavor protease in the magnetic nanocarrier immobilized complex enzyme being 1:1-2:1, and the total added amount being 1%-4% of the substrate protein mass based on the complex enzyme.

[0008] In a preferred embodiment, in step S4, trypsin and a composite enzyme immobilized on a magnetic nanocarrier are prepared by co-precipitation of Fe3O4 magnetic nanoparticles, which are then coated with SiO2 and activated with glutaraldehyde. These nanoparticles are then immobilized with a mixed enzyme solution of trypsin, neutral protease, and flavor protease at 4°C for 12-24 hours. After the first and second enzymatic hydrolysis are completed, the immobilized composite enzyme is separated and recovered by an external magnetic field.

[0009] In a preferred embodiment, in step S5, the centrifugation speed is 10,000 rpm, the centrifugation time is 15-30 minutes, the microfiltration membrane pore size is 0.22-0.45 μm, and the ultrafiltration membrane molecular weight cutoff is 3-10 kDa.

[0010] In a preferred embodiment, in step S6, the macroporous adsorption resin is DA201-C type resin, the volume fraction of the ethanol eluent is 10%-30%, the temperature of vacuum concentration is 40-50℃, and the concentration is reduced to 1 / 10-1 / 5 of the original volume.

[0011] In a preferred embodiment, in step S7, the freeze-drying protectant is a mixture of mannitol and trehalose in a mass ratio of 1:1, and the total amount added is 5% of the mass of the concentrate.

[0012] In a preferred embodiment, in step S8, the freeze-drying process is as follows: pre-freezing at -40°C to -50°C for 4-8 hours, drying for 12-24 hours at -25°C to -15°C and a vacuum of 10-30Pa, and drying for 4-8 hours at 20-30°C and a vacuum of 5-15Pa.

[0013] In a preferred embodiment, in step S8, the obtained lyophilized peptide powder has a peptide content of ≥65%, more than 80% of the peptides have a molecular weight between 500-3000 Da, a DPPH free radical scavenging rate of ≥75%, an ABTS free radical scavenging rate of ≥70%, and a water content of ≤3%, wherein the test concentrations for the DPPH free radical scavenging rate and the ABTS free radical scavenging rate are both 1 mg / mL.

[0014] The technical effects and advantages of this invention are as follows: 1. This invention combines ultrasonic treatment with high-pressure micro-jet homogenization to form a pre-treatment for crushing, and then combines it with repeated freeze-thaw cycles to form a three-stage progressive synergistic cell-wall breaking system of "ultrasound-high-pressure micro-jet homogenization-freeze-thaw". The three processes work sequentially and progressively, ensuring the cell-wall breaking effect while reducing the number of freeze-thaw cycles, thereby shortening the process cycle and improving the production efficiency of placental polypeptide freeze-dried powder. 2. This invention uses magnetic nanocarriers to immobilize trypsin, neutral protease and flavor protease respectively. By applying an external magnetic field, the immobilized enzymes can be rapidly separated and recovered and the precise switching between steps can be achieved. This avoids thermal damage to peptide activity caused by heating inactivates the enzymes, enables the enzymes to be reused, and thus reduces production costs. 3. This invention achieves targeted enrichment of antioxidant active peptides and effective protection of their active conformation through the synergistic effect of DA201-C type macroporous adsorption resin chromatography enrichment and mannitol / trehalose freeze-drying protectant. The molecular weight of peptide 8 in the product is concentrated between 500-3000 Da, and the peptide content is higher, which leads to the improvement of product purity and antioxidant activity while ensuring the long-term storage stability of the product. Attached Figure Description

