Low-salt full-water-soluble intestinal membrane peptide powder as well as preparation method and application thereof

Abstract

The invention relates to the technical field of intestinal membrane peptide preparation, and provides low-salt full-water-soluble intestinal membrane peptide powder and a preparation method and application thereof.The preparation method comprises the steps that an intestinal protein matrix and water are mixed, and an enzymolysis substrate is obtained; alkaline protease is added into the enzymolysis substrate for first-stage enzymolysis, and the pH value of the system is maintained to be 8-10 with calcium hydroxide; after the first-stage enzymolysis is completed, an enzyme preparation is directly added for second-stage enzymolysis without inactivation treatment, the pH value of the system is maintained to be 8-10 with calcium hydroxide, and the enzyme preparation comprises one or more of papain, animal protease and flavor enzyme; and after the enzymolysis is finished, inactivating, adding phosphoric acid to adjust the pH value to 6.7-7.2, precipitating, filtering, collecting a liquid phase, and spray-drying to obtain intestinal membrane peptide powder. By means of the technical scheme, the problems that raw material pretreatment is complex when intestinal membrane peptide powder is prepared in the prior art, and the intestinal membrane peptide powder is low in protein content, high in ash content and poor in water solubility are solved.

Description

Technical Field

[0001] This invention relates to the field of intestinal membrane peptide preparation technology, specifically to a low-salt, fully water-soluble intestinal membrane peptide powder, its preparation method, and its application. Background Technology

[0002] Intestinal membrane peptides are protein raw materials rich in small molecule peptides, obtained from the byproducts of heparin extraction from porcine small intestinal mucosa (hereinafter referred to as intestinal protein matrix) through enzymatic hydrolysis, separation, and other processes. They possess functions such as easy absorption, growth promotion, and enhanced immunity. Currently, they are widely used in high-end feed and pet food, and also show considerable promise in the field of functional protein raw materials.

[0003] At present, the production process of intestinal membrane peptide powder in the industry generally has the following defects: (1) In order to ensure the efficiency of enzymatic hydrolysis, the raw material intestinal protein matrix needs to undergo pretreatment processes such as filtration, drying and pulverization. The steps are complicated and the energy consumption and labor costs are high. (2) In order to achieve the most suitable reaction conditions for different proteases, in the multi-step enzymatic hydrolysis process, it is often necessary to heat up and inactivate after each enzymatic hydrolysis step before proceeding to the next enzymatic hydrolysis step. This process is time-consuming and energy-intensive, and high temperature can easily lead to the inactivation of active peptides. (3) Intestinal membrane peptide powder has poor water solubility and low clarification rate, which limits its application in liquid feed or high transparency products. (4) Intestinal membrane peptide powder has high salt content. In order to reduce its ash content, it is also necessary to use ion exchange resin, molecular biomembrane and other methods to desalinate, which increases equipment investment and production costs. Summary of the Invention

[0004] This invention proposes a low-salt, fully water-soluble intestinal membrane peptide powder, its preparation method, and its application, thereby solving or at least alleviating one or more of the above-mentioned problems and other problems existing in the prior art.

[0005] The technical solution of the present invention is as follows: This invention proposes a method for preparing low-salt, fully water-soluble intestinal membrane peptide powder, comprising: Preparation of enzymatic hydrolysis substrate: Mix the intestinal protein matrix with water to obtain the enzymatic hydrolysis substrate; First stage of enzymatic hydrolysis: Alkaline protease is added to the enzymatic hydrolysis substrate to carry out the first stage of enzymatic hydrolysis. During the first stage of enzymatic hydrolysis, calcium hydroxide is used to maintain the pH of the enzymatic hydrolysis system at 8-10. Second-stage enzymatic hydrolysis: After the first-stage enzymatic hydrolysis is completed, without inactivation treatment, the enzyme preparation is directly added for the second-stage enzymatic hydrolysis. During the second-stage enzymatic hydrolysis, calcium hydroxide is used to maintain the pH of the enzymatic hydrolysis system at 8-10. The enzyme preparation includes one or more of papain, animal protease, and flavor enzyme. Neutralization and filtration: After the second stage of enzymatic hydrolysis, the enzyme is inactivated, and phosphate is added to adjust the pH to 6.7-7.2. The precipitate is then filtered and the liquid phase is collected. Spray drying: The liquid phase is spray dried to obtain intestinal membrane peptide powder.

