Lactoferrin antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a lactoferrin antibacterial peptide as well as a preparation method and application thereof. The invention provides a preparation method of lactoferrin antibacterial peptide with improved heat resistance and antibacterial property, which comprises the following steps: a preparation step: dissolving lactoferrin in a solvent to prepare a protein solution; an enzymolysis step: carrying out step-by-step enzymolysis on the protein solution, firstly adding trypsin to carry out a first enzymolysis reaction, and then adding flavourzyme to carry out a second enzymolysis reaction; and an ultrafiltration step: performing ultrafiltration on the product obtained in the second enzymolysis reaction step by using a filter membrane, and retaining the filtrate, the purity of the lactoferrin being more than 80% by mass. Through a specific composite enzymolysis process, the prepared lactoferrin antibacterial peptide has specific molecular weight distribution, and the antibacterial property of the lactoferrin antibacterial peptide is still kept after heat treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactoferricin, its preparation method and application, and belongs to the field of proteolysis, more specifically, the present application relates to a lactoferricin with high heat resistance, its preparation method and application. BACKGROUND

[0002] Lactoferricin is an important natural immune protein, which is widely present in the milk of mammals, especially colostrum. It not only has the ability to bind and transport iron, but also releases lactoferricin peptides with stronger biological activity after protease hydrolysis, which has attracted much attention. Lactoferricin peptides, especially antibacterial peptides derived from their N-terminus, have a wide range of physiological functions such as broad-spectrum antibacterial, antiviral, and immune regulation, and have extremely important nutritional value for the infant population with immature immune system. However, the direct application of lactoferricin peptides, especially their antibacterial active fragments, in infant formula milk powder and other heat-processed dairy products faces key technical bottlenecks in the prior art.

[0003] The prior art faces two key technical bottlenecks:

[0004] First, the heat resistance of natural lactoferricin peptides is poor. In the production process of infant formula milk powder, heat treatment such as spray drying and high-temperature sterilization is essential. These high-temperature environments can easily lead to the destruction of the spatial structure of natural lactoferricin peptides and the inactivation of active sites, greatly reducing or even completely losing their antibacterial and immune regulation functions, making it difficult to achieve the expected efficacy in the final product.

[0005] Second, the traditional enzymatic hydrolysis process lacks directionality and efficiency. In the prior art, single proteases (such as pepsin and trypsin) are often used to hydrolyze lactoferricin. This method has problems such as single enzyme cleavage site and strong randomness in the hydrolysis process, making it difficult to efficiently and specifically release target antibacterial peptide fragments (such as lactoferricin peptides). Meanwhile, the composition of the hydrolysis product is complex, the yield of active peptides is low, and bitter peptides may be produced, which affects the flavor of the product, which is particularly important for infants who are picky about taste.

[0006] The prior art attempts to optimize, for example:

[0007] Reference Document 1 provides a heat-resistant lactoferricin active peptide and its preparation method and application, which uses neutral protease and flavor protease to achieve a specific degree of hydrolysis interval, and finally obtains a peptide segment that can withstand high-temperature treatment. However, this method still has problems. The degree of hydrolysis is a measure of the results of enzymatic hydrolysis, which has a lag in actual production, making it difficult to achieve real-time and precise end-point control, which may lead to unstable quality between batches. This method also lacks post-processing steps such as purification of the product, debittering, and enrichment of target peptides, and only a complex mixture of peptides is obtained directly, rather than the final product.

[0008] Reference 2 provides a method and application for preparing lactoferrin bioactive peptides through dual enzymatic hydrolysis. It uses pepsin and trypsin for enzymatic hydrolysis to obtain lactoferrin bioactive peptides. The heat resistance and flavor of the product were not investigated.

[0009] Therefore, there is an urgent need in this field for a method to prepare a complex enzyme containing highly heat-resistant lactoferrin antimicrobial peptides and for their application in dairy products.

[0010] References

[0011] Reference 1 CN103014110A

[0012] Reference 2 CN118979089A Summary of the Invention

[0013] Problem to be solved by the invention

[0014] In existing technologies, natural lactoferrin antimicrobial peptides exhibit poor thermal stability. The prepared lactoferrin antimicrobial peptides are prone to denaturation and inactivation under common heat processing methods in dairy products (such as pasteurization and UHT sterilization) or high-temperature environments, leading to the loss of their antimicrobial function and significantly limiting their application in heat-treated dairy products. Therefore, it is necessary to overcome the shortcomings of insufficient heat resistance in existing lactoferrin antimicrobial peptides and to develop a composite enzyme preparation method that can significantly improve the thermal stability of lactoferrin antimicrobial peptides, thereby expanding their application in processed dairy products.

[0015] Therefore, this invention provides a composite bio-enzyme catalysis method that enables specific enzymatic hydrolysis and targeted modification of lactoferrin substrates, efficiently generating the target antimicrobial peptide under mild conditions. Simultaneously, enzymatic modification imparts high heat resistance and a specific molecular weight, resulting in a lactoferrin antimicrobial peptide possessing both high heat resistance and strong antimicrobial activity, along with a specific molecular weight. This peptide has been successfully applied in various processed dairy products. Furthermore, this invention addresses the functional stability of lactoferrin antimicrobial peptides in complex dairy systems and optimizes the process for large-scale production, reducing production costs.

[0016] Solution to the problem

[0017] [1]. A method for preparing lactoferrin antimicrobial peptides with improved heat resistance and antibacterial properties, wherein the method is as follows:

[0018] The preparation step involves dissolving the lactoferrin in a solvent to prepare a protein solution;

[0019] The enzymatic hydrolysis step involves stepwise enzymatic hydrolysis of the protein solution, first adding trypsin for the first enzymatic hydrolysis reaction, and then adding flavor protease for the second enzymatic hydrolysis reaction;

[0020] In the ultrafiltration step, the product obtained from the second enzymatic hydrolysis step is subjected to ultrafiltration using a filter membrane, and the filtrate is retained.

[0021] The purity of the lactoferrin is above 80% by mass.

[0022] [2]. According to the preparation method described in [1], in the enzymatic hydrolysis step, the amount of trypsin added is 0.1%-5% of the lactoferrin mass; the amount of flavor protease added is 0.1%-5% of the lactoferrin mass.