[0015] Figure 1 This is a flowchart illustrating the preparation process of the polypeptide lyophilized powder according to the present invention. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. In addition, the morphology of each structure described in the following embodiments is merely illustrative. The preparation process of placental polypeptide freeze-dried powder using enzymatic hydrolysis involved in the present invention is not limited to the structures described in the following embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Reference Figure 1 This invention provides a process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis, comprising the following steps: Step S1: Raw material pretreatment. Remove the fascia and blood vessels from the animal placenta, wash it, cut it into small pieces, add physiological saline or deionized water to homogenize it, and obtain placental homogenate. Step S2, pre-treatment of crushing: The placental homogenate is subjected to ultrasonic treatment and high-pressure micro-jet homogenization treatment in sequence. Step S3: Repeated freeze-thaw cycle to break the cell wall. The placental homogenate after the crushing and pretreatment in step S2 is frozen at -20℃ to -40℃ for 12-24 hours, and then frozen and thawed repeatedly 2-4 times. Step S4: Stepwise enzymatic hydrolysis of immobilized enzyme. The trypsin immobilized on the magnetic nanocarrier is added to the placental homogenate obtained in step S3 after freeze-thaw to carry out the first enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the immobilized trypsin is separated and recovered by an external magnetic field. The composite enzyme of neutral protease and flavor protease immobilized on the magnetic nanocarrier is added to the recovered enzymatic hydrolysate to carry out the second enzymatic hydrolysis. Step S5: Centrifugation and membrane separation. Centrifuge the enzymatic hydrolysate after step S4, collect the supernatant, and filter it through a microfiltration membrane and an ultrafiltration membrane in sequence. Collect the ultrafiltration permeate. Step S6: Chromatographic enrichment. The ultrafiltration permeate obtained in step S5 is passed through a macroporous adsorption resin column for chromatographic separation. The ethanol eluent is collected and concentrated under reduced pressure to obtain placental polypeptide concentrate. Step S7: Add freeze-drying protectant. Add freeze-drying protectant to the placental polypeptide concentrate obtained in step S6. Step S8: Freeze-drying. The concentrate obtained in step S7 is freeze-dried to obtain placental polypeptide freeze-dried powder.

[0018] In this embodiment, a three-stage progressive synergistic cell disruption system is formed by ultrasonic and high-pressure microfluidic homogenization pretreatment and repeated freeze-thaw cycles. This system is then combined with stepwise enzymatic hydrolysis and magnetic separation technology using magnetic nanocarriers to achieve efficient cell disruption of placental cells and directional degradation of proteins. This reduces the number of subsequent freeze-thaw cycles and ensures the quality of the produced placental polypeptide freeze-dried powder.

[0019] Reference Figure 1 In step S1, the animal placenta is any one of sheep placenta, human placenta, bovine placenta, and camel placenta. In step S1, homogenization is carried out at 4-10℃, and washing is performed by repeatedly washing with 0.9% physiological saline 2-4 times. The placenta is then cut into small pieces of 0.5cm-2cm×0.5cm-2cm.

[0020] In this embodiment, by limiting the diversity of placental sources, this application demonstrates that the process has good raw material versatility; low-temperature homogenization at 4-10℃ effectively prevents protein enzymatic degradation and denaturation during homogenization; repeated washing with 0.9% saline 2-4 times thoroughly removes surface blood and microorganisms; and cutting into 0.5-2cm pieces ensures the uniformity of subsequent homogenization and the effectiveness of the crushing pretreatment.

[0021] Reference Figure 1 In step S2, during the crushing pretreatment, the placental homogenate is first ultrasonically treated for 10-30 minutes under ultrasonic power of 200-500W and frequency of 20-40kHz, and then subjected to high-pressure micro-jet homogenization treatment under pressure of 100-200MPa, and the cycle is repeated 2-4 times.

[0022] In this embodiment, the application optimizes the specific parameters of ultrasound and high-pressure microfluidic homogenization to maximize the synergistic cell wall disruption effect of both. Ultrasound pre-loosens the cell wall, and high-pressure microfluidic homogenization further breaks it down on the basis of pre-damage, resulting in a better cell wall disruption effect, reducing the number of subsequent freeze-thaw cycles, and thus improving the overall processing efficiency.