[0006] As a further technical solution, the weight fraction of the enzymatic hydrolysis substrate is 4% to 8%.

[0007] As a further technical solution, the weight of the enzymatic hydrolysate is used as the basis; The amount of alkaline protease added is 0.1% to 0.3%; and / or The amount of enzyme preparation added is 0.01% to 0.03%.

[0008] As a further technical solution, the temperature for the first stage of enzymatic hydrolysis is 50~60℃ and the time is 3~4h; and / or The second stage of enzymatic hydrolysis is carried out at a temperature of 50-60℃ for 3-4 hours.

[0009] As a further technical solution, during the precipitation process, a filter aid is added, which includes diatomaceous earth or perlite.

[0010] As a further technical solution, the filtration includes coarse filtration and fine filtration.

[0011] As a further technical solution, the fine filtration is performed by sequentially using a microfiltration membrane and a nanofiltration membrane.

[0012] As a further technical solution, the pore size of the microfiltration membrane is ≤0.22μm, and the molecular weight cutoff of the nanofiltration membrane is 80~100Da.

[0013] This invention also proposes a low-salt, fully water-soluble intestinal membrane peptide powder, which is prepared by the aforementioned preparation method.

[0014] The present invention also proposes the application of the intestinal membrane peptide powder prepared by the preparation method described herein, or the intestinal membrane peptide powder described herein, in the preparation of feed, food, or functional protein raw materials.

[0015] The beneficial effects of this invention are as follows: The enzymatic hydrolysis, neutralization, and filtration steps in the preparation process of this invention have a synergistic effect. Through specific enzyme combinations and stepwise enzymatic hydrolysis, and by combining the alkaline calcium hydroxide environment of the enzymatic hydrolysis system with the acidic phosphoric acid environment of the neutralization step, the preparation process can be simplified, production costs reduced, and the intestinal membrane peptide powder can achieve low ash content and high water solubility. Specifically: (1) In the stepwise enzymatic hydrolysis strategy of the present invention, alkaline protease can first degrade macromolecular proteins, liquefying the original solid-liquid mixture and reducing viscosity, thus providing a good system environment for subsequent enzymatic hydrolysis. In addition, under specific enzyme combinations, there is no need for any pretreatment of the raw material intestinal protein matrix, nor is there a need to inactivate the alkaline protease after the first stage of enzymatic hydrolysis. The second stage of enzymatic hydrolysis can be carried out directly, and the subsequent enzyme preparation can still play a highly efficient role. This not only eliminates the pretreatment process such as raw material impurity removal in the prior art, but also reduces the energy consumption and time of repeated heating and cooling caused by repeated inactivation in the stepwise enzymatic hydrolysis in the prior art, shortens the production cycle, reduces production costs, and avoids damage to the activity of intestinal membrane peptide powder by frequent heat treatment, thereby improving the enzymatic hydrolysis efficiency and the quality of intestinal membrane peptide powder.

