A method for preparing a product enriched in highly active lactoferrin

Abstract

The application discloses a preparation method of a high-activity lactoferrin product, and belongs to the technical field of food processing. The application adopts high-voltage pulse electric field technology to replace traditional heat sterilization technology to process lactoferrin, and avoids the damage of heat effect on the activity of lactoferrin; when the electric field intensity is below 30 kV / cm, the lactoferrin retention rate can reach more than 95%; meanwhile, the method can improve the iron binding capacity, heat stability, foaming property and stability and emulsifying property and stability of lactoferrin. The application can effectively kill pathogenic microorganisms in drinks, prolong the shelf life of products, and ensure the safety of food microorganisms; and the combined treatment of lactoferrin and pulse electric field can enhance the sterilization effect, so that a small part of sublethal microorganisms generated by single pulse electric field treatment can be further killed. The application widens the application range of lactoferrin, and provides a new way for the processing of lactoferrin products.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and specifically relates to a method for preparing a product rich in highly active lactoferrin. Background Technology

[0002] Lactoferrin (LF), often referred to as "milk gold" in cow's milk, is an important iron-binding glycoprotein primarily found in various exocrine secretions of mammals, such as milk, tears, and saliva. Lactoferrin possesses many powerful functional properties. First, it acts as an iron carrier, binding iron ions and promoting iron absorption and utilization, making it potentially valuable in the prevention and treatment of iron-deficiency anemia. Furthermore, it competitively deprives bacteria of the iron they need for growth, directly binding to microbial cell membranes and causing their rupture; therefore, lactoferrin also exhibits antibacterial, antiviral, antifungal, and antiparasitic effects. Another important function of lactoferrin is the regulation of the immune system. It can bind to receptors on the surface of immune cells, exerting immunomodulatory functions, while simultaneously regulating the levels of cytokines in the body, thus playing a role in antitumor, immunomodulatory, and antioxidant activity.

[0003] Human milk contains approximately 7 g / L of lactoferrin, while bovine milk contains approximately 0.3-2.1 g / L. Bovine lactoferrin (bLF) and human lactoferrin (hLF) share 69% amino acid sequence homology, exhibiting high similarity in biological activity and physicochemical properties. Therefore, the abundance of bovine milk resources and related basic research on bovine lactoferrin have attracted significant attention. Global bovine lactoferrin production has rapidly increased from less than 80 tons per year in 2006 to over 400 tons in 2023. Due to the huge demand in infant nutrition, immune health, sports nutrition, cosmetics, livestock products, and neonatal nutrition, the bovine lactoferrin market is projected to grow at a rate of 10% to 12% annually in the future.

[0004] However, lactoferrin is a heat-sensitive protein, and traditional pasteurization and ultra-high temperature (UHT) sterilization can lead to varying degrees of loss. Traditional heat sterilization techniques suffer from the inherent contradiction between long shelf life and high lactoferrin retention rates, making it difficult to reconcile these factors. Pasteurization faces challenges such as incomplete microbial inactivation, short shelf life (7-14 days), and stringent storage requirements (2-6°C), while still resulting in some loss of active proteins. Although UHT sterilization can achieve commercial sterility, excessive heat treatment causes almost complete denaturation and inactivation of heat-sensitive active substances like lactoferrin in milk, and it easily leads to Maillard reactions, severely impacting its nutritional and flavor profiles. Therefore, the bottleneck restricting the sustainable development of lactoferrin is the lack of a stable lactoferrin raw material supply chain supported by modern food processing technologies.

[0005] Currently, CN117918423A discloses a high-activity protein content, long-shelf-life milk and its production method. This invention uses raw milk as raw material and, through rapid preheating, high-voltage pulsed electric field, and aseptic homogenization, can retain the flavor and content of active substances in the milk. However, this method cannot solve the problems of maintaining the stability of lactoferrin activity and improving its activity. Although the pulsed electric field treatment of raw milk has a certain sterilization effect, it still requires preheating to 50-65℃ to ensure a reduction in colony count (reaching 3 lg). Heating will inevitably lead to an increase in solution conductivity, and the electric field will generate more Joule heat, thus raising the actual temperature of the material. It is known that the N-lobule denaturation temperature of lactoferrin is around 58℃, so the retention rate and activity of lactoferrin obtained by this treatment will be greatly reduced. Secondly, this method does not use the new national standard GB 5009.299-2024, "National Food Safety Standard - Determination of Lactoferrin in Food," for the determination of lactoferrin. The old method would detect inactivated lactoferrin, while the new standard method uses a selective heparin column, which effectively retains active lactoferrin, making it more targeted. Therefore, how to improve the retention rate and activity of lactoferrin while ensuring sterilization effect is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for preparing beverages rich in highly active lactoferrin. This processing method yields lactoferrin with high retention rates, low energy consumption, and high efficiency, while ensuring the product's microbial safety, extending its shelf life, and enhancing the iron-binding capacity, thermal stability, foaming properties and stability, and emulsifying properties and stability of lactoferrin, resulting in higher and more stable lactoferrin activity. Furthermore, lactoferrin itself possesses antibacterial properties, and the synergistic effect of lactoferrin with a pulsed electric field enhances its bactericidal effect. This not only significantly broadens the application range of lactoferrin but also makes a valuable contribution to its sustainable development.