[0023] Reference Figure 1In step S4, the enzymatic hydrolysis conditions for the first stage are: pH 7.0-8.5, hydrolysis temperature 35-45℃, and hydrolysis time 2-6 hours. The amount of trypsin immobilized on the magnetic nanocarrier added is 1%-5% of the substrate protein mass, calculated as trypsin. The enzymatic hydrolysis conditions for the second stage are: pH 6.0-7.5, hydrolysis temperature 45-55℃, and hydrolysis time 2-4 hours. The mass ratio of neutral protease to flavor protease in the magnetic nanocarrier immobilized complex enzyme is 1:1-2. :1, The total amount added is 1%-4% of the substrate protein based on the complex enzyme. In step S4, trypsin and complex enzyme are immobilized on magnetic nanocarriers. Fe3O4 magnetic nanoparticles are prepared by co-precipitation method. After being coated with SiO2 and activated with glutaraldehyde, they are immobilized with a mixed enzyme solution of trypsin solution, neutral protease and flavor protease at 4°C for 12-24 hours. After the first and second enzymatic hydrolysis are completed, the immobilized complex enzyme is separated and recovered by an external magnetic field.

[0024] In this embodiment, by optimizing the parameters of the immobilized enzyme's stepwise enzymatic hydrolysis, trypsin first cuts large protein molecules into medium-sized polypeptide fragments, exposing more cleavage sites. After separating trypsin by a magnetic field, neutral protease and flavor protease are further hydrolyzed in the second stage, producing abundant short peptides and small-molecule active peptides. This achieves precise, gradual, and directional degradation by first cutting large molecules and then breaking them down into smaller ones. The stepwise differential setting of pH and temperature ensures that each enzyme exerts its maximum activity under its optimal conditions. Through the preparation method of immobilized enzymes using magnetic nanocarriers and the magnetic separation and recovery method, rapid enzyme separation and recovery and precise switching between steps are achieved. Low-temperature immobilization at 4°C effectively protects the active site of the enzyme. With an external magnetic field of 0.5T, enzyme separation can be completed within minutes, avoiding the thermal damage to polypeptide activity caused by traditional heat inactivation of enzymes. After processing using this method, the DPPH removal rate of the magnetic separation group is higher than that of the heat-inactivated enzyme group. Furthermore, the immobilized enzyme can be recovered after use and retains its enzyme activity, enabling enzyme reuse and reducing production costs.

[0025] Reference Figure 1 In step S5, the centrifugation speed is 10,000 rpm, the centrifugation time is 15-30 minutes, the microfiltration membrane pore size is 0.22-0.45 μm, and the ultrafiltration membrane molecular weight cutoff is 3-10 kDa.

[0026] In this embodiment, by optimizing centrifugation and membrane separation parameters, cell debris, insoluble impurities, and large molecular proteins in the enzymatic hydrolysate are effectively removed, and an ultrafiltration permeate rich in target active peptides is collected. Microfiltration at 0.22-0.45 μm removes fine particulate matter, and ultrafiltration at 3-10 kDa ensures efficient recovery of peptides within the target molecular weight range, providing high-quality samples for subsequent chromatographic enrichment.

[0027] Reference Figure 1 In step S6, the macroporous adsorption resin is DA201-C type resin, the volume fraction of ethanol eluent is 10%-30%, the temperature of vacuum concentration is 40-50℃, and the concentration is reduced to 1 / 10-1 / 5 of the original volume.

[0028] In this embodiment, the present application uses DA201-C type macroporous adsorption resin chromatography and 10%-30% ethanol gradient elution. Based on the hydrophobicity and molecular size difference of peptides, peptides with specific antioxidant active structural domains are effectively separated from inactive impurities, achieving targeted enrichment of antioxidant active peptides. Low temperature vacuum concentration at 40-50℃ effectively prevents the thermal degradation of peptides. More than 85% of the peptides in the product have molecular weights concentrated between 500-3000 Da.

[0029] Reference Figure 1 In step S7, the freeze-drying protectant is a mixture of mannitol and trehalose in a mass ratio of 1:1, and the total amount added is 5% of the mass of the concentrate.

[0030] In the embodiments of this application, the freeze-drying protectant is selected from one or more of mannitol, trehalose, and glycine. During the freeze-drying process, it replaces water molecules in binding with peptides through hydrogen bonding, effectively preventing ice crystal formation and damage to the active conformation of peptides by the vacuum environment, and ensuring the high activity of the product during freeze-drying and long-term storage.