[0016] (2) In the enzymatic hydrolysis stage, calcium hydroxide is used to adjust the pH value of the system. After the enzymatic hydrolysis is completed, phosphoric acid is added to neutralize the calcium hydroxide, which can generate calcium phosphate precipitate. This precipitate is removed in the subsequent filtration process. On the one hand, this transforms the soluble salt (such as sodium chloride) in the prior art into a removable insoluble precipitate, thereby reducing the ash content of the intestinal membrane peptide powder from the source. On the other hand, during the neutralization process, some acid-soluble but water-insoluble protein components will precipitate and co-precipitate along with the formation of calcium phosphate, and thus be removed in the filtration step. This ensures that the small molecule peptides retained in the liquid phase are all water-soluble components, thus guaranteeing the fully water-soluble characteristics of the intestinal membrane peptide powder. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] It should be understood that, unless the context clearly indicates otherwise, the terms "comprising," "including," or "having" as used herein refer to the presence of a particular element, but do not exclude the presence or addition of one or more other elements. Furthermore, as used herein, "comprising" and / or "including" specify the presence of shapes, numbers, steps, operations, members, elements, and / or combinations thereof, and do not exclude the presence or addition of one or more other shapes, numbers, operations, elements, and / or combinations thereof.

[0019] In this invention, the numerical range indicated by "~" refers to the range of values ​​specified as the lower and upper limits, respectively, before or after the term. When multiple values ​​for the upper or lower limit of any numerical range are mentioned, the range disclosed herein can be understood as a range with any one of the mentioned upper limits as its upper limit and any one of the mentioned lower limits as its lower limit.

[0020] Chinese patent application CN105779546A discloses a method for preparing intestinal membrane peptides. The method involves pretreatment steps such as filtration, deodorization, and molecular biomembrane desalination of the intestinal membrane stock solution, followed by enzymatic hydrolysis and evaporation concentration to obtain the intestinal membrane peptide product. Testing revealed that the ash content of the intestinal membrane peptide product obtained by this method is capped at 20%. This not only reduces the content of effective components such as protein in the product but also affects palatability when used in animal feed. This preparation method suffers from drawbacks including complex raw material pretreatment steps, high production costs, and failure to significantly reduce the upper limit of ash content in the intestinal membrane peptide product. Furthermore, this method does not address the water solubility of the intestinal membrane peptide product, limiting its application in high-transparency products.

[0021] Based on this, the present invention provides a method for preparing intestinal membrane peptide powder that can simultaneously achieve simplified process flow, low production cost, low ash content, and high water solubility by using a specific enzymatic hydrolysis system, a stepwise enzymatic hydrolysis strategy, and the synergistic effect between the enzymatic hydrolysis step and the neutralization and filtration step.

[0022] The following will describe in detail a low-salt, fully water-soluble intestinal membrane peptide powder according to embodiments of the present invention, its preparation method, and its application.

[0023] According to one aspect of the present invention, a method for preparing low-salt, fully water-soluble intestinal membrane peptide powder is provided, comprising the following steps: S1. Preparation of enzymatic hydrolysis substrate: Mix the intestinal protein matrix with water to obtain the enzymatic hydrolysis substrate; S2, First stage of enzymatic hydrolysis: Alkaline protease is added to the enzymatic hydrolysis substrate to carry out the first stage of enzymatic hydrolysis. During the first stage of enzymatic hydrolysis, calcium hydroxide is used to maintain the pH of the enzymatic hydrolysis system at 8~10. S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. Instead, enzyme preparations are directly added for the second stage of enzymatic hydrolysis. During the second stage of enzymatic hydrolysis, calcium hydroxide is used to maintain the pH of the enzymatic hydrolysis system at 8-10. The enzyme preparations include one or more of papain, animal protease, and flavor enzymes. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the enzyme is inactivated, and phosphate is added to adjust the pH to 6.7~7.2. The precipitate is then filtered and the liquid phase is collected. S5. Spray drying: The liquid phase is spray dried to obtain intestinal membrane peptide powder.