[0007] To achieve the above effects, the first objective of this invention is to provide a method for preparing a product rich in lactoferrin, the product including but not limited to beverages, and the method comprising the following steps:

[0008] (1) Raw material processing

[0009] Lactoferrin powder is added to raw milk or sterile water to obtain a solution rich in lactoferrin.

[0010] (2) Degassing treatment

[0011] The lactoferrin-rich solution obtained in step (1) was degassed.

[0012] (3) Pulsed electric field sterilization treatment:

[0013] The degassed solution obtained in step (2) is subjected to pulsed electric field sterilization treatment. The electric field strength is set to 10-45kV / cm, the pulse width is set to 1-3 us, the pulse frequency is set to 50-150 Hz, and the total treatment time is set to 200-400us. At the same time, the temperature inside the pulsed electric field equipment pipeline is controlled not to exceed 20 ℃. A beverage rich in lactoferrin can then be obtained.

[0014] Furthermore, the content of lactoferrin powder in fresh milk or sterile water in step (1) is 0.5-5%.

[0015] Furthermore, the electric field strength in step (3) is set to 10-30 kV / cm.

[0016] Furthermore, the method for controlling the temperature inside the pipeline to not exceed 20 ℃ in step (3) is to place the pipeline under a 0-4 ℃ water bath condition.

[0017] Furthermore, when the electric field strength is between 10-45 kV / cm, the lactoferrin retention rate in the lactoferrin product of the present invention can reach more than 90%.

[0018] Preferably, when the electric field strength is below 30 kV / cm, the retention rate of lactoferrin of the present invention is above 95% in all four systems.

[0019] It was also found that lactoferrin treated by the method of this invention, when combined with pulsed electric field treatment, can produce a synergistic effect to enhance the bactericidal effect, further killing a small number of sublethal microorganisms produced by single pulsed electric field treatment. This is of great value in ensuring food microbial safety.

[0020] A second objective of this invention is to provide a method for increasing the content, activity, relative iron-binding capacity, thermal stability, or antibacterial ability of lactoferrin in a beverage, wherein the method comprises the following steps:

[0021] (1) Raw material processing

[0022] Add lactoferrin powder to fresh milk or sterile water to make a lactoferrin-rich solution.

[0023] (2) Degassing treatment

[0024] The lactoferrin-rich solution obtained in step (1) was degassed.

[0025] (3) Pulsed electric field sterilization treatment:

[0026] The degassed solution obtained in step (2) is subjected to pulsed electric field sterilization treatment. The electric field strength is set to 10-45kV / cm, the pulse width is set to 1-3 us, the pulse frequency is set to 50-150 Hz, and the total treatment time is set to 200-400us. At the same time, the temperature inside the pulsed electric field equipment pipeline is controlled not to exceed 20 ℃. A beverage rich in lactoferrin can then be obtained.

[0027] Furthermore, the content of lactoferrin powder in fresh milk or sterile water in step (1) is 0.5-5%.

[0028] Furthermore, the electric field strength in step (3) is set to 10-30 kV / cm.

[0029] Furthermore, the method for controlling the temperature inside the pipeline to not exceed 20 ℃ in step (3) is to place the pipeline under a 0-4 ℃ water bath condition.

[0030] Furthermore, when the electric field strength is between 10-45 kV / cm, the lactoferrin retention rate in the lactoferrin product of the present invention can reach more than 90%.

[0031] Preferably, when the electric field strength is below 30 kV / cm, the retention rate of lactoferrin of the present invention is above 95% in all four systems.

[0032] It was also found that lactoferrin treated by the method of this invention, when combined with pulsed electric field treatment, can produce a synergistic effect to enhance the bactericidal effect, further killing a small number of sublethal microorganisms produced by single pulsed electric field treatment. This is of great value in ensuring food microbial safety.

[0033] The third objective of this invention is to provide a product rich in highly active lactoferrin prepared by the method of this invention.

[0034] Furthermore, the products rich in highly active lactoferrin include, but are not limited to, beverages.

[0035] Furthermore, the product rich in highly active lactoferrin refers to a lactoferrin product in which the lactoferrin retention rate can reach more than 90%; when the electric field strength in the method of the present invention is below 30 kV / cm, the lactoferrin retention rate in the lactoferrin product reaches more than 95%.