[0023] [3]. According to the preparation method described in [1] or [2], wherein, in the enzymatic hydrolysis step, the mass ratio of the trypsin to the flavor protease is 1:1.2 to 1:5.

[0024] [4]. The preparation method according to any one of [1] to [3], wherein, before the ultrafiltration step, the product obtained from the second enzymatic hydrolysis step is subjected to enzyme inactivation treatment.

[0025] [5]. The preparation method according to any one of [1] to [4], wherein the enzyme inactivation treatment is performed at an enzyme inactivation temperature of 85-100°C and an enzyme inactivation time of 5-20 minutes.

[0026] [6]. The preparation method according to any one of [1] to [5], wherein the filtrate is post-treated after the ultrafiltration step;

[0027] The post-processing includes drying.

[0028] [7]. A lactoferrin antimicrobial peptide, wherein it is prepared by any one of the preparation methods described in [1] to [6].

[0029] [8]. The antimicrobial peptide according to [7], wherein, according to gel permeation chromatography (GPC), the protein component of the lactoferrin antimicrobial peptide has a molecular weight of less than 5 kDa of more than 85% by mass.

[0030] [9]. According to the antimicrobial peptide described in [7] or [8], the lactoferrin antimicrobial peptide is heated in a water bath at 100°C for 15 minutes and then cooled. Its antimicrobial activity is determined by the inhibition zone method with Staphylococcus aureus as the indicator bacterium, and its antimicrobial activity retention rate is more than 80%.

[0031]

[10] . The use of lactoferrin antimicrobial peptide prepared according to any one of the preparation methods described in [1] to [6] or according to any one of the preparation methods described in [7] to [9] in the preparation of food or health products.

[0032]

[11] . A formulated dairy product, wherein the formulated dairy product comprises:

[0033] (A) Lactoferrin antimicrobial peptide prepared according to any one of [1] to [6] or according to any one of [7] to [9], and one or more of the following additional components:

[0034] (B) Functional protein components, (C) Carbohydrate components, (D) Fats and oils, (E) Mineral salts, and (F) Other nutritional supplement components.

[0035]

[12] . The formula dairy product according to

[10] , wherein the lactoferrin antimicrobial peptide is present in the formula dairy product at a mass percentage of 0.01%-1%.

[0036] Effects of the invention

[0037] The present invention has the following beneficial effects:

[0038] 1. The present invention provides a method for preparing a complex enzyme of high heat-resistant lactoferrin antimicrobial peptide and its application in dairy products. The method uses a complex enzyme system of trypsin and flavor protease. The protease first specifically cleaves lactoferrin under alkaline conditions to expose more cleavage sites. Subsequently, the flavor protease further hydrolyzes the lactoferrin, which can not only efficiently release the target antimicrobial peptide fragment, but also cleave the terminal hydrophobic amino acids, thereby improving the flavor of the product.

[0039] 2. The present invention uses a specific compound enzymatic hydrolysis process to obtain a product with a specific molecular weight distribution, and the antibacterial properties after heat treatment are significantly higher than those of natural lactoferrin and peptides obtained without compound enzymatic hydrolysis.

[0040] 3. The antimicrobial peptide powder of this invention can be added in the final dry mixing process of milk powder production, which perfectly avoids the destruction of its activity by high-temperature steps such as spray drying, and ensures the activity and content of functional ingredients in the final product.

[0041] 4. The final product of this invention not only retains the core antibacterial and immune-enhancing functions of lactoferrin peptides, making it easy for infants to accept, but also provides core raw materials and technical support for the development of a new generation of high-end functional infant formula. Attached Figure Description

[0042] Figure 1 This is a simplified flowchart of the method of the present invention. Detailed Implementation

[0043] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0044] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0045] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0046] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0047] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., mean that a specific element (e.g., feature, structure, property, and / or characteristic) related to that embodiment is included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0048] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0049] In this invention, “Da” is used to represent the unit of molecular weight, “Dalton”.

[0050] In this invention, the term "infant" is used to refer to the human group aged 0 to 6 months.

[0051] In this invention, the term "older infant" refers to the human group aged 6 to 12 months.

[0052] In this invention, the term "infant" is used to refer to the human group aged 12 to 36 months.

[0053] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0054] In this invention, the term "children" refers to a group of human beings who are older than 3 years and younger than 12 years and are in the growth and development stage.

[0055] In this manual, the term "adult" refers to a person who is 18 years of age or older.

[0056] In this manual, the term "teenager" refers to people aged 7 to 40.

[0057] In this manual, the term "middle-aged person" refers to a person aged 41 to 65.

[0058] In this manual, the term "elderly" or "senior citizen" refers to a person who is 65 years of age or older.

[0059] In this specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0060] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0061] All unit names used in this manual are international standard unit names.

[0062] First aspect

[0063] The first aspect of the present invention provides a method for preparing lactoferrin antimicrobial peptides with improved heat resistance and antibacterial properties, the method comprising the following steps: a preparation step in which the lactoferrin is dissolved in a solvent to prepare a protein solution; an enzymatic hydrolysis step in which the protein solution is subjected to stepwise enzymatic hydrolysis, firstly by adding trypsin for a first enzymatic hydrolysis reaction, and then by adding flavor protease for a second enzymatic hydrolysis reaction; and an ultrafiltration step in which the product obtained in the second enzymatic hydrolysis reaction is ultrafiltered using a filter membrane, and the filtrate is retained, wherein the purity of the lactoferrin is 80% by mass or higher.

[0064] The lactoferrin antimicrobial peptides prepared by the method provided by this invention have antimicrobial and immune functions, and also improve flavor, making them easier for infants to accept, thus achieving a better enzymatic hydrolysis effect.

[0065] In this manual, lactoferrin antimicrobial peptide is also referred to as lactoferrin hydrolysate or hydrolysate.

[0066] (Lactoferrin)

[0067] In this specification, lactoferrin (LF) is a multifunctional glycoprotein that is naturally found in mammalian milk (especially colostrum) and other exocrine secretions.

[0068] In some embodiments, the sources of lactoferrin include, but are not limited to: natural proteins isolated from the milk of mammals such as cow's milk and sheep's milk, or recombinant proteins obtained through recombinant expression by microorganisms (such as Pichia pastoris).

[0069] In some implementations, the purity of lactoferrin is not less than 80%.

[0070] In some preferred embodiments, the purity of lactoferrin is not less than 85%.