[0024] In step S1 of this invention, the initial raw material for the intestinal membrane peptide powder is a byproduct of heparin extraction from porcine small intestinal mucosa (hereinafter referred to as intestinal protein matrix). The intestinal protein matrix is ​​directly mixed with water to obtain the enzymatic hydrolysis substrate, and enzymatic hydrolysis can begin immediately without any pretreatment steps. This not only simplifies the process but also significantly reduces water consumption, energy consumption, and equipment investment, resulting in a substantial reduction in production costs. The weight fraction of the enzymatic hydrolysis substrate is 4% to 8%, for example, including but not limited to any value within 4%, 5%, 6%, 7%, and 8%, and the range between any two values. Within this range, the enzyme and substrate can fully contact and react, facilitating the effective action of each enzyme, ensuring enzymatic hydrolysis efficiency and product quality, while also considering production costs. On the one hand, it avoids insufficient enzyme utilization, low production efficiency, and increased costs due to an excessively low substrate weight fraction; on the other hand, it avoids excessive viscosity and uneven enzymatic hydrolysis due to an excessively high substrate weight fraction.

[0025] In step S2 of this invention, the amount of alkaline protease added is 0.1% to 0.3% based on the weight of the enzymatic hydrolysis substrate. For example, it includes, but is not limited to, any value from 0.1%, 0.2%, and 0.3%, or a range between any two values. Within this range, the alkaline protease can react appropriately with the enzymatic hydrolysis substrate, effectively degrading large protein molecules initially, reducing system viscosity, creating a favorable environment for subsequent enzymatic hydrolysis, and improving enzymatic hydrolysis efficiency and the quality of intestinal membrane peptide powder. On the one hand, it avoids insufficient degradation of large protein molecules due to too low an addition, resulting in high system viscosity and difficulty in efficient subsequent enzymatic hydrolysis; on the other hand, it avoids over-hydrolysis due to too high an addition, which would damage the quality of the intestinal membrane peptide powder. The temperature for the first stage of enzymatic hydrolysis is 50-60℃, and the time is 3-4 hours. For example, the temperature can be any value from 50℃, 55℃, and 60℃, or a range between any two values; the time can be any value from 3 hours, 3.5 hours, and 4 hours, or a range between any two values. Within this range, the alkaline protease activity can be fully utilized, efficiently degrading large protein molecules and optimizing the enzymatic hydrolysis system environment, laying a good foundation for subsequent enzymatic hydrolysis. On the one hand, it avoids the limitations of enzyme activity and insufficient degradation of large protein molecules caused by excessively low temperatures or short times, which would prevent the system viscosity from being effectively reduced and affect the subsequent enzymatic hydrolysis process. On the other hand, it also avoids premature enzyme inactivation and over-hydrolysis caused by excessively high temperatures or long times, which would damage the quality of the intestinal membrane peptide powder.

[0026] In step S3 of this invention, during the second-stage enzymatic hydrolysis, the enzyme preparation includes one or more of papain, animal protease, and flavor enzyme. The amount of enzyme preparation added, based on the weight of the hydrolysate, is 0.01% to 0.03%, for example, including but not limited to any value among 0.01%, 0.02%, and 0.03%, and a range between any two such values. Within this range, the enzyme preparation can fully interact with the substrate hydrolyzed in the first stage, further enhancing the hydrolysis effect and optimizing the quality of the intestinal membrane peptide powder. On the one hand, this avoids insufficient hydrolysis due to excessively low addition, which would prevent the enzyme preparation from fully exerting its further degradation effect on the substrate; on the other hand, it avoids over-hydrolysis due to excessive addition, which could alter the molecular structure of the intestinal membrane peptide powder and affect its quality. The temperature for the second-stage enzymatic hydrolysis is 50-60℃, and the time is 3-4 hours. For example, the temperature can be any value among 50℃, 55℃, and 60℃, and a range between any two such values; the time can be any value among 3 hours, 3.5 hours, and 4 hours, and a range between any two such values. Within this range, suitable reaction conditions can be provided for the enzyme preparation, promoting a full reaction with the substrate, effectively improving the enzymatic hydrolysis effect, and ensuring the quality of the intestinal membrane peptide powder. On the one hand, it can avoid excessively low temperatures or short times, which would lead to insufficient enzyme activity, incomplete substrate hydrolysis, and failure to effectively exert the enzyme preparation's function; on the other hand, it can also avoid excessively high temperatures or long times, which would reduce or even deactivate the enzyme preparation.