[0031] Reference Figure 1 In step S8, the freeze-drying process is as follows: pre-freezing at -40℃ to -50℃ for 4-8 hours, drying at -25℃ to -15℃ and vacuum degree of 10-30Pa for 12-24 hours, and drying again at 20-30℃ and vacuum degree of 5-15Pa for 4-8 hours.

[0032] In this embodiment, the application optimizes the three-stage freeze-drying process (pre-freezing, primary drying, and secondary drying) to ensure the scientific nature and controllability of the freeze-drying process. Rapid pre-freezing at -40°C to -50°C forms fine and uniform ice crystals, reducing mechanical damage to the peptide structure. Primary drying effectively removes most of the free water under low temperature (-25°C to -15°C) and high vacuum (10-30Pa) conditions. Secondary drying removes bound water under suitable temperature (20-30°C) and high vacuum (5-15Pa), resulting in a product with a water content as low as 1.8%, exhibiting a loose and porous structure that facilitates resolvation and absorption.

[0033] Reference Figure 1In step S8, the obtained lyophilized peptide powder has a peptide content of ≥65%, more than 80% of the peptides have a molecular weight between 500-3000 Da, a DPPH free radical scavenging rate of ≥75%, an ABTS free radical scavenging rate of ≥70%, and a water content of ≤3%. The test concentrations for the DPPH free radical scavenging rate and the ABTS free radical scavenging rate are both 1 mg / mL.

[0034] In this application embodiment, the product quality and efficacy are ensured by defining key performance indicators: peptide content ≥65% to ensure the content of effective ingredients; more than 80% of peptides have molecular weights between 500-3000 Da to ensure a molecular weight distribution dominated by small molecule active peptides, which is conducive to human absorption and utilization; DPPH scavenging rate ≥75% and ABTS scavenging rate ≥70% to ensure the high antioxidant activity of the product; and water content ≤3% to ensure the long-term storage stability of the product, which is convenient for long-distance transportation and commercial application.

[0035] Example 2

[0036] A process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis is described below: (1) Raw material pretreatment: Take fresh sheep placenta, remove fascia and blood vessels, wash repeatedly with 0.9% physiological saline 3 times, cut into small pieces of about 1cm×1cm, mix the cut placental tissue with physiological saline at a ratio of 1:3 (g / mL), place in a tissue homogenizer and homogenize for 10 minutes at 4℃ to obtain placental homogenate. (2) Crushing pretreatment: Place the placental homogenate in an ultrasonic processor and ultrasonically treat it for 20 minutes at an ultrasonic power of 350W and an ultrasonic frequency of 28kHz. During the ultrasonic treatment, the temperature is controlled not to exceed 25℃. Then, place the ultrasonically treated placental homogenate in a high-pressure micro-jet homogenizer and cycle it 3 times at a pressure of 150MPa.

[0037] (3) Repeated freeze-thaw cell breaking: The placental homogenate after crushing and pretreatment is frozen at -30℃ for 18 hours, then thawed naturally at room temperature, and the freeze-thaw cycle is repeated twice.

[0038] (4) Stepwise enzymatic hydrolysis of immobilized enzymes (41) First enzymatic hydrolysis: The placental homogenate after freezing and thawing was adjusted to pH 8.0 with 0.5 mol / L NaOH solution, and trypsin immobilized on magnetic nanocarrier was added. The mixture was enzymatically hydrolyzed in a constant temperature water bath at 40℃ for 4 hours with continuous stirring. After the enzymatic hydrolysis was completed, a magnetic field (magnetic induction intensity 0.5T) was applied to the outside of the reaction vessel to make the immobilized trypsin adsorb onto the container wall. The immobilized enzyme was separated and recovered, and the hydrolysate was collected.