[0071] In some specific implementations, the lactoferrin has a purity of 95%, which can further ensure hydrolysis efficiency and product yield.

[0072] (preparation)

[0073] In some embodiments, the lactoferrin described above is dissolved in a solvent to prepare a protein solution.

[0074] In some implementations, the solvent may be water or a buffer solution.

[0075] In some specific implementations, the buffer solution is a phosphate buffer solution.

[0076] In some embodiments, the protein solution concentration is 1%-10% (w / v).

[0077] In some specific implementations, 50.0 g of lactoferrin powder is accurately weighed and gradually added to 1000 mL of phosphate buffer under slow stirring (magnetic stirrer, 300-500 rpm).

[0078] In some specific implementations, stirring is continued for 60 minutes to ensure that the lactoferrin is completely dissolved and evenly dispersed, forming a clear or microemulsified protein solution with a concentration of 3%-8% (w / v), or more specifically, a clear or microemulsified protein solution with a concentration of 5% (w / v).

[0079] In some specific implementations, the pH of the protein solution is precisely adjusted to 7.2.

[0080] (Enzymatic hydrolysis)

[0081] In some embodiments, the protein solution is subjected to stepwise enzymatic hydrolysis, first by adding trypsin for a first enzymatic hydrolysis reaction, and then by adding flavor protease for a second enzymatic hydrolysis reaction.

[0082] In some embodiments, during the enzymatic hydrolysis step, the amount of trypsin added is 0.1%-5% of the lactoferrin mass; the amount of flavor protease added is 0.1%-5% of the lactoferrin mass.

[0083] In some embodiments, during the enzymatic hydrolysis step, the mass ratio of the trypsin to the flavor protease is 1:1.2 to 1:5.

[0084] In some preferred embodiments, during the enzymatic hydrolysis step, the mass ratio of the trypsin to the flavor protease is 1:1.2 to 1:4.

[0085] In some more preferred embodiments, during the enzymatic hydrolysis step, the mass ratio of the trypsin to the flavor protease is 1:1.5 to 1:4.5.

[0086] In some further preferred embodiments, the mass ratio of the trypsin to the flavor protease is 1:1.5 to 1:3, for example, 1:1.5, 1:1.6, 1:1.7, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:2.9, 1:3, etc.

[0087] In some further preferred embodiments, the mass ratio of the trypsin to the flavor protease is 1:1.6, 1:3, etc.

[0088] First enzymatic reaction

[0089] In some implementations, trypsin is added first to perform a first enzymatic hydrolysis reaction, wherein the amount of trypsin added is 0.1%-5% of the lactoferrin mass.

[0090] In some preferred embodiments, during the enzymatic hydrolysis step, the amount of trypsin added is 0.1%-3% of the lactoferrin mass.

[0091] Trypsin is commercially available to those skilled in the art. In some preferred embodiments, the trypsin of this invention is food-grade trypsin with an enzyme activity ≥500 U / g. Regarding the dosage of trypsin, in some preferred embodiments, the amount of trypsin added is 0.5%-2% of the lactoferrin mass, for example: 0.5%, 1%, 1.4%, 1.5%, 1.6%, 2%, etc. For example, the amount of trypsin added is 1.5% of the lactoferrin mass.

[0092] In this invention, the pH of the casein solution can be adjusted by adding a pH adjuster.

[0093] In some embodiments of the present invention, the pH adjuster is selected from food-grade sodium bicarbonate, calcium hydroxide, potassium hydroxide, sodium hydroxide or any combination thereof, preferably a sodium hydroxide or potassium hydroxide solution (e.g., an aqueous solution) with a concentration of 0.1-1M, more preferably a sodium hydroxide solution with a concentration of 0.5M.

[0094] In some preferred embodiments, during the first enzymatic hydrolysis reaction, the hydrolysis temperature is between 30-50°C, the pH is between 6-9, and the hydrolysis time is between 20-100 minutes.

[0095] In some more preferred embodiments, the first enzymatic hydrolysis reaction is carried out at a temperature of 37-45°C, a pH of 7.5-8.5, and a time of 30-90 minutes.

[0096] The above-mentioned time, pH and / or temperature for enzymatic hydrolysis, used alone or in combination, further enhance the effect of enzymatic hydrolysis.

[0097] In some specific implementation schemes, during the first enzymatic hydrolysis reaction, trypsin (accounting for 0.5%-2% of the lactoferrin content) is weighed and dissolved in a small amount of ultrapure water (about 5 mL). The dissolved trypsin solution is then added to the protein solution. A constant temperature water bath is used to maintain the reaction system temperature at 37-45°C. A pH automatic titrator or manual intermittent detection is used to continuously add 0.5M NaOH solution to dynamically stabilize the pH of the reaction system at 7.5-8.5. The reaction lasts for 30-90 minutes. Gentle stirring (200 rpm) is maintained throughout the process.

[0098] In some preferred embodiments, during the first enzymatic hydrolysis reaction, 1.5% of trypsin is weighed and dissolved in a small amount of ultrapure water (about 5 mL). The dissolved trypsin solution is then added to the protein solution, and the reaction system is maintained at 40℃±1℃. 0.5M NaOH solution is continuously added dropwise to dynamically stabilize the pH of the reaction system at 7.5-8.5, specifically 8.0±0.1. The reaction lasts for 60 minutes, with gentle stirring (200 rpm) maintained throughout.

[0099] After the first enzymatic hydrolysis reaction described above, the reaction solution (or product) gradually changes from slightly viscous to thin and its transparency increases.

[0100] Second enzymatic reaction

[0101] In some embodiments, during the enzymatic hydrolysis step, the amount of flavor protease added is 0.1%-5% of the lactoferrin mass.

[0102] In some preferred embodiments, the amount of flavor protease added is 0.5%-4% of the amount of lactoferrin.

[0103] Flavor proteases are commercially available to those skilled in the art. In some preferred embodiments, the flavor protease of the present invention is a food-grade flavor protease with an enzyme activity ≥500 U / g. Regarding the dosage of the flavor protease, in some preferred embodiments, the amount added is 1%-3% of the lactoferrin mass, for example: 1%, 1.5%, 2%, 2.4%, 2.5%, 2.6%, 3%, etc. For example, the amount added is 2.5% of the lactoferrin mass.