[0036] Beneficial effects

[0037] (1) The present invention uses a pulsed electric field treatment method to treat a solution containing lactoferrin. By controlling the temperature during the pulsed electric field treatment process to be below 20°C, the lactoferrin structure is not damaged while the activity of lactoferrin is enhanced, and the lactoferrin retention rate in the raw milk, aqueous solution, raw milk and bovine colostrum obtained after treatment is high.

[0038] (2) Increased lactoferrin activity

[0039] Heat treatment-induced protein denaturation, inactivation, and aggregation often lead to a decrease in the performance of lactoferrin. However, this invention controls the temperature during the pulsed electric field treatment process to below 20°C. On the one hand, after pulsed electric field treatment, the lactoferrin structure can be appropriately expanded, improving its iron-binding capacity, thermal stability, foaming properties and foaming stability, emulsifying properties and emulsifying stability, thereby further improving the quality of the beverage prepared by this invention. On the other hand, lowering the temperature of the system during the pulsed electric field treatment process can reduce the electrical conductivity of the material, which not only helps to avoid the generation of electric sparks or arcs in the pulsed electric field equipment, thus preventing the equipment from stopping suddenly and maintaining stable operation, but also extends the service life of the equipment.

[0040] (3) High retention rate of lactoferrin

[0041] After pasteurization at different intensities, the retention rate of lactoferrin is usually between 50% and 70%, while after ultra-high temperature instantaneous sterilization, lactoferrin is almost completely inactivated. However, after treatment with a pulsed electric field (electric field strength 10-45 kV / cm) below 20°C according to this invention, the retention rate of lactoferrin can reach more than 90%. When the electric field strength is below 30 kV / cm, the retention rate of lactoferrin in the four systems is more than 95%.

[0042] (4) The process is simple, efficient, energy-saving, has a good sterilization effect, and a long shelf life.

[0043] This invention employs pulsed electric field technology, which can instantly cause irreversible "electroporation" of the cell membranes and cell walls of bacteria in solution within microseconds, disrupting their metabolism, causing intracellular solutions to leak out, and thus killing the microorganisms. Moreover, the pulsed electric field can act on a wide range of microorganisms, and has a good killing effect on both Gram-negative and Gram-positive bacteria. This invention can reduce the total bacterial count of raw milk and aqueous solutions rich in lactoferrin, as well as raw milk and bovine colostrum, by 3-5 logs, thereby significantly extending their shelf life.

[0044] (5) The synergistic bactericidal effect of lactoferrin and pulsed electric field is enhanced.

[0045] Lactoferrin itself has good antibacterial properties. When a pulsed electric field is applied to a system rich in lactoferrin, the bactericidal effect is enhanced, causing the small number of sublethal microbial cells generated by the pulsed electric field to die further. Attached Figure Description

[0046] Figure 1 The sterilization effect of a pulsed electric field with an electric field strength of 10-45 kV / cm on raw milk, raw milk with 0.5% lactoferrin, 5% lactoferrin aqueous solution and bovine colostrum.

[0047] Figure 2 The effect of pulsed electric field treatment with an electric field strength of 10-45 kV / cm on the relative iron-binding capacity of lactoferrin.

[0048] Figure 3 The effect of pulsed electric field treatment with an electric field strength of 10-45 kV / cm on the foaming properties and foaming stability of lactoferrin was investigated.

[0049] Figure 4 The effect of pulsed electric field treatment with an electric field strength of 10-45 kV / cm on the emulsifying properties and emulsion stability of lactoferrin was investigated.

[0050] Figure 5 The effect of pulsed electric field treatment with electric field strength of 15-45 kV / cm on the thermal stability of lactoferrin (N leaf) was investigated.

[0051] Figure 6 The effect of pulsed electric field treatment with electric field strength of 15-45 kV / cm on the thermal stability of lactoferrin (C leaf) was investigated.

[0052] Figure 7 This invention illustrates the changes in the secondary structure of lactoferrin under different electric field intensities.

[0053] Figure 8 This invention illustrates the changes in the tertiary structure of lactoferrin under different electric field intensities. Detailed Implementation

[0054] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0055] The test methods involved in the following embodiments are as follows:

[0056] I. Methods for determining colony count:

[0057] The national standard GB 4789.2-2022, "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count," was adopted.

[0058] II. Methods for determining lactoferrin content:

[0059] The national standard GB 5009.299-2024, "National Food Safety Standard - Determination of Lactoferrin in Food," was adopted.

[0060] III. Methods for determining relative iron bonding ability:

[0061] (1) Dissolve lactoferrin in Tris-HCl buffer (containing 0.15 mol / L NaCl) at pH 7.55 mmol / L and prepare a lactoferrin solution with a mass concentration of 2 mg / mL.

