A method for preparing a hybrid loach animal protein hydrolysis enzyme processing active peptide

Abstract

The application belongs to the technical field of active peptide preparation, and discloses a preparation method of hybrid loach animal protein hydrolysis enzyme processing active peptide, which comprises the following steps: step S1, raw material pretreatment, freezing and crushing cell walls of fresh loach meat, and pre-enzymolysis treatment; and step S2, composite enzymolysis, adding and mixing protein liquid, the raw material pretreatment link is treated by freezing at-18 DEG C for 24 hours and crushing cell walls by 300MPa ultrahigh pressure microjet, meanwhile, papain and alkaline protease are added in a mass ratio of 1:2 for pre-enzymolysis at 4 DEG C for 1.5-2h, and the protein liquid is treated by 20kHz and 40kHz dual-frequency ultrasonic waves at 300W for 15 minutes, compared with the problem that heat-sensitive active ingredients are inactivated due to traditional high-temperature degreasing, the low-temperature pre-enzymolysis and the combined use of ultrahigh pressure and ultrasonic waves can not only avoid the damage of high temperature to active peptide precursors, but also efficiently crush cell structures, so that the protein extraction rate is greater than or equal to 92%, and sufficient and high-activity protein raw materials are provided for the subsequent enzymolysis link.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, specifically a method for preparing bioactive peptides from hybrid loach animal protein hydrolysates. Background Technology

[0002] With the rapid development of the functional food and health product industry, animal-derived bioactive peptides have become a research hotspot in food science due to their excellent bioactivities, such as antioxidant, blood pressure-lowering, and immune-enhancing effects. Hybrid loach, a high-protein, low-fat freshwater aquaculture species, has muscle protein rich in high-quality amino acids, making it an ideal raw material for preparing bioactive peptides. Currently, the industry primarily uses traditional processes for preparing loach protein bioactive peptides. These processes mainly include high-temperature defatting of raw materials, isothermal enzymatic hydrolysis with a single exogenous protease, activated carbon decolorization or macroporous resin adsorption purification, and hot air drying. These methods have achieved a certain scale of industrial production in practical applications, providing a foundation for the supply of animal-derived bioactive peptides. However, traditional preparation processes have significant technical bottlenecks that restrict the quality improvement and industrialization efficiency of hybrid loach active peptides. Specifically, traditional processes often use high-temperature treatment at 60-80℃ to achieve defatting and protein denaturation, which leads to the inactivation of a large number of heat-sensitive active ingredients in loach protein, such as some antioxidant peptide precursors. As a result, the bioactivity of the final product, such as DPPH free radical scavenging rate and ACE inhibition rate, is generally low. In the enzymatic hydrolysis stage, traditional methods often use a single protease, such as alkaline protease or papain, and maintain isothermal hydrolysis. Since different proteases have different optimal temperatures and action sites, a single enzyme system is difficult to efficiently decompose large molecular weight proteins. This not only results in a long hydrolysis cycle, but also easily generates large molecular weight peptides with a molecular weight >3000Da, which require complex subsequent purification steps to remove. It is also easy to generate bitter peptides, affecting the product flavor. In the purification and drying stages, the traditional activated carbon decolorization process adsorbs the target active peptides at different angles, causing loss of active ingredients. The hot air drying inlet temperature further aggravates the oxidation of active peptides, resulting in decreased product solubility and difficulty in controlling the moisture content to a low level. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing bioactive peptides from hybrid loach animal protein hydrolysates, in order to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing bioactive peptides from hybrid loach animal protein hydrolysates processed by enzymes, comprising the following steps: Step S1, raw material pretreatment, involves freezing, breaking cell walls, and pre-enzymatic hydrolysis of fresh loach meat; Step S2, compound enzymatic hydrolysis, involves adding and mixing the protein solution; Step S3: Purification and drying, and effective filtration of the enzyme hydrolysate.

[0005] Preferably, the detailed process of raw material pretreatment in step S1 is as follows: Step S101: Freeze the fresh meat of hybrid loach at -18℃ for 24 hours; Step S102: Cell walls are broken by ultra-high pressure microjet at 300 MPa pressure, while 0.2% of a complex protease by substrate mass is added. The complex protease is a mixture of papain and alkaline protease at a mass ratio of 1:2, and pre-enzymatic hydrolysis is performed at 4°C. Step S103: Use dual-frequency ultrasound (20kHz and 40kHz, power 300W) to treat the pre-enzymatically hydrolyzed protein solution for 15 minutes.