[0104] In this invention, the pH of the casein solution can be adjusted by adding a pH adjuster.

[0105] In some embodiments of the present invention, the pH adjuster is selected from food-grade dilute hydrochloric acid, dilute sulfuric acid, citric acid or any combination thereof, preferably a dilute hydrochloric acid solution (e.g., an aqueous solution) with a concentration of 0.1-1M, and more preferably a dilute hydrochloric acid solution with a concentration of 0.5M.

[0106] In some preferred embodiments, the second enzymatic hydrolysis reaction is carried out at a temperature between 40-60°C, a pH between 6-7, and a time between 50-150 minutes.

[0107] In some more preferred embodiments, the second enzymatic hydrolysis reaction is carried out at a temperature of 45-55°C, a pH of 6.0-7.0, and a time of 60-120 minutes.

[0108] The above-mentioned time, pH and / or temperature for enzymatic hydrolysis, used alone or in combination, further enhance the effect of enzymatic hydrolysis.

[0109] In some implementations, after the first enzymatic hydrolysis reaction is completed, there is no need to heat up for inactivation or separation, and the second enzymatic hydrolysis reaction can be carried out directly.

[0110] In some specific implementation schemes, a flavor protease solution (the amount of flavor protease added is 1%-3% of the lactoferrin content) is added to the above hydrolysate, the pH of the reaction system is adjusted to 6.0-7.0 with 0.5M HCl, the water bath temperature is raised to 45-55℃, and the reaction is continued for 60-120 minutes.

[0111] In some preferred embodiments, a flavor protease solution (the amount of flavor protease added is 2.5% of the lactoferrin content) is added to the above hydrolysate, the pH of the reaction system is adjusted to 6.5±0.1 with 0.5M HCl, the water bath temperature is increased to 50±1℃, and the reaction is continued for 90 minutes.

[0112] In some specific implementation schemes, during the second enzymatic hydrolysis reaction, it is not necessary to strictly control the pH; allowing it to fluctuate naturally can achieve better enzymatic hydrolysis results.

[0113] This invention uses flavor protease to further cleave medium- and long-chain peptides produced by trypsin, which on the one hand releases more target small-molecule antimicrobial peptides, and on the other hand improves the product flavor by removing the terminal hydrophobic amino acids.

[0114] The product obtained by the above two enzymatic hydrolysis reactions has a good molecular weight distribution, and the antibacterial effect of the product is significantly improved after heating, achieving a better enzymatic hydrolysis effect.

[0115] (Enzyme inactivation treatment)

[0116] In some embodiments, the product obtained from the second enzymatic hydrolysis step is subjected to enzyme inactivation treatment prior to the ultrafiltration step.

[0117] In some embodiments, the enzyme inactivation treatment is performed at a temperature between 85-100°C for 5-20 minutes.

[0118] The enzyme inactivation treatment provided by this invention can achieve the following effects:

[0119] a) Completely inactivate the activity of trypsin and flavor protease, thus terminating the hydrolysis reaction;

[0120] b) Pasteurize the hydrolysate to reduce the microbial load;

[0121] c) Perform preliminary thermal stabilization treatment on hydrolyzed peptides to induce conformational changes or precipitation of heat-sensitive peptides, thereby enriching the population of naturally heat-resistant peptides.

[0122] In some specific implementation schemes, the hydrolysate obtained in the second step is rapidly transferred to a constant temperature water bath at 85-95°C and maintained at this temperature for 10-15 minutes.

[0123] In some preferred embodiments, the hydrolysate obtained in the second step is rapidly transferred to a constant temperature water bath at 90°C and maintained at this temperature for 15 minutes.

[0124] In some implementations, the hydrolysate after enzyme inactivation treatment is enriched.

[0125] Enriching the products after enzyme inactivation can remove a small amount of insoluble precipitate or heat-denatured material, thus achieving a better enzymatic hydrolysis effect.

[0126] In some specific implementations, the hydrolysate after heat treatment (enzyme inactivation) is rapidly cooled to room temperature (below 25°C) using an ice-water bath. Subsequently, it is centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant is collected, discarding any small amount of insoluble precipitate or heat-denatured material.

[0127] (Ultrafiltration)

[0128] In some specific implementations, the product after enzyme inactivation is subjected to ultrafiltration, wherein the product obtained from the second enzymatic hydrolysis step is ultrafiltered using a filter membrane, and the filtrate is retained.

[0129] In some specific implementations, a tangential flow ultrafiltration system is used, with a membrane membrane molecular weight cutoff of 5 kDa.

[0130] In some specific implementations, the supernatant is introduced into an ultrafiltration system and filtered under operating pressure of 0.2-0.4 MPa and temperature of 25°C. The permeate is collected, which is the component rich in target antimicrobial peptides (mainly lactoferrin peptides) with a molecular weight of less than or equal to 5 kDa. The retained solution (with a molecular weight greater than 5 kDa) can be recycled or discarded.

[0131] (Post-processing)

[0132] In some implementations, the filtrate is post-treated after the ultrafiltration step.

[0133] In some implementations, the post-processing includes a drying process.

[0134] In some preferred embodiments, the drying process is vacuum freeze drying.

[0135] This invention uses vacuum freeze drying to maximize the protection of peptide activity and facilitate storage, thereby achieving better product results.

[0136] In some specific implementations, the ultrafiltration permeate is dispensed into freeze-drying trays, pre-frozen to -40°C and held for 4 hours. The freeze dryer is then started, and under a vacuum of less than 10 Pa, primary drying (plate temperature slowly increased from -30°C to 0°C, approximately 20 hours) and desorption drying (plate temperature increased to 25-30°C, approximately 6 hours) are performed. The product is then discharged, yielding a white to slightly yellow, fluffy, porous solid.

[0137] In some implementations, the drying process is followed by sieving.

[0138] In some specific implementations, the freeze-dried solid is pulverized in a dry environment with a relative humidity of <20%, passed through an 80-mesh sieve, and a uniform, highly heat-resistant lactoferrin antimicrobial peptide powder is obtained, which is then sealed and stored away from light.

[0139] The product prepared by the preparation method described in this invention not only retains the core antibacterial and immune-enhancing functions of lactoferrin peptides, but is also easily accepted by infants.

[0140] In some implementations, based on the above preparation method, the final product yield is greater than 5%.