[0062] (2) Take 0.9 mL of this solution and mix it with 0.1 mL of freshly prepared 0.1 mL of NaHCO3 solution. After mixing thoroughly, react with 1.0 mL of 0.6 mmol / L Fe(NH4)2(SO4)2·6H2O solution (containing 0.01 mol / L HCl). React at 25℃ for 10 min, and then measure the absorbance at 465 nm. The absorbance of 10 mg / mL iron-saturated lactoferrin (Fe-LF) at 465 nm is 0.57.

[0063] (3) The formula for calculating the relative iron bonding capacity is as follows:

[0064]

[0065]

[0066] IV. Methods for determining foaming properties and foam stability:

[0067] Homogenize the lactoferrin solution at 10,000 rpm for 2 minutes until it becomes foamy. Quickly transfer the foam to a 50 mL graduated cylinder. The volume of foam after standing for 2 minutes is labeled V2, and the volume of foam after standing for 30 minutes is labeled V. 30 ;

[0068] Foaming property (FA) and foaming stability (FS) are calculated using the following formulas:

[0069]

[0070]

[0071] Where Vs is the initial liquid volume.

[0072] V. Methods for determining emulsifying properties and emulsion stability:

[0073] Mix 10 mL of lactoferrin aqueous solution (5%) with 3 mL of soybean oil, homogenize at 10000 rpm for 2 min, take 3 μL of the emulsion from the bottom of the beaker, and mix thoroughly with 3.0 mL of 0.1% (m / v) SDS solution. Use a UV spectrophotometer to record the emulsion at 0 min (A0) and 10 min (A1) after preparation. 10 The absorbance at 500 nm was measured at that time.

[0074] The equations for calculating EAI and ESI are as follows:

[0075]

[0076]

[0077] Where c is the protein concentration (0.05 g / mL), D is the dilution factor (1000), and α is the oil phase volume fraction (20%).

[0078] VI. Methods for determining thermal stability:

[0079] The thermal stability of lactoferrin was analyzed using a Nano-DSC differential scanning calorimeter: a certain amount of lactoferrin solution was taken, with ultrapure water as the baseline, the temperature scanning range was 40~100 ℃, the heating rate was 1 ℃ / min, and the data were read using a Nano-DSC differential scanning calorimeter from TA Instruments-Waters LLC to study the thermal stability of lactoferrin.

[0080] Raw materials and equipment involved in the embodiments:

[0081] The lactoferrin powder, raw milk, and bovine colostrum involved in the following examples all came from Yili Dairy Research Institute. The purity of the lactoferrin powder was ≥95%, and the iron content of the lactoferrin was 5.4 mg / kg. The lactoferrin content in the raw milk was approximately 120 mg / kg, and the lactoferrin content in the bovine colostrum was approximately 5180 mg / kg.

[0082] The pulsed electric field device involved in the following embodiments is the OSU-4L model from Ohio State University, USA. The pulse polarity is unipolar or bipolar pulse, the waveform is rectangular, sawtooth or exponential decay type, and the processing cavity is a continuous processing system.

[0083] The aseptic filling laminar flow hoods involved in the following embodiments were purchased from Shenzhen Sannuo Instrument Equipment Factory, model SW-CJ-1D;

[0084] The 4°C refrigerator involved in the following embodiments was purchased from Haier Group, model BCD-478WGHTD5DDYU1;

[0085] The tube sheet type combined ultra-high temperature sterilizer involved in the following embodiments was purchased from Powerpoint International, Japan, model PT-20C-R.

[0086] The culture medium components involved in the following examples are as follows:

[0087] The non-selective culture medium was BHI brain heart infusion broth agar medium.

[0088] The selective medium was VRBA crystal violet neutral red bile salt agar.

[0089] Example 1:

[0090] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0091] (1) Raw material processing

[0092] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0093] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0094] (2) Degassing treatment

[0095] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0096] (3) Equipment disinfection:

[0097] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0098] (4) Pulsed electric field sterilization treatment:

[0099] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 10 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0100] (5) Canned storage:

[0101] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0102] Example 2:

[0103] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0104] (1) Raw material processing

[0105] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0106] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0107] (2) Degassing treatment

[0108] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0109] (3) Equipment disinfection:

[0110] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0111] (4) Pulsed electric field sterilization treatment:

[0112] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 15 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0113] (5) Canned storage:

[0114] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0115] Example 3:

[0116] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0117] (1) Raw material processing

[0118] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0119] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0120] (2) Degassing treatment

[0121] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0122] (3) Equipment disinfection:

[0123] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0124] (4) Pulsed electric field sterilization treatment:

[0125] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 20 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0126] (5) Canned storage:

[0127] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0128] Example 4:

[0129] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0130] (1) Raw material processing

[0131] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0132] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0133] (2) Degassing treatment

[0134] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0135] (3) Equipment disinfection:

[0136] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0137] (4) Pulsed electric field sterilization treatment:

[0138] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 25 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0139] (5) Canned storage:

[0140] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0141] Example 5:

[0142] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0143] (1) Raw material processing

[0144] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0145] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0146] (2) Degassing treatment