[0006] Preferably, the detailed process of the complex enzymatic hydrolysis in step S2 is as follows: Step S201: Add a complex enzyme system to the pretreated protein solution for gradient temperature-controlled enzymatic hydrolysis; Step S202: The complex enzyme system consists of hybrid loach endogenous protease, flavor protease and neutral protease in an activity unit ratio of 3:2:1, and the total amount added is 1.2% of the substrate mass.

[0007] Preferably, the detailed process for purification and drying in step S3 is as follows: S301 involves sequentially passing the enzymatic hydrolysate through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane for multi-stage separation. The microfiltration membrane has a pore size of 0.22 μm, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa, and the nanofiltration membrane has a molecular weight cutoff of 1 kDa. A cross-flow filtration method is used. S302, add 0.5% maltodextrin by mass of the purified active peptide solution as a protective agent, and use low-temperature vacuum spray drying with an inlet air temperature of 65-70℃, an outlet air temperature of 35-40℃, and a vacuum degree of -0.09MPa to obtain active peptides.

[0008] Preferably, the gradient temperature-controlled enzymatic hydrolysis is divided into three stages: First stage: Enzymatic hydrolysis for 2 hours at 50℃ and pH 7.0-7.5; Second stage: Enzymatic hydrolysis for 3 hours at 45℃ and pH 7.0-7.5; Third stage: Enzymatic hydrolysis for 1 hour at 40℃ and pH 7.0-7.5.

[0009] Preferably, the activity units of the endogenous protease in the intestinal tract of the hybrid loach in step 101 need to be ≥500U / mg, and the pre-enzymatic hydrolysis time at 4℃ in step 102 is 1.5-2h to ensure that the protein extraction rate is ≥92%.

[0010] Preferably, in step 201, the gradient temperature-controlled enzymatic hydrolysis involves peptides with a molecular weight ≤1000 Da accounting for ≥85%.

[0011] Preferably, the operating pressure of the multi-stage membrane separation in step 301 is controlled at 0.3-0.5 MPa, the membrane surface flow rate is controlled at 1.5-2.0 m / s, and the backwashing cycle of the cross-flow filtration in step 301 is 30 min / time, with a backwashing pressure of 1.2 times the operating pressure, in order to reduce the membrane fouling rate.

[0012] Preferably, the moisture content of the active peptide product in step 302 is ≤3%, the solubility at 25°C is ≥98%, and the feed rate of the low-temperature vacuum spray drying in step 302 is controlled at 15-20 mL / min to ensure that the oxidation rate of the active peptide is ≤2%. The beneficial effects of this invention are as follows: 1. This invention employs a raw material pretreatment process involving freezing at -18℃ for 24 hours combined with 300MPa ultra-high pressure micro-jet to disrupt cell walls. Simultaneously, papain and alkaline protease are added in a 1:2 mass ratio for pre-enzymatic hydrolysis at 4℃ for 1.5-2 hours, followed by dual-frequency ultrasonic treatment at 20kHz and 40kHz at 300W for 15 minutes. Compared to the problem of heat-sensitive active ingredients being inactivated due to traditional high-temperature defatting, the combination of low-temperature pre-enzymatic hydrolysis and ultra-high pressure and ultrasound avoids the destruction of active peptide precursors by high temperatures and efficiently disrupts cell structures, ensuring a protein extraction rate of ≥92%. This provides sufficient and highly active protein raw materials for subsequent enzymatic hydrolysis.