[0027] In this invention, the amount of enzyme preparation added is significantly lower than that added with alkaline protease. This is because alkaline protease is mainly responsible for the initial degradation of large protein molecules, breaking down complex protein structures, reducing system viscosity, and creating favorable conditions for subsequent enzymatic hydrolysis. This process requires a relatively large amount of alkaline protease to ensure the degradation effect. The enzyme preparation, on the other hand, further degrades the already fragmented protein based on the initial degradation by alkaline protease. After the first stage of enzymatic hydrolysis, the substrate structure is more easily hydrolyzed, so only a small amount of enzyme preparation is needed to function efficiently. Excessive enzyme preparation can lead to over-hydrolysis, which may damage the beneficial peptide chain structure in the intestinal membrane peptide powder, altering its molecular properties and affecting its water solubility.

[0028] In step S4 of this invention, calcium phosphate precipitate is generated by adding phosphoric acid and calcium hydroxide, avoiding the introduction of large amounts of soluble inorganic salts and the resulting increase in ash content of the intestinal membrane peptide powder caused by repeated acid-base adjustments in the prior art. Furthermore, the desalination steps using ion exchange resins or molecular biomembranes, as in the prior art, are omitted, saving production costs. In this invention, maintaining the pH value of the enzymatic hydrolysis system with calcium hydroxide refers to maintaining the pH value of the enzymatic hydrolysis system using calcium hydroxide, calcium hydroxide solution, or calcium hydroxide suspension. This invention does not require a specific mass fraction of calcium hydroxide solution or calcium hydroxide suspension; those skilled in the art can adjust it as needed. Preferably, a 30% mass fraction calcium hydroxide suspension is used to maintain the pH value of the enzymatic hydrolysis system. In this invention, the mass fraction of phosphoric acid is not required; those skilled in the art can adjust it as needed. Preferably, a 20% mass fraction phosphoric acid is used. During precipitation, a filter aid can be added to promote rapid precipitation. The filter aid can be any commonly used filter aid in the art, preferably diatomaceous earth or perlite.

[0029] In one embodiment of the invention, filtration includes coarse filtration and fine filtration.

[0030] Current technologies typically employ only coarse filtration methods, such as plate and frame filtration or centrifugal filtration. This approach is insufficient to remove microorganisms and viruses, posing biosafety risks. Furthermore, unremoved fats are prone to oxidative rancidity during storage, affecting the flavor and shelf life of the intestinal membrane peptide powder. Additionally, it can impact the water solubility of the intestinal membrane peptide powder. Combining coarse and fine filtration can effectively address these issues. Moreover, directly performing fine filtration without coarse filtration is prone to clogging and is extremely inefficient.

[0031] In one embodiment of the present invention, fine filtration is performed by sequentially using a microfiltration membrane and a nanofiltration membrane.

[0032] This invention achieves both filtration and concentration through membrane filtration. In existing technologies, carriers such as soybean meal, wheat bran, and starch are added to achieve better concentration. The addition of these carriers dilutes the protein content of the intestinal membrane peptide powder; furthermore, their poor water solubility or presence of insoluble components severely affects the water solubility and clarity of the intestinal membrane peptide powder. The liquid phase concentration after membrane filtration in this invention already meets the requirements for spray drying, thus eliminating the need for any carriers. This not only ensures the high protein content of the intestinal membrane peptide powder but also guarantees its high water solubility.

[0033] In one embodiment of the present invention, the pore size of the microfiltration membrane is ≤0.22μm, and the molecular weight cutoff of the nanofiltration membrane is 80~100Da.