[0147] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0148] (3) Equipment disinfection:

[0149] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0150] (4) Pulsed electric field sterilization treatment:

[0151] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 30 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0152] (5) Canned storage:

[0153] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0154] Example 6:

[0155] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0156] (1) Raw material processing

[0157] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0158] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0159] (2) Degassing treatment

[0160] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0161] (3) Equipment disinfection:

[0162] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0163] (4) Pulsed electric field sterilization treatment:

[0164] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 35 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0165] (5) Canned storage:

[0166] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0167] Example 7:

[0168] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0169] (1) Raw material processing

[0170] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0171] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0172] (2) Degassing treatment

[0173] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0174] (3) Equipment disinfection:

[0175] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0176] (4) Pulsed electric field sterilization treatment:

[0177] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 40 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0178] (5) Canned storage:

[0179] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0180] Example 8:

[0181] The pulsed electric field processing method of the present invention comprises the following specific steps:

[0182] (1) Raw material processing

[0183] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0184] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0185] (2) Degassing treatment

[0186] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0187] (3) Equipment disinfection:

[0188] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0189] (4) Pulsed electric field sterilization treatment:

[0190] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 45 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20℃ by using a 4℃ water bath.

[0191] (5) Canned storage:

[0192] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0193] Example 9

[0194] The experimental method for treating bacteria with a combination of lactoferrin and an electric field involves the following steps:

[0195] 1. Escherichia coli ( E. coliSuspension preparation:

[0196] Escherichia coli was transferred from nutrient agar slant medium to nutrient broth medium and cultured at 37°C and 200 rpm for 12 h to reach the logarithmic growth phase. The E. coli culture was centrifuged at 4°C and 6000 rpm for 5 min, washed twice with sterile PBS buffer, and the bacterial culture was resuspended in PBS buffer.

[0197] 2. Contains Escherichia coli (E. coli) E. coli Preparation of lactoferrin aqueous solution:

[0198] The pulsed electric field processing method in this embodiment is the same as in embodiment 1, except that step (1) is adjusted as follows: lactoferrin powder is dissolved in sterile pure water and magnetically stirred at 500 rpm until it is uniform and fully hydrated, so as to obtain aqueous solutions of lactoferrin with concentrations of 5 mg / mL, 10 mg / mL and 20 mg / mL respectively.

[0199] The above-mentioned Escherichia coli ( E. coli The suspension was mixed with aqueous solutions of lactoferrin at a ratio of 1:10 to obtain bacterial concentrations of 10. 6 -10 8 A CFU / mL aqueous solution of lactoferrin. Store this aqueous solution at 4 °C before processing.

[0200] 3. Combined treatment of E. coli with lactoferrin and pulsed electric field E. coli :

[0201] Separately, containing E. coli ( E. coli Lactoferrin aqueous solutions of 5 mg / mL, 10 mg / mL, and 20 mg / mL were introduced into the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment. The electric field strength was set to 20, 30, or 40 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs. At the same time, the temperature inside the pipeline was controlled to not exceed 20 ℃ by using a 4 ℃ water bath. Finally, the colony count was performed by selective and non-selective culture media (where A is a non-selective culture medium, which is rich in nutrients and has a good repair effect on damaged microbial cells; B is a selective culture medium, which can screen out completely healthy microorganisms). E. coli cell)

[0202] The results of this embodiment are as follows:

[0203] In the group without added lactoferrin, as the electric field strength increased, the pulsed electric field had a greater effect on Escherichia coli (E. coli). E. coliThe kill log value of the suspension gradually increased, and there was a difference of 0.3-0.6 lg in selective and non-selective media, indicating that a single pulse electric field failed to kill a small number of microorganisms completely, and some sublethal microbial cells may be produced (i.e., damaged cells that can recover their vitality under suitable conditions).

[0204] In the group without applied pulsed electric field, with increasing lactoferrin concentration, in selective and non-selective culture media... E. coli The kill logarithm increases, but the bactericidal effect is limited (less than 1 lg) in selective culture media. E. coli The high kill log number may be due to the increased concentration of lactoferrin leading to the death of some sublethal microorganisms.

[0205] In the group where a pulsed electric field and lactoferrin were applied simultaneously, as the electric field strength and lactoferrin concentration increased, the effect on... E. coli The kill logs increased accordingly, and the difference between selective and non-selective culture media gradually decreased, indicating that the combined treatment with lactoferrin and pulsed electric field can cause further death of sublethal microbial cells generated by single pulsed electric field treatment, which is of great value for ensuring food microbial safety.

[0206] Table 1 Synergistic effect of lactoferrin and pulsed electric field treatment E. coli The effect of kill log

[0207]

[0208] Note: A is a non-selective culture medium, and B is a selective culture medium for culturing.