[0013] 2. This invention employs a complex enzymatic hydrolysis process, selecting hybrid loach endogenous protease, flavor protease, and neutral protease in an activity ratio of 3:2:1, with a total addition of 1.2% of the substrate mass. The hydrolysis is performed in three stages with gradient temperature control: 50℃ to 45℃ and then back to 40℃, with a pH of 7.0-7.5 throughout, for a total duration of 6 hours. Compared to traditional single-protease isothermal hydrolysis, this complex enzyme system synergistically decomposes large protein molecules through different action sites. The gradient temperature control optimizes the reaction conditions for each enzyme, effectively shortening the hydrolysis cycle and reducing the formation of bitter peptides. Simultaneously, it ensures that small molecule peptides with a molecular weight ≤1000 Da account for ≥85%, improving the product's bioactivity and palatability. 3. This invention employs a multi-stage cross-flow filtration process using 0.22μm microfiltration membrane, 10kDa ultrafiltration membrane, and 1kDa nanofiltration membrane in the purification and drying stage. The operating pressure is 0.3-0.5MPa, the membrane surface flow rate is 1.5-2.0m / s, and the backwashing cycle is 30min / time. This is combined with low-temperature vacuum spray drying with an inlet air temperature of 65-70℃, an outlet air temperature of 35-40℃, a vacuum degree of -0.09MPa, and a feed rate of 15-20mL / min. 0.5% maltodextrin is added as a protective agent. Compared with the problems of traditional activated carbon decolorization leading to loss of active peptides and hot air drying exacerbating oxidation, multi-stage membrane separation can accurately retain impurities without adsorption of active ingredients. Low-temperature vacuum drying can significantly reduce the oxidation rate of active peptides to ≤2%. The final product has a moisture content of ≤3% and a solubility of ≥98% at 25℃, ensuring the purity, stability, and solubility of the active peptides. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the raw material pretreatment process of the present invention; Figure 2 This is a schematic diagram of the compound enzymatic hydrolysis process of the present invention; Figure 3 This is a schematic diagram of the purification and drying process of the present invention; Figure 4 This is a schematic diagram of the overall process of the present invention. Detailed Implementation

[0015] 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.

[0016] like Figures 1 to 4 As shown in the figure, this embodiment of the invention provides a method for preparing bioactive peptides processed by hybrid loach animal protein hydrolysate enzyme, comprising the following steps: Step S1, raw material pretreatment, involves freezing, breaking cell walls, and pre-enzymatic hydrolysis of fresh loach meat; Step S2, compound enzymatic hydrolysis, involves adding and mixing the protein solution; Step S3: Purification and drying, and effective filtration of the enzyme hydrolysate.

[0017] Through the continuous operation of step S1 (raw material pretreatment, freezing, cell wall disruption, pre-enzymatic hydrolysis), S2 (compound enzymatic hydrolysis, addition, mixing), and S3 (purification and drying), and effective filtration, a complete preparation process from raw materials to finished products is established. This solves the problems of active ingredient loss, low enzymatic hydrolysis efficiency, and complex purification in traditional processes, and provides a basic framework for the efficient preparation of highly active hybrid loach peptides.

[0018] The detailed process for raw material pretreatment in step S1 is as follows: Step S101: Freeze the fresh meat of hybrid loach at -18℃ for 24 hours; Step S102: Cell walls are broken by ultra-high pressure microjet at 300 MPa pressure, while 0.2% of the substrate mass of a complex protease is added. The complex protease is a mixture of papain and alkaline protease at a mass ratio of 1:2, and pre-enzymatic hydrolysis is carried out at 4°C. Step S103: Use dual-frequency ultrasound (20kHz and 40kHz, power 300W) to treat the pre-enzymatically hydrolyzed protein solution for 15 minutes.

[0019] By utilizing the ultrasonic cavitation effect to remove impurities such as mucoprotein and fat residue, and simultaneously relaxing protein molecules to increase the sites for subsequent enzymatic hydrolysis, the three factors synergistically improve the raw material processing effect.

[0020] The detailed process of step S2, compound enzymatic hydrolysis, is as follows: Step S201: Add a complex enzyme system to the pretreated protein solution for gradient temperature-controlled enzymatic hydrolysis; Step S202: The complex enzyme system consists of hybrid loach endogenous protease, flavor protease and neutral protease in an activity unit ratio of 3:2:1, and the total amount added is 1.2% of the substrate mass.

[0021] Step S2 involves compound enzymatic hydrolysis, which is achieved by adding a compound enzyme system in S201 with gradient temperature control and a compound enzyme system in S202 with an activity unit ratio of endogenous protease: flavor protease: neutral protease = 3:2:1, and a total addition amount of 1.2% of substrate mass. The endogenous protease is adapted to the characteristics of loach protein and works synergistically with the exogenous protease to cover more protein degradation sites. Gradient temperature control provides suitable reaction conditions for different enzymes, reduces the limitations of single enzymatic hydrolysis, and improves enzymatic hydrolysis efficiency and peptide quality.