[0034] In this invention, a combined fine filtration process using a microfiltration membrane with a pore size ≤0.22μm and a nanofiltration membrane with a pore size of 80~100Da is employed. On the one hand, this process can replace evaporation concentration. Specifically, the 80~100Da nanofiltration membrane can effectively retain target peptide molecules while allowing water molecules and some small salt molecules to pass through, thereby achieving physical concentration at room temperature. This significantly reduces energy consumption while avoiding the destruction of heat-sensitive peptides. On the other hand, it can also ensure biosafety. Specifically, the microfiltration membrane with a pore size ≤0.22μm can effectively retain bacteria or viruses such as Escherichia coli and Staphylococcus aureus, improving the biosafety of the intestinal membrane peptide powder.

[0035] According to another aspect of the present invention, a low-salt, fully water-soluble intestinal membrane peptide powder is prepared by the above-described preparation method.

[0036] According to another aspect of the present invention, the present invention also proposes the application of the intestinal membrane peptide powder prepared by the above-described preparation method or the intestinal membrane peptide powder prepared by the above-described preparation method in the preparation of feed, food or functional protein raw materials.

[0037] The present invention will now be described in detail with reference to examples. The embodiments of the invention described below can be modified in various ways, and therefore the scope of the invention should not be construed as limited to the embodiments described in detail below. Examples are provided to help those skilled in the art to more readily understand the invention.

[0038] In the following examples and comparative examples, the enzyme activity of alkaline protease was 200,000 U / g, the enzyme activity of papain was 100,000 U / g, the enzyme activity of animal protease was 100,000 U / g, and the enzyme activity of flavor enzyme was 1,000 U / g. The gut-derived protein matrix is ​​a byproduct of heparin extraction from porcine small intestinal mucosa using conventional methods; Protein content was tested according to the Kjeldahl method in GB5009.5-2025, and the results were rounded to two decimal places. Ash content was tested according to GB 5009.4-2016, and the results were based on the mass of the sample without magnesium acetate solution and were retained to two decimal places. Moisture content was tested according to the direct drying method in GB 5009.3-2016, and the results were retained to two decimal places. Water solubility was characterized by the transmittance of a 1 wt% intestinal membrane peptide powder solution. The transmittance was tested using a commercially available spectrophotometer, and the results were rounded to two decimal places.

[0039] Example 1 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 6%, heat and stir at the same time, and raise the temperature to 60℃. S2, First stage of enzymatic hydrolysis: Adjust the pH of the system to 10 with a 30% calcium hydroxide suspension, add 0.2% alkaline protease based on the weight of the enzymatic hydrolysate, and enzymatically hydrolyze for 3 hours at 60°C; S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. The pH of the system is maintained at 10 using a 30% calcium hydroxide suspension. Based on the weight of the enzymatic hydrolysate, 0.02% papain is directly added to carry out the second stage of enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 60℃ for 3 hours. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 20% phosphoric acid is added to adjust the pH to 7.2, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S5. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 83.41% protein, 6.99% ash, 3.42% moisture, and 91.00% water solubility.

[0040] Example 2 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 4%, heat and stir at the same time, and raise the temperature to 50°C. S2, First stage of enzymatic hydrolysis: Adjust the pH of the system to 8 with a 30% calcium hydroxide suspension, add 0.1% alkaline protease based on the weight of the enzymatic hydrolysate, and enzymatically hydrolyze at 50℃ for 4 hours; S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. The pH of the system is maintained at 8 using a 30% calcium hydroxide suspension. Based on the weight of the enzymatic hydrolysate, 0.01% animal protease is directly added to carry out the second stage of enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 50℃ for 4 hours. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 20% phosphoric acid is added to adjust the pH to 6.7, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S5. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 86.63% protein, 5.38% ash, 4.12% moisture, and 92.27% water solubility.