[0209] Comparative Example 1:

[0210] This comparative example demonstrates the method without applying an electric field. The specific steps are as follows:

[0211] (1) Raw material processing

[0212] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0213] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0214] (2) Degassing treatment

[0215] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0216] (3) Equipment disinfection:

[0217] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0218] (4) No pulsed electric field treatment:

[0219] The degassed solution was passed into the cleaned and sterilized pulse electric field equipment pipeline, but no electric field was applied. At the same time, the temperature inside the pipeline was controlled to not exceed 20°C by using a 4°C water bath.

[0220] (5) Canned storage:

[0221] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0222] Comparative Example 2:

[0223] This comparative example demonstrates a pasteurization treatment method, with the specific steps as follows:

[0224] (1) Raw material processing

[0225] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0226] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0227] (2) Degassing treatment

[0228] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0229] (3) Pasteurization treatment

[0230] The degassed solutions were then passed into a sterilized tube-plate combined ultra-high temperature sterilizer (72 ℃, 15s) for pasteurization.

[0231] (4) The solutions obtained after pasteurization were aseptically bottled and stored in a refrigerator at 4 ℃.

[0232] Comparative Example 3

[0233] The comparative pulsed electric field processing method is the same as in Example 5, except that the 4 ℃ water bath control of the pipe temperature in step (4) is not performed. The specific steps are as follows:

[0234] (1) Raw material processing

[0235] Dissolve 1 g of lactoferrin powder in 200 ml of raw milk and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain raw milk rich in lactoferrin.

[0236] Dissolve 10 g of lactoferrin powder in 200 mL of sterile pure water and stir magnetically at 500 rpm until it is homogeneous and fully hydrated to obtain an aqueous solution rich in lactoferrin.

[0237] (2) Degassing treatment

[0238] Raw milk, bovine colostrum, raw milk rich in lactoferrin obtained in step (1), and aqueous solution rich in lactoferrin were degassed under a pressure of 600 mm Hg.

[0239] (3) Equipment disinfection:

[0240] The pulse electric field equipment pipeline was thoroughly disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence.

[0241] (4) Pulsed electric field sterilization treatment:

[0242] The degassed solution obtained in step (2) was placed into the pulse electric field equipment pipeline after disinfection and cleaning in step (3) for pulse electric field sterilization treatment. The electric field strength was set to 30 kV / cm, the pulse width was set to 2 μs, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 μs.

[0243] (5) Canned storage:

[0244] The solutions obtained from the pulsed electric field treatment in step (4) were aseptically packaged and stored in a refrigerator at 4 ℃.

[0245] Examples 1-8 and Comparative Examples 1-3: Experimental Results

[0246] 1. Results of lactoferrin retention rate

[0247] The results are shown in Table 2:

[0248] Table 2. Retention rate of lactoferrin in different systems under different treatment conditions

[0249]

[0250] (1) By Figure 1 It can be seen that, Figure 1 The effect of pulsed electric field (PEF) treatment on the total bacterial count in raw milk, 0.5% lactoferrin-containing raw milk, 5% lactoferrin aqueous solution, and bovine colostrum was shown at electric field strengths of 10–45 kV / cm, treatment time of 300 μs, and water bath temperature of 4 °C. The figure shows that the total bacterial count decreases with increasing electric field strength, indicating a better sterilization effect. When the electric field strength is increased to 45 kV / cm, the total bacterial count in raw milk, 0.5% lactoferrin-containing raw milk, 5% lactoferrin aqueous solution, and bovine colostrum decreases by 5.54, 5.70, 5.95, and 5.15 log values, respectively. This demonstrates that pulsed electric field treatment (PEF) has a good bactericidal effect on microorganisms.

[0251] (2) As can be seen from Table 2, the retention rate of lactoferrin decreases slightly with the increase of electric field strength. When the electric field strength is 30 kV / cm (sterilization effect reaches 3-5), the retention rate of lactoferrin in the four systems is above 95%. If the electric field strength is further increased to 45 kV / cm, the loss rate of lactoferrin is still less than 10%. After pasteurization (72℃, 15s), the retention rates of lactoferrin in raw milk, 0.5% lactoferrin raw milk, 5% lactoferrin aqueous solution and bovine colostrum are 75.7%, 68.6%, 74.9% and 70.3%, respectively, which are significantly lower than the retention rate of lactoferrin after pulsed electric field treatment. This indicates that pulsed electric field treatment has less damage to lactoferrin and can better maintain the content of lactoferrin.