[0141] In some specific implementation schemes, based on the above preparation method, the final product yield is 7-9%.

[0142] Second aspect

[0143] A second aspect of the present invention provides a lactoferrin antimicrobial peptide, which is prepared by the preparation method described in the first aspect.

[0144] In some embodiments, the protein components of the lactoferrin antimicrobial peptide, according to gel permeation chromatography (GPC), comprise at least 85% by mass of protein components with a molecular weight less than 5 kDa.

[0145] In some preferred embodiments, the protein components of the lactoferrin antimicrobial peptide, according to gel permeation chromatography (GPC), comprise between 85-98% by mass of protein components with a molecular weight less than 5 kDa.

[0146] In some preferred embodiments, the protein components of the lactoferrin antimicrobial peptide, according to gel permeation chromatography (GPC), comprise between 90-97% by mass of protein components with a molecular weight less than 5 kDa.

[0147] In some specific implementation schemes, gel permeation chromatography (GPC) analysis showed that the proportion of components with a molecular weight of less than 5 kDa in the product was 94.3%, among which the characteristic peak of the target lactoferrin peptide (Lfcin) was significant.

[0148] In some embodiments, after the lactoferrin antimicrobial peptide is heated in a water bath at 100°C for 15 minutes and then cooled, its antimicrobial activity is determined using the inhibition zone method with Staphylococcus aureus as the indicator bacterium, and the retention rate of its antimicrobial activity is above 80%.

[0149] In some specific embodiments, 100 mg of the lactoferrin antimicrobial peptide powder is dissolved in 10 mL of ultrapure water, heated in a water bath at 100°C for 15 minutes, and then cooled. Its antimicrobial activity is determined using the inhibition zone method (using Staphylococcus aureus as the indicator bacterium).

[0150] In some preferred embodiments, the lactoferrin antimicrobial peptide is determined using the inhibition zone method, and its antimicrobial activity is not less than 85%.

[0151] In some specific implementations, the retention rate of antimicrobial activity of lactoferrin antimicrobial peptides determined by the inhibition zone method is between 85.2% and 87.5%.

[0152] The present invention provides a method for preparing lactoferrin antimicrobial peptides using a complex enzyme system of trypsin and flavor protease. The protease first specifically cleaves lactoferrin under alkaline conditions, exposing more cleavage sites. Subsequently, the flavor protease further hydrolyzes the lactoferrin, which can efficiently release the target antimicrobial peptide fragments and cleave the terminal hydrophobic amino acids, thereby improving the flavor. The lactoferrin hydrolysate obtained by the present invention through a specific complex enzymatic hydrolysis process has significantly higher thermal stability than natural lactoferrin and peptides obtained without complex enzymatic hydrolysis.

[0153] Third aspect

[0154] The third aspect of the present invention provides the use of lactoferrin antimicrobial peptides prepared by the preparation method according to the first aspect of the present invention or lactoferrin antimicrobial peptides according to the second aspect of the present invention in the preparation of food or health products.

[0155] In some embodiments, the food or health product of the present invention includes beverages, desserts, and dairy products. In some embodiments, the food is a solid dairy product or a liquid dairy product; for example, milk powder, cheese, ice cream, yogurt, liquid milk, etc.

[0156] The specific form of the food / health food described in this invention is not particularly limited. It can be solid (powder or block, etc.), semi-solid (soft, paste or thick substance, etc.) containing the product of this invention, or liquid containing the product of this invention.

[0157] In some exemplary embodiments, the present invention also provides a formulated dairy product and a method for preparing the same. The formulated dairy product of the present invention can be in solid, liquid, or semi-solid form; preferably, the dairy product of the present invention can be in powder form.

[0158] In some specific implementations, the formula dairy product is formula milk powder, such as formula milk powder for infants, adults, and the elderly, or formula milk powder for pregnant women and postpartum women.

[0159] Specifically, the dairy products of the present invention include:

[0160] (A) Lactoferrin antimicrobial peptide prepared according to the preparation method described in the first aspect or according to the lactoferrin antimicrobial peptide described in the second aspect, and one or more of the following additional components (i.e., these components are not derived from the lactoferrin hydrolysate):

[0161] (B) Functional protein components, (C) Carbohydrate components, (D) Fats and oils, (E) Mineral salts, and (F) Other nutritional supplement components.

[0162] (Functional protein components)

[0163] There are generally no special restrictions on the functional protein ingredients that can be used; for example, various whey proteins and immunoglobulins are acceptable. There are also no special restrictions on the amount of these ingredients added, as long as they comply with laws and regulations.

[0164] (Carbohydrate composition)

[0165] There are no particular restrictions on the type or source of carbohydrates added to the formula dairy products of this invention; those carbohydrates commonly used in formula dairy products in this field can be used.

[0166] In some specific embodiments, the carbohydrate component described in this invention mainly refers to sugars. These sugars are typically a general term for polyhydroxy aldehydes or polyhydroxy ketones and their condensation polymers and certain derivatives, generally composed of carbon, hydrogen, and oxygen. All sugars can be written with the empirical molecular formula: Cn(H₂O). n .

[0167] In this invention, the carbohydrates typically include monosaccharides, disaccharides, polysaccharides, or oligosaccharides.

[0168] Monosaccharides can mainly include glucose, fructose, etc.

[0169] Disaccharides, polysaccharides, or oligosaccharides can include: sucrose, lactose, fructotriose, fructotetraose, fructopentose, fructooligosaccharides, glucosamine, (maltodextrin), and various forms of human milk oligosaccharides.

[0170] In some specific implementations, the carbohydrates may be added in the form of dietary fiber. Examples of dietary fiber substances include one or more of the following: inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, maltodextrin, resistant dextrin, and soybean fiber.

[0171] Furthermore, there is no particular limitation on the total carbohydrate content in the formula dairy products of the present invention, and it can be formulated with reference to the carbohydrate content commonly used in infant formula milk powder products for the middle-aged and elderly in the art.

[0172] (Oil and fat components)

[0173] There are no particular restrictions on the types of oils added to the formula dairy products of this invention. Fatty acid glycerides commonly used in infant formula and formula milk powder products for the middle-aged and elderly can be used.