[0041] Example 3 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 8%, heat and stir at the same time, and raise the temperature to 55℃. S2, First stage of enzymatic hydrolysis: Adjust the pH of the system to 8.5 with a 30% calcium hydroxide suspension, add 0.3% alkaline protease based on the weight of the enzymatic hydrolysate, and enzymatically hydrolyze at 55℃ for 3.5h; S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. The pH of the system is maintained at 8.5 using a 30% calcium hydroxide suspension. Based on the weight of the enzymatic hydrolysate, 0.03% of flavor enzyme is directly added to carry out the second stage of enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 55℃ for 3.5 hours. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 20% phosphoric acid is added to adjust the pH to 7.0, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S5. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 85.71% protein, 5.20% ash, 4.18% moisture, and 91.77% water solubility.

[0042] Example 4 The only difference between this embodiment and Embodiment 1 is that, in this embodiment, during filtration, plate and frame coarse filtration and membrane fine filtration are performed sequentially. Specifically, the coarse filtration is first performed by a plate and frame filter press, and then fine filtration is performed by pumping in a 0.22μm microfiltration membrane system and a 100Da nanofiltration membrane system sequentially. Tests showed that the intestinal membrane peptide powder contained 88.05% protein, 2.77% ash, 2.35% moisture, and 96.35% water solubility.

[0043] Comparative Example 1 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 6%, heat and stir at the same time, and raise the temperature to 60℃. S2. Enzymatic hydrolysis: Adjust the pH of the system to 10 with a 30% calcium hydroxide suspension. Add 0.2% alkaline protease and 0.02% papain by weight of the substrate and hydrolyze at 60°C for 6 hours. S3, Neutralization and Filtration: After enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 20% phosphoric acid is added to adjust the pH to 7.2, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S4. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 67.38% protein, 8.95% ash, 2.50% moisture, and 92.16% water solubility.

[0044] Comparative Example 2 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 6%, heat and stir at the same time, and raise the temperature to 60℃. S2, First stage of enzymatic hydrolysis: Adjust the pH of the system to 10 with a 30% calcium hydroxide suspension, add 0.2% papain based on the weight of the enzymatic hydrolysate, and enzymatically hydrolyze at 60℃ for 3 hours; S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. The pH of the system is maintained at 10 using a 30% calcium hydroxide suspension. Based on the weight of the enzymatic hydrolysate, 0.02% alkaline protease is added directly to carry out the second stage of enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 60℃ for 3 hours. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 20% phosphoric acid is added to adjust the pH to 7.2, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S5. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 59.81% protein, 5.70% ash, 2.46% moisture, and 91.45% water solubility.

[0045] Comparative Example 3 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 6%, heat and stir at the same time, and raise the temperature to 60℃. S2, First stage of enzymatic hydrolysis: Adjust the pH of the system to 10 with a 30% calcium hydroxide suspension, add 0.2% alkaline protease based on the weight of the enzymatic hydrolysate, and enzymatically hydrolyze for 3 hours at 60°C; S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. The pH of the system is maintained at 10 using a 30% calcium hydroxide suspension. Based on the weight of the enzymatic hydrolysate, 0.02% papain is directly added to carry out the second stage of enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 60℃ for 3 hours. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 30% hydrochloric acid is added to adjust the pH to 7.2, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S5. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 77.77% protein, 13.16% ash, 3.64% moisture, and 92.28% water solubility.