[0252] (3) If, as shown in Comparative Example 3, the temperature inside the pulse electric field pipeline is not controlled by a 4 ℃ water bath, the pipeline temperature cannot be controlled below 20 ℃ during pulse electric field treatment. The Joule heat generated inside the pipeline will accumulate continuously, causing the pipeline to heat up rapidly, and ultimately the retention rate of lactoferrin after electric field treatment will decrease significantly (as shown in Table 2). At the same time, not controlling the temperature inside the pulse electric field pipeline by a 4 ℃ water bath will increase the conductivity of the solution inside the pipeline and the current inside the pipeline will increase continuously. When it reaches a certain level, the pulse electric field pipeline may generate electric sparks and arcs, and may even cause the equipment to be forcibly shut down and stop, and cannot work normally. It can be seen that if the temperature inside the pulse electric field pipeline is not controlled by a 4 ℃ water bath, it will cause great damage to the lactoferrin content and lactoferrin activity after pulse electric field treatment, and will also be detrimental to extending the service life of the equipment.

[0253] 2. Enhances the relative iron-binding capacity of lactoferrin.

[0254] The results are as follows Figure 2 , Figure 7, Figure 8 As shown in Table 3:

[0255] Table 3. Secondary structure of lactoferrin under different electric field intensities.

[0256]

[0257] Table 4. Relative iron-binding capacity of lactoferrin under different electric field intensities.

[0258]

[0259] Depend on Figure 2 , Figure 7 , Figure 8 As shown in Table 3, after pulsed electric field treatment, the spatial structure of lactoferrin was appropriately expanded, the α-helix structure increased slightly, the β-sheet structure decreased slightly, and more iron-binding sites were exposed; the activity of lactoferrin, i.e. the iron-binding ability, was enhanced, and the relative iron-binding ability of lactoferrin exceeded 140% when treated with electric field strength of 15-30 kV / cm; while the relative iron-binding ability of the pasteurization group (72 ℃, 15 s) was only 87.7%, indicating that heat treatment not only caused a decrease in the content of lactoferrin, but also caused a certain degree of damage to the structure of lactoferrin, thereby reducing the activity of lactoferrin; (3) When, as shown in Comparative Example 3, the temperature inside the pulsed electric field pipe was not controlled by a 4 ℃ water bath, the temperature of the pulsed electric field pipe could not be controlled below 20 ℃, and the heating of the pipe would cause a large degree of damage to the relative iron-binding ability of lactoferrin, which was only 61.8%.

[0260] Under moderate electric field strength, lactoferrin exposes more iron-binding sites, enhancing its iron-binding reactivity. Further increasing the field strength decreases the relative iron-binding capacity of lactoferrin, but it remains higher than the untreated group, the pasteurized (72 ℃, 15 s) group, and the group without temperature control within the pulsed electric field pipeline at 4 ℃. When the pulsed electric field strength reaches a certain value, the bilobal ring structure of lactoferrin tends to loosen, causing a decrease in its iron-binding capacity. However, overall, pulsed electric field treatment has a positive effect on the iron-binding capacity of lactoferrin.

[0261] 3. Improves the foaming properties and foaming stability of lactoferrin.

[0262] Foaming property refers to the ability of a protein to form foam under stirring or mechanical action, while foam stability refers to the ability of foam to maintain its structure and volume within a certain period of time. Protein molecules are amphiphilic, thus reducing the surface tension of water and exhibiting good foaming property during vigorous stirring. The determination of the foaming property and foam stability of lactoferrin is of great significance to the food industry. These characteristics determine the effectiveness of lactoferrin in food processing, especially in products requiring foam structure (such as ice cream, cakes, and fermented dairy products), providing a scientific basis for food formulation and process optimization.

[0263] The results are as follows Figure 3 As shown:

[0264] (1) By Figure 3 The results showed that the foaming property of lactoferrin initially increased with increasing electric field strength, but gradually decreased after reaching 25 kV / cm. Lactoferrin exhibited the best foaming property at an electric field strength of 25 kV / cm, representing 133.6% of its untreated value. At 45 kV / cm, its foaming property decreased to 92.3% of its untreated value.

[0265] (2) Pulsed electric field treatment has a similar effect on the foaming stability of lactoferrin. The foaming stability is more sensitive to the pulse conditions. The foaming stability is best when the field strength is 20-30 kV / cm. If the electric field strength reaches 45 kV / cm, its foaming stability is 89.4% of that before treatment.

[0266] The improved foaming properties of lactoferrin are mainly the result of protein structure improvement under the action of pulsed electric field. Under the action of electric field, the protein unfolds, which is conducive to the exposure of its internal groups and allows it to fully exert its foaming properties. However, when the electric field is too strong and the structural change is too drastic, the unfolded protein molecules interact to form aggregates, which reduces protein solubility and decreases the foaming properties of lactoferrin.

[0267] 4. Improves the emulsifying properties and emulsion stability of lactoferrin.