[0039] (42) Second stage of enzymatic hydrolysis (immobilized complex enzyme): Adjust the pH of the hydrolysate recovered in step (41) to 7.0, add the magnetic nanocarrier immobilized complex enzyme (neutral protease to flavor protease mass ratio 1.5:1, total addition amount is 2.5% of substrate protein mass), and continue enzymatic hydrolysis at 50℃ for 3 hours. After the enzymatic hydrolysis is completed, apply a magnetic field (magnetic induction intensity 0.5T) to the outside of the reaction vessel, separate and recover the immobilized complex enzyme, and collect the hydrolysate.

[0040] (5) Centrifugation and membrane separation: Centrifuge the enzyme hydrolysate at 10,000 rpm for 20 minutes, collect the supernatant, and filter the supernatant sequentially through a microfiltration membrane with a pore size of 0.22-0.45 μm and an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and collect the ultrafiltration permeate.

[0041] (6) Chromatographic enrichment: The ultrafiltration permeate was loaded onto a DA201-C macroporous adsorption resin column, washed with 2 column volumes of deionized water, and then eluted with 20% ethanol solution. The ethanol eluent was collected and concentrated under reduced pressure at 45°C to 1 / 8 of the original volume to obtain placental polypeptide concentrate.

[0042] (7) Add freeze-drying protectant: add mannitol and trehalose (mass ratio 1:1) to the concentrate, with a total addition of 5% of the concentrate mass.

[0043] (8) Freeze-drying: The concentrated liquid with added freeze-drying protectant is dispensed into freeze-drying trays and placed in a vacuum freeze dryer. The pre-freezing temperature is -45℃ and the time is 6 hours; the first drying temperature is -20℃ and the vacuum degree is 20Pa and the time is 18 hours; the second drying temperature is 25℃ and the vacuum degree is 10Pa and the time is 6 hours. After freeze-drying, it is passed through a 100-mesh sieve to obtain sheep placental polypeptide freeze-dried powder.

[0044] The generated lyophilized sheep placental polypeptide powder was analyzed. The polypeptide content was 71.6% using the biuret method. In terms of molecular weight distribution, more than 88.2% of the peptides had a molecular weight between 500 and 3000 Da. The DPPH free radical scavenging rate was 86.4%, the ABTS free radical scavenging rate was 80.2%, and the water content was 1.8%. The recovered immobilized trypsin and immobilized complex enzyme were reused 5 times each, and the enzyme activity retention rate after each use was measured. The results showed that the enzyme activity retention rate of the immobilized trypsin after 5 uses was 82.3%.

[0045] The lyophilized sheep placental polypeptide powder prepared according to Example 2 has a polypeptide content as high as 71.6%, which is significantly better than the existing technology level. Moreover, more than 85% of the peptide molecular weights are concentrated between 500-3000 Da, mainly small molecule active peptides, which are conducive to human absorption and utilization. The DPPH free radical scavenging rate reaches 86.4% and the ABTS free radical scavenging rate reaches 80.2%, which shows high antioxidant activity, proving that magnetic separation instead of heating to inactivate enzymes effectively protects the active conformation of polypeptides. The water content is only 1.8%, and the addition of mannitol and trehalose lyophilization protectants ensures the stability of the product during long-term storage. The immobilized trypsin has an enzyme activity retention rate of 82.3% after 5 uses, and the immobilized composite enzyme has a retention rate of 78.6% after 5 uses, realizing enzyme reuse and reducing production costs. This application can achieve the above excellent indicators with only 2 freeze-thaw cycles, compared with the existing technology which requires 3-4 freeze-thaw cycles, shortening the process cycle by about 30%-50% and reducing energy consumption.

[0046] Example 3

[0047] The basic process is the same as in Example 2, except that: (1) Fresh bovine placenta is used as the raw material; (2) In the crushing pretreatment, the ultrasonic treatment conditions are: ultrasonic power 400W, frequency 40kHz, ultrasonic treatment for 15 minutes; the high pressure micro-jet homogenization treatment conditions are: pressure 120MPa, 4 cycles. (3) The number of freeze-thaw cycles is 1; (4) First stage of enzymatic hydrolysis conditions: pH 8.2, temperature 45℃, enzymatic hydrolysis for 3 hours, and the amount of immobilized trypsin added is 2.5% of the substrate protein mass; (5) Second stage of enzymatic hydrolysis conditions: pH 7.2, temperature 50℃, enzymatic hydrolysis for 3.5 hours, the mass ratio of neutral protease to flavor protease in the immobilized complex enzyme is 1.8:1, and the total amount added is 3.0% of the substrate protein mass.