[0174] These glycerides can be obtained from plant extraction or through artificial synthesis (transesterification). Transesterification can be achieved by esterification of glycerol with fatty acids in the presence of a catalyst, or by transesterification of triglycerides and fatty acids from various existing sources in the presence of a (specific) catalyst (enzyme). For plant oils extracted from plants, the desired fatty acid glycerides can be obtained by mixing one or more of the following plant oils: rapeseed oil, soybean oil, sunflower oil, olive oil, sesame oil, corn oil, flaxseed oil, and camellia seed oil.

[0175] The fatty acid glycerides described in this invention are primarily triglycerides. Examples of such glycerides include OPL, OPO, MLCT, OOL, OPP, OLO, OLL, LPL, LPLn, OPLn, LPCa, and OPCa.

[0176] The term "MLCT structured mixed ester" refers to a structurally mixed ester whose main components are medium- to long-chain fatty acid triglycerides. "MLCT" stands for Middle to Long Chain Triglycerides, a structural ester whose glycerol backbone contains both medium-chain fatty acids (M) and long-chain fatty acids (L). Medium-chain fatty acids are those containing 6 to 12 carbon atoms, while long-chain fatty acids are those with more than 12 carbon atoms. MLCTs possess the characteristics of both medium- and long-chain fatty acids, offering the advantages of supplementing essential fatty acids, providing rapid energy, and not causing fat accumulation. Common MLCTs include OPLa, LPLa, OPCa, OLaO, OLaL, OMLa, SLaL, and SLaO.

[0177] (Mineral salt components)

[0178] The mineral salt components that can be added to the dairy products formulated in this invention are mainly used to introduce micronutrients, including iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.

[0179] Furthermore, regarding the content of mineral salts in formula dairy products, as long as it complies with the provisions of laws and regulations, it is acceptable.

[0180] (Other nutritional supplement ingredients)

[0181] There are no particular restrictions on other nutritional supplement ingredients that can be added to the dairy products formulated according to the present invention, and they can be prepared in accordance with existing methods in the art.

[0182] In some specific implementations, these supplement ingredients include one or more selected from vitamins, probiotics, and unsaturated fatty acids.

[0183] Examples of vitamins include one or more of the following: vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, and biotin.

[0184] For probiotics, for example, Bifidobacteria probiotics that are beneficial to the gut.

[0185] Examples of unsaturated fatty acids include arachidonic acid and docosahexaenoic acid.

[0186] Furthermore, there are no particular restrictions on the source of each of the components (B) to (F) above; for example, they can be introduced by mixing with animal milk or other nutritional additives.

[0187] (use)

[0188] The lactoferrin antimicrobial peptides obtained by the preparation method of the present invention can be used to prepare a variety of food products, including infant food, milk powder, and health products.

[0189] In some embodiments, the lactoferrin antimicrobial peptide is present in the formulated dairy product at a mass percentage of 0.01%-1%.

[0190] In some embodiments, the lactoferrin antimicrobial peptide is present in the formulated dairy product at a mass percentage of 0.01%-1%.

[0191] In some embodiments, the lactoferrin antimicrobial peptide is added to infant formula at an amount of 0.3% (w / w).

[0192] In some embodiments, the lactoferrin antimicrobial peptide is mixed with other ingredients during the formulation dairy product manufacturing process.

[0193] In some preferred embodiments, the production process is a dry mixing process.

[0194] In some specific implementation schemes, all dry ingredients, such as the formula dairy product base (spray-dried whey protein, lactose, vegetable oil microcapsules, etc.), vitamins, minerals, DHA / ARA microcapsules, and probiotics (such as Bifidobacterium animalis Bb-12), are mixed with 0.3% of lactoferrin antimicrobial peptide powder. A small amount of the formula dairy product base is first premixed with the entire amount of lactoferrin hydrolysate powder in a container, and then this premix is ​​further mixed.

[0195] The antimicrobial peptide powder of this invention can be added in the final dry mixing process of milk powder production, perfectly avoiding the destruction of its activity by high-temperature steps such as spray drying, and ensuring the activity and content of functional components in the final product; the final product of this invention not only retains the core antimicrobial and immune-enhancing functions of lactoferrin peptides, but is also easily accepted by infants, providing core raw materials and technical support for the development of a new generation of high-end functional infant formula milk powder.

[0196] The following is an exemplary preparation method of the present invention:

[0197] Reference Figure 1 The preparation method of the complex enzyme of highly heat-resistant lactoferrin antimicrobial peptides is as follows:

[0198] Step 1: Preparation of Substrate Solution Raw Materials and Reagents:

[0199] Raw material: food-grade or higher purity lactoferrin powder, with a purity of ≥95%.

[0200] Solvent: Ultrapure water.

[0201] Buffer salts: disodium hydrogen phosphate, sodium dihydrogen phosphate.

[0202] Solution preparation:

[0203] Weigh out a certain amount of disodium hydrogen phosphate and sodium dihydrogen phosphate, dissolve them in ultrapure water, and prepare a phosphate buffer solution with a concentration of 50 mM and a pH of 6.5-7.5. Preferably, the pH of the phosphate buffer solution is 7.0.

[0204] Accurately weigh 50.0 g of lactoferrin powder and gradually add it to 1000 mL of the above-mentioned phosphate buffer solution while slowly stirring (magnetic stirrer, 300-500 rpm). Continue stirring for 60 minutes to ensure that the lactoferrin is completely dissolved and evenly dispersed, forming a clear or microemulsified lactoferrin solution with a concentration of 3%-8% (w / v), specifically a 5% (w / v) clear or microemulsified lactoferrin solution. Fine-tune the pH of the solution to 7.2 with 1M NaOH or 1M HCl.

[0205] Step 2: Stepwise synergistic hydrolysis by complex enzymes

[0206] First stage enzymatic digestion - trypsin-specific cleavage:

[0207] Enzyme preparation: Food-grade trypsin (enzyme activity ≥2500U / g).

[0208] Enzyme addition: Weigh out trypsin (0.5%-2% of lactoferrin, specifically 1.5%) and dissolve it in a small amount of ultrapure water (about 5 mL).

[0209] Reaction initiation: Add all the dissolved trypsin solution to the protein solution prepared in step one.

[0210] Reaction condition control:

[0211] Temperature: A constant temperature water bath is used to maintain the reaction system temperature at 37-45℃, specifically 40℃±1℃.