[0022] The detailed process for step S3, purification and drying, is as follows: S301 involves sequentially passing the enzymatic hydrolysate through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane for multi-stage separation. The microfiltration membrane has a pore size of 0.22 μm, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa, and the nanofiltration membrane has a molecular weight cutoff of 1 kDa. A cross-flow filtration method is used. S302, add 0.5% maltodextrin by mass of the purified active peptide solution as a protective agent, and use low-temperature vacuum spray drying with an inlet air temperature of 65-70℃, an outlet air temperature of 35-40℃, and a vacuum degree of -0.09MPa to obtain active peptides.

[0023] The purification and drying process in step S3 involves sequentially passing the solution through a 0.22μm microfiltration membrane, a 10kDa ultrafiltration membrane, and a 1kDa nanofiltration membrane with cross-flow filtration in step S301. The microfiltration membrane removes impurities from the enzymatic hydrolysate, the ultrafiltration membrane retains large protein molecules, and the nanofiltration membrane enriches the target active peptides. Cross-flow filtration reduces membrane clogging. In step S302, 0.5% maltodextrin is added, and the solution is spray-dried at a low temperature with an inlet air temperature of 65-70℃, an outlet air temperature of 35-40℃, and a vacuum degree of -0.09MPa. The maltodextrin protects the structure of the active peptides, and the low-temperature vacuum environment reduces the oxidation of the active peptides and the inactivation of heat-sensitive components, ultimately yielding a high-purity active peptide product.

[0024] Gradient-temperature controlled enzymatic hydrolysis is divided into three stages: First stage: Enzymatic hydrolysis for 2 hours at 50℃ and pH 7.0-7.5; Second stage: Enzymatic hydrolysis for 3 hours at 45℃ and pH 7.0-7.5; Third stage: Enzymatic hydrolysis for 1 hour at 40℃ and pH 7.0-7.5.

[0025] Gradient temperature-controlled enzymatic hydrolysis is employed in three stages: 50℃ / pH 7.0-7.5 / 2h, 45℃ / pH 7.0-7.5 / 3h, and then 40℃ / pH 7.0-7.5 / 1h. This approach optimizes the temperature of different enzymes within the complex enzyme system, enabling the efficient, phased decomposition of large protein molecules. It first breaks down long protein chains and then gradually refines them into smaller peptides, optimizing peptide distribution and avoiding the problem of insufficient activity of some enzymes at a single temperature, thus improving the thoroughness of enzymatic hydrolysis.

[0026] In step 101, the activity units of endogenous protease in the intestinal tract of hybrid loach must be ≥500U / mg, and in step 102, the pre-enzymatic hydrolysis time at 4℃ is 1.5-2h to ensure a protein extraction rate ≥92%.

[0027] By limiting the activity of endogenous protease in the intestine of hybrid loach to ≥500 U / mg, sufficient catalytic activity of endogenous protease is ensured, guaranteeing its synergistic decomposition effect with exogenous protease. By limiting the pre-enzymatic hydrolysis time at 4℃ to 1.5-2h, the protein extraction efficiency and retention of active ingredients are balanced, ultimately achieving a protein extraction rate of ≥92%, providing sufficient high-quality protein raw materials for subsequent enzymatic hydrolysis.

[0028] In step 201, the gradient temperature-controlled enzymatic hydrolysis results in peptides with a molecular weight ≤1000 Da accounting for ≥85%.

[0029] By limiting the proportion of peptides with a molecular weight ≤1000Da to ≥85% after gradient temperature-controlled enzymatic hydrolysis, the enzymatic hydrolysis products are mainly small molecule active peptides. These peptides are more easily absorbed by the human body and can better retain their antioxidant, blood pressure-lowering and other biological activities, thereby enhancing the functional value of the finished product.

[0030] In step 301, the operating pressure of the multi-stage membrane separation is controlled at 0.3-0.5 MPa, the membrane surface flow rate is controlled at 1.5-2.0 m / s, and the backwashing cycle of the cross-flow filtration in step 301 is 30 min / time, with the backwashing pressure being 1.2 times the operating pressure, in order to reduce the membrane fouling rate.