[0046] Comparative Example 4 A method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder includes the following steps: S1. Preparation of enzymatic hydrolysis substrate: Add 200 kg of intestinal protein matrix into the enzymatic hydrolysis tank, add water until the weight fraction of the enzymatic hydrolysis substrate is 6%, heat and stir at the same time, and raise the temperature to 60℃. S2, First stage of enzymatic hydrolysis: Adjust the pH of the system to 10 with a 20% sodium hydroxide solution, add 0.2% alkaline protease based on the weight of the enzymatic hydrolysate, and enzymatically hydrolyze for 3 hours at 60°C; S3. Second stage of enzymatic hydrolysis: After the first stage of enzymatic hydrolysis is completed, no inactivation treatment is performed. The pH of the system is maintained at 10 using a 20% sodium hydroxide solution. Based on the weight of the enzymatic hydrolysate, 0.02% papain is directly added to carry out the second stage of enzymatic hydrolysis. The enzymatic hydrolysis is carried out at 60℃ for 3 hours. S4, Neutralization and Filtration: After the second stage of enzymatic hydrolysis, the temperature is raised to 90℃ for inactivation. 20% phosphoric acid is added to adjust the pH to 7.2, causing precipitation. 20kg of food-grade diatomaceous earth is added as a filter aid and the mixture is filtered through a plate and frame filter press. The liquid phase is collected. S5. Spray drying: The liquid phase is fed into a spray drying tower and spray dried to obtain intestinal membrane peptide powder; Tests showed that the intestinal membrane peptide powder contained 76.37% protein, 15.42% ash, 2.40% moisture, and 91.93% water solubility.

[0047] The comparison between Example 1 and Comparative Examples 1-4 shows that the present invention achieves the preparation of intestinal membrane peptide powder with high protein content, low ash content and high water solubility by using a specific enzyme combination and stepwise enzymatic hydrolysis, and by combining the alkaline environment of calcium hydroxide in the enzymatic hydrolysis system with the acidic environment of phosphoric acid in the neutralization step.

[0048] The above are merely preferred embodiments of the present invention and are 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 method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder, characterized in that, include: Preparation of enzymatic hydrolysis substrate: Mix the intestinal protein matrix with water to obtain the enzymatic hydrolysis substrate; First stage of enzymatic hydrolysis: Alkaline protease is added to the enzymatic hydrolysis substrate to carry out the first stage of enzymatic hydrolysis. During the first stage of enzymatic hydrolysis, calcium hydroxide is used to maintain the pH of the enzymatic hydrolysis system at 8-10. Second-stage enzymatic hydrolysis: After the first-stage enzymatic hydrolysis is completed, without inactivation treatment, the enzyme preparation is directly added for the second-stage enzymatic hydrolysis. During the second-stage enzymatic hydrolysis, calcium hydroxide is used to maintain the pH of the enzymatic hydrolysis system at 8-10. The enzyme preparation includes one or more of papain, animal protease, and flavor enzyme. Neutralization and filtration: After the second stage of enzymatic hydrolysis, the enzyme is inactivated, and phosphate is added to adjust the pH to 6.7-7.

2. The precipitate is then filtered and the liquid phase is collected. Spray drying: The liquid phase is spray dried to obtain intestinal membrane peptide powder.

2. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 1, characterized in that, The weight fraction of the enzymatic hydrolysate is 4% to 8%.

3. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 1, characterized in that, Based on the weight of the enzymatically hydrolyzed substrate; The amount of alkaline protease added is 0.1% to 0.3%; and / or The amount of enzyme preparation added is 0.01% to 0.03%.

4. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 1, characterized in that, The first stage of enzymatic hydrolysis is carried out at a temperature of 50-60℃ for 3-4 hours; and / or The second stage of enzymatic hydrolysis is carried out at a temperature of 50-60℃ for 3-4 hours.

5. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 1, characterized in that, During the precipitation process, a filter aid is added, which may include diatomaceous earth or perlite.

6. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 1, characterized in that, The filtration includes coarse filtration and fine filtration.

7. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 6, characterized in that, The fine filtration process involves sequentially using a microfiltration membrane and a nanofiltration membrane.

8. The method for preparing a low-salt, fully water-soluble intestinal membrane peptide powder according to claim 7, characterized in that, The microfiltration membrane has a pore size of ≤0.22μm, and the nanofiltration membrane has a molecular weight cutoff of 80~100Da.

9. A low-salt, fully water-soluble intestinal membrane peptide powder, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The intestinal membrane peptide powder prepared by the preparation method according to any one of claims 1 to 8 or the intestinal membrane peptide powder according to claim 9, in the preparation of feed, food or functional protein raw materials.