[0212] pH: Since the pH will decrease during the enzymatic hydrolysis process, use an automatic pH titrator or manual interval detection to continuously add 0.5M NaOH solution to stabilize the pH of the reaction system at 7.5-8.5, specifically 8.0±0.1.

[0213] Time and stirring: The reaction lasts for 30-90 minutes (specifically 60 minutes); keep the stirring gentle throughout (200 rpm).

[0214] Process observation: The reaction solution gradually changed from slightly viscous to thin and the transparency increased.

[0215] Second-stage enzymatic hydrolysis - flavor protease debittering and refining:

[0216] Enzyme preparation: Food-grade flavored protease (enzyme activity ≥500U / g).

[0217] System conversion: After the first stage reaction is completed, there is no need to heat up for inactivation or separation; proceed directly to the next step.

[0218] Enzyme addition: Accurately weigh 1.25 grams of flavor protease (accounting for 2.5% of the lactoferrin content) and dissolve it in a small amount of ultrapure water.

[0219] Conditioning and Response:

[0220] Add the flavor protease solution (the amount of flavor protease added is 1%-3% of the lactoferrin content) to the above hydrolysate.

[0221] Adjust the pH of the reaction system to 6.0-7.0 using 0.5M HCl, specifically to 6.5±0.1.

[0222] Raise the water bath temperature to 45-55℃, specifically 50±1℃.

[0223] Under these conditions, continue the reaction for 60-120 minutes (specifically 90 minutes). Strict pH control is unnecessary; allow it to fluctuate naturally.

[0224] Preferably, the mass ratio of trypsin to flavor protease is 1:1.2 to 1:5.

[0225] Step 3: Heat treatment and termination of enzyme activity

[0226] Procedure: Quickly transfer the hydrolysate from step two to a constant temperature water bath at 85-95℃ (specifically 90℃) and maintain it at this temperature for 10-15 minutes (15 minutes is optional).

[0227] Step 4: Separation and Enrichment

[0228] Cooling and pretreatment: The heat-treated hydrolysate was rapidly cooled to room temperature (below 25°C) using an ice-water bath. Subsequently, it was centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant was collected, discarding any small amount of insoluble precipitate or heat-denatured material.

[0229] Ultrafiltration:

[0230] A tangential flow ultrafiltration system was used, with a membrane membrane molecular weight cutoff of 5 kDa. The supernatant was introduced into the ultrafiltration system and filtered under operating conditions of 0.2-0.4 MPa and 25°C. The permeate was collected; this fraction is rich in target antimicrobial peptides (mainly lactoferrin peptides) with a molecular weight of 5 kDa or less. The retained fraction (molecular weight greater than 5 kDa) can be recycled or discarded.

[0231] Step 5: Drying and Powdering

[0232] Method selection: To maximize the protection of peptide activity and facilitate storage, vacuum freeze drying is preferred.

[0233] process:

[0234] The ultrafiltration permeate was dispensed into freeze-drying trays and pre-frozen to -40°C for 4 hours. The freeze dryer was then started, and primary drying (plate temperature slowly increased from -30°C to 0°C, approximately 20 hours) and desorption drying (plate temperature increased to 25-30°C, approximately 6 hours) were performed under a vacuum of less than 10 Pa. The product was discharged, yielding a white to slightly yellow, fluffy, porous solid.

[0235] Subsequent processing: The freeze-dried solid was pulverized in a dry environment with a relative humidity of <20%, passed through an 80-mesh sieve, and a uniform, heat-resistant lactoferrin antimicrobial peptide powder was obtained. It was then sealed and stored away from light.

[0236] Example

[0237] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0238] Example 1

[0239] 1. Materials and Equipment

[0240] Ingredients: 50.0g food-grade lactoferrin (purity ≥95%)

[0241] Enzyme preparations: trypsin (2500 U / g), flavor protease (500 U / g)

[0242] Buffer: 50mM, pH 7.0 phosphate buffer (PBS)

[0243] Equipment: Precision electronic balance, magnetic stirrer and water bath, precision pH meter, high-speed centrifuge, tangential flow ultrafiltration system (5kDa membrane pack), vacuum freeze dryer.

[0244] 2. Operating Procedures

[0245] Substrate preparation: Add 1000 mL of PBS buffer to a reactor and slowly add 50.0 g of lactoferrin powder while stirring at 300 rpm. Continue stirring for 60 minutes until completely dissolved, and adjust the final pH to 7.2 precisely with a trace amount of NaOH solution.

[0246] First stage of enzymatic hydrolysis: The reaction system temperature was raised to 40℃. 0.75g of trypsin (1.5% of lactoferrin) was weighed, dissolved in 5mL of ultrapure water, and added to the reaction system. The pH was dynamically stabilized at 8.0 by adding 0.5M NaOH dropwise. The reaction was carried out for 60 minutes under these conditions.

[0247] Second stage enzymatic hydrolysis: Weigh 1.25g of flavor protease (2.5% of lactoferrin), dissolve it in 5 mL of ultrapure water, and add it directly to the above reaction solution. Adjust the pH to 6.5 with 0.5M HCl, and simultaneously raise the temperature to 50℃. Continue the reaction under these conditions for 90 minutes.

[0248] Heat treatment: Quickly transfer the hydrolysate to a 90°C water bath and maintain this temperature for 15 minutes.

[0249] Separation and enrichment: The heat-treated hydrolysate was immediately cooled to 25°C in an ice bath, and then centrifuged at 4°C and 8000 rpm for 15 minutes. The supernatant was collected, and the permeate was collected using an ultrafiltration system with a molecular weight cutoff of 5 kDa.

[0250] Drying and powdering: The permeate was subjected to vacuum freeze drying (pre-freezing at -40℃, main drying and desorption drying for a total of about 26 hours) to obtain a white fluffy powder. After pulverizing, it was passed through an 80-mesh sieve and weighed to obtain 4.1g of the finished product.

[0251] 3. Results and Detection

[0252] Yield: Based on the amount of lactoferrin fed, the yield of the finished powder was 8.2%.

[0253] Heat resistance test: 100 mg of the finished powder was dissolved in 10 mL of ultrapure water, heated in a water bath at 100 °C for 15 minutes, and then cooled. The antibacterial activity was determined using the inhibition zone method (with Staphylococcus aureus as the indicator bacterium). The results showed that the antibacterial activity retention rate of the heat-treated sample was 87.5%.