[0031] By limiting the operating pressure of multi-stage membrane separation to 0.3-0.5 MPa and the membrane surface flow rate to 1.5-2.0 m / s, the membrane separation process is ensured to be stable and efficient, avoiding slow separation due to excessively low pressure or damage to the membrane structure due to excessively high pressure. By limiting the backwashing cycle of cross-flow filtration to 30 min / time and the backwashing pressure to 1.2 times the operating pressure, impurities attached to the membrane surface are effectively removed, reducing the membrane fouling rate, extending the membrane life, and ensuring the continuous stability of the purification process.

[0032] In step 302, the moisture content of the active peptide product is ≤3%, the solubility at 25℃ is ≥98%, and the feed rate of the low-temperature vacuum spray drying in step 302 is controlled at 15-20mL / min to ensure that the oxidation rate of the active peptide is ≤2%.

[0033] By limiting the moisture content of the finished active peptide product to ≤3%, mold growth due to excessive moisture is prevented, thus improving storage stability. By limiting the solubility to ≥98% at 25℃, the finished product is ensured to dissolve easily in subsequent applications such as functional foods and health products, facilitating processing and use. By limiting the feeding rate of low-temperature vacuum spray drying to 15-20mL / min, the heating time of the active peptides during the drying process is controlled. Combined with the low-temperature environment, the oxidation rate of the active peptides is ensured to be ≤2%, maximizing the preservation of biological activity.

[0034] Working principle and usage process: In the raw material pretreatment stage, fresh hybrid loach meat is first frozen at 18℃ for 24 hours to form ice crystals in the cells, destroying the basic cell structure. Then, the cell walls are broken up using a 300MPa ultra-high pressure micro-jet. At the same time, a complex protease consisting of papain and alkaline protease in a mass ratio of 1:2 (0.2% of the substrate mass) is added and pre-enzymatically hydrolyzed at 4℃ for 1.5-2 hours. The low temperature environment avoids the inactivation of heat-sensitive active ingredients. The complex protease initially decomposes the hydrogen bonds between protein molecules. Ultra-high pressure helps cell breakage and improves protein release efficiency. Finally, the mixture is treated with dual-frequency ultrasound at 20kHz and 40kHz at a power of 300W for 15 minutes. The ultrasonic cavitation effect removes mucoprotein and fat residues, while also allowing protein molecules to expand and increasing the sites for subsequent enzymatic hydrolysis, ensuring a protein extraction rate of ≥92%. In the complex enzymatic hydrolysis stage, a complex enzyme system consisting of hybrid loach endogenous protease, flavor protease, and neutral protease with an activity unit ≥500U / mg in an activity unit ratio of 3:2:1 was added to the pretreated protein solution. The total amount added was 1.2% of the substrate mass. Then, a three-stage gradient temperature-controlled enzymatic hydrolysis was performed. In the first stage, enzymatic hydrolysis was carried out at 50℃ and pH 7.0-7.5 for 2 hours. This temperature was used to adapt the optimal reaction conditions of some enzymes in the enzyme system to decompose large molecular weight proteins. In the second stage, enzymatic hydrolysis was carried out at 45℃ and pH 7.0-7.5 for 3 hours. This temperature was used to adapt the optimal temperature of other enzymes to further decompose proteins into small molecular weight peptides. In the third stage, enzymatic hydrolysis was carried out at 40℃ and pH 7.0-7.5 for 1 hour to optimize peptide distribution. Finally, the proportion of peptides with a molecular weight ≤1000Da was ≥85%. The complex enzyme system reduced the formation of bitter peptides through the synergistic effect of different action sites, and the gradient temperature control shortened the enzymatic hydrolysis cycle. In the purification and drying stage, the enzymatic hydrolysate is first sequentially passed through a 0.22 μm microfiltration membrane, a 10 kDa ultrafiltration membrane, and a 1 kDa nanofiltration membrane for multi-stage separation using a cross-flow filtration method. The operating pressure is controlled at 0.3-0.5 MPa, the membrane flow rate is controlled at 1.5-2.0 m / s, and the backwash cycle is 30 min / time, with a backwash pressure of 1.2 times the operating pressure. The microfiltration membrane removes impurity particles, the ultrafiltration membrane retains large protein molecules, and the nanofiltration membrane enriches the target active peptides. Cross-flow filtration and backwashing... The washing process reduces membrane fouling. Then, 0.5% maltodextrin by weight of the purified active peptide solution is added. Low-temperature vacuum spray drying is then used, with the inlet air temperature controlled at 65-70℃, the outlet air temperature at 35-40℃, the vacuum degree at 0.09MPa, and the feed rate at 15-20mL / min. The maltodextrin protects the active peptides, and the low-temperature vacuum environment reduces the oxidation of the active peptides. Finally, an active peptide product with a moisture content ≤3%, a solubility ≥98% at 25℃, and an oxidation rate ≤2% is obtained.