[0048] The obtained bovine placental polypeptide lyophilized powder had a polypeptide content of 69.8%, a DPPH free radical scavenging rate of 83.6%, and an ABTS free radical scavenging rate of 77.1%.

[0049] Example 4

[0050] The basic process is the same as in Example 2, except that: (1) The raw material is fresh human placenta (delivered at full term by healthy mothers and qualified by pathogen testing). (2) In the crushing pretreatment, the ultrasonic treatment conditions are: ultrasonic power 300W, frequency 20kHz, ultrasonic treatment for 25 minutes; the high pressure micro-jet homogenization treatment conditions are: pressure 180MPa, 2 cycles. (3) First stage of enzymatic hydrolysis conditions: pH 7.8, temperature 37℃, enzymatic hydrolysis for 5 hours, and the amount of immobilized trypsin added is 4% of the substrate protein mass; (4) Second stage enzymatic hydrolysis conditions: pH 6.8, temperature 48℃, enzymatic hydrolysis for 4 hours, the mass ratio of neutral protease to flavor protease in the immobilized complex enzyme is 1.2:1, and the total amount added is 3.5% of the substrate protein mass; (5) The freeze-drying protectant is trehalose (6% added).

[0051] The obtained human placental polypeptide lyophilized powder had a polypeptide content of 72.4%, a DPPH free radical scavenging rate of 87.2%, and an ABTS free radical scavenging rate of 81.5%.

[0052] In Example 3 of this application, bovine placenta was used as the raw material, and in Example 4, human placenta was used as the raw material. The lyophilized polypeptide powders prepared under the same process conditions both achieved excellent results. The bovine placenta group had a polypeptide content of 69.8%, and DPPH and ABTS free radical scavenging rates of 83.6% and 77.1%, respectively. The human placenta group had a polypeptide content of 72.4%, and DPPH and ABTS free radical scavenging rates of 87.2% and 81.5%, respectively. These results are highly consistent with the sheep placenta group in Example 2 (polypeptide content 71.6%, DPPH scavenging rate 86.4%, ABTS scavenging rate 80.2%). This fully demonstrates that the process of this invention has good raw material universality and does not depend on a specific placental source. Both methods can stably prepare placental polypeptide lyophilized powder products with high purity (polypeptide content ≥69.8%) and high activity (DPPH scavenging rate ≥83.6%, ABTS scavenging rate ≥77.1%).

[0053] The above embodiments can be implemented, in whole or in part, by software, hardware, firmware, or any other combination thereof. When implemented in software, the above embodiments can be implemented, in whole or in part, as a computer program product. The units and algorithm steps of the various examples described in the embodiments can be implemented in electronic hardware or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0054] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection between apparatuses or units may be electrical, mechanical, or other forms.

[0055] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0056] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis, characterized in that: Includes the following steps: Step S1: Raw material pretreatment. Remove the fascia and blood vessels from the animal placenta, wash it, cut it into small pieces, add physiological saline or deionized water to homogenize it, and obtain placental homogenate. Step S2, pre-treatment of crushing: The placental homogenate is subjected to ultrasonic treatment and high-pressure micro-jet homogenization treatment in sequence. Step S3: Repeated freeze-thaw cycle to break the cell wall. The placental homogenate after the crushing and pretreatment in step S2 is frozen at -20℃ to -40℃ for 12-24 hours, and then frozen and thawed repeatedly 2-4 times. Step S4: Stepwise enzymatic hydrolysis of immobilized enzyme. The trypsin immobilized on the magnetic nanocarrier is added to the placental homogenate obtained in step S3 after freeze-thaw to carry out the first enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the immobilized trypsin is separated and recovered by an external magnetic field. The composite enzyme of neutral protease and flavor protease immobilized on the magnetic nanocarrier is added to the recovered enzymatic hydrolysate to carry out the second enzymatic hydrolysis. Step S5: Centrifugation and membrane separation. Centrifuge the enzymatic hydrolysate after step S4, collect the supernatant, and filter it through a microfiltration membrane and an ultrafiltration membrane in sequence. Collect the ultrafiltration permeate. Step S6: Chromatographic enrichment. The ultrafiltration permeate obtained in step S5 is passed through a macroporous adsorption resin column for chromatographic separation. The ethanol eluent is collected and concentrated under reduced pressure to obtain placental polypeptide concentrate. Step S7: Add freeze-drying protectant. Add freeze-drying protectant to the placental polypeptide concentrate obtained in step S6. Step S8: Freeze-drying. The concentrate obtained in step S7 is freeze-dried to obtain placental polypeptide freeze-dried powder.

2. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S1, the animal placenta is any one of sheep placenta, human placenta, bovine placenta, and camel placenta. In step S1, homogenization is carried out at 4-10℃, and washing is performed by repeatedly washing with 0.9% physiological saline 2-4 times. The placenta is then cut into small pieces of 0.5cm-2cm×0.5cm-2cm.

3. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S2, during the crushing pretreatment, the placental homogenate is first ultrasonically treated for 10-30 minutes at an ultrasonic power of 200-500W and a frequency of 20-40kHz, and then subjected to high-pressure micro-jet homogenization treatment at a pressure of 100-200MPa, and the cycle is repeated 2-4 times.

4. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S4, the enzymatic hydrolysis conditions for the first stage are: pH 7.0-8.5, hydrolysis temperature 35-45℃, and hydrolysis time 2-6 hours. The amount of trypsin immobilized on the magnetic nanocarrier added is 1%-5% of the substrate protein mass, calculated as trypsin. The enzymatic hydrolysis conditions for the second stage are: pH 6.0-7.5, hydrolysis temperature 45-55℃, and hydrolysis time 2-4 hours. The mass ratio of neutral protease to flavor protease in the magnetic nanocarrier immobilized complex enzyme is 1:1-2:1, and the total added amount is 1%-4% of the substrate protein mass, calculated as complex enzyme.

5. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 4, characterized in that: In step S4, trypsin and composite enzyme are immobilized on magnetic nanocarriers. Fe3O4 magnetic nanoparticles are prepared by co-precipitation. After being coated with SiO2 and activated with glutaraldehyde, they are immobilized with a mixed enzyme solution of trypsin solution, neutral protease and flavor protease at 4°C for 12-24 hours. After the first and second enzymatic hydrolysis are completed, the immobilized composite enzyme is separated and recovered by an external magnetic field.

6. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S5, the centrifugation speed is 10,000 rpm, the centrifugation time is 15-30 minutes, the microfiltration membrane pore size is 0.22-0.45 μm, and the ultrafiltration membrane molecular weight cutoff is 3-10 kDa.

7. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S6, the macroporous adsorption resin is DA201-C type resin, the volume fraction of the ethanol eluent is 10%-30%, the temperature of vacuum concentration is 40-50℃, and the concentration is reduced to 1 / 10-1 / 5 of the original volume.

8. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S7, the freeze-drying protectant is a mixture of mannitol and trehalose in a mass ratio of 1:1, and the total amount added is 5% of the mass of the concentrate.

9. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S8, the freeze-drying process is as follows: pre-freezing at -40℃ to -50℃ for 4-8 hours, drying at -25℃ to -15℃ and vacuum degree of 10-30Pa for 12-24 hours, and drying again at 20-30℃ and vacuum degree of 5-15Pa for 4-8 hours.

10. The process for preparing placental polypeptide lyophilized powder using enzymatic hydrolysis according to claim 1, characterized in that: In step S8, the obtained lyophilized peptide powder has a peptide content of ≥65%, more than 80% of the peptides have a molecular weight between 500-3000 Da, a DPPH free radical scavenging rate of ≥75%, an ABTS free radical scavenging rate of ≥70%, and a water content of ≤3%. The test concentrations for the DPPH free radical scavenging rate and the ABTS free radical scavenging rate are both 1 mg / mL.

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