[0254] Molecular weight distribution: Gel permeation chromatography (GPC) analysis showed that the proportion of components with a molecular weight of less than 5 kDa in the product was 94.3%, among which the characteristic peak of the target lactoferrin peptide (Lfcin) was significant.

[0255] Example 2

[0256] 1. Materials and equipment are the same as in Example 1.

[0257] 2. Operating Procedures

[0258] Substrate preparation: exactly the same as in Example 1.

[0259] First stage of enzymatic hydrolysis: The amount of trypsin added was adjusted to 0.5g (1.0%), the reaction conditions were 42℃, pH 8.1, and the reaction time was shortened to 50 minutes.

[0260] Second stage of enzymatic hydrolysis: The amount of flavor protease added was increased to 1.5g (3.0%), the reaction conditions were 51℃, pH 6.4, and the reaction time was extended to 100 minutes.

[0261] Subsequent steps: heat treatment, separation and enrichment, and drying are exactly the same as those in Example 1.

[0262] 3. Results and Detection

[0263] Yield: The yield of the finished powder was 7.8%.

[0264] Heat resistance test: The same method was used to determine that the antibacterial activity retention rate was 85.2% after heat treatment at 100℃ for 15 minutes, indicating that the heat resistance was still excellent.

[0265] Comparative Example 1:

[0266] It is basically the same as Example 1, but without performing the second stage of enzymatic hydrolysis.

[0267] Comparative Example 2:

[0268] It is basically the same as Example 1, but the first stage of enzymatic hydrolysis is not performed.

[0269] Comparative Example 3:

[0270] It is basically the same as Example 1, but the second stage of enzymatic hydrolysis is performed first, followed by the first stage of enzymatic hydrolysis.

[0271] Furthermore, molecular weight distribution analysis was performed on Example 1 and Comparative Examples 1-3. The analysis showed that there were significant differences in the peak shape distribution of molecular weight distribution between the examples and the comparative examples. In addition, the characteristic peak of the target lactoferrin peptide (Lfcin) was significantly blunted in the comparative examples.

[0272] Meanwhile, the heat resistance of the products of Comparative Examples 1-3 was tested, and the antibacterial activity retention rate was less than 80% after heat treatment at 100°C for 15 minutes.

[0273] Application Example 1: Application of High Heat-Resistant Lactoferrin Antimicrobial Peptides in Infant Formula

[0274] Activity assays showed a significant increase in the content of the target antimicrobial peptide (lactoferrin peptide) in the powder. The recommended addition level in infant formula is 0.3% (w / w).

[0275] Added process:

[0276] Timing: Add during the dry mixing process of milk powder production.

[0277] Specific operations:

[0278] a. Add all dry ingredients, such as milk powder base (spray-dried whey protein, lactose, vegetable oil microcapsules, etc.), vitamins, minerals, DHA / ARA microcapsules, and probiotics (such as Bifidobacterium animalis Bb-12), into a three-dimensional motion mixer or a V-type mixer.

[0279] b. Accurately weigh 0.3% of the total feed amount of lactoferrin antimicrobial peptide powder.

[0280] c. Use the equal incremental mixing method: First, take a small amount of milk powder base and the total amount of antimicrobial peptide powder and premix them evenly in a container. Then, add this premix to the main material of the mixer.

[0281] d. Start the mixer and mix for 30-45 minutes to ensure absolute homogeneity.

[0282] e. After mixing evenly, package directly to obtain the final product – functional infant formula milk powder rich in highly heat-resistant lactoferrin antimicrobial peptides.

[0283] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0284] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing lactoferrin antimicrobial peptides with improved heat resistance and antibacterial properties, characterized in that, The method is as follows: The preparation step involves dissolving the lactoferrin in a solvent to prepare a protein solution; The enzymatic hydrolysis step involves stepwise enzymatic hydrolysis of the protein solution, first adding trypsin for the first enzymatic hydrolysis reaction, and then adding flavor protease for the second enzymatic hydrolysis reaction; In the ultrafiltration step, the product obtained from the second enzymatic hydrolysis step is subjected to ultrafiltration using a filter membrane, and the filtrate is retained. The purity of the lactoferrin is above 80% by mass.

2. The preparation method according to claim 1, characterized in that, In the enzymatic hydrolysis step, the mass ratio of the trypsin to the flavor protease is 1:1.2 to 1:

5.

3. The preparation method according to claim 1 or 2, characterized in that, Prior to the ultrafiltration step, the product obtained from the second enzymatic hydrolysis step is subjected to enzyme inactivation treatment. Preferably, during the enzyme inactivation treatment, the enzyme inactivation temperature is between 85-100℃, and the enzyme inactivation time is between 5-20 minutes.

4. The preparation method according to any one of claims 1 to 3, characterized in that, After the ultrafiltration step, the filtrate is post-treated; The post-processing includes drying.

5. A lactoferrin antimicrobial peptide, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 4.

6. The antimicrobial peptide according to claim 5, characterized in that, Of the protein components of the lactoferrin antimicrobial peptide, according to gel permeation chromatography (GPC), the protein components with a molecular weight of less than 5 kDa account for more than 85% by mass.

7. The antimicrobial peptide according to claim 5 or 6, characterized in that, The lactoferrin antimicrobial peptide was heated in a water bath at 100°C for 15 minutes and then cooled. Its antimicrobial activity was determined by the inhibition zone method using Staphylococcus aureus as the indicator bacterium, and the retention rate of its antimicrobial activity was over 80%.

8. The use of the lactoferrin antimicrobial peptide prepared by the preparation method according to any one of claims 1 to 4, or the lactoferrin antimicrobial peptide according to any one of claims 5 to 7, in the preparation of food or health products.

9. A formula dairy product, characterized in that, The formulated dairy products include: (A) The lactoferrin antimicrobial peptide prepared according to any one of claims 1 to 4, or the lactoferrin antimicrobial peptide according to any one of claims 5 to 7, and one or more of the following additional components: (B) Functional protein components, (C) Carbohydrate components, (D) Fats and oils, (E) Mineral salts, and (F) Other nutritional supplement components.

10. The formulated dairy product according to claim 9, characterized in that, The lactoferrin antimicrobial peptide is present in the formula dairy product at a mass percentage of 0.01%-1%.

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