[0268] Because lactoferrin possesses an amphiphilic structure (hydrophilic and lipophilic groups), in oil-water mixtures, protein molecules tend to diffuse to the oil-water interface, with the hydrophobic portion facing the lipid phase and the hydrophilic portion facing the aqueous phase. This reduces interfacial tension and promotes film formation, thus exhibiting emulsifying properties. Due to its unique functionalities, such as antibacterial and immunomodulatory effects, lactoferrin is frequently added to various foods to enhance their nutritional value and functionality. Therefore, the determination of lactoferrin's emulsifying properties and emulsifying stability is of great significance to the food industry. Emulsifying stability is an important component of food quality control, helping to ensure that dairy products maintain their sensory and nutritional value during storage and shelf life. By measuring the emulsifying stability of lactoferrin, the stability of dairy products during food processing and storage, as well as the quality of the final product, can be assessed.

[0269] The results are as follows Figure 4 As shown:

[0270] Depend on Figure 4 It was found that lactoferrin exhibited the highest emulsifying property at an electric field strength of 25 kV / cm, reaching 173.3% of its untreated value. At an electric field strength of 45 kV / cm, its emulsifying property decreased to 95.3% of its untreated value. Pulsed electric field treatment had a similar effect on the emulsifying stability of lactoferrin. After pulsed electric field treatment, the spatial structure of lactoferrin expanded, which not only increased the surface area of ​​lactoferrin in contact with the interface but also exposed the internal groups of lactoferrin, enhancing surface hydrophobicity and improving emulsifying performance. However, when the pulsed electric field exceeded a certain threshold, hydrophobic collapse and protein aggregation may occur, reducing the emulsifying performance of lactoferrin. In summary, pulsed electric field treatment has a positive effect on the emulsifying performance of lactoferrin under appropriate conditions. 5. Improving the thermal stability of lactoferrin

[0271] The results are as follows Figure 5 and Figure 6 As shown, after treatment with a pulsed electric field, the thermal stability of lactoferrin improved, with the denaturation temperatures represented by its N-lobes and C-lobes increasing by 1-2 °C each. However, after treatment at 72 °C (15 s), the denaturation temperatures represented by its N-lobes and C-lobes decreased by 0.18 °C and 1.90 °C, respectively. The improved thermal stability under the action of a pulsed electric field may be related to the changes in its secondary structure under the pulsed electric field treatment. The physical modification of the lactoferrin structure under moderate pulsed electric field conditions makes its structure more stable, thus making lactoferrin more heat-resistant. Lactoferrin itself is relatively sensitive to heat. Treatment at 72 °C (15 s) makes the structure of lactoferrin more loose, which has caused some damage to the structure of lactoferrin, thus reducing its thermal stability.

[0272] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a product enriched in highly active lactoferrin, characterized in that, The product includes a beverage, and the method comprises the following steps: (1) raw material treatment Lactoferrin powder is added into fresh milk or sterile water to obtain a lactoferrin-rich solution; (2) degassing treatment The lactoferrin-rich solution obtained in step (1) is subjected to degassing treatment; (3) pulse electric field sterilization treatment: The degassed solution obtained in step (2) is subjected to pulse electric field sterilization treatment, the electric field strength is set to 15-30 kV / cm, the pulse width is set to 1-3 μs, the pulse frequency is set to 50-150 Hz, and the total treatment time is set to 200-400 μs; at the same time, the temperature in the pipeline of the pulse electric field equipment is controlled to be not higher than 20 ℃; thus, a product rich in high-activity lactoferrin is obtained; In step (1), the content of the lactoferrin powder in the fresh milk or sterile water is 0.5-5 %; In step (3), the method for controlling the temperature in the pipeline to be not higher than 20 ℃ is to place the pipeline in a 0-4 ℃ water bath.

2. A method of increasing the content of lactoferrin in a beverage, or increasing the activity of lactoferrin, or increasing the relative iron binding capacity of lactoferrin, or increasing the thermal stability of lactoferrin, characterised in that, The method comprises the following steps: (1) raw material treatment Lactoferrin powder is added into fresh milk or sterile water to obtain a lactoferrin-rich solution; (2) degassing treatment The lactoferrin-rich solution obtained in step (1) is subjected to degassing treatment; (3) pulse electric field sterilization treatment: The degassed solution obtained in step (2) is subjected to pulse electric field sterilization treatment, the electric field strength is set to 15-30 kV / cm, the pulse width is set to 1-3 μs, the pulse frequency is set to 50-150 Hz, and the total treatment time is set to 200-400 μs; at the same time, the temperature in the pipeline of the pulse electric field equipment is controlled to be not higher than 20 ℃; In step (1), the content of the lactoferrin powder in the fresh milk or sterile water is 0.5-5 %; In step (3), the method for controlling the temperature in the pipeline to be not higher than 20 ℃ is to place the pipeline in a 0-4 ℃ water bath.

3. A lactoferrin-rich product rich in high-activity lactoferrin prepared by the preparation method of claim 1.

4. The high activity lactoferrin-enriched product of claim 3, wherein, The lactoferrin-rich product rich in high-activity lactoferrin refers to a lactoferrin product in which the lactoferrin retention rate can reach 90 % or more.

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