[0035] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0036] 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 bioactive peptides from hybrid loach animal protein hydrolysates using enzymes, characterized in that, Includes the following steps: Step S1, raw material pretreatment, involves freezing, breaking cell walls, and pre-enzymatic hydrolysis of fresh loach meat; Step S2, compound enzymatic hydrolysis, involves adding and mixing the protein solution; Step S3: Purification and drying, and effective filtration of the enzyme hydrolysate.

2. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 1, characterized in that: The detailed process for raw material pretreatment in step S1 is as follows: Step S101: Freeze the fresh meat of hybrid loach at -18℃ for 24 hours; Step S102: Cell walls are broken by ultra-high pressure microjet at 300 MPa pressure, while 0.2% of a complex protease by substrate mass is added. The complex protease is a mixture of papain and alkaline protease at a mass ratio of 1:2, and pre-enzymatic hydrolysis is performed at 4°C. Step S103: Use dual-frequency ultrasound (20kHz and 40kHz, power 300W) to treat the pre-enzymatically hydrolyzed protein solution for 15 minutes.

3. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 1, characterized in that: The detailed process for step S2, compound enzymatic hydrolysis, is as follows: Step S201: Add a complex enzyme system to the pretreated protein solution for gradient temperature-controlled enzymatic hydrolysis; Step S202: The complex enzyme system consists of hybrid loach endogenous protease, flavor protease and neutral protease in an activity unit ratio of 3:2:1, and the total amount added is 1.2% of the substrate mass.

4. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 1, characterized in that: The detailed process for purification and drying in step S3 is as follows: S301 involves sequentially passing the enzymatic hydrolysate through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane for multi-stage separation. The microfiltration membrane has a pore size of 0.22 μm, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa, and the nanofiltration membrane has a molecular weight cutoff of 1 kDa. A cross-flow filtration method is used. S302, add 0.5% maltodextrin by mass of the purified active peptide solution as a protective agent, and use low-temperature vacuum spray drying with an inlet air temperature of 65-70℃, an outlet air temperature of 35-40℃, and a vacuum degree of -0.09MPa to obtain active peptides.

5. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 3, characterized in that: The gradient temperature-controlled enzymatic hydrolysis is divided into three stages: First stage: Enzymatic hydrolysis for 2 hours at 50℃ and pH 7.0-7.5; Second stage: Enzymatic hydrolysis for 3 hours at 45℃ and pH 7.0-7.5; Third stage: Enzymatic hydrolysis for 1 hour at 40℃ and pH 7.0-7.

5.

6. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 2, characterized in that: The activity units of the endogenous protease in the intestinal tract of the hybrid loach mentioned in step 101 need to be ≥500U / mg, and the pre-enzymatic hydrolysis time at 4℃ in step 102 is 1.5-2h to ensure that the protein extraction rate is ≥92%.

7. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 3, characterized in that: In step 201, the gradient temperature-controlled enzymatic hydrolysis results in peptides with a molecular weight ≤1000 Da accounting for ≥85%.

8. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 4, characterized in that: In step 301, the operating pressure of the multi-stage membrane separation is controlled at 0.3-0.5 MPa, and the membrane surface flow rate is controlled at 1.5-2.0 m / s. In step 301, the backwashing cycle of the cross-flow filtration is 30 min / time, and the backwashing pressure is 1.2 times the operating pressure, in order to reduce the membrane fouling rate.

9. The method for preparing bioactive peptides from hybrid loach animal protein hydrolysates according to claim 4, characterized in that: The moisture content of the active peptide product described in step 302 is ≤3%, and the solubility at 25℃ is ≥98%. The feed rate of the low-temperature vacuum spray drying in step 302 is controlled at 15-20mL / min to ensure that the oxidation rate of the active peptide is ≤2